s2fieldsofstudy: Biology Medicine Chemistry

text:
Epigenetics and Regulation of Oxidative Stress in Diabetic Retinopathy

Purpose Oxidative stress plays a central role in the development of diabetic retinopathy, and in the pathogenesis of this blinding disease, activation of NADPH oxidase 2 (Nox2)-mediated cytosolic reactive oxygen species (ROS) production precedes mitochondrial damage. The multicomponent cytosolic Nox2 has an obligatory component, Ras-related C3 botulinum toxin substrate 1 (Rac1); in diabetes, Rac1 is functionally and transcriptionally active. Diabetes also facilitates many epigenetic modifications, and activates both DNA methylating (Dnmts) and hydroxymethylating (Tets) enzymes. Our aim was to investigate the role of epigenetics in Rac1 regulation in diabetes. Methods Using human retinal endothelial cells, exposed to high glucose, 5-methyl cytosine (5mC) and 5-hydroxy methyl cytosine (5hmC) levels, and binding of Dnmt and Tets were quantified at the Rac1 promoter. The effect of inhibition of Dnmts/Tets (pharmacological inhibitors or short interfering RNA [siRNA]) on glucose-induced activation of Rac1-ROS production was evaluated. Results were confirmed in retinal microvessels from streptozotocin-induced diabetic mice receiving intravitreally Dnmt1-siRNA. Results Despite high glucose-induced increased binding of Dnmt1, 5mC levels remained subnormal at Rac1 promoter. But, at the same site, 5hmC levels and transcription factor nuclear factor (NF)-kB binding were increased. Inhibition of Dnmts/Tets prevented increase in 5hmC and NF-kB binding, and attenuated Rac1 activation. Similarly, in mouse retinal microvessels, Dnmt1-siRNA ameliorated diabetes-induced increase in Rac1 transcripts and activity, and decreased ROS levels. Conclusions Thus, despite Dnmts activation, concomitant increase in Tets rapidly hydroxymethylates 5mC, allowing NF-κB to bind and activate Rac1. These results imply a critical role of an active DNA methylation in cytosolic ROS regulation in the development of diabetic retinopathy.

D iabetic retinopathy, a slow progressing complication, is the leading cause of blindness in working age adults. The pathology of the disease is complex and involves many interrelated metabolic abnormalities initiated by persistent high circulating glucose. 1 Increase in cytosolic reactive oxygen species (ROS) in the retina and its vasculature is an early event in the development of diabetic retinopathy. 2 In the cytosol, ROS are mainly generated by NADPH oxidases (Nox), and among this family of enzymes, Nox2 is one of the isomers activated in the retina in diabetes. 3 Nox2 is a multicomponent enzyme, and for its stabilization, a small molecular weight GTPase, Rasrelated C3 botulinum toxin substrate 1 (Rac1), 4 plays an important role. Experimental models have shown that the activation of Rac1-Nox2-ROS precedes mitochondrial damage, and the development of diabetic retinopathy. 5 Functional activation of Rac1 requires specific guanine exchange factors, including Tiam1 and Sos1, and in diabetes, Tiam1-mediated activation of Rac1 is implicated in retinal Nox2-ROS production. 2,5 Hyperglycemic milieu also increases Rac1 transcription, and our recent work has shown that hyperglycemia promotes this transcriptional activation by increasing the binding of nuclear transcriptional factor-jB (NF-jB, p65 subunit) at the Rac1 promoter. 6 However, the putative mechanism underlying increased transcription factor binding remains to be explored.
Epigenetic modifications, the modifications that can alter gene expression without affecting the DNA sequence, play an important role in regulating the binding of the transcription factors in many disease conditions. 7 Many epigenetic modifications, including DNA methylation and histone acetylation, are shown to play a role in the development of diabetic complications, including retinopathy. [8][9][10][11][12] Addition of a methyl group to the cytosine by DNA methyl transferases (Dnmts) forms 5-methylcytosine (5mC), and methylated cytosine impedes transcriptional factor binding, resulting in gene repression. However, 5mC can be rapidly oxidized by Ten-Eleven Translocation dioxygenases (Tets), and the resulting formation of 5-hydroxymethylcytosine (5hmC) stimulates gene expres-sion. [13][14][15][16] In diabetes, both Dnmts and Tets are activated in the retina and its vasculature, and among these two families of enzymes, Dnmt1 and Tet2 are the only respective isoforms that are upregulated. 9 How these opposing enzymes regulate DNA methylation status of Rac1 is not clear.
The aim of this study was to investigate the role of epigenetic modifications in the transcriptional regulation of Rac1. Using human retinal endothelial cells (HRECs), we investigated the effect of high glucose on DNA methylation status of the Rac1 promoter. The role of DNA methylation in Rac1 transcriptional activation was further established using pharmacological inhibitors and specific small interfering RNAs (siRNAs) of Dnmt1 and Tet2. Key parameters were confirmed in an in vivo model using retinal microvessels from streptozotocin-induced diabetic mice, receiving intravitreal administration of Dnmt1-siRNA or negative control siRNA.

Mice
Seven-to 8-week-old C57BL/6J mice (either sex) were made diabetic by streptozotocin injection (55 mg/kg body weight for 4 consecutive days). Mice with blood glucose more than 250 mg/dL 2 days after the last injection were considered diabetic. 20 Soon after establishment of diabetes, a group of diabetic mice received intravitreal administration of 2-lg Dnmt1-siRNA (ID: MSS203624; Thermo Fisher Scientific, Waltham, MA, USA); this stealth Dnmt1 siRNA is a combination of sequences targeting three positions at exon 4. Dnmt1-siRNA was suspended in 2-lL nuclease-free water and mixed with Invivofectamine (catalog no. IVF 3001; Invitrogen, Carlsbad, CA, USA). The right eye received Dnmt1-siRNA and the left eye received a medium GC content negative control siRNA (catalog no. 12935-300; Thermo Fisher Scientific). Mice were anesthetized by intraperitoneal injection of ketamine/xylazine (100 mg/kg ketanest and 12 mg/kg xylazine), and using a 32-gauge needle attached to a 5-lL glass syringe (Hamilton), intravitreal injections were performed under a dissecting microscope. Care was taken to position the needle 1 mm posterior to the limbus, and siRNA mixture was slowly injected into the vitreous chamber. 21 Four weeks after administration of siRNA, the mice were killed, and the retina was quickly isolated. Agematched normal mice and diabetic mice, without receiving any siRNA, served as controls. The treatment of animals conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and was approved by the Wayne State University's Institutional Animal Care and Use Committee.
Freshly isolated retina was used to prepare microvessels by incubating it in 5-mL de-ionized water for 60 minutes in a shaking water bath at 378C. The nonvascular tissue was gently removed under the microscope. 6 Quantification of 5mC and 5hmC Genomic DNA was isolated using Qiagen DNA isolation kit (Qiagen, Valencia, CA, USA), and was immunoprecipitated with 5mC or 5hmC antibodies. The levels of 5mC and 5hmC were quantified using methylated or hydroxymethyldated DNA Immunoprecipitation (MeDIP)/(hMeDIP) kits, respectively (Cat. Nos. P-1015 and P-1038, respectively; EPIGENTEK, Farmingdale, NY, USA). The enrichment of 5mC or 5hmC at the Rac1 promoter was analyzed by qRT-PCR using specific primers. 9 Chromatin Immunoprecipitation (ChIP) The binding of DNA modifying enzymes, or of the transcription factor NF-jB (p65 subunit), at the Rac1 promoter was determined by ChIP assay using cross-linked samples sonicated in ChIP lysis buffer. Protein-DNA complex (100 lg) was immunoprecipitated with Dnmt1, Tet2, or p65 antibody (Cat. Nos. ab13537, ab135087, and ab7970, respectively; Abcam, Cambridge, MA, USA). Each experiment included antibody control, where the samples were immunoprecipitated with IgG (catalog no. ab171870, Abcam). The immunoprecipitated complex was captured using Protein A Agarose/Salmon Sperm DNA (catalog no. 16157; EMD Millipore, Temecula, CA, USA), washed and de-crosslinked at 658C for 6 hours, followed by DNA isolation with phenol:choloroform:isoamylalcohol, using the methods reported previously. 8 Relative binding of Dnmt1/Tet2/p65 at the promoter was quantified by qRT-PCR using Rac1 promoter specific primer (Table). The target values were normalized to the input controls respectively to obtain fold change.

Gene Transcripts
Gene expression was quantified using gene/species-specific primers by qRT-PCR (Table), and the specific products were confirmed by SYBR green single melt curve analysis. The results were normalized to the expression of the housekeeping gene b-actin (human) or 18S (mice), and the relative fold change was calculated using delta delta Ct method. 8

Rac1 Activation
Active Rac1 was measured by G-LISA colorimetric assay kit (catalog no. BK-128; Cytoskeleton, Denver, CO, USA) in 20-to 25-lg protein, as described previously. 2 Fold change was calculated considering the values obtained from cells in normal glucose as one.

Reactive Oxygen Species
Total ROS levels were quantified using 2 0 ,7 0 -dichlorofluorescein diacetate (DCFH-DA; catalog no. D6883; Sigma-Aldrich Corp.). Briefly, 5-lg protein was incubated with 4-lM DCFH-DA for 20 minutes, and the fluorescence was measured at 485nm excitation wavelength and 535-nm emission wavelengths. Fold change was calculated as percent change with respect to values obtained from cells in normal glucose. 22

Statistical Analysis
Data are presented as mean 6 SD. Comparison between groups was made using 1-way ANOVA followed by Dunn's ttest; P < 0.05 was considered significant.

RESULTS
Rac1 is activated in hyperglycemic milieu in the retina and its vasculature and its gene transcripts are increased 2,22 ; to investigate the role of DNA methylation in Rac1 transcriptional regulation, 5mC levels at the Rac1 promoter were quantified in the cells exposed to high glucose for 96 hours, a duration when cell apoptosis is detected. 23 Figure 1a shows that high glucose decreased 5mC levels by 50%. Because 5mC is associated with transcriptional repression, 24 and hyperglycemic milieu activates both Dnmts and Tets, 9 the effect of high glucose on hydroxymethylation of 5mC was investigated; compared with cells in normal glucose, cells incubated in high glucose had 2-fold higher 5hmC levels at Rac1 promoter (Fig.  1b). To investigate the mechanism responsible for decrease in 5mC at Rac1 promoter, despite activation of Dnmts, in hyperglycemic milieu, 9 the binding of Dnmt1 was investigated. Compared with the cells in normal glucose, high glucose exposure for 96 hours increased Dnmt1 binding by more than 1.5-fold, and in the same samples, Tet2 binding at Rac1 promoter was also elevated by more than 2-fold (Figs. 2a, 2b).
To confirm the role of active DNA methylation-hydroxymethylation in the regulation of Rac1 promoter methylation status, the effect of inhibition of Dnmt by its chemical (5-Aza) or molecular (Dnmt1-siRNA) inhibitors was analyzed; both 5-Aza and Dnmt1-siRNA prevented glucose-induced increase in Dnmt1 binding at Rac1 promoter (Fig. 2a). However, these inhibitors had no effect on Dnmt1 binding in the normal glucose conditions. To further understand the role of 5mC hydroxymethylation in Rac1 transcriptional regulation, the effect of Tet regulation on its binding at Rac1 promoter was investigated. Figure 2b shows that Tet2 inhibition either by 2-HG or Tet2-siRNA significantly attenuated glucose-induced increase in Tet2 binding. In the same cell preparations, inhibition of Dnmts or Tets also ameliorated glucose-induced increase in 5hmC levels at Rac1 promoter (Fig. 1b). Untransfected cells or scrambled RNA transfected cells, incubated in high glucose for 96 hours, had similar values, and also values from cells in 20-mM L-glucose or in normal glucose were not different from each other, confirming the use of 20-mM L-glucose as a valid osmotic/metabolic control.
Inhibition of DNA methylation-hydroxymethylation machinery also attenuated glucose-induced increase in p65 binding at Rac1 promoter, and the values obtained from cells in high glucose with Dnmt or Tet inhibitors were not significantly different from the cells in normal glucose (Fig. 3a). Consistent with the p65 binding, these inhibitors ameliorated glucoseinduced increase in Rac1 gene transcripts and activity, and ROS levels (Figs. 3b-d). The role of NF-jB in Rac1 transcriptional activation was confirmed by inhibiting glucose-induced NF-jB activation by SN50, and as shown in Figure 3b, SN50 ameliorated glucose-induced increase in Rac1 transcription.
Rac1-Nox2-ROS signaling is one of the early events in the pathogenesis of diabetic retinopathy; in retinal endothelial cells it can be seen within 6 hours of high glucose insult. 2 To investigate if DNA-methylation-hydroxymethylation is playing any role in the early activation of Rac1, DNA methylation status of Rac1 promoter was analyzed in HRECs exposed to high glucose for 6 to 48 hours. Compared with cells in normal glucose, while at 6 hours of high glucose both 5mC levels and Dnmt1 binding were decreased, at 24 hours and beyond, despite increased Dnmt1 binding, 5mC levels remained subnormal (Fig. 4a). Consistent with this, although Dnmt1 GCCCTGTAATTGGAATGAGTCCACTT CTCCCCAAGATCCAACTACGAGCTTT transcripts were elevated by approximately 2-fold at 24 hours (and beyond) of exposure of high glucose, at 6 hours Dnmt1 expression was significantly decreased (Fig. 4b). In the same samples, 5hmC levels at Rac1 promoter were, however, not affected, but within 24 hours, >50% increase in 5hmC was observed. In accordance, 6 hours of high glucose also failed to affect Tet2 binding at Rac1 promoter, and the gene transcripts of Tet2 were also not different from those obtained from cells in normal glucose. But, at 24 hours (and beyond) of high glucose incubation, 5hmC levels, Tet2 binding, and its gene transcripts were significantly elevated, and they remained elevated at 48 to 96 hours of high glucose exposure (Figs. 4c,  4d). Cells in high glucose for 6 hours and beyond, as expected, 6,22 had an approximately 2-fold increase in Rac1 gene transcripts (Fig. 4e). Values obtained from cells in 20-mM L-glucose were not different from those obtained from cells in 5-mM glucose.
To confirm the key parameters in an in vivo model, DNA methylation status of the Rac1 promoter was investigated in retinal microvessels obtained from diabetic mice. Compared with normal mice, although diabetic mice had decreased 5mC levels at the Rac1 promoter, 5hmC levels were significantly elevated (Figs. 5a, 5b). The role of DNA methylation in Rac1 transcriptional regulation was further confirmed in the mice receiving intravitreal administration of Dnmt1-siRNA. Although due to some technical issues, Dnmt1 silencing could not be confirmed in the target cells, and off-target effects of the Dnmt1-siRNA could not be investigated, administration of siRNA ameliorated diabetes-induced increase in gene transcripts and activity of Rac1 and ROS levels; the values obtained from the retinal microvessels prepared from the diabetic mouse eye receiving Dnmt1-siRNA were significantly different from those obtained from the eye receiving siRNA negative control (Figs. 5c, 5d).

DISCUSSION
Diabetic retinopathy has a complex underlying pathophysiology, and in spite of cutting edge research in the field, molecular events contributing to its development remain unclear. Among many abnormalities implicated in the development of diabetic retinopathy, experimental models have documented oxidative stress to be at a central place. 1 Although mitochondria are the major source of ROS, ROS are also produced in the cytosol by Nox enzymes, 25 and our previous study has shown that in the pathogenesis of diabetic retinopathy, activation of cytosolic Nox2 precedes mitochondrial damage. 2,5 Rac1, an obligatory component of the multicomponent Nox2, is activated in hyperglycemic milieu in the retina and its vasculature, and its gene transcripts are elevated. 2,6,22 The data provided in the current report suggest the role of epigenetic modifications in Rac1 transcriptional regulation. DNA at the Rac1 promoter undergoes active methylation-hydroxymethylation; in the initial stages of hyperglycemic insult (6 hours of high glucose), due to inhibition of Dnmts, the promoter is hypomethylated. However, at 24 hours, although the binding of Dnmt1 is increased, concomitant activation of Tets hydroxymethylates 5mC to 5hmC, resulting in Rac1 activation. Role of active DNA methylation-hydroxymethylation in regulation of Rac1 transcription is further confirmed by our results showing increased 5hmC levels at the Rac1 promoter in the retinal microvessels from mice diabetic for 2 weeks, and amelioration of diabetes-induced activation of Rac1 (transcription and activity) by Dnmt1-siRNA. These results have significant implications for diabetic retinopathy because, although the appearance of retinal histopathology, characteristic of diabetic retinopathy, in rodent models is visible only after 6 to 8 months of diabetes, Rac1-Nox2-ROS signaling is an early event, which can be seen within 2 weeks after induction of diabetes.
Addition of a methyl group to carbon-5 of a cytosine base, forming 5mC, is considered as a fundamental epigenetic mediator, and is an important component in many cellular processes, including genomic imprinting and chromosome stability. 26 DNA methylation is mainly associated with repression of gene expression by changing the chromatin structure and restricting access of the transcription factors to the gene promoter. 24 However, errors in DNA methylation are observed with many genes in chronic diseases including cancer and diabetes. 9,27-29 Human Rac1 promoter has four CpG islands within À1200 bases upstream of transcription start site, and the binding site for the transcription factors lies in the CpG island close to the transcription start site between À317 to 147 bp. 30 Our previous work has shown that in diabetes, the binding of transcriptional factor NF-jB at Rac1 promoter is increased in the retinal vasculature, 6 and here we show that at a duration of high glucose insult when mitochondrial damage and apoptosis are also observed (96 hours), 5mC levels are also decreased. As mentioned above, Dnmts catalyze the transfer of a methylation residue from Sadenosyl-L-methionine to cytosine, and among these, Dnmt3a and Dnmt3b are the de novo methyltransferases, Dnmt1 is considered as the maintenance methyltransferase and recognizes hemimethylated sites restoring symmetric methylation during replication. 31 In diabetes, Dnmts are activated, and among this family of enzymes, only Dnmt1 is upregulated in the retina and its vasculature. 9 At 96 hours, despite decrease in 5mC levels, binding of Dnmt1 at Rac1 promoter is increased, suggesting that the 5mC formed by Dnmt1 could be demethylated. The results from the in vivo model further FIGURE 5. Effect of diabetes on Rac1 promoter DNA methylation. Retinal microvessels from diabetic mice were analyzed for (a) 5mC and (b) 5hmC levels using IgG as an antibody control. Retinal microvessels from the eye of diabetic mice receiving intravitreal administration of Dnmt1-siRNA or negative control siRNA were analyzed for (c) Rac1 gene transcripts and activation, and (d) ROS levels. Values are represented as mean 6 SD from five to six mice in each group. Norm and Diab indicate normal and diabetic mice, respectively; D-si and NC indicate retinal microvessels from the eye of diabetic mice receiving Dnmt1-siRNA or negative control siRNA, respectively. *P < 0.05 compared with normal; #P < 0.05 compared with diabetes or NC. support the role of active DNA methylation in Rac1 activation, as administration of Dnmt1-siRNA ameliorates both Rac1 transcription and activation, and decreases ROS levels, seen in the retinal microvasculature of diabetic rodents.
DNA is a dynamic structure, and the methylated cytosine can be rapidly demethylated. 13 Demethylation of cytosine is considered necessary for epigenetic reprogramming of genes, and is directly involved in many diseases including tumor progression. 14 Demethylation of DNA can either be passive or active, or a combination of both, and the enzymebased oxidation of 5mC to 5hmC promotes DNA demethylation by binding to CpG-rich regions to prevent unwanted Dnmt activity. 13 Retinal Tets are activated in diabetes, and among this family of enzymes, Tet2 is the only member with upregulated gene transcripts. 9 The results presented here clearly show that, although Dnmt1 binding at Rac1 promoter is increased in diabetes, due to increased Tet2 binding, 5hmC levels are elevated, suggesting a rapid hydroxymethylation of 5mC formed by Dnmt1. This increase in 5hmC allows binding of the transcription factor, increasing Rac1 expression. Further, we show that inhibition of Tets, in addition to regulating DNA methylation status of Rac1 promoter, also inhibits NF-jB binding and Rac1 transcription, further confirming the role of active DNA methylation in Rac1 activation. In support, diabetes modulates transcription of matrix metalloproteinase MMP-9, an enzyme implicated in mitochondrial damage, via dynamic DNA methylation-hydroxymethylation of its promoter. 9 Moreover, hyperglycemia has a direct impact on changes in the epigenome, and high DNA methylation and hydroxymethylation levels are observed in poorly controlled diabetic patients compared with healthy individuals. 32 We recognize that Tets can further oxidize 5hmC to 5-formylcytosine, and 5-formylcytosine to 5-carboxylcytosine 33 ; their role in regulation of Rac1 transcription cannot be ruled out. Although methylated CpGs are considered to influence transcription factor binding at their recognition site, and DNA methylation at promoter generally locks genes in a stable silent state, many transcription factor recognition sites do not possess CpGs in their recognition sequence, 10 and their role in maintaining Rac1 gene transcription remains to be examined. However, amelioration of diabetes-induced increased NF-jB binding at the Rac1 promoter by both Dnmt and Tet inhibitors, observed in the present study, clearly suggests that dynamic CpG methylation is playing a critical role in the regulation of binding of this transcription factor.
Diabetic retinopathy is a progressive disease, and activation of Rac1-Nox2 is one of the early events in its pathogenesis. Rac1-Nox2-mediated cytosolic ROS play a major role in damaging the mitochondria, leading to the development of histopathology characteristic of diabetic retinopathy. 2 DNA methylation varies in a global and local context, and active changes in DNA methylation are observed during aging. [34][35][36][37] Site-specific hypermethylation are observed predominantly in promoter regions, 38,39 and methylation marks are also considered to predict the age of the organism. 37,40 Our data show that in the early stages of diabetic retinopathy (6 hours of high glucose exposure), although Tet2 is not affected, Dnmt1 is repressed, and promoter DNA is hypomethylated, resulting in Rac1 transcriptional activation. However, as the disease progresses, the two enzymes with opposing functions, Dnmts and Tets, are both activated. Although there could be increase in 5mC at the promoter, activated Tets overpower Dnmts activation, and hydroxymethylate 5mC to 5hmC, transcriptionally activating Rac1.
There are some limitations of our study, including the following: Retinal endothelial cells are not the only cells that are lost in the early stages of diabetic retinopathy; instead, pericytes of the vasculature also undergo accelerated apoptosis before any pathology, characteristic of diabetic retinopathy, can be observed, and in diabetes, photoreceptors are also a major source of ROS. [41][42][43] The possibility of a similar mechanism(s) operating in retinal pericytes, and in other nonvascular cells, cannot be ruled out. Diabetic retinopathy is a slow progressing disease, mitochondrial damage and capillary cell apoptosis precedes the development of microvascular histopathology, and the histopathology not visible until 6 to 8 months of diabetes in rodents, 23,42,44,45 raising a possibility that the in vitro model might not fully reproduce the in vivo model. However, most of the mechanism-based data are comparable between the in vitro and in vivo models. 2,5,9,17 As mentioned above, high glucose-induced increase in Rac1-ROS can be seen within 6 hours in retinal endothelial cells and within 15 days of diabetes in rodents, and increase in mitochondrial damage and capillary cell apoptosis within 96 hours in cells and approximately 6 months of diabetes in mice. However, our retinal microvessel Rac1-ROS data, presented here, are from diabetic mice receiving Dnmt1-siRNA for only 4 weeks. In support of a possible link between early changes in DNA methylation and the onset of diabetic retinopathy, our previous work has shown that inhibition of the enzymes responsible for maintaining DNA methylation ameliorates mitochondrial damage and capillary cell apoptosis, the events that precede retinal microvascular pathology in the in vivo models of diabetic retinopathy. 9,46 Because epigenetic modifications are influenced by external factors, and in vitro culture of endothelial cells may introduce additional epigenetic modifications, this may interfere with the true recapitulation of the disease state. To alleviate the effect of cell passaging, the present study compared Rac1 promoter DNA methylation in high glucose and normal glucose conditions using HRECs from the same passage (and the same batch). Moreover, experiments in in vitro models for many chronic diseases. including diabetic retinopathy, using appropriate controls, have provided consistent results as seen in the in vivo disease models. 8,10,47,48 In summary, despite the limitations stated above, our convincing data clearly demonstrate a role of active DNA methylation-demethylation in the regulation of Rac1 in the development of diabetic retinopathy. Although DNA methylation is an epigenetic repressive mark, hydroxymethylation of 5mC is also now considered as an independent epigenetic marker; imbalance in genomic 5mC/5hmC levels are associated with aberrant gene activation and oncogenic transformation. 49,50 Thus, despite increased 5mC formed at the Rac1 promoter due to activation of Dnmt1, concomitant increase in Tet2 hydroxymethylate it to 5hmC. This allows the transcription factor to bind, and activate Rac1 transcription. Rac1, via Nox2, elevates cytosolic ROS levels, and ROS damage mitochondria and increase capillary cell apoptosis, which, ultimately, results in diabetic retinopathy (Fig. 6). These data clearly imply a critical role of DNA methylation in diabetic retinopathy, and demonstrate the importance of DNA methylation/hydroxymethylation machinery in regulation of Rac1mediated oxidative stress. This opens up the possibility of using this machinery as a target to regulate ROS production during early stages of the disease, which could help diabetic patients from facing the devastating consequences of this progressing disease, and protect them from losing their vision.


extfieldsofstudy: Chemistry Medicine

text:
Evaluation of In Vitro Antioxidant and Anticancer Properties of the Aqueous Extract from the Stem Bark of Stryphnodendron adstringens

Stryphnodendron adstringens (Mart.) Coville (Fabaceae) is a tree species native to the Brazilian Cerrado commonly known as barbatimão. In traditional medicine, decoctions or infusions of the stem bark of this plant are used in the treatment of several diseases. The objective of this study was to analyze the chemical composition of Stryphnodendron adstringens aqueous extracts (SAAE) prepared from the stem bark to assess their antioxidant activity and anticancer effects as well as characterize cell death mechanisms against murine B16F10Nex-2 melanoma cells. From the SAAE, gallic acid, gallocatechin, epigallocatechin, dimeric and trimeric proanthocyanidins mainly composed of prodelphinidin units and the isomeric chromones C-hexosyl- and O-pentosyl-5,7-dihydroxychromone were identified. The SAAE showed antioxidant activity through direct free-radical scavenging as well as through oxidative hemolysis and lipid peroxidation inhibition in human erythrocytes. Furthermore, SAAE promoted apoptosis-induced cell death in melanoma cells by increasing intracellular reactive oxygen species (ROS) levels, inducing mitochondrial membrane potential dysfunction and activating caspase-3. Together, these data show the antioxidant and anticancer effects of Stryphnodendron adstringens. These results open new perspectives for studies against other tumor cell lines and in vivo models as well as for the identification and isolation of the chemical constituents responsible for these effects.

Introduction
Brazilian biodiversity has numerous plant species with bioactive compounds among their constituents with potential for the development of new drugs. Among them, Stryphnodendron adstringens (Mart.) Coville (Fabaceae) is a tree species native to the Brazilian Cerrado, commonly

Identification of the Constituents from the SAAE by LC-DAD-MS/MS
The chemical constituents from the Stryphnodendron adstringens aqueous extracts (SAAE) were identified based on UV, accurate MS and MS/MS data compared to spectral data reported in the literature, and some compounds could be confirmed by analyses of authentic standards. All identified compounds and spectral data are summarized on Table 1.  The chromatographic peaks ( Figure 1) 1-9 and 12-13 showed an intense band at ≈280 nm (λ max ) in the UV spectrum, which are assignable to gallic acid and flavan-3-ol units, including the proanthocyanidins (condensed tannins) ( Figure 2). The compounds 2, 6 and 8 exhibited deprotonated ions at m/z 169.0140, 305.0673 and 305.0673, respectively, which are compatible with the molecular formulas C 7 H 6 O 5 and C 15 H 14 O 7 , and these compounds were confirmed to be gallic acid, gallocatechin and epigallocatechin by the injection of standards. Their fragmentation profiles were compatible with published data [27], and they had already been reported from S. adstringens [8,28].   Thus, the dimers PDE-PDE, PCY-PDE and PRO-PDE could be  identified in the extract, and their spectral data are compatible with the published data [27]. In the same way, the trimer of prodelphinidin (1) and the hydroxy-benzoyl dimer of prodelphinidin, which already were isolated from S. adstringens [8], were identified. Compounds 10 and 11 showed similar UV spectra with bands at wavelengths of approximately 257, 290 and 325 nm (shoulder), which are compatible with chromone compounds [29] (90 u) and water molecules (18 u). The losses of 120 and 90 u are produced from the fragmentation of the sugar ring and suggest a C-hexoside group. All of these losses were confirmed by the calculation of the accurate masses of product ions. Thus, substances 10 and 11 were putatively identified as C-hexosyland O-pentosyl-5,7-dihydroxychromone, and their spectral data are similar to their reported data [30,31].

Chemical Composition
The concentration of total phenols was 195.16 ± 0.94 mg GAE/g SAAE, and the concentration of flavonoids was 2.87 ± 0.08 mg QE/g SAAE.

ABTS and DPPH Free-Radical Scavenging
The 50% inhibitory concentration (IC 50 ) of 2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free-radicals as well as the maximum activity of the SAAE are outlined in Table 2. The SAAE had IC 50 results similar to those of the antioxidant control, ascorbic acid, in both assays. Conversely, the necessary concentrations of SAAE to reach the maximum free-radical inhibition were two and five times higher than those of ascorbic acid in the ABTS and DPPH assays, respectively. Results are expressed as the mean ± SEM (n = 3).

Protective Effect of the SAAE Against Oxidative Hemolysis
Throughout the experimental period, the SAAE showed no hemolytic activity in human erythrocytes at any time and concentration tested ( Figure 3A-D). When in contact with the oxidizing agent 2,2 -Azobis (2-methylpropionamidine) dihydrochloride (AAPH), all of the SAAE concentrations tested were able to protect the erythrocytes against hemolysis during the 240 min incubation in a concentration-and time-dependent manner ( Figure 4A-D). This result was better than that of the antioxidant control, ascorbic acid, which lost its anti-hemolytic activity at concentrations of 50 and 75 µg/mL after 240 min of incubation ( Figure 4D).

Malondialdehyde (MDA) Dosage
The ability of the SAAE to protect erythrocytes incubated with the oxidizing agent AAPH against lipid peroxidation was assessed by MDA quantification. All of the SAAE concentrations were able to reduce the MDA levels after 240 min of incubation. Erythrocyte treatment with the SAAE at concentrations of 50 and 75 µg/mL reduced the MDA levels by 59.9% and 62.0%, respectively, compared with the control AAPH. These results were better than those assessed with the control ascorbic acid, which showed similar reductions (52.8% and 62.3%) only at concentrations of 100 and 125 µg/mL, respectively ( Figure 5). The higher doses SAAE were not so effective and a pro-oxidant effect probably occurs.

Cell Cycle Phases
The histogram shows the distribution of the cell cycle phases of control (untreated) and treated (65 µg/mL SAAE for 24 h) B16F10Nex-2 cells ( Figure 8A). No significant differences were observed between cells from the control and SAAE treatment group, thus showing that treatment with the SAAE had no effect on the distribution of cell cycle phases of this cell line ( Figure 8B

Reactive Oxygen Species (ROS) Levels
Cells treated with the SAAE (65 µg/mL) had increased ROS levels, as shown by the right-shifted fluorescence levels in the histogram ( Figure 9A). The ROS levels increased by approximately 75% in B16F10Nex-2 cells treated with the SAAE in comparison to untreated cells ( Figure 9B).

Mitochondrial Membrane Potential
B16F10Nex-2 cells treated with the SAAE showed decreased mitochondrial membrane potential after 24 h of treatment ( Figure 10A). The mitochondrial membrane potentials of cells treated with the positive control CCCP (30 µM) and the SAAE (65 µg/mL) decreased by 63.1 ± 2.4% and 38.7 ± 2.9%, respectively, in comparison with the untreated control cells ( Figure 10B).

Caspase-3 Activation
The assessment of the effects of the SAAE on caspase-3 in B16F10Nex-2 cells showed a right shift in the fluorescence levels in the histogram ( Figure 11A), with an approximately 40% increase in the cleaved caspase-3 labeling intensity compared with untreated control cells ( Figure 11B).

Discussion
Medicinal plants have been used for centuries by humanity in the prevention and treatment of various diseases. However, the chemical constituents and biological properties of a wide variety of endemic plants in poorly studied biomes remain unidentified and uncharacterized.
In this context, Brazil has been highlighted for its rich plant diversity with the potential for the development of new antioxidant and antitumor drugs, which are important for preventing oxidative stress-related diseases and different types of cancer [13].
Stryphnodendron adstringens is one of the species found in the Brazilian Cerrado that shows potential for the development of new drugs considering its chemical composition and biological activities previously described in the literature [3,5,6]. Previous phytochemical studies show that S. adstringens stem bark has proanthocyanidins [7,32], chalcones and triterpene compounds [4]. Those compounds are described for their antioxidant and anticancer activities [33][34][35][36].
In recent years, natural antioxidants have gained importance for their prophylactic and therapeutic potential for many diseases as an effective tool in scavenging reactive species responsible for inducing oxidative stress [14,17,38]. The SAAE prepared from stem bark showed antioxidant activity by scavenging ABTS and DPPH free-radicals. This property is related to the concentration of phenolic compounds present in the SAAE, as these compounds are considered excellent natural antioxidants. These compounds are mainly described for their redox activity, acting as reducing agents, hydrogen donors and singlet oxygen scavengers [39], thereby preventing several diseases associated with oxidative stress [40]. Among the phenolic compounds identified in the SAAE, tannins are described as key antioxidant agents [41]. Furthermore, Luiz et al. [7] identified proanthocyanidins in S. adstringens stem bark, which are a class of polyphenols also known as condensed tannins. Dimeric procyanidins are the most active compounds of the proanthocyanidin class in scavenging free-radicals because they have a high molecular weight and a high degree of hydroxylation in the aromatic ring [41].
In addition to the direct effect on free-radical scavenging, the SAAE also protected human erythrocytes against damage caused by the antioxidant agent AAPH, resulting in oxidative hemolysis inhibition and reduced MDA levels. Antioxidant activity assessments in cellular models are highly important for studying the mechanisms of action of different compounds, including natural products. The antioxidant property observed in erythrocytes was not due to gene expression regulation because this cell model is anucleate, although it may occur through permeation of the compounds through the cellular membrane and interaction with endogenous antioxidant systems [42].
The ROS act as inducers of cell membrane lipid oxidation, a metabolic process that releases several by-products, such as malondialdehyde, which promotes DNA damage and may significantly contribute to cancer development [43].
The antioxidant capacity observed in erythrocytes may be related to the chemical compounds identified in the SAAE. Among the compounds identified, gallic acid is a well-described phenolic compound for its antioxidant and anti-hemolytic activities in human erythrocytes [14,44]. Procyanidins are also excellent antioxidants [45] capable of protecting erythrocytes from oxidative hemolysis and AAPH-induced lipid peroxidation [46,47]. Furthermore, flavonoids known as catechins are among the most abundant and important chemical constituents of green tea [48,49] and other plant species [13], and they are described as lipid peroxidation inhibitors [50].
Several studies have shown that extracts from natural products with high concentrations of phenolic compounds inhibit lipid peroxidation in human erythrocytes, thus decreasing the production of malondialdehyde [14,15] and showing cytotoxic effects on tumor cells [12,16,51].
The growing search for natural products with antioxidant properties and selective cytotoxic effects on tumor cells is among the strategies for discovering new anti-cancer drugs. Accordingly, this study aimed to investigate the cytotoxic activity of the SAAE against the B16F10Nex-2 melanoma line and to identify its mechanisms of action. The SAAE showed cytotoxic effects on melanoma cells, inducing cell death by apoptosis. Those effects may be attributed to the phenolic composition of the SAAE because these compounds have already been described for their cytotoxic effects on other tumor cell lines [52][53][54]. In vitro models are readily available and economically practical. In addition, they have been widely applied, including for studies related to discovery, therapeutic efficacy, and identification of molecular mechanism, optimizing the obtention of results. Despite the beneficial use of B16 lineage, melanoma cells isolated from human donors also are important to reveal the real clinical relevance of antitumor compounds [54].
Melanoma is among the cancers described for its chemoresistance to antitumor agents [55,56]. The search for derivatives of natural products has identified compounds with promising results in the treatment of this type of cancer. Among them, the chloroform fraction of Anthelminticum centratherum fruit seeds [57] and lectins extracted from Polygonatum curtonema rhizomes [58] showed cytotoxic effects via apoptosis in A375 melanoma cells resulting from increased ROS production, decreased mitochondrial membrane potential, cytochrome C release and caspase activation.
Apoptosis is a programed cell death process essential for maintaining homeostasis; however, this is the mechanism of action of some antitumor drugs, such as doxorubicin [59], cisplatin [60] and vinblastine [61]. This mechanism is characterized by a series of biochemical and morphological changes, including an increase in ROS levels [62], caspase activation [63], cell shrinkage, chromatin condensation, DNA fragmentation and the formation of apoptotic bodies [64]. ROS are chemically reactive molecules produced in the mitochondria that present essential functions in cellular respiration, acting in the maintenance of homeostasis in normal cells. In tumor cells, the ROS production imbalance may be irreversible, making the cell vulnerable to increased oxidative stress by promoting cellular apoptosis [65]. Agents such as procarbazine [66] and various phytochemical compounds have shown cytotoxic effects in tumor cells by increasing the intracellular ROS production [57,58,67]. The increase in the ROS promotes mitochondrial dysfunction, resulting in the loss of the mitochondrial membrane potential [68]. In this study, the SAAE increased the production of ROS, which may have triggered the dysfunction in mitochondrial membrane potential and apoptotic cell death observed in melanoma cells. Singh et al. [69] showed that proanthocyanidins extracted from cranberries have a cytotoxic effect on neuroblastoma cells by increasing ROS production and depolarizing the mitochondrial membrane potential. Furthermore, other extracts and natural compounds have already been described as promising in cancer treatment because they increase ROS and depolarize the mitochondrial membrane potential [70][71][72][73]. Excessive ROS production contributes to apoptotic cell death by releasing pro-apoptotic factors, including caspases [74]. The SAAE increased the caspase-3 activity, thus highlighting that the activation of this enzyme is directly involved in the apoptosis of B16F10Nex-2 melanoma cells.
Caspases are a family of proteases considered to be central regulators of apoptosis [75]. They act as a key apoptotic-signaling factor, triggered by the expression of proteins of the Bcl-2 family and by cytochrome C release from the internal mitochondrial membrane into the cytosol [76]. Caspases can be activated through two pathways, the extrinsic and intrinsic pathways [77]. The extrinsic pathway begins after extracellular signaling with death receptors [78], whereas the intrinsic pathway is initiated by intracellular stimuli that trigger mitochondrial membrane permeabilization, cytochrome C release into the cytosol, and subsequent caspase-9 activation, which activates caspase-3, the key enzyme for apoptosis [77,79,80].
Among the compounds identified, gallic acid shows cytotoxic effects against various tumor cell lines, including basal-like breast [81], non-small cell lung [82], oral squamous cell carcinoma [83] and hepatocellular cancer cell lines, showing no cytotoxic effects on normal cells [84]. In murine B16F10 melanoma cells, this compound is described for inducing apoptosis through the mitochondrial pathways by promoting the overexpression of the enzymes caspase-3, caspase-9 and PARP-1 as well as the pro-apoptotic proteins Bax and Bad; it also promotes the under-expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL [85]. In human melanoma cells (A375.S2), in addition to the involvement of caspases and pro-and anti-apoptotic proteins, an increase in ROS production and a decrease in mitochondrial membrane potential are observed [86]. However, Badhani et al. [87] show that the anticancer potential of gallic acid is not related to its antioxidant activity but instead to its pro-oxidant activity.
Prodelphinidins, whose subunits contain gallocatechin and epigallocatechin, inhibit cell proliferation and induce apoptosis in cancer cells by caspase-dependent extrinsic and intrinsic apoptotic pathways [88] as well as cell cycle arrest [89]. Procyanidins, the most abundant class of proanthocyanidins found in plants [88], show no cytotoxic effects on normal cells [90], although they are able to inhibit cell proliferation and induce apoptotic death in cancer cells by increasing the ROS levels [91], decreasing the mitochondrial membrane potential and activating caspase-3 [92]. Chromones and their derivatives are heterocyclic compounds distributed throughout the plant kingdom [93] known for presenting various pharmacological activities, including antioxidant and anticancer activities, especially against multiple drug-resistant tumor cell lines [94,95].

Aqueous Extract Preparation
Approximately 300 g of fresh bark was subjected to maceration in distilled water (2 L) for 48 h at room temperature. The macerate was filtered through Whatman No. 1 filter paper, frozen at −20 • C and subsequently lyophilized to prepare the Stryphnodendron adstringens aqueous extract (SAAE). The dry extract yield was 2.3%, calculated using the following formula: extraction yield (%) = (W DE /W FB ) × 100, where W DE is the dry extract weight (g) and W FB is the fresh bark weight (g). The SAAE was stored at −20 • C and protected from light.

Identification of Constituents by LC-DAD-MS
A chromatographic system UFLC Shimadzu Prominence coupled to a diode array detector (DAD) and a mass spectrometer was used to analyze and identify the compounds from SAAE extract. The mass spectrometer was a MicrOTOF-Q III (Bruker Daltonics, Billerica, MA, USA), composed by an electrospray ionization source and quadrupole time-of-flight analyzers. The MS analyses were performed on negative and positive ion mode. The applied chromatographic and mass spectrometric parameters were the same described by Nocchi et al. [27]. The SAAE extract was solubilized in methanol and ultrapure water (6:4, v/v) at 1 mg/mL, filtered (PTFE membrane, 0.22 µm, Millex ® , Millipore Corporation, Bedford, MA, USA) and 2 µL was injected on the chromatographic column.

Phenolic Compounds
To determine the total phenolic content present in the SAAE, the Folin-Ciocalteu colorimetric method was performed [96]. Thus, 2.5 mL of Folin-Ciocalteu reagent (1:10 v/v, diluted in distilled water) were added to 0.5 mL SAAE (at concentration 200 µg/mL). This solution was incubated in the dark for 5 min. Subsequently, 2.0 mL of 14% aqueous sodium carbonate (Na 2 CO 3 ) were added and incubated at room temperature for 120 min and protected of light. The absorbance was measured at 760 nm using a T70 UV/Vis spectrophotometer (PG Instruments Limited, Leicestershire, UK). To construct a calibration curve, gallic acid (0.04-200 µg/mL) was used as standard. The concentration of phenolic compounds present in SAAE was expressed in mg equivalent to gallic acid (GAE)/g of SAAE. The assay was performed in triplicate.

Total Flavonoids
Total flavonoid content in SAAE was determined as described by Liberio et al. [97]. A methanolic solution of 2% aluminum chloride hexahydrate (AlCl 3 ·6H 2 O) (4.5 mL) was added to 0.5 mL of SAAE (at concentration 200 µg/mL) and this solution was kept in the dark for 30 min at room temperature. Subsequently, the absorbances were measured at 415 nm (T70 UV/Vis spectrometer, PG Instruments Limited, Leicestershire, UK) and the flavonoid quercetin (00.4-200 µg/mL) was used to construct a calibration curve. The total flavonoid content was expressed in mg equivalent of quercetin (QE)/g extract. The assay was performed in triplicate.

Antioxidant Activity
The antioxidant activities were determined by different methods and the doses were defined by previously assays performed, as well as based on data reported from species of Cerrado Biome [12,[14][15][16].

ABTS •+ Radical Discoloration Assay
The discoloration test of 2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS + ) radical was performed as described by Re et al. [98]. The ABTS •+ radical was prepared with 5 mL of ABTS •+ aqueous solution (7 mM) and 88 µL of potassium persulfate solution (140 mM). The solution was incubated for 12-16 h in dark at room temperature and then diluted in absolute ethanol (50 mL) to obtain an absorbance of 0.70 nm ± 0.05 units at 734 nm applying a T 70 UV/Vis spectrophotometer (PG Instruments Limited, Leicestershire, UK). A volume of 20 µL from SAAE solution (0.1-500 µg/mL) was mixed with 1980 µL of the ABTS •+ radical solution, incubated for 6 min (in the dark at room temperature) and the absorbances were measured at 734 nm. Ascorbic acid (AA) were used as a reference antioxidant and controls with the extract was performed for each concentration evaluated. Three independent assays were performed in triplicate. The percentage inhibition of the ABTS •+ radical was calculated according to the following equation, where Abs control is the absorbance of ABTS •+ radical without the tested sample: Inhibition of the radical ABTS •+ (%) = ((Abs control − Abs sample )/Abs control ) × 100

DPPH Free Radical Capture Activity
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical capture activity was evaluated as described by Gupta and Gupta [99]. For the assay, 0.2 mL of SAAE extract (0.1-500 µg/mL) was added to 1,800 µL of DPPH solution (0.11 mM, 80% ethanol) and maintained for 30 minutes at room temperature in the dark. Thereafter, the absorbances at 517 nm were measured by a T 70 UV/VIS spectrophotometer (PG Instruments Limited, Leicestershire, UK). Ascorbic acid (AA) were used as a reference antioxidant and the controls of the extract were also acquired for each concentration evaluated. Three independent experiments were performed in triplicate. The inhibition was determined according to the following equation, where Abs control is the absorbance of the unsampled DPPH solution: Capture activity of DPPH (%) = (1 − Abs sample /Abs control ) × 100

Erythrocyte Suspension Preparation
The experiments were approved by the Research Ethics Committee (Comitê de Ética em Pesquisa, CEP) of the Federal University of Grande Dourados, (UFGD, Brazil (CEP process number: 2.407.793)). The human erythrocytes were obtained from peripheral blood of healthy donors. The collected blood was added in tubes containing the anticoagulant sodium citrate and they were centrifuged (400× g for 10 min). Subsequently, the plasma was removed, and the erythrocytes were washed three times with 0.9% sodium chloride solution (NaCl). The erythrocytes were resuspended in 0.9% NaCl solution, obtaining a final concentration of 2.5%.

Hemolytic Activity and Oxidative Hemolysis Inhibition
The evaluations of hemolytic activity and lipid peroxidation were determined in human erythrocytes according to the method described by Campos et al. [100]. The erythrocytes were preincubated with SAAE or ascorbic acid (50-125 µg/mL) at 37 • C for 30 min. Afterwards, 0.5 mL of 0.9% NaCl was added to evaluate the hemolytic activity of treatments or 0.5 mL of the oxidizing agent, 2,2 -azobis-(2-amidinopropane) dihydrochloride (AAPH), at 50 mM was added to evaluate the protection against oxidative hemolysis. As control of basal hemolysis, erythrocytes were incubated with 0.9% NaCl, while the erythrocytes were incubated with distilled water for total hemolysis control. The treatments were maintained for 240 min at 37 • C under constant agitation and analyzed each 60 min. To evaluate the hemolysis, the respective tubes (each time) were centrifuged at 700× g for 5 min and the supernatants (0.2 mL) were separated, added to 1800 µL of 0.9% NaCl and measured at 540 nm using a T 70 UV/VIS spectrophotometer (PG Instruments Limited, Leicestershire, UK). The percentage of hemolysis was calculated by the formula below; where A is the absorbance of sample and B the absorbance of the total hemolysis. Three independent experiments were performed in duplicate.

Malondialdehyde (MDA) Dosage
The ability of SAAE to protect against lipid peroxidation in human erythrocytes was determined by dosage of malondialdehyde (MDA) according to the methodology described by Campos et al. [100]. Erythrocytes were preincubated with SAAE or ascorbic acid (50-125 µg/mL) at 37 • C for 30 min. Then, 0.5 mL of the oxidizing solution AAPH (50 mM) was added in each treatment and incubated at 37 • C for 240 min with constant stirring. After 60, 120, 180 and 240 min, the tubes were centrifuged at 700× g for 5 min, and then 0.5 mL of supernatant was transferred into tubes containing 1 mL of 10 nM thiobarbituric acid (TBA) and incubated at 96 • C for 45 min. Soon after, the samples were cooled, added n-butyl alcohol (4 mL) and centrifuged at 1600× g for 5min. The supernatant was measured at the wavelength 532 nm by a T 70 UV/VIS spectrophotometer (PG Instruments Limited, Leicestershire, UK). The control was prepared by the mixture of TBA (1 mL) and 20 mM MDA solution (0.5 mL). Two independent experiments were performed in duplicate. The MDA levels were expressed in nM/mL, according to the following formula: MDA (nmol/mL) = Abssample × 20 × 220, 32 AbsstandardMDA

Cell Cultures
Human peripheral blood from healthy donors was collected in tubes containing the anticoagulant sodium citrate. Peripheral blood mononuclear cells (PBMC) was isolated by Ficoll Histopaque-1077 (1.077 g/cm 3 ) (Gold Analisa Diagnóstica, Belo Horizonte, MG, Brasil) following the manufacturer's instructions, the blood was centrifuged at 400× g for 30 min separating mononuclear cells into easily differentiated interfaces. The use of human samples was approved by the local Ethical Committee of the Federal University of Grande Dourados (protocol number 2.407.793) and signing written informed consent by donors.
The B16F10 cell line was originally provided by the Ludwig Institute for Cancer Research (LICR), São Paulo, Brazil. B16F10Nex-2, a sub-line isolated at the Department of Experimental Oncology, Federal University of São Paulo (UNIFESP), has the same characteristics as the original tumor cell line with moderate in vivo aggressiveness. Human peripheral blood mononuclear B16F10Nex-2 cells were grown in RPMI 1640 media (Gibco/Invitrogen, Minneapolis, MN, USA) supplemented with 10 nM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), 24 nM sodium bicarbonate and 10% fetal bovine serum (FBS), all from Gibco/Invitrogen, as well as with 40 mg/mL gentamicin (Hipolabor Farmacêutica, Sabará, Minas Gerais, MG, Brazil). All cells were kept in an incubator with a humidified atmosphere containing 5% CO 2 at 37 • C.

Cell Death Profile
The cell death profile was evaluated according to the methods described by Paredes-Gamero et al. [101], with minor modifications. B16F10Nex-2 cells were grown on 48-well cell culture plates (1 × 10 4 cells/mL) in RPMI medium containing 10% FBS for 24 h. After this period the cells were stimulated with IC50 of SAAE (65 µg/mL) and 100 µg/mL. Then, the cells were retired and washed with PBS. Afterward, the cells were resuspended in annexin labeling buffer (0.14 M NaCl, 0.01 M Hepes, and 2.5 Mm CaCl 2 , pH 7.4). The cells were labeled with annexin V-FITC and Propidium iodide (PI) (Becton Dickinson, San Jose, CA, USA) according to the manufacturer's instructions for 15 min at room temperature. A total of 10,000 events were acquired per sample through analysis in an Accuri C6 flow cytometer (Becton Dickinson). The analysis was performed using FlowJo v10.2 LCC software (Ashland, OR, USA).

Cell Cycle Phases
The cell cycle was profiled according to the method described by Paredes-Gamero et al. [101]. B16F10Nex-2 cells were plated in 24-well microtiter plates (6 x 10 4 cells/mL) and grown in RPMI 1640 media supplemented with 10% FBS with or without SAAE (65 µg/mL) for 24 h at 37 • C. After this period, the cells were fixed, permeabilized as previously described and incubated with 4 mg/mL RNAse (Sigma-Aldrich) for 1 h at 37 • C. To label cellular DNA, the cells were incubated with 5 µg/mL SytoxGreen (Molecular Probes Inc., Eugene, OR, USA). The percentage of cells in each cell cycle phase (G0/G1, S and G2/M) was determined by flow cytometry, in an Accuri™ C6 cytometer (Becton Dickinson). A total of 20,000 events were acquired per sample. The analysis was performed using FlowJo v10.2 LCC software.

Assessment of Reactive Oxygen Species (ROS) Levels
Intracellular ROS levels were assessed by flow cytometry using the dye 2 ,7dichlorodihydrofluorescein diacetate (CM-H 2 DCFDA) (Molecular Probe-Life Technologies, Carlsbad, CA, USA). B16F10Nex-2 cells were plated in 24-well microtiter plates (6 × 10 4 cells/mL) were treated with SAAE (65 µg/mL) for 24 h at 37 • C. Subsequently, the cells were trypsinized and incubated with 10 µM H 2 DCFDA for 30 min at room temperature and protected from light. The fluorescence of the ROS was assessed in an Accuri™ C6 flow cytometer (Becton Dickinson, San Jose, CA, USA). A total of 10,000 events were acquired per sample. The analysis was performed using FlowJo v10.2 LCC software.

Assessment of the Mitochondrial Membrane Potential
To assess the possible effect of the SAAE on the mitochondrial membrane potential, B16F10Nex-2 cells were incubated with the fluorescent dye JC-1 (5,5',6,6 -tetrachloro-1,1 ,3,3tetraethylbenzimidazolylcarbocyanine iodide; Molecular Probes, Eugene, OR, USA) according to the method described by Moraes et al. [102]. The JC-1 probe accumulates in mitochondria depending on the potential. Viable cells have a high mitochondrial membrane potential and are labeled in red. When the mitochondrial membrane potential decreases, cells are labeled in green. In this assay, cells were plated in 24-well microtiter plates (6 × 10 4 cells/mL) containing RPMI media supplemented with 10% FBS and treated with the positive control carbonyl cyanide m-chlorophenylhydrazone (CCCP, 30 µM) or with SAAE (65 µg/mL) for 24 h at 37 • C. After this period, the cells were trypsinized, centrifuged and incubated with JC-1 (1 µg/mL) for 15 min at room temperature. The fluorescence was analyzed in an Accuri™ C6 flow cytometer (Becton Dickinson). A total of 10,000 events were acquired per sample. The analysis was performed using FlowJo v10.2 LCC software.

Caspase-3 Activity
Caspase-3 activation was assessed by flow cytometry according to the method described by Moraes et al. [102], with small modifications. B16F10Nex-2 cells were plated in 24-well plates (6 × 10 4 cells/mL) containing RPMI media supplemented with 10% FBS and treated with SAAE (65 µg/mL) for 24 h at 37 • C. Then, cells were fixed with 2% paraformaldehyde in PBS for 30 min and permeabilized in 0.01% saponin for 20 min at room temperature. Subsequently, the cells were incubated with the antibody conjugated to the cleaved caspase-3 fluorophore (Asp175), Alexa Fluor 488, at room temperature and protected from light. After incubation, the fluorescence was analyzed in an Accuri™ C6 flow cytometer (Becton Dickinson, San Jose, CA, USA). A total of 10,000 events were acquired per sample. The analysis was performed using FlowJo v10.2 LCC software.

Statistical Analysis
Data are expressed as the mean ± standard error of the mean (SEM). Significant differences between groups were determined using the Student's t-test for comparison between two groups and analysis of variance (ANOVA) followed by Dunnett's test for comparison of two or more groups using the GraphPad Prism 5 software (San Diego, CA, USA). The results were considered significant when p < 0.05.

Conclusions
In conclusion, the results from the present study show that the SAAE from stem bark had a high concentration of phenolic compounds (flavan-3-ols, gallic acid, proanthocyanidins and chromones), which may be related to its antioxidant activities and anticancer effects on B16F10Nex-2 melanoma cells. Furthermore, the SAAE promoted cell death by apoptosis by increasing the intracellular ROS levels, mitochondrial membrane potential dysfunction and cleaved caspase-3 activation. These results open new perspectives for studies on these effects on other tumor cell lines and in vivo cancer models.


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Understanding the Role of Gui-Zhi-Fu-Ling-Capsules (Chinese Medicine) for Treatment of Endometriosis in the Rat Model: Using NMR Based Metabolomics

The objective of this study is to identify the changes of metabolites in the rat endometriosis models treated with Gui-Zhi-Fu-Ling-capsules (GZFLC), a classic Chinese medicinal formula, and to explore the effects of GZFLC on the serum levels of transforming growth factor-β1 (TGF-β1) and the mRNA expression levels of vascular endothelial growth factor (VEGF) and glucose transporter 4 (GLUT-4) in the endometriotic tissues. Forty female Wistar rats were randomly divided into the sham-operation group (Normal group), Model group, Danazol group, and GZFLC group. The serum levels of TGF-β1 were measured using enzyme-linked immune-sorbent assay (ELISA). The mRNA expression levels of VEGF and GLUT-4 in the endometriotic tissue of the rat endometriosis models were measured using real-time quantitative PCR. The metabolites in urine were detected by 1H NMR method. Eight identified metabolites of the NMR resonance were involved in the glycolysis metabolism. Among the 8 metabolites, Lactate, Acetate, TMA, and Formate were downregulated with GZFLC. Citrate, TMAO, Taurine, and Hippurate were unregulated with GZFLC. The serum levels of TGF-β1 in the Danazol and GZFLC groups were significantly higher than those of Normal group and significantly lower than the Model group. GZFLC treatment significantly decreased the GLUT-4 and VEGF mRNA expression levels in the endometriotic tissues of the endometriosis rats (P < 0.05). GZFLC significantly decreased the GLUT-4 mRNA expression levels in rats of GZFLC group compared with Danazol group. It is through regulating the metabolites changes of glycolysis or gluconeogenesis that GZFLC significantly affected the expression levels of TGF-β1, GLUT-4, and VEGF of the model rats with endometriosis.

Introduction
Endometriosis is the most common gynecological disease, which is characterized by the growth of endometrial tissue at ectopic locations [1]. It is a chronic condition characterized by the presence of endometrial tissue outside the uterine cavity, most commonly on the pelvic peritoneum and ovaries [2,3]. Endometriosis can result in substantial morbidity, including pelvic pain, multiple operations, and infertility [3]. Endometrial tissue is characterized by an exaggerated inflammatory environment around ectopic tissues [4,5]. Endometriosis affects approximately 10% of women of reproductive age [3,6]. About 30% of the patients are asymptomatic and the incidence of infertility among women suffering from endometriosis ranges from 30%-40% [6]. Increased peritoneal fluids concentrations of cytokines that lead to the migration, proliferation, and activation of macrophages have been reported in patients with endometriosis [7,8]. Surgery provides relief to women in pain, but symptoms recur in 75% of cases within 2 years [9].
Although surgical and hormonal treatment are applied as the common interventions, the unpleasant side effects and high rates of relapse limited the range of these interventions [10,11]. Some traditional Chinese medicinal herbs, such as Bupleurum (Chinese name, Chai hu) and Chinese angelica (Chinese name, Dang Gui), have significant curative effects in treating endometriosis [12,13]. For traditional Chinese medicinal formulas, 2
Gui-Zhi-Fu-Ling-Wan (GZFLW) is a classic Chinese medicinal formula, which was firstly described in Essential Prescriptions from the Golden Cabinet (Jingui Yaolue) by an ancient Chinese doctor, Zhong-Jing Zhang, Han Dynasty (300 A.D.) [15][16][17]. GZFLW is one of the top ten most frequently prescribed CHPs used to relieve endometriosisrelated symptoms [14]. GZFLW consists of five herbs: Ramulus Cinnamomi, Poria, Semen Persicae, Radix Paeoniae Rubra or Radix Paeoniae Alba, and Cortex Moutan. The traditional effects of GZFLW are invigorating blood, dissolving stasis, and resolving masses [15] and it has sedative and antiinflammatory effects on endometriosis-related symptoms [14]. Gui-Zhi-Fu-Ling Capsules (GZFLC) originate from the classic Chinese medicinal formula, GZFLW, which is more convenient to be taken and easily accepted by majority of the patients.
The metabolic disorder has been found to play an important role in the development of endometriosis, and rats' urinary metabolomes have revealed the potential roles of CHPs in management of metabolic disorder [18,19]. Metabonomics is a scientific discipline that can be used to study changes in the metabolite ensembles associated with disease pathophysiology [20]. 1H-NMR based metabolomics, a rapid and noninvasive approach, to identify metabolic changes associated with endometriosis in urine samples, is useful to get a better understanding of the pathogenesis of endometriosis, thus providing support to the noninvasive diagnosis of this pathology [21]. Few studies have been conducted on the mechanism and the functions of GZFLC in relation to the metabolic changes in pelvic endometriosis. Therefore, the present study was then designated to identify the changes of metabolites in the rat endometriosis models treated with GZFLC and to explore the effects of GZFLC on the levels of TGF-1 and the mRNA expression levels of vascular endothelial growth factor (VEGF) in the endometriotic tissues.  23, revised 1996). We attempt to minimize suffering of animals and reduce the numbers of animal in the study. Among the 40 rats, 10 rats were randomly taken as the Normal control group. The method of establishing the model rats with endometriosis was based on our previous studies [22][23][24][25]. Four weeks after the endometriosis model were made, the 30 model rats were randomly divided into three groups (n=10 in each group) including model control group (Model group), GZFLC group, and Danazol group. After all the treatment ended, the 40 rats were all sacrificed.

Group and Administration.
The 40 rats were divided into four groups: GZFLC group (the rats were orally administrated with the extracts of GZFLC at 40 mg/kg once daily for 30 consecutive days), Danazol group (the rats were orally administrated with Danazol at 36 mg/kg once daily for 30 consecutive days), Model Control group (Model group, the rats were orally administrated with saline at 8ml/kg once daily for 30 consecutive days), and Normal group (the rats were orally administrated with saline at 8ml/kg once daily for 30 consecutive days). After all the treatment ended, the rats were sacrificed and the samples of endometriotic tissue and the serum were taken. A total of 0.1g endometriotic tissue was collected and stored in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and stored frozen at −80 ∘ C until being used for further evaluation. The endometriotic tissue was homogenized in the tissue homogenizer. Then CCL 4 was added and centrifuged at 12,000 rpm for 7 min at 4 ∘ C. The supernatants were collected and stored frozen at −80 ∘ C until being used for further evaluation. After GZFLC o.a. for 30 consecutive days, at day 31, urine samples were collected in metabolism cages overnight from all groups and then frozen at −20 ∘ C and stored until analysis. The levels of Lactate in urine were detected by NMR. The serum levels of transforming growth factor-1 (TGF-1) were detected with ELISA. The mRNA expression levels of vascular endothelial growth factor (VEGF) and glucose transporter 4 (GLUT-4) were detected with qPCR.

Rat Urine Sample Preparation.
Urine samples were collected in metabolism cages overnight from all groups. Then, urine samples were frozen at −20 ∘ C and stored until analysis. A 1ml aliquot of each of the urine samples was taken for 1 H-NMR spectroscopy. To reduce pH induced chemical shift variability in the urine samples, 200 L of phosphate buffer (0.2 mol/L KH 2 PO 4 , 0.8 mol/L KH 2 PO 4 ) were added to 500 L urine then centrifuged at 8000x g for 5 min. 10 L of sodium trimethylsilyl propionate (TSP) and 50 L of deuterium oxide (D 2 O) were added to 550 L of the supernatant. TSP was used as a chemical shift reference. 10% D 2 O was used as a lock solvent for high resolution for NMR Evidence-Based Complementary and Alternative Medicine 3 spectrum. Push solvent (1g NaN 3 in 1000 ml dH 2 O) was used for NMR automation [26].

1 NMR Analysis of Metabolites in Urine.
All the 1 H NMR spectra were acquired from Bruker 600 MHz spectrometer (Bruker BioSpin, Germany). Free induction decay was zero filled to 64 K points then multiplied with an exponential function to 1.0 Hz line-broadening factor before Fourier transformation. The 1 H NMR spectra between 0.5 and 10.0 were segmented into consecutive nonoverlapping regions of 0.002 chemical shift bins. We chose the bin size of 0.002 as the primary chemical shift width for the identification of metabolites and integrated the spectral area in each bin, which can avoid the encompassment of several peaks and have clear peak multiplicities. The water resonance of 4.50-5.23 and urea resonance of 5.46-6.25 were excluded to eliminate baseline effects. We use the division of integrated segment with the total area of the spectrum to compensate the differences of concentration [27]. The peak area of each bin was calculated.

Multivariate Statistical Techniques of 1 NMR Data.
Multivariate analysis of 1 H NMR data was analyzed with SIMCA-P+ (version 11.5.0.0; Umetrics AB, Umeå, Sweden). Mean centered integral values were used for the principal component analysis (PCA), partial least square discriminant analysis (PLS-DA), and OPLS-DA analysis. Unsupervised PCA was used for the data of initial visualization. PLS-DA was used for the further trends of the data. The quality of PLS-DA models was analyzed using R 2 and Q 2 , where R 2 is the estimate of goodness-of-fit of model to the data, while Q 2 is the estimate of goodness of prediction.

2.7.
Determination of TGF-1 in Serum. 2 ml of blood was drown in polypropylene tubes and was allowed to clot for 30 minutes before centrifugation. The serum was obtained by centrifugation for 10 minutes at approximately 1000 g. The samples were stored at -70 ∘ C before the measurements (not longer than 3 months). Serum TGF-1 concentrations were measured using commercially available ELISA kit (Neobioscience, Shen-zhen, China).

Detection of GLUT-4 and VEGF mRNA Expression Levels in Endometriotic Uterus Tissue.
A total of 0.1g endometriotic tissue was collected and stored in TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The tissue was homogenized in the tissue homogenizer (30 s at 4.0 m/s). The relative expression was determined by quantitative real-time PCR (Q-RT PCR) in an ABI Step-one Sequence Detection System (Applied Biosystems, Forrest City, CA, USA). The total RNA was isolated with RNAiso6 reagent (Takara Biotechnology, Dalian, China) according to the instructions of the manufacturer. The purity and concentration of the RNAs were determined by OD260/280 readings with a NanoDrop5 ND-100 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of 1.8-2.0 is generally accepted as pure for RNA. The cDNA was prepared from 500 ng total RNA by reverse transcription (RT) with the PrimeScript6 RT Reagent kit (Perfect Real Time; Takara Biotechnology). The cDNA samples were then diluted in DNase-and RNase-free water at a proportion of 1:3 prior to further analysis. For the mRNA quantification, the normalized Ct of each gene was compared with the endometriotic tissue of control group mRNA. The rat GLUT-4 and VEGF gene-specific primers were provided by Sangon Biological Engineering Technology (Shanghai, China). PCR reactions were performed in a final volume of 25 l containing 50 ng of templates complementary DNA (cDNA) and 2.5 l of 10× PCR buffer real-time polymerase chain reaction (PCR) amplifications for GLUT-4 and VEGF were conducted using One-Step SYBR PrimeScript RT-PCR Kit according to the manufacturer's instructions under the following conditions: 95 ∘ C for 5min, followed by 40 cycles of 95 ∘ C for 5s and 60 ∘ C for 10s. Polymerase chain reaction products of -actin primer gene were used as an internal standard. Specificity of amplification was confirmed by melt curve analysis. After normalization against -actin mRNA, the comparative threshold (CT) method (i.e., ΔΔCT method) was used to quantify the relative expression levels of the samples. Fold changes were calculated using the equation 2-ΔΔCT. Q-RT PCR experiments were repeated three times independently. Data are represented as the mean ±SD.

Statistical Analysis.
Processing of the NMR spectra was performed by comparing spectral overlays using Topspin 3.0 (Bruker Biospin). The shifting signals were aligned in MAT-LAB 7.13 (MathWorks, Inc., Natick, MA, USA). Regular and dynamic bins were applied. Dynamic bins were determined with MATLAB for the Dynamic Adaptive Binning (DAB) Algorithm (Anderson et al., 2011). The statistical analysis was conducted using the Simca-P+ software (Umetrics, Umeå, Sweden). Data were analyzed using the Statistical Package for Social Sciences (SPSS 22.0 for Windows). The comparisons of different groups were performed with one-way analysis of variance (ANOVA) and multiple comparison tests with Bonferroni correction procedure. For all the hypothesis tests, significance level was set at P=0.05 and two-tailed tests were used.

1 NMR Metabolomic Analysis of Urine Samples.
The urine samples were subjected to 1 H NMR analysis to investigate the metabolic changes in the urine caused by GZFLC treatment. All the rats were divided randomly into two groups for NMR analysis, with 8 rats in each group: A, control group (endometriosis model rats without any treatment; B, GZFLC group, endometriosis model rats with GZFLC treatment. Representative 600MHz 1H NMR spectra ( 0.5-4.7, 5.0-9.6) from group A and group B were shown in Figure 1. Data obtained from the 1 H NMR spectra were analyzed. The data were subsequently analyzed using multivariate statistics (PCA, PLS-DA, and OPLS-DA). The metabolites were identified based on data obtained from database of Human Metabolome Database (HMDB). The PCA scores plots of 1 H NMR data in Figure 2 give an overview of the profiles for the respective treatments. The PCA and PLS-DA score plots were visualized with the first principal component (PC) and the second principal component (PC2). Based on R 2 X=82.2%, Q 2 =0.54 (Figure 2), the mathematical model is effective. The clustering phenomenon between the two groups is obvious. There is significant difference between the control group and the GZFLC group.

Discrimination between the Control and GZFLC Groups
Using 1 NMR. The PLS-DA was based on a unit variance scaling strategy. A 10-fold cross-validation was used to validate the PLS-DA model, which was performed to evaluate the quality of the model by parameters R 2 X, R 2 Y, and Q2. R 2 X represents the total variation in X and describes the optimization of the analytical model. R 2 Y represents the variation in the response variable Y. Q 2 represents the predictive ability of the model and the authenticity of the predicted results. On the scores plot, each point represented an individual sample. The center and the margin of each ellipse indicate mean and standard deviation, respectively. A permutation test from the verification plots was performed to validate the degree of overfitting for the PLS-DA model. The correlation coefficient between the original Y and the permutated Y was plotted against the cumulative R 2 and Q 2 ; a regression line was calculated with the R 2 and Q 2 intercept limits. These tests compared the goodness-of-fit of the original model with the goodness-of-fit of several models based on data in which the order of the Y observations was randomly permutated while the X matrix remained constant. In the verification plots, the Y-axis represents R 2 (green triangle) and Q 2 (blue box) and the X-axis designates the Pearson correlation coefficient between the original and permutated rank-ripeness. When the Q 2 regression line had a negative intercept and R 2 values on the left were lower than the original points on the right, the validation plots of the PLS-DA model were effective. The scores plots and validation plots of PLS-DA are shown in Figure 3. Based on the PLS-DA models in Figure 3, group A and B were discriminated with an R 2 X of 0.448, an R 2 Y of 0.959, and a Q 2 of 0.904. The results showed that the original PLS-DA models were effective. As shown in Figure 3, there is significant difference between the rats of the control group and the GZFLC group.
OPLS-DA plots were visualized with the first principal component (t [1]) and the orthogonal component (t [2]). The parameters Q 2 and R 2 X were computed to test the validity of the model against overfitting, where R 2 X is the total variation explained in the data and Q 2 is the cross-validated explained variation with increasing reliability as Q 2 approaches 1. The sixfold cross-validation method and permutation test for 500 times with the first component were carried out to measure  the robustness of the model, where if Q 2 (max) obtained from permutation test is less than or equal to Q 2 (cum) obtained from OPLS-DA, the established OPLS-DA model is robust. Based on the OPLS-DA models, the rats in the control and GZFLC group were discriminated with R 2 X = 44.8% and Q 2 = 0.904. In OPLS-DA scores plots, significant biochemical differences were also observed between the two groups ( Figure 4).
By assessing the coefficient plots of OPLS-DA in Figure 4, the main metabolites contributing to the separation of the two groups were shown in Table 1. Among the 8 metabolites, Lactate, Acetate, TMA, and Formate were downregulated with GZFLC. Citrate, TMAO, Taurine, and Hippurate were upregulated with GZFLC. At the end of the study, the two groups were subjected to NMR metabolomic studies. 8 metabolites were identified by fully mapping of chemical shifts, coupling patterns, and coupling constants to previously reported data. In Table 1, discriminating metabolites (with respect to the control group) of the group treated with GZFLC were presented. Eight identified metabolites of the NMR resonance, including Lactate, Acetate, Citrate, TMA (Trimethylamine N), TMAO (Trimethylamine N-oxide), Taurine, Hippurate, and Formate are involved in glycolysis and other metabolisms.

Identification of Predicative Metabolite Pathways.
To determine metabolic pathways which were affected by GZFLC, the metabolites Lactate, Acetate, Citrate, TMA (Trimethylamine N), TMAO (Trimethylamine N-oxide), Taurine, Hippurate, and Formate (Table 1) were mapped with Simca-P+ software. The metabolic pathways were presented graphically as a bubble plot in Figure 5. In Table 2 Figure 6, the serum levels of TGF-1 in Danazol group and GZFLC group were significantly higher than those of Normal group (P<0.05) and significantly lower than the Model group (P<0.05). No marked difference existed between the GZFLC and Danazol groups (P>0.05).

GLUT-4 and VEGF Gene Expression Levels in the Endometriotic Tissues.
For the GLUT-4 mRNA expression levels, it was significantly higher in the Danazol and the Model groups compared with the Normal group (P<0.05) ( Figure 5). For the GLUT-4 and VEGF mRNA expression levels, there were no significant differences between the

Discussion
The present study focuses on the use of proton nuclear magnetic resonance spectroscopy based targeted metabolite profiling approach to explore metabolites changes expression for the rats with endometriosis treated with Chinese formula GZFLC. Furthermore, association of glycolysis with the metabolite ensembles was also investigated. Xia Liu et al. found Herba Rhodiolae, a different traditional Chinese medicine, has similar infect on the glycolysis metabolisms [28]. In the present study, we identified eight metabolites of the NMR resonance involved in the glycolysis and other metabolisms, which were treated with different medicine Gui-Zhi-Fu-Ling-capsules (GZFLC). Among the 8 metabolites, Lactate, Acetate, TMA, and Formate were downregulated with GZFLC. Citrate, TMAO, Taurine, and Hippurate were unregulated with GZFLC. Based on the KEGG databases and the pathways and functional analysis of metabolites, glycolysis or gluconeogenesis has the most total numbers of compounds in the pathway. Glycolysis is an energy-producing mechanism that occurs in almost all cells and requires an adequate uptake of glucose mediated by glucose transporter (GLUT) proteins [29]. Increased glucose metabolism and defects in the mitochondrial respiratory system are suggested to be the possible sources of excessive reactive oxygen species generation in endometriosis [20]. Endometriotic lesions induced glycolysis, but limited metabolites related researches have been focused on this. Little is known about GLUT proteins expression either in the endometrium or the endometriotic lesions.
Via glycolysis pathway, TGF-induces the metabolic conversion of glucose to Lactate, a process referred to as the "Warburg effect" [30]. Overproduction of Lactate increases cell invasion, angiogenesis, and immune suppression, all crucial steps in the development known regulators of endometriosis [31]. TGF-is believed to play a major role in the etiology of peritoneal endometriosis [9]. Also, TGF-1 stimulated glucose transport and induces changes in the metabolic phenotype of peritoneal mesothelial cells from women with endometriosis.
Adequate glucose uptake and metabolism are essential for the proper differentiation of the uterine endometrium toward a receptive state capable of supporting embryo implantation. The inducible nature of GLUT-4 and its limited cellular expression may make GLUT-4 an attractive target for nonhormone-based treatments of endometriosis [29]. TGF-beta and VEGF are released by inflammatory process cells which may serve as the source of wide spectrum of inflammatory mediators and growth factors [32].
In the proton NMR study, based on the KEGG databases and the pathways and functional analysis of metabolites using, glycolysis or gluconeogenesis has the most total numbers of compounds in the pathway. At the meanwhile, we found that the serum levels of TGF-1 in the Danazol and GZFLC groups were significantly higher than those of Normal group and significantly lower than the Model group. GZFLC treatment significantly decreased the GLUT-4 and VEGF mRNA expression levels in the endometriotic tissues of the endometriosis rats. Compared with Danazol group, GZFLC significantly decreased the GLUT-4 mRNA expression levels in rats of GZFLC group.
To conclude, GZFLC significantly affected the expression levels of TGF-1, GLUT-4, and VEGF of the model rats with endometriosis, accompanied by regulating the metabolites 8 Evidence-Based Complementary and Alternative Medicine changes of glycolysis. We hypothesized that GZFLC regulates endometriosis by effect on glycolysis or gluconeogenesis. However, more studies should be conducted to explore the underling molecular and metabolism mechanism further.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare no conflicts of Interest.