extfieldsofstudy: Medicine Chemistry

text:
Validation and evaluation of a common biomarker in human cancers sera protein detected by a monoclonal antibody UNIVmAb

Objective Management and diagnosis of multiple human cancers remains a challenge and search for a common biomarker is still debatable. In this manuscript we have evaluated the use of monoclonal antibody UNIVmAb, to detect the protein (H11) as a common biomarker for all cancers irrespective of the grade and origin. We have shown by both ELISA and Western Blot that the H11 protein, is a unique hyaluronan binding protein that has not been detected earlier. H11 protein was fractionated in an anion exchange column followed by cibacron blue gel exclusion chromatography. Hyaluronan binding H11 protein reacted with Monoclonal antibody UNIVmAb and b-HA inspite of b-Hyaluronan (biotinylated Hyaluronan) interaction and HA-Oligo (Hyaluronan oligosaccharides) competition from various grades of Human cancers sera. Results ELISA, Western blot and b-Hyaluronan interactions clearly showed an over-expression of UNIVmAb reacted H11 protein in all fifty cancer’s sera when compared with seventy normal sera. UNIVmAb reactive H11 protein can be used as a common biomarker. We believe, UNIVmAb detected H11 protein, is a unique hyaluronan binding protein, that can be used as a common biomarker for all cancers.

Introduction
The appearance of cancer serum biomarker is a molecular event that indicates the pathological changes that happen in a particular tissue or cell type during cancer development. The most important part of screening is the ability for an early cancer diagnosis. Cancer diagnosis based on the quantification or localization of a particular antigen in cells, tissues or body fluids [1][2][3][4][5] is a topic of interest with human body fluids (serum) as an important source of minimal invasive biomarkers. The widely used blood test for early detection of cancer of the prostate is PSA (prostate-specific antigen) and the proper use of this test is highly debatable [6]. However, diagnosis using circulatory serum antigens makes the detection of biomarkers more feasible, and it is relatively a non-invasive method to acquire experimental samples [4,5,[7][8][9][10][11].
The development of monoclonal antibodies (mAb's) identifying specific antigens expressed by cancer cells offers a diagnostic technique for early cancer detection [12]. mAb's are widely used reagents in the clinical diagnostic laboratories to develop sensitive immunoassays for the detection of their target antigen in circulation, their levels relate with the disease progression [11,[13][14][15][16]. (Drugs/Guidance Compliance RegulatoryI nformation/ Guidances CM070107.pdf. Analysis of human embryonic cells, adult tissues and human serum has revealed the presence of hyaluronan (HA) and its receptors. Hyaluronan is a nonsulfated, high molecular weight glycosaminoglycan consisting of the d-glucuronic acid and N-acetyl d-glucosamine [17]. HA is present in the extracellular matrix of most tissues and is enriched in many tumours [17,18]. It is well documented that HA and its receptors, known as hyaladherins (hyaluronan binding proteins) are involved in matrix regulation, cell proliferation, migration and malignant tumour progression [18]. Hyaladherins not only interact with hyaluronan at the matrix proper but also with hyaluronan in the plasma membrane as cell surface receptors and thus influence cell physiology, including secretion of this protein into the circulatory system [19][20][21]. Previously, we have identified H11B2C2 clones reactive hyaluronan binding protein and revealed its association with tumour progression by immunohistochemistry [13]. The antigen expression increases during tumour progression irrespective of cancer origin. Antibody produced by the clones, detected the antigen in multiple human cancer tissues/serum and we renamed the antibody UNIVmAb. We assume that UNIVmAb reactive H11 is a protein biomarker in various tumour tissues that may give us crucial diagnostic information in early cancer detection. We have described the procedures for fractionation of serum proteins, mainly hyaluronic acid binding protein (HABP), from various cancer patient's samples and healthy subjects using single dimension electrophoresis. The detection of H11 antigen using UNIVmAb in multiple cancer's led us to think that H11 antigen can be a common biomarker for multiple cancers.

Sample collection and preparation
The study consisted of 70 normal subjects and 50 cancer patients (Lymphoma (2 patient samples), Salivary samples), Urinary bladder (2 patient samples), Prostate (4 patient samples), ovary (5 patient samples), endometrial (4 patient samples) and cervix (2 patient samples). Serum samples from normal subjects and cancer patients were accessed from cancer hospitals in Mysore, India (Preethi Centre for oncology, and KR Hospital, Logic and Clue Diagnostic center) and the protocols were approved by the ethical review committee (IHEC-UOM NO 35) and the patient's consent were taken. Blood samples were collected from each patient before any treatment. Samples were centrifuged at 2000×g for 30 min at room temperature and the separated sera were stored at − 80 °C. The H&E stained tumour sections of patients were obtained from hospitals and were graded using the TNM grading system. Serum samples treated with 4× lysis buffer, containing 0.2 M Tris-HCl (pH 8.0), 80 mM EDTA, 4 mM PMSF, 4 mM Benzamidine-HCl and 2% Triton X 100 plus protease inhibitor cocktails were centrifuged at 10,000×g for 30 min at 4 °C. The supernatant was stored at − 80 °C until further analysis. The protein estimation was done at UV 280 nm and Bradford reagent assay using Bovine serum Albumin (BSA) as standard.
Biotinylated hyaluronic acid was prepared according to Boregowda et al. [13] and Srinivas et al. [24]. In brief, HA dissolved in PBS-A was dialyzed in MES buffer and reacted with biotin_LC-hydrazide and EDC in DMSO. This was Incubated for 16 h and then dialyzed against PBS-A and stored in glycerol at -20 °C.

Production of monoclonal antibody UNIVmAb
Hybridoma and the antibody were prepared according to Boregowda et al. [18,22,23]. In brief, the hybridoma was grown in DMEM with human serum (pathogen and complement free) that were received from the hospitals. The antibody production in the presence of human serum (any blood groups) did not affect UNIVmAb recogntion of the human H11 antigen. The clones were grown in DMEM containing 10% (v/v) inactivated human serum. After 21 days, the media was collected and precipitated with cold saturated ammonium sulphate solution (final 50%) at 4 °C overnight and centrifuged at 12,000×g for 30 min. The pellet was dissolved in PBS and dialyzed against PBS.

Statistical analysis
Statistical differences between groups from ELISA were analyzed using graphpad prism version 5 software. Results are expressed as the mean ± SD. A diiference with P values is defined as follows: P < 0.001 = extremely significant. For westerblot, image analysis was done using Image J software.

Detection of H11 antigen by ELISA using UNIVmAb
MaxiSorp flat-bottom high protein binding capacity polystyrene-96 well plates were used. Serum samples were diluted with 0.05 M carbonate-bicarbonate buffer pH 9.6 to obtain a final concentration of 1 µg/ml. 100 µl of samples in triplicate were plated on to the 96well plate and incubated overnight at 4 °C. Following day the plate was blocked with skimmed milk (prepared in PBS) for 1 h and incubated with UNIVmAb at 1:10,000 overnight at 4 °C. Following day the plate was washed with 0.2% Tween-PBS followed by incubation with b-goat anti-mouse antibody at 1:20,000 for 1 h and reacted with streptavidin-peroxidase at 1:50,000 for one hour. Plate was washed with 0.2% Tween-PBS and 100 µl of ABTS (1.0 mg/mL) in 0.1 M citrate buffer at pH 4.0 and 5%. Hydrogen peroxide. The reactions were stopped after one hour with 0.2 M citric acid, and the absorbance was measured at 405 nm Fig. 1. Experiments were repeated at least three times. Protein levels were measured by quantitative ELISA.

Western blot analysis of serum according to Boregowda et al. [15] and Fekry et al. [16]
50 μg proteins from serum lysate were resolved on 10% SDS-PAGE, transferred to PVDF membrane and reacted with UNIVmAb (1:1000 dilution) or anti HCAM mAb (1:1000 dilution) or with bHA probe (1:100 dilution) overnight at 4 °C. Following day, the blot was developed and the proteins were detected using Enhanced ChemiLuminescence (ECL). Since the isolation of antigen is vital to understand its property, we used purified circulating antigen by antibody conjugated CNBR activated Sepharose and Cibacron blue affinity purification method. The purified H11 protein was reacted with UNI-VmAb and was also cross reacted with bHA and HAoligo (500 µg Oligo) competition was also performed. This showed that H11 antigen is a Hyaladherin.

Results and discussion
We validated the clones H11B2C2 reactive novel HABP in human cancer tissues. HABP overexpression was related to poor tumour outcomes [13,25]. The antibody produced by the clones detected the antigen in all human cancer tissues and cancers sera; we renamed the antibody as UNIVmAb. We have also identified soluble hyaluronan binding proteins in colon cancer serum with a molecular mass of 57 and 30 kDa [14]. We believed that the soluble 57 kDa H11 protein from colon cancer might have relation with multiple cancer samples. UNIVmAb reactive H11 proteins maybe a new class of hyaluronan binding protein and we did a further investigation in the present study to understand its expression and the nature of H11 protein in various cancer's sera to further characterize by biochemical analysis and its application as a common cancer biomarker. Table 1 shows the detailed results of H11 expression in various cancers sera.
From ELISA experiment (Fig. 1), normal sera showed a mean O.D: 0.175 from triplicates, However sera from  UNIVmAb, similar results were also observed with the remaining cancer samples. Fig. 2 To detect whether UNIVmAb reactive antigens were present in circulation, we conducted western blot analysis of normal and various cancers sera samples using UNI-VmAb. The UNIVmAb showed reduced H11 reactivity in normal sera at 57 kDa (Fig. 2 a), the intensity of reaction is enhanced in tumour samples of high-grade cancers ( Fig. 2d vs b, c). We compared this with 70 normal samples and 50 cancer patient's samples of different grade. We have shown H11 expression in 12 different healthy individuals sera samples in Fig. 2 (panel a lanes 1 to 12). The UNIVmAb reaction with serum proteins of six different cancer sera of different cancer patients are shown in panel b, c and d. The presence of UNIVmAb reactive protein in normal and eighteen cancer subjects suggests that H11 protein might have several biological functions. Similar results were observed in the tumour/normal tissues [13]. Figure 3 Scion image analysis of the western blot showed that UNIVmAb reacted antigen overexpression is real and accepted when tested with all other cancers sera samples.

UNIVmAb reactive H11 proteins were overexpressed in human cancer serum shown in
Western blot analysis with bHA probe showed that the detected serum HABP expression is H11 antigen (Fig not shown) and HA-Oligo competition showed 80%

Table 1 Evaluation of UNIVmAb reacted H11 expression in multiple tumours
We also detected H11 expression from astrocytoma glioblastoma and medulloblastoma not included into the  These results indicate that the 57 kD, named H11 antigen is a hyaluronan binding protein (data not shown). We performed immunoprecipitation and affinity purification of normal and cancer patient's serum proteins with mAb to prove that mAb is reacting with 57 kDa protein with high specificity. As expected the normal sera showed low levels of circulatory 57 kDa by western blot, whereas, advanced cancer sera showed overexpression of 57 kDa antigen. To show that the cancer antigen 57 kDa is associated with carrier protein such as Albumin we used Cibacron blue gel exclusion method. Eluted fractions were tested for reactivity with b-HA and UNIVmAb, showing a strong reaction at 57 kDa (data not shown). To investigate UNIVmAb reactive cancer antigen may be related to a known hyaladherin, CD44 (std); Whether, UNIVmAb immunoprecipitated protein can pull down CD44 reactive protein, we used stomach grade lll cancer sample, which overexpressed for mAb reacted H11 antigen and normal sera were tested. We find no reaction to HCAM antibody to the H11 antigen (data not shown).

Conclusion
Blood-borne metastasis is the greatest obstacle to cure patients with cancers. The abundant blood proteins, such as albumin, immunoglobulin, etc.; may mask the less abundant proteins, which are usually potential markers. There are many known specific serum markers but not a common biomarker for various cancers sera. UNIVmAb detected the overexpression of serum specific H11 antigen in various cancer's sera, offers significant advantages. In the present study, screening serum samples from 70 normal and 50 cancer patients samples (18 different cancer types of various grade) of different grades we predicted that the UNIVmAb might be used as a antibody to detect a common cancer biomarker. This Mab detected a protein H11 and is unique HABP not identified earlier in serum and serum albumin may be the carrier protein of H11 antigen. Still unanswered question is, what is the nature of 57 kDa H11 antigen. We investigated the H11 antigen by proteomic analysis from grade ll adenocarcinoma of the colon. We observed the presence of IgGH1, member of immunoglobulin super family (data not shown). The present data showed that UNI-VmAb detected the H11 might be a unique hyaladherin and can be used as a common biomarker for progressive human cancers sera.

Limitations
At present, there is no common biomarker for multiple cancer detection. The present study could reflect the UNIVmAb reactive serum antigen as a common biomarker for multiple cancers. In addition, future experiment on the proteomic analysis will help us identify the nature of the antigen as a potential common biomarker.


Association between miRNA‐196a2 rs11614913 T>C polymorphism and Kawasaki disease susceptibility in southern Chinese children

Abstract Background miRNAs play important roles in a variety of diseases. Thus, the association between miRNA‐196a2 rs11614913 T>C polymorphism and Kawasaki disease susceptibility is still unknown. Methods We included 532 children with Kawasaki disease and 623 healthy children from South China, and their DNA was extracted for genotyping by TaqMan methodology. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the strength of association. Results No significant associations were observed between the miRNA‐196a2 rs11614913 T>C polymorphisms and Kawasaki disease risk (TC vs TT: adjusted OR = 1.04, 95% CI = 0.79‐1.37; CC vs TT: adjusted OR = 0.87, 95% CI = 0.63‐1.21; dominant model: adjusted OR = 0.99, 95% CI = 0.76‐1.27; and recessive model: adjusted OR = 0.85, 95% CI = 0.64‐1.13). There was also no significant correlation found in stratified analyses. Conclusion This study suggests that miRNA‐196a2 rs11614913 T>C may not be associated with Kawasaki disease susceptibility in a southern Chinese population. Larger, multicenter studies are needed to confirm our conclusions.

| Study subjects
A total of 532 unrelated Chinese children who went to the Guangzhou Women and Children's Medical Center were diagnosed with Kawasaki disease from January 2012 to January 2017. The control group was composed of 623 healthy Chinese children who went to the hospital for physical examination in the same period. [16][17][18] Peripheral venous blood was taken from each child to extract DNA.
In addition, personal information such as gender, age, and coronary diameter of cases and controls was collected. This study obtained the consent of the Guangzhou Women and Children Medical Center Ethics Committee and the guardian of each child (ethics number: 2014073009).

| DNA extracting and genotyping
We used a Qiagen kit (Qiagen Inc) to extract genomic DNA from blood and stored in a −80°C freezer and genotyped this SNP using the TaqMan real-time PCR method with an ABI-Q6 instrument (Applied Biosystems), as described previously. [21][22][23] For accuracy and reliability, each plate was provided with an eight-well blank control, including equal amounts of distilled water but no DNA.

| Statistical analysis
The chi-square test was used to assess significant differences between the case group and the control group, the risk factors, and the distribution of this SNP in the control and case groups, including additive, dominant, and recessive models. Logistic regression analysis was used to calculate the original odds ratio (OR) value and 95% confidence interval (CI). Stratified analysis was used to analyze subgroups of data, such as gender, age, and coronary artery outcomes.
Data were analyzed using SAS 9.1 software, and P values <0.05 were considered indicative of statistical significance.

| Research population characteristics
The basic information of the case group and the control group is shown in Table S1. The study included 532 KD children and 623 healthy children matched by age (P = 0.602) and gender (P = 0.143).
The incidence of coronary artery aneurysm and coronary artery lesions was 9.59% and 31.58%, respectively. Table 1 shows the distribution of genotypes in the case and control groups. We can see that the genotype distribution in the two groups is equivalent, so there is no significant difference (TT, TC, and CC in the two groups are 28

| Stratification analysis
The results for the stratified analysis are shown in Table 2. No significant difference was found in the stratified analyses of age, gender, and coronary artery outcome.

| DISCUSS IONS
In this study, we found that the miRNA-196a2 rs11614913 T>C may not be associated with Kawasaki disease susceptibility in a southern Chinese population. Over the past 50 years, researchers have tried to find the cause of Kawasaki disease, but it has not been revealed so far. The genetic susceptibility of Kawasaki disease has driven us to study the relationship between miRNA polymorphisms and Kawasaki disease. Our previous study reported that miRNA-146a rs2910164 C>G may not be associated with Kawasaki disease susceptibility in children from South China. 18 We found that miRNA-137 rs1625579 T>G is associated with the onset of Kawasaki disease in the same population in children younger than 1 year old. 16 To our knowledge, this report is the first on the association of miRNA-196a2 rs11614913 T>C with Kawasaki disease susceptibility among children in South China.
Due to the high conservation and tissue specificity of miRNAs, many studies report them as diagnostic markers of diseases such as cancer, 24,25 nervous system disease, 26 immune disease, 27 and cardiovascular disease. 28 miRNA-196a was identified more than a decade ago. 29 SNPs can alter miRNA function by activating miRNA transcription and processing miRNA precursors to alter miRNA expression. 30  lial cell proliferation and prostaglandin production, and plays a protective role in the anti-inflammatory process. 39,40 Annexin A1 is expressed by inflammation-related cells such as white blood cells, mast cells, and vascular endothelial cells. 41 In neutrophils, Annexin A1 is mainly distributed in the cytoplasm. 42 The expression of Annexin A1 in circulating neutrophils was higher in inflammatory cells than that in neutrophils, indicating that Annexin A1 was lost during neutrophil adhesion and migration. 43 In vitro, neutrophils interact with endothelial cells to stimulate Annexin A1 externalization through intercellular adhesion molecules and platelet adhesion molecules. 43 Externalized Annexin A1 binds to formyl peptide receptors to exert anti-inflammatory effects, 44  In our study, it was revealed that miRNA-196a2 rs11614913 T>C may not be associated with Kawasaki disease susceptibility in a southern Chinese population and that there was no significant association in the stratified analysis. The advantage of this study is that it is the first report involving miRNA-196a2 rs11614913 T>C and Kawasaki disease susceptibility. Due to the relatively large number of subjects, the effectiveness of the experimental results is guaranteed. There are also some limitations. First, due to a lack of data, our study examined the incidence of coronary lesions only in children with Kawasaki disease during hospitalization and failed to have long-term follow-up. In the future, we will continue to study and consider these patients. Second, this is a single-center, single-ethnicity study, and as explained in the Discussion, the heterogeneity of the population may produce different results.
Overall, we failed to find any significant association between miRNA-196a2 rs11614913 T>C polymorphism and Kawasaki disease susceptibility in a southern Chinese population. Larger multicenter, multiethnic research is needed to validate our results.

ACK N OWLED G M ENTS
We


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

text:
Intrapancreatic Ganglia and Neural Regulation of Pancreatic Endocrine Secretion

Extrapancreatic nerves project to pancreatic islets directly or converge onto intrapancreatic ganglia. Intrapancreatic ganglia constitute a complex information-processing center that contains various neurotransmitters and forms an endogenous neural network. Both intrapancreatic ganglia and extrapancreatic nerves have an important influence on pancreatic endocrine function. This review introduces the histomorphology, innervation, neurochemistry, and electrophysiological properties of intrapancreatic ganglia/neurons, and summarizes the modulatory effects of intrapancreatic ganglia and extrapancreatic nerves on endocrine function.

INTRODUCTION
Intrapancreatic ganglia were first described by Langerhans in 1869 and have been studied over several decades (Sha et al., 1995(Sha et al., , 1996(Sha et al., , 1997(Sha et al., , 2001bLiu and Kirchgessner, 1997;Love and Szebeni, 1999b;Sha and Szurszewski, 1999;Yi et al., 2005;Shen et al., 2016). While intrapancreatic ganglia have been traditionally considered to be simple parasympathetic relays, growing evidence suggests that, similar to the enteric nervous system (ENS), intrapancreatic ganglia may constitute a complex information-processing center containing various neurotransmitters and form an endogenous neural network (Gylfe and Tengholm, 2014;Shen et al., 2016;Tang et al., 2018b). Although the influence of extrapancreatic nerves, especially autonomic nerves, on pancreatic endocrine function has been extensively studied, the role of intrapancreatic ganglia in endocrine and exocrine functions has garnered far less attention (Sha et al., 2001b;Love et al., 2007;Gylfe and Tengholm, 2014;Rodriguez-Diaz and Caicedo, 2014). In this review, we will focus on research studies from the past 30 years. We will introduce the histomorphology, innervation, neurochemistry, and electrophysiological properties of intrapancreatic ganglia/neurons, and summarize the modulatory effects of intrapancreatic ganglia and extrapancreatic nerves on endocrine function.

ANATOMY AND MORPHOLOGY OF INTRAPANCREATIC GANGLIA
Intrapancreatic ganglia are composed of pancreatic neurons, glial cells, and extrinsic and intrinsic nerve fibers. They are located over or alongside nerve trunks in the interlobular, acinar, or insular connective tissues, or within lobules and islets (Liu and Kirchgessner, 1997;Tang et al., 2018a). Intrapancreatic ganglia can be oval, round, or polygonal in shape, and vary in size (Shen et al., 2016). Most intrapancreatic ganglia are found alongside or inside nerve trunks that are branches of larger nerve trunks traveling along the pancreaticoduodenal artery in the head of the pancreas (Liu and Kirchgessner, 1997). Ganglia are also found along nerve bundles from the celiac and superior mesenteric plexuses, and the splenic nerves in the body and tail of the pancreas (Love et al., 2007). The ganglia in the head are larger and contain more neurons than those in the body and tail of the pancreas (Sha et al., 1996). In the rabbit pancreas, the larger ganglia (≥6 neurons) often appear to be encapsulated and connect to larger nerve trunks, while the smaller ganglia are similar to grape clusters; single pancreatic neurons are also found within islets (Love and Szebeni, 1999b). The peri-lobular ganglia are larger than intra-parenchymal ganglia. The ganglia of patients with type 2 diabetes are larger than those of individuals without diabetes (Tang et al., 2018a). The number of neurons in each pancreatic ganglion is different in different species. For example, rabbit ganglia contain 1 to 35 neurons per ganglion [mean ± standard error of the mean (SEM), 4 ± 1] (Love and Szebeni, 1999b), while rat ganglia contain 2-24 neurons per ganglion (mean ± SEM, 8.8 ± 0.5) (Shen et al., 2016). Although the neurons are typically oval or round, triangular neurons are also found. Most of the neurons are bipolar or multipolar, and unipolar neurons are rare. While the dendritic neuropil of neurons is variable in shape, most of the dendrites are typically short and stubby (Sha et al., 1996). Most nerve fibers of intrapancreatic ganglia are unmyelinated. The unmyelinated nerve bundles have a fine axonal structure, with a large number of microtubules present in the center. Some axons contain both small, clear vesicles and large dense-cored vesicles, while others appear to contain only small, clear vesicles (Love et al., 2007). The axons of pancreatic neurons project to all pancreatic effector cells, including islets, pancreatic ducts, acini, blood vessels, and other intrapancreatic ganglia, indicating that intrapancreatic ganglia may play an important role in pancreatic functions, including endocrine function (Niebergall-Roth and Singer, 2001;Love et al., 2007).

Sympathetic Efferent Fibers
Preganglionic efferent sympathetic fibers project from cell bodies in the lateral horn of the spinal cord (C 8 -L 3 ) to paravertebral and prevertebral sympathetic ganglia (Llewellyn-Smith, 2009; Figure 1). The fibers projecting from the prevertebral ganglia (the celiac and superior mesenteric ganglia) enter the pancreas directly or with other autonomic nerves (Ahrén, 2000;Gilon and Henquin, 2001;Love et al., 2007). The celiac plexus mainly includes four branches: the anterior hepatic plexus, posterior hepatic plexus, splenic plexus, and plexus accompanying the transverse pancreatic artery (Dolensek et al., 2015). These branches are distributed in different locations of the human pancreas. The head of the pancreas receives the anterior and posterior hepatic plexuses, which are distributed along the hepatic artery and located in the dorsal aspect of the portal vein.
The body and tail of the pancreas are innervated by the other two plexuses. Some nerves originate from the superior mesenteric ganglia and enter the uncinate process of the pancreas along the inferior pancreaticoduodenal artery. There also exist plexuses that surround the main pancreatic duct, but pass through the pancreas independently of the blood vessels (Yi et al., 2003). The postganglionic sympathetic fibers project to intrapancreatic ganglia, islets, ducts, and blood vessels, and release several neurotransmitters such as norepinephrine (NE), galanin, and neuropeptide Y (NPY) (Ahrén, 1999;Di Cairano et al., 2016). Preganglionic sympathetic fibers also enter the pancreas directly and terminate at the intrapancreatic ganglia (Havel et al., 1989).

Parasympathetic Efferent Fibers
Most preganglionic parasympathetic fibers supplying the pancreas originate from the dorsal motor nucleus of the vagus (DMV) in the medulla, and their cell bodies are rarely observed in the nucleus ambiguous (Love et al., 2007;Chandra and Liddle, 2013;Rodriguez-Diaz and Caicedo, 2014;Ma and Vella, 2018; Figure 1). Vagus nerve fibers pass through the hiatus of the esophagus into the abdomen and are divided into five distinct branches that subsequently innervate different organs. The vagus nerve fibers that innervate the pancreas include the hepatic and bilateral gastric branches (primary), and bilateral celiac branches (secondary) (Niebergall-Roth and Singer, 2001;Love et al., 2007;Chandra and Liddle, 2014).

Enteropancreatic Plexus
The ENS within the wall of the gastrointestinal tract is often considered to be the "local brain" that regulates gastrointestinal functions. Enteric neurons in the myenteric plexus of the gastric antrum and proximal duodenum also project out of the gut to the intrapancreatic ganglia (Kirchgessner and Gershon, 1990;Chandrasekharan and Srinivasan, 2007). Cholinergic enteropancreatic neurons form excitatory nicotinic synapses on pancreatic neurons. In addition, some enteropancreatic nerves, such as the PACAP-containing nerves, are peptidergic. PACAP is often considered as a neuromodulator that strengthens pancreatic secretion (Kirchgessner and Liu, 2001). The fibers of serotonergic enteropancreatic neurons form inhibitory axoaxonic synapses to inhibit amylase secretion (Kirchgessner and Gershon, 1995). Enteropancreatic neurons form nicotinic synapses in intrapancreatic ganglia; however, other excitatory pathways remain unknown. The role of enteric nerves in controlling the endocrine functions of the pancreas has not been FIGURE 1 | Innervation of intrapancreatic ganglia. Intrapancreatic ganglia receive inputs from sympathetic fibers (red), parasympathetic fibers (green), enteric plexus (brown), and nerves of other intrapancreatic ganglia. The parasympathetic efferent fibers (green solid line) originate from the dorsal motor nucleus of the vagus (DMV) and ambiguous nucleus, whereas the cell bodies of sympathetic efferent fibers (red solid line) project from the lateral horn of the spinal cord to the celiac ganglia (CG), superior mesenteric ganglia (SMG), or paravertebral ganglia (not show in the Figure). The cell bodies of parasympathetic (green dashed line) and sympathetic (red dashed line) afferents are positioned in the nodose ganglia (NG) and the dorsal root ganglia (DRG), respectively.
completely elucidated. The ENS may be a therapeutic target for the regulation of glucose metabolism (Abot et al., 2018a,b).

Afferent Nerve Fibers
Intrapancreatic ganglia receive both sympathetic and parasympathetic afferents, most of which are sensitive to capsaicin. These afferent fibers transmit sensory information from the pancreas to the central nervous system (Dolensek et al., 2015).
Sympathetic afferents are thought to exit the pancreas along the postganglionic sympathetic fibers within the splanchnic nerves and the celiac plexus to the dorsal root ganglia (DRG, T 6 -L 2 ) (Love et al., 2007;Rodriguez-Diaz and Caicedo, 2014;Dolensek et al., 2015). Pancreatic parasympathetic afferents originate from the nodose ganglia (NG) (Lartigue, 2016). While pancreatic parasympathetic afferent fibers primarily supply the blood vessels, ducts, acini, and islets, few parasympathetic afferents are found in intrapancreatic ganglia (Love et al., 2007). Although the central targets of vagal afferents remain largely unclear, the nucleus of the solitary tract (NTS) is considered to be the main relay center for vagal afferent nerves (Renehan et al., 1995;Dolensek et al., 2015). Sympathetic and parasympathetic afferent fibers contain either substance P (SP) or calcitonin gene-related peptide (CGRP), or both (Rodriguez-Diaz and Caicedo, 2014).

NEUROTRANSMITTERS OF INTRAPANCREATIC GANGLIA
A variety of neurotransmitters can be identified in the intrapancreatic ganglia (Figure 2). Studies show that choline FIGURE 2 | Neurotransmitters are released from both extrinsic nerves and intrinsic intrapancreatic ganglia. Neurotransmitters are, respectively released from parasympathetic fibers, sympathetic fibers, sensory fibers, enteric fibers, the intrapancreatic ganglionic fibers. ACh, acetylcholine; GRP, gastrin-releasing peptide; VIP, vasoactive intestinal peptide; NO, nitric oxide; NE, noradrenaline; NPY, neuropeptide Y; PACAP, pituitary adenylate cyclase activating polypeptide; 5-HT, serotonin; CGRP, calcitonin gene-related peptide; SP, substance P. acetyltransferase (CHAT)-immunopositive neuronal cell bodies and nerve fibers are present in the pancreas of many species (Liu et al., 1998;Love and Szebeni, 1999a;Wang et al., 1999), suggesting that ACh is not only released from parasympathetic efferents and enteropancreatic fibers, but also from the neurons in the intrapancreatic ganglia. However, intrapancreatic ganglia are not innervated by cholinergic nerve fibers in the sheep pancreas (Arciszewski and Zacharko-Siembida, 2007), which suggests that the cholinergic innervation of islets from pancreatic neurons is species-dependent.
It has been shown that intrinsic noradrenaline-containing neurons are present in the pancreases of rats and newborn guinea pigs. Dense catecholamine-containing nerves with few cell bodies are observed in the intrapancreatic ganglia of rabbits. Moreover, high performance liquid chromatography (HPLC) reveals higher levels of NE in the ganglia of the head and neck regions than in those of the body of the pancreas (Yi and Love, 2005a). It has also been reported that CHAT is colocalized with dopamine-β-hydroxylase (DβH), indicating that ACh and NE can be released from the same neurons. However, the origin of these fibers that release both ACh and NE remains unknown (Liu et al., 1998). Recent studies have also shown an increased density of noradrenergic nerve fibers in the islets of obese mice compared to control mice (Giannulis et al., 2014).
Apart from ACh and NE, many neuropeptides are also released from the intrapancreatic ganglia (De Giorgio et al., 1992a). VIP and GRP are located in both neuronal cell bodies and fibers of the intrapancreatic ganglia (De Giorgio et al., 1992bMyojin et al., 2000). VIP is present in most intrapancreatic ganglionic cell bodies, while the number of neuronal cell bodies containing GRP is much lower (De Giorgio et al., 1992b). External PACAP originates from vagal nerves, sensory nerves, or enteropancreatic nerves, while intrinsic PACAP has been observed in the neurons of intrapancreatic ganglia (Hannibal and Fahrenkrug, 2000;Kirchgessner and Liu, 2001). PACAP is co-localized with VIP in the neurons of the rat pancreas (Hannibal and Fahrenkrug, 2000). PACAP is also co-localized with galanin, SP, and corticotrophin-releasing factor (CRF) in the intrapancreatic ganglia of the sheep pancreas (Arciszewski et al., 2015). Moreover, both galanin and NPY can be observed in intrapancreatic ganglia of the bovine pancreas (Furuzawa et al., 1996;Myojin et al., 2000;Adeghate et al., 2001), with galanin being more frequently observed than NPY (Myojin et al., 2000).
Nicotinamide adenine dinucleotide phosphate (NADPH)diaphorase (d) and nitric oxide synthase (NOS) levels have been used as markers of NO production in the intrapancreatic ganglia (Sha et al., 1995;Liu and Kirchgessner, 1997). This demonstrates that apart from enteric and vagal nerve fibers, nitrinergic nerves are also present in the intrapancreatic ganglia (Wang et al., 1999;Chaudhury, 2014). Most NADPH-d-positive neurons show immunoreactivity for VIP or galanin in the chicken pancreas (Hiramatsu et al., 1994). Moreover, ACh is co-localized with VIP or NOS or both in most cholinergic neurons (Myojin et al., 2000).

ELECTROPHYSIOLOGICAL PROPERTIES AND SYNAPTIC POTENTIALS OF PANCREATIC NEURONS
The electrophysiological properties of intrapancreatic ganglionic neurons have been characterized through intracellular recording in guinea pigs, rabbits, cats, and dogs (King et al., 1989;Sha et al., 1996;Liu and Kirchgessner, 1997;Sha and Szurszewski, 1999;Love, 2000). The resting membrane potentials (RMP), input resistances (IR), action potential (AP) thresholds, AP amplitudes, time constant, and after-spike hyperpolarization (ASH) durations are similar in different species, as are the electrophysiological properties of pancreatic neurons in different regions of the pancreas (Sha et al., 1996). Electrophysiological properties of pancreatic neurons are summarized in Table 1. Pancreatic neurons have higher IR than other autonomic neurons. Most of the pancreatic neurons are classified as neurons with phasic firing patterns. A small number (9%) of rabbit pancreatic neurons show spontaneous low amplitude oscillations that resemble pacemaker potentials (Love, 2000).
Pancreatic neurons exhibit both fast and slow excitatory postsynaptic potentials (fEPSPs and sEPSPs). Stimulation of nerve bundles attached to intrapancreatic ganglia evokes multiple fEPSPs (Sha et al., 1996(Sha et al., , 1997Liu and Kirchgessner, 1997;Love, 2000) that are blocked by hexamethonium in 75% of the neurons, while non-cholinergic fEPSPs are observed in 25% of the neurons (Love, 2000). Repetitive stimulation evokes muscarinic and non-cholinergic sEPSPs, whose duration and amplitude are positively related to the stimulus trains; hexamethonium has no impact on the sEPSPs (Sha et al., 1996;Liu and Kirchgessner, 1997). sEPSPs are evoked by repetitive stimulation using atropine or BRL24924, a 5-HT 1p receptor antagonist (Sha et al., 1996(Sha et al., , 1997. Spontaneous fEPSPs are usually observed at low frequency, and action potentials (APs) are also observed due to summation of subthreshold fEPSPs (King et al., 1989). Approximately 41% of the intrapancreatic neurons in the head and body of the pancreas show spontaneous fEPSPs and APs, while only 31% of the intrapancreatic neurons in the pancreatic tail show spontaneous fEPSPs and APs (Sha et al., 1996). Pancreatic neurons are capable of coordinating intrinsic activity owing to the rhythmic bursts of fEPSPs and APs in interconnected ganglia (Love, 2000). Inhibitory postsynaptic potentials (IPSPs) have not been reported yet. Intrapancreatic ganglia of rabbits in all three regions of pancreas exhibit paired-pulse facilitation (PPF) or depression (PPD) of fEPSPs. PPF peaking and disappearing have shorter interstimulus intervals than PPD. PPF is observed mainly in the pancreatic head and neck (60%), while PPD is observed in the body of the pancreas. In the head/neck region, facilitation during the initial 1-2 s of train stimulation is reduced by midtrain depression (2-5 s) of a greater magnitude. The frequency of postsynaptic APs is inversely related to the magnitude of mid-train depression, suggesting that the electrophysiological activities of pancreatic neurons can regulate synaptic strength. Train stimulation in the head or neck of the pancreas is seen to be followed by brief post-train augmentation of fEPSPs (Sha et al., 1996;Love, 2000;Yi and Love, 2005b). Presynaptic post-train depression results in a decrease in ACh release. Regional differences in electrophysiological activities may reflect regional differences in pancreatic function (Yi and Love, 2005b;Love et al., 2007).
Neurotransmitters of external or internal origin are observed to affect ganglionic transmission. In cats, ACh evokes fast depolarization with a decrease in IR, an effect mediated by nicotinic receptors. Most fEPSPs are activated by nicotinic receptor subunits in ganglionic neurons and few fEPSPs are not sensitive to hexamethonium, indicating that there exist noncholinergic receptors in the ganglia. The slow transmission is mediated by M 1 receptors through an increase in the IR (Sha et al., 1997). However, muscarine can convert synaptic APs of rabbit pancreatic neurons into fEPSPs without any changes in the RMP or IR (Love, 2000). The electrical activity of intrapancreatic ganglia has a vital influence on amplitude regulation of pulsatile insulin secretion by isolated ganglia together with the adjacent pancreatic parenchyma (Sha et al., 2001b).
A higher percentage of neurons respond to adrenergic agonists in the pancreatic head than in the body or the tail of the pancreas, a finding consistent with the fact that the density of noradrenergic innervation to intrapancreatic ganglia in the pancreas head is higher than in the body or tail (Yi et al., 2004). A predominance of inhibitory α 2 receptors in the body of the pancreas contributes to the inhibition of ganglionic transmission, and excitatory α 1 receptors in the ganglia from the head/neck regions participate in the facilitation of ganglionic transmission. Presynaptic post-train depression (PTD) is mediated by NE and other unknown neurotransmitters (Yi and Love, 2005a).
PACAP depolarizes pancreatic neurons via the activation of ganglionic PAC 1 receptors and may enhance endocrine secretion (Kirchgessner and Liu, 2001). Guinea pigs Liu and Kirchgessner (1997) Rabbits Love (2000) Dogs Sha and Szurszewski (1999) Cats King et al. (1989) Region of the cat pancreas Sha et al. (1996)  RMP, resting membrane potentials; IR, input resistances; AP, action potential; ASH, after-spike hyperpolarization. All the values represent mean ± standard error of the data.
While NO evokes membrane hyperpolarization in neurons of the cat pancreas, the membrane IR does not markedly change. Inhibition of NO production may enhance release of ACh to evoke fEPSPs and increase the amplitude of sEPSPs (Sha et al., 1995).
Serotonergic nerve fibers are found in the intrapancreatic ganglia of guinea pigs and cats, but are rare or absent in rabbits (Ma and Szurszewski, 1996b;Liu and Kirchgessner, 1997;Yi et al., 2004). No 5-HT immunoreactive neurons are observed in cat intrapancreatic ganglia (Sha et al., 1996). Neurons in the pancreatic head have a higher excitability in response to 5-HT than those in the tail. The responses of neurons of the intrapancreatic ganglia to 5-HT with fast and slow depolarization are mediated by 5-HT 3 and 5-HT 1 p receptors, respectively (Ma and Szurszewski, 1996b;Sha et al., 1996).
Cholecystokinin (CCK) has been shown to be present in nerve terminals which surround but are not in the intrapancreatic ganglia. The vagal and sympathetic nerve fibers may be the source of CCK in the pancreas. In cats, cholecystokinin octapeptide (CCK-8) can evoke slow depolarization by acting on CCK B postsynaptic receptors, and amplify nicotinic transmission by facilitating the release of ACh from preganglionic nerve terminals or by increasing the sensitivity of postsynaptic membranes to ACh (Cantor and Rehfeld, 1984;Ma and Szurszewski, 1996a).
Investigation of electrophysiological effects of gammaaminobutyric acid (GABA) on cat pancreatic neurons indicates that GABA acts on the ganglia through GABA A receptors, which can cause depolarization and inhibit fEPSPs (Sha et al., 2001a). While endogenous GABA has been shown to be stored in and released from the ganglionic glial cells, the origin of the GABA remains unclear (Sha et al., 2001a). Leptin promotes fast synaptic transmission by acting on presynaptic nerve terminals of intrapancreatic ganglia in dogs (Sha and Szurszewski, 1999).

NEURAL REGULATION OF ISLETS
Intrapancreatic ganglia are the integration center for extrinsic nerve inputs and intrinsic neuronal inputs. This integration center plays an important role in regulating pancreatic endocrine function. The extrinsic nerves modulate pancreatic endocrine function either directly at the islet level or by going through the integration center (Gylfe and Tengholm, 2014;Di Cairano et al., 2016 ; Figure 3).

Innervation of Islets
The modes of innervation of pancreatic islets vary across species. In mice, the postganglionic parasympathetic fibers contact all types of endocrine cells (α, β, δ, and PP cells) in the islets, while the postganglionic sympathetic fibers innervate α cells and smooth muscle cells of the blood vessels, but not the β cells (Dolensek et al., 2015). The islets are sparsely innervated in adult humans. Parasympathetic postganglionic fibers are observed to contact exocrine tissue preferentially. Most sympathetic nerves innervate smooth muscle cells of the blood vessels in the adult human pancreas. However, the human fetal pancreas is richly innervated (Taborsky, 2011;Krivova et al., 2016). Islet innervation is correlated with age and environmental factors in humans and rats (Proshchina et al., 2014;Mundinger et al., 2016;Tang et al., 2018b). The integration between epithelial cells FIGURE 3 | Effects of neurotransmitters on insulin secretion. Neurotransmitters modulate the insulin secretion by activating different receptors both directly at the islets and indirectly at the ganglionic level. ACh, acetylcholine; GRP, gastrin-releasing peptide; VIP, vasoactive intestinal peptide; NO, nitric oxide; NE, noradrenaline; NPY, neuropeptide Y; PACAP, pituitary adenylate cyclase activating polypeptide; 5-HT, serotonin; CCK, cholecystokinin; CGRP, calcitonin gene-related peptide; SP, substance P. and components of the nervous system may form the neuroinsular complexes seen during cellular differentiation (Krivova et al., 2016). In addition, the composition and cytoarchitecture of endocrine cells are also different in humans and rats (Rodriguez-Diaz and Caicedo, 2014;Thorens, 2014;Dolensek et al., 2015;Da Silva Xavier, 2018). Current findings indicate that different species may have different mechanisms for neural regulation of pancreatic endocrine function.

Intrapancreatic Ganglionic Regulation of Islets
As the integrated innervation enters the pancreas, intrapancreatic ganglia directly innervate islets, thereby modulating islet secretion.
Tract-tracing of intrapancreatic ganglionic neurons shows that intrapancreatic ganglia send nerve fibers directly to the islets (Kirchgessner and Pintar, 1991). This ganglionislet association has been recently demonstrated by a threedimensional panoramic histology study (Tang et al., 2018b). In vivo ganglionic nicotinic activation stimulates insulin and glucagon secretion in the mouse pancreas . Cholinergic nerves of the intrapancreatic ganglia regulate the oscillatory pattern of basal endocrine secretion Fendler et al., 2009). Activation of postganglionic cholinergic nerves by GRP regulates insulin secretion in vivo ). An electrophysiological study in a ganglion-islet attached preparation setting provides direct evidence for the modulation of insulin secretion by the intrapancreatic ganglia. The activity of intrapancreatic ganglionic neurons directly stimulates insulin secretion, while blockage of this activity removes the modulation (Sha et al., 2001b).

Sympathetic Regulation of Islets
Sympathetic nerves regulate pancreatic endocrine secretion with multiple direct and indirect mechanisms through their actions on islets, blood vessels, and intrapancreatic ganglia (Yi et al., 2005). Electrical stimulation of sympathetic nerves induces NE release and has an inhibitory effect on basal and glucose-stimulated insulin release by acting on the α 2 -adrenoceptor of β cells in dogs and calves. Sympathetic nerves do not participate in the inhibition of basal insulin secretion in rats, pigs, and humans (Ahrén, 2000). NE can also stimulate insulin secretion by acting on the β 2 -adrenoceptor of β cells or by acting on the α 2 -and β 2 -adrenoceptors of α cells, where glucagon secretion leads to an increase in insulin secretion (Ahrén, 2000). In general, the overall effect of NE is lowering the plasma insulin concentration (Rodriguez-Diaz and Caicedo, 2013). In humans, the primary targets of the sympathetic fibers are vascular smooth muscle cells, which can reduce blood flow to regulate secretion (Rodriguez-Diaz and Caicedo, 2013;Dolensek et al., 2015). Patients with type 1 diabetes show a severe loss of islet sympathetic nerves. Moreover, rats with short-term hyperglycemia show a dysfunction in the islet sympathetic innervation from the celiac ganglia, while hyperphagic weaning mice show markedly increased sympathetic innervation compared to control mice (Mundinger et al., 2015(Mundinger et al., , 2016Tang et al., 2018b).
Activation of the sympathetic pathway potentiates glucagon secretion and inhibits somatostatin secretion. However, the effects of sympathetic nerve activity on pancreatic polypeptide (PP) secretion are different across species, in that it enhances PP secretion in pig and human pancreases, but inhibits PP secretion in the dog pancreas (Ahrén, 1999;Gilon and Henquin, 2001;Rodriguez-Diaz and Caicedo, 2014).
Both NPY and galanin are known to mediate insulin secretion (Chandra and Liddle, 2013). NPY inhibits glucose-induced insulin secretion in rats and mice, but has no effect in dogs (Dunning et al., 1987;Pettersson et al., 1987;Morgan et al., 1998). Galanin inhibits insulin secretion in dogs but not in humans, as the innervation of galanin-containing nerves is abundant in dog islets, but rare in human pancreatic islets (Ahrén and Lindskog, 1992;Ahrén, 2000). Interestingly, both NPY and galanin have an insulinotropic effect on the pig pancreas (Sheikh et al., 1988). NPY binds to the Y1 receptor subtype, while galanin interacts with GA1R1 receptors in β cells to mediate insulin secretion (Parker et al., 1995;Morgan et al., 1998;Chandra and Liddle, 2009). Oral administration of galanin has been shown to improve insulin sensitivity by decreasing duodenal contraction in diabetic mice (Abot et al., 2018b).

Parasympathetic Regulation of Islets
Insulin secretion is stimulated by the electrical activation of parasympathetic fibers and inhibited by vagotomy (Ahrén, 2000;Teff, 2008;Balbo et al., 2016). The density of parasympathetic axons is lower in the exocrine tissue of the pancreas in individuals with new-onset type 1 diabetes (Lundberg et al., 2017) than in control individuals. ACh released by postganglionic nerve fibers acts on muscarinic receptors of β-cells and stimulates insulin secretion directly ( Van der Zee et al., 1992). The M 3 receptor subtype plays an important role in regulating cholinergic nerves to induce secretion of islet hormones (Karlsson and Ahrén, 1993;Gautam et al., 2006;Miranda et al., 2016). ACh stimulates glucagon and PP secretion that can be blocked by atropine, and the effect of ACh on somatostatin secretion varies across species (Gilon and Henquin, 2001). VIP, PACAP, and GRP can stimulate the secretion of glucagon and insulin. Given their similar structures, VIP and PACAP have common receptors called VIP 1 and VIP 2 receptors, whereas the PAC 1 receptor is specific for PACAP (Kirchgessner and Liu, 2001). GRP directly acts on the islets via the GRP receptors . As the nerves containing VIP are found very close to α cells in dogs, α cells are more sensitive to VIP than β cells (Havel et al., 1997).

Sensory Nerve-Mediated Regulation of Islets
Sensory nerves regulate endocrine secretion through two mechanisms: (a) direct action on the islet receptors, and (b) formation of neural circuits with adrenergic nerves (Ahrén, 2000). CGRP inhibits glucose-induced insulin release and stimulates glucagon secretion Silvestre et al., 1996), but the specific CGRP receptor that mediates these processes has not yet been identified. There is considerable variation in reports of the effects of SP on insulin release (Hermansen, 1980;Chiba et al., 1985;Shen et al., 2016). SP induces a marked increase in insulin secretion from the pancreas of healthy rats, but inhibits insulin secretion from the pancreas of diabetic rats, indicating that it may play a role in the onset of diabetes (Adeghate et al., 2001;Shen et al., 2016). It is generally considered that SP interacts with neurokinin G-protein-coupled receptors (NK-R) on islets. In rats, it is suggested that the role of vagal afferents is to suppress insulin secretion (Meyers et al., 2016). The role of sensory nerves in mediating glucose homeostasis remains unexplored.

CONCLUSION AND PERSPECTIVES
Intrapancreatic ganglia constitute endogenous neural networks and release various neurotransmitters. They integrate both intrinsic and external nerve inputs to play an important role in pancreatic endocrine secretion. The mechanisms underlying the role of intrapancreatic ganglia in islet function remain relatively unclear. Further studies are needed to elucidate the complete mechanisms underlying the functions of intrapancreatic ganglia in normal physiological and disease states of the pancreas. This may provide us with novel ways to treat diseases of the pancreas.

AUTHOR CONTRIBUTIONS
WL researched the articles and wrote the manuscript. GY, YL, and LS revised the draft before submission. All authors have made direct and intellectual contribution to the work, and approved it for publication.


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Isolated Silymarin Flavonoids Increase Systemic and Hepatic Bilirubin Concentrations and Lower Lipoperoxidation in Mice

Bilirubin is considered to be one of the most potent endogenous antioxidants in humans. Its serum concentrations are predominantly affected by the activity of hepatic bilirubin UDP-glucuronosyl transferase (UGT1A1). Our objective was to analyze the potential bilirubin-modulating effects of natural polyphenols from milk thistle (Silybum marianum), a hepatoprotective herb. Human hepatoblastoma HepG2 cells were exposed to major polyphenolic compounds isolated from milk thistle. Based on in vitro studies, 2,3-dehydrosilybins A and B were selected as the most efficient compounds and applied either intraperitoneally or orally for seven days to C57BL/6 mice. After, UGT1A1 mRNA expression, serum, intrahepatic bilirubin concentrations, and lipoperoxidation in the liver tissue were analyzed. All natural polyphenols used increased intracellular concentration of bilirubin in HepG2 cells to a similar extent as atazanavir, a known bilirubinemia-enhancing agent. Intraperitoneal application of 2,3-dehydrosilybins A and B (the most efficient flavonoids from in vitro studies) to mice (50 mg/kg) led to a significant downregulation of UGT1A1 mRNA expression (46 ± 3% of controls, p < 0.005) in the liver and also to a significant increase of the intracellular bilirubin concentration (0.98 ± 0.03vs.1.21 ± 0.02 nmol/mg, p < 0.05). Simultaneously, a significant decrease of lipoperoxidation (61 ± 2% of controls, p < 0.005) was detected in the liver tissue of treated animals, and similar results were also observed after oral treatment. Importantly, both application routes also led to a significant elevation of serum bilirubin concentrations (125 ± 3% and 160 ± 22% of the controls after intraperitoneal and oral administration, respectively, p < 0.005 in both cases). In conclusion, polyphenolic compounds contained in silymarin, in particular 2,3-dehydrosilybins A and B, affect hepatic and serum bilirubin concentrations, as well as lipoperoxidation in the liver. This phenomenon might contribute to the hepatoprotective effects of silymarin.

Introduction
Bilirubin, the end product of heme catabolism in the systemic circulation, is a potent antioxidant substance [1]. Despite the fact that for decades bilirubin has been considered a toxic catabolic waste product and an ominous sign of liver dysfunction, its role as a powerful protective molecule has increasingly been recognized [2]. In vitro and in vivo studies have shown that bilirubin may suppress the oxidation of lipids [1] and has anti-inflammatory [3], antiproliferative [4], antigenotoxic [5], antimutagenic [6], or even anti-aging properties [7]. Interestingly, bilirubin has been reported as a potent peroxisome proliferator-activated receptor-α (PPARα) agonist, thus acting as a real endocrine molecule, with all potential clinical consequences [8]. The clinical evidence is even more important. Bilirubin, when only mildly elevated, has been demonstrated to protect from a wide array of oxidative stress-related diseases, including cardiovascular diseases, certain cancers, and autoimmune or neurodegenerative diseases [2,9]. In fact, substantial protective effects of mild unconjugated hyperbilirubinemia, as seen in subjects with Gilbert syndrome (benign hyperbilirubinemia), have been reported for atherosclerotic diseases in particular [10].
Bilirubin production is dependent on heme oxygenase (HMOX) activity, but systemic bilirubin concentrations are predominantly affected by hepatic bilirubin UDP-glucuronosyl transferase (UGT1A1), its biotransforming enzyme [11]. Partial inhibition of bilirubin glucuronosylation was proposed as a wise strategy used to induce "iatrogenic" Gilbert syndrome [12]. This indeed was demonstrated as a "side effect" of several drugs used in various indications, typically for atazanavir, whose administration is often associated with mildly elevated concentrations of unconjugated bilirubin. Surprisingly, hyperbilirubinemia induced by atazanavir was reported to decrease markers of oxidative stress [13] and cardiometabolic risk factors [14], as well as endothelial functions [15], but safer approaches are certainly needed.
Since xenobiotics used in clinical medicine are often associated with potentially severe side effects, natural compounds interfering with the UGT1A1 hepatic biotransformation system seem a better strategy to induce mild hyperbilirubinemia. Based on the scarce data reported in the literature (mainly as the result of investigating potential nutraceuticals/herb-drug interactions), it seems that flavonoids from silymarin might modulate this enzyme. This assumption is based on the fact that many herbal extracts including silymarin, a seed extract of milk thistle (Silybum marianum (L.) Gaertn.), are rich in phenolic phytochemicals that are substrates for UGT1A1 or even exhibit UGT1A1-inhibiting activities [16][17][18]. Indeed, therapy for prostate cancer patients with high doses of silybin (silibinin) has been associated with unconjugated hyperbilirubinemia, which was considered by the authors as an adverse effect of such treatment [19]. Similar findings were also reported in hepatitis C patients receiving silybin therapy [20][21][22][23]. Although most of the experimental reports as well as some clinical data suggest its beneficial role, silymarin is generally considered to have a negligible importance clinically [24]. There are many possible reasons: one of them being the poorly defined content of the active ingredients and also the improperly characterized biological properties of individual pure flavonoids in the silymarin complex [25,26]. Silymarin is a mixture of 5 major flavonolignans (silybins A and B, isosilybin A, silychristin A, and silydianin) plus their precursor taxifolin, as well as other minor polyphenolic compounds ( Figure 1) [27]. Among them, the 2,3-dehydroflavonolignans such as 2,3-dehydrosilybins A and B possess potent biological activities [28][29][30][31][32].

Cell
Cultures. The HepG2 human hepatoblastoma cell line was used for the in vitro studies (ATCC, Manassas, VA, USA). Cells were cultured in MEM Eagle medium, containing 10% of fetal bovine serum, in 75 cm 2 culture flasks, at 37°C, in a 5% CO 2 atmosphere. For an estimation of the intracellular bilirubin concentration, the cells were plated in 10 cm Petri dishes and treated with natural polyphenols dissolved in DMSO (vehicle; 0.66%, v/v) for 24 h. For qPCR measurement, the cells were cultured in 24-well plates and treated with natural flavonoids dissolved in DMSO (vehicle; 0.66%, v/v) for 4 h. The cell lines were authenticated at ATCC by STR profiling before distribution and also reauthenticated at the end of the study by an external laboratory (Generi Biotech, Hradec Králové, Czech Republic).

MTT Cytotoxicity
Assays. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay in a 96-well format was used for the determination of the cytotoxicity of the compounds tested. HepG2 cells were seeded into a 96-well plate at a density of 1 × 10 5 cells/well.   Table 1), and 6 μL of a 1/5 dilution of the RT products. qPCR for the liver tissue was performed using a TaqMan® Gene Expression Assay Kit (Life Technologies). Threshold cycle (Ct) values were analyzed using the comparative Ct (ΔΔCt) method as recommended by the manufacturer (Applied Biosystems, Foster City, CA, USA). The data were normalized to the expression of hypoxantin phosphoribosyl transferase (HPRT) and expressed as the multiplicity change from the control levels.
2.6. Determination of Bilirubin. For the determination of bilirubin in HepG2 cells and liver tissue, the biological materials were sonicated on ice and then extracted with methanol/ chloroform/hexane 10/5/1 (v/v/v) against PBS buffer (pH 6.2). The lower organic phase was subsequently extracted into 50 μL of carbonate buffer (pH 10) in hexane. The resulting polar droplet was loaded onto a C-8 reverse phase column (Luna 3 μm, 150 × 4 6 mm, Phenomenex, Torrance, CA, USA), and bilirubin was determined using an HPLC Agilent 1200 with a diode array detector (Agilent, Santa Clara, CA, USA) as described earlier [36,37]. The concentration of bilirubin was calculated in nmol/g of wet tissue or pmol/mg of protein for tissue samples or cell cultures, respectively.
For determination of bilirubin in serum, the LC-MS/MS method was used. Ten μL of serum was mixed with 2.5 μL of internal standard (mesobilirubin, c = 20 μmol/L) and deproteinized by 50 μL of 1% BHT in methanol, 50 μL of 0.4% ascorbic acid in methanol, and 200 μL of methanol (pH 11). Five μL of the resulting supernatant was loaded on a Poroshell 120 EC-C18 2.1 μm 3 0 × 50 mm column (Agilent), with a gradient of 1 mM NH 4 F in water (A) and methanol (B) as follows: 60% of B was changed in 5 minutes to 100% of B, with a flow rate of 0.4 mL/min and was kept at 100% of B for 3 minutes, with the gradient of flow rate at 0.5 mL/min. The flow was then maintained at 0.5 mL/min until the end of the gradient program. Phase B was changed back to 60% in 0.1 minutes and was kept at 60% until the end of the program at 10 minutes. The mass spectra were recorded on an Agilent 6470 LC/QQQ (Agilent) LC-MS/MS device with electrospray ionization and multiple reaction monitoring in a positive mode. Conditions of the electrospray ionization source, gas flows, and potentials of the mass spectrometer were manually tuned for high sensitivity and a low signal-to-noise ratio of the analytes: ion spray voltage, 3000 V; source temperature, 250°C; heater gas, 8 L/min; nebulizer, 300 psi; sheath gas temperature, 400°C; sheath gas flow, 12 L/min; and nozzle voltage, 500 V. Within the total scan time of 10 min, m/z 585.3 → 299.1, 16 eV collision energy (CE) at 5.96 min was monitored for bilirubin and m/z 589.3 → 301.2, 20 eV CE at 6.084 min for mesobilirubin.

HMOX Activity Assay.
Twenty μL of liver sonicate (10%) was incubated for 15 min at 37°C in CO-free septum-sealed vials containing 20 μL of 150 μM heme and 20 μL of 4.5 mM NADPH, as previously described [38]. Blank reaction vials contained potassium phosphate buffer in place of NADPH. The amount of CO generated by the reaction and released into the vial headspace was quantified by gas chromatography (GC) with a reduction gas analyzer (Peak Laboratories, Mountain View, CA, USA). HMOX activity was calculated as pmol CO/h/mg fresh weight and expressed as percentage of the control.
2.8. UGT1A1 Inhibition Assay. The UGT activity in the microsomal samples was determined by a UGT-Glo™ assay kit according to the manufacturer's instructions (Promega, Madison, WI, USA). Briefly, microsomes with recombinant UGT1A1 (0.0125 mg/mL) were incubated with 16 mM UDPGA and 20 μM multienzyme substrate with increasing concentrations of 2,3-dehydrosilybins A and B at 37°C for 30 min. Then, 40 μL of reconstituted luciferin detection reagent containing D-cysteine was added, and the luminescent signal was allowed to stabilize for 20 min at room temperature. Luminescence was read on a Synergy II plate reader (BioTek, VT, USA). The data were analyzed using the curve fitting method with GraphPad Prism (GraphPad Software, San Diego, CA).
2.9. Malondialdehyde Determination. Malondialdehyde (MDA) in the tissue homogenates was measured according to the method described by Wills [39], with some modifications. A 200 μL aliquot of 10% tissue homogenate was mixed with 2 mL of the thiobarbituric acid-trichloroacetic acid reagent (0.375 and 15%, respectively). The mixture was heated on a water bath at 95°C for 20 min. The solution was then cooled to room temperature. The reaction product (thiobarbituric acid-MDA complex) was extracted by adding 3 mL of n-butanol to the above solution. The absorbance of the pink-colored extract in n-butanol was measured at 532 nm using a spectrophotometer (Sunrise, Tecan, USA). The amount of MDA was calculated using the molar extinction coefficient of 1 56 × 10 5 M −1 × cm −1 and expressed as a percentage of the control.
2.10. 2,3-Dehydrosilybin Determination in Sera. 2,3-Dehydrosilybin concentrations in serum samples were determined after deconjugation of phase II conjugates (sulfates and glucuronide). The activity of the deconjugation enzymes (β-glucuronidase and sulfatase) was checked on the selected glucuronides and sulfates prior to analysis. All the samples were measured in duplicate.
The conjugates in serum samples were deconjugated in acetate buffer (50 mM, pH 5.0) by adding β-glucuronidase (440 U) with sulfatase (35 U), incubated for 2 h at 37°C and 600 rpm. The reaction mixture was then freeze-dried, and an internal standard (2,3-dehydrosilychristin, 4.8 μg/mL, 150 μL, in acetonitrile/DMSO 1 : 1) was added. Samples were incubated (1 h, 37°C, 600 rpm) and centrifuged (15 min), and the supernatant was injected into a LC-MS (injection volume 25 μL). 2.11. Animal Studies. Female C57BL/6 mice (n = at least 6 in each group, 8 w old) obtained from Velaz (Prague, Czech Republic) had access to both water and a standard diet ad libitum. An equimolar mixture of dehydrosilybins A and B dissolved in DMSO (vehicle; 5%, v/v) was applied intraperitoneally, and individual dehydrosilybins were applied orally for seven days at a dose of 50 mg/kg b.wt. After 24 h of the last application, the mice were sacrificed and blood from their superior vena cava was collected for further analyses. The livers were cleaned and stored as pieces in nitrogen until used. For RNA analysis, 100 mg of each tissue was immediately placed in 1.5 mL microfuge tubes containing RNAlater and stored following the manufacturer's protocol until the RNA isolation was performed.
All animal studies met the criteria for the care and use of animals in experiments and were approved by the Animal Research Committee of the 1st Faculty of Medicine, Charles University, Prague.
2.12. Statistical Analyses. All data are expressed as mean ± SEM. Depending on their normality, the data were analyzed either by the Student t-test or the Mann-Whitney rank sum test and Kruskal-Wallis ANOVA with Dunn's correction. Differences were considered statistically significant when P values were less than 0.05.

The Effect of Silymarin Flavonoids and Related
Compounds on HMOX and UGT1A1 Expressions and Activities in HepG2 Cells. In our in vitro screening studies, focused on evaluation of the possible modulating effect of silymarin flavonoids and related compounds on HMOX1 and UGT1A1 expressions, a wide array of compounds were used (Figure 1). Based on the MTT assays (Table 1), HMOX1 and UGT1A mRNA expression analyses were performed with the nontoxic plus 1/2 of nontoxic concentrations for HepG2 cells.
Significant HMOX1 mRNA overexpression was observed for quercetin and isoquercitrin; on the other hand, HMOX1 mRNA downregulation was present after exposure to 2,3-dehydrosilybins A and B (Figure 2). More importantly, the activity of HMOX was upregulated not only by quercetin and isoquercitrin but also after exposure to the silymarin complex per se, as well as silybins A and B. Downregulation of HMOX1 mRNA expression by 2,3-dehydrosilybins A and B (Figure 2) was also reflected by decreased HMOX activity (Figure 3(a)).

The Effect of Silymarin Flavonoids and Related
Compounds on Intracellular Concentrations of Bilirubin in HepG2 Cells. To investigate whether the effects of silymarin flavonoids and related compounds on HMOX and UGT1A1 are translated into phenotypic changes, we analyzed bilirubin concentrations within the HepG2 cells after 24 h exposure to individual compounds.
Most of silymarin flavonoids and related compounds significantly elevated intracellular bilirubin ( Figure 4) in a dose-dependent manner (data not shown). Interestingly, the increase of intracellular bilirubin concentrations caused by exposure of both 2,3-dehydrosilybins A and B was even higher than that caused by atazanavir, a known inhibitor of UGT1A1 [40] (Figure 4).
Based on these in vitro results, 2,3-dehydrosilybins A and B were selected for the in vivo studies.

The Effect of 2,3-Dehydrosilybins A and B on Bilirubin Metabolism in Mice
3.3.1. Concentrations of 2,3-Dehydrosilybins in Sera. Since 2,3-dehydrosilybins have a low solubility in water and the poor oral bioavailability might contribute to the low clinical efficiency of silymarin in humans, we first measured concentrations of 2,3-dehydrosilybins in sera to verify whether they pass into the systemic circulation. Use of both application routes (i.e., intraperitoneal as well as oral administration of 2,3-dehydrosilybins (50 mg/kg b.wt.)), after 24 h, resulted in a substantial appearance in the systemic blood, reaching serum concentrations up to 300 ng/mL (0.62 μmol/L) ( Figure 5).

Hepatic HMOX1 and UGT1A1 mRNA Expressions.
Then we analyzed the effect of 2,3-dehydrosilybin administration on HMOX1 and UGT1A1 mRNA expressions in the livers of treated mice. Treatment with an equimolar mixture of 2,3-dehydrosilybins A and B (50 mg/kg b.wt.) administered intraperitoneally for 7 days led to a significant downregulation of UGT1A1 mRNA expression in the liver (57 ± 19% of the controls) with no change in HMOX1 expression ( Figure 6(a)). Similar results were observed for oral treatment with 2,3-dehydrosilybin A (downregulation of UGT1A1 mRNA expression to 55 ± 26% of the control, Figure 6(b)); while no effect was demonstrated for 2,3-dehydrosilybin B. None of the individual dehydrosilybins had any effect on HMOX1 expression (Figure 6(b)).
Neither intraperitoneal nor oral administration of 2,3-dehydrosilybins caused any increase in markers of liver damage (ALT, AST, and ALP activities; data not shown).

Hepatic
Lipoperoxidation. Treatment of mice with 2,3-dehydrosilybins had consistent inhibitory effects on lipid peroxidation in the liver tissue. MDA concentrations in the liver tissue were significantly reduced after intraperitoneal   application of the mixture of 2,3-dehydrosilybins (to 70 ± 7% of the control; Figure 7(a)), and the same effect was also observed after oral administration of both 2,3-dehydrosilybins A and B (61 ± 15% and 64 ± 13% of the control, respectively; Figure 7(b)).

Discussion
Bilirubin, a bile pigment, for decades considered only an ominous sign of liver diseases, has been revisited as a potent antioxidant, immunosuppressive, and cytoprotective agent [2,9,10,41]. Even single micromolar elevations of systemic concentrations of bilirubin, still within the physiological range, are associated with a substantial decreases of the risks of cardiovascular, cancer, and inflammatory diseases [42][43][44], and this association is clearly evident in subjects with Gilbert syndrome, characterized with mild systemic elevations of unconjugated bilirubin [9,45]. This led to the suggestion to induce Gilbert syndrome iatrogenically in order to suppress the development of oxidative stress-related diseases [12].
As reported in our recent study, due to a dynamic equilibrium, a correlation between systemic and intracellular concentrations of bilirubin exists in the liver (and also probably in other organs and tissues) [37]. Thus, it is important to monitor intrahepatic concentrations of bilirubin when assessing potential hepatoprotective effects, in particular when considering the fact that hepatic UGT1A1 is the major enzyme affecting systemic concentrations of bilirubin [11]. Hepatic UGT1A1 is a biotransforming enzyme important not only for bilirubin but also for a variety of other endogenous substances as well as xenobiotics, including natural agents often used as nutraceuticals, including the flavonolignans and flavonols of the silymarin complex. In fact, inhibitory effects towards UGT1A1 were reported for silybin, a major flavonoid constituent of milk thistle extract [46]; indeed, the therapy with this substance has also been associated with marked unconjugated hyperbilirubinemia [19]. A significant increase in serum bilirubin levels was also reported in other human studies on patients with chronic hepatitis C receiving silybin therapy [20][21][22][23]. These clinical data are consistent with our findings, i.e., increased intracellular as well as systemic concentrations of bilirubin upon exposure to silymarin flavonoids. It is important to emphasize that these effects differed substantially among individual flavonoids-dehydrosilybins A and B being the most efficient.
Despite generally low bioavailability of silymarin flavonoids, effective serum concentrations of both 2,3-dehydrosilybins A and B were detected in our study (up to 300 ng/mL ( Figure 5), corresponding to concentrations of 0.62 μmol/L). This value entirely fits to the mean peak plasma concentration of silybin, reached after oral administration of a 700 mg dose of silymarin (containing approximately 250 mg of silybin), which was 0.6 μmol/L [46]. Very similar plasma silybin concentrations were reported in another human study (0.4, 1.4, and 4 μmol/L after 360, 720, or 1440 mg of silybin administered daily for 7 days), with corresponding concentrations in the liver tissue [47], as well as in the studies by Barzaghi [49].
In addition, silybin was demonstrated to potently inhibit UGT1A1, with the IC 50 value correlating with the clinically relevant plasma concentrations [46]. Similar inhibitory concentrations were also reported by others for other silymarin flavonoids, although 2,3-dehydrosilybins were not tested in these studies [50]. These data are consistent with our findings, with the UGT1A1 IC 50 value for 2,3-dehydrosilybin A = 2 1 μmol/L and the mean serum concentration of 0.62 μmol/L. Importantly, we found the UGT1A1 IC 50 value for 2,3-dehydrosilybin A even lower than that reported for atazanavir (2.3 μmol/L), the well-known hyperbilirubinemia-inducing drug [40] indicating the high bilirubinemia-enhancing potential of this flavonolignan.
Although the UGT1A1 IC 50 values of 2,3-dehydrosilybin A are slightly higher than those reported for silybin, 2,3-dehydrosilybin A most efficiently increased both the hepatic intracellular and systemic concentrations of bilirubin. The reason for this might be that 2,3-dehydrosilybin A also affects other mechanisms implicated in hepatic bilirubin metabolism. Such an example may include the modulation of the basolateral bilirubin organic anion-transporting proteins OATP1B1/3, which are also inhibited by silymarin flavonoids [51].
2,3-Dehydrosilybins, together with other 2,3-dehydroflavonolignans, belong to the minor flavonoids of the silymarin complex, usually accounting for 1-2% of all flavonolignans (1.8% in the preparation used in the present work; Supplementary Figure 3). Their biological effects, however, might be of real clinical importance [28]. This is supported by a recent observation that 2,3-dehydrosilybins A/B significantly suppressed oxidative stress in C. elegans  resulting in lifespan extension [32]. Although the effect on bilirubin metabolism was not investigated in this study, it is important to note that the antiaging effects of bilirubin have been demonstrated in another of our studies [7]. In this respect, it is also important to emphasize the antioxidant effects of 2,3-dehydrosilybins observed in our study. Indeed, decreased MDA concentrations in the livers of mice treated with 2,3-dehydrosilybins might have been mediated, at least partially, via increased intracellular bilirubin concentrations. It is thus likely that these antioxidant effects are not directly related to antioxidant activities of the flavonolignans per se, but rather parahormetic action is more important [52].

Conclusions
Natural silymarin flavonolignans contained in milk thistle and related flavonols, in particular 2,3-dehydrosilybins A and B, affect hepatic and serum bilirubin concentrations, as well as lipoperoxidation in the liver. This phenomenon might contribute to the hepatoprotective effects of silymarin observed in many, although not all clinical studies. Modulation of bilirubin metabolism by well-defined natural polyphenols can represent a safe chemopreventive approach against oxidative stress-mediated diseases including atherosclerosis, cancer, diabetes, or inflammatory diseases.