Effect of Modified Da Chaihutang on FXR/FGF15/FGFR4 Signaling Pathway in Mice with Cholesterol Gallstone Differentiated into Damp-Heat Syndrome / 中国实验方剂学杂志

ObjectiveTo observe the effect of modified Da Chaihutang on cholesterol gallstone （CS） in mice due to damp-heat based on the farnesoid X receptor （FXR）/fibroblast growth factor 15 （FGF15）/fibroblast growth factor receptor 4 （FGFR4） pathway and explore the molecular biological mechanisms of CS differentiated into damp-heat syndrome from the perspective of correspondence between prescription and syndrome. MethodForty-eight six-week-old mice were randomly divided into the blank group， model group， modified Da Chaihutang （23.4 g·kg-1） group， and ursodeoxycholic acid （0.12 g·kg-1） group， with 12 mice in each group. The ones in the latter three groups were exposed to "internal dampness + external dampness + high-cholesterol diet" for 12 weeks for inducing CS due to damp-heat. Mice in the modified Da Chaihutang group and ursodeoxycholic acid group were gavaged with the corresponding drugs， while those in the model and blank groups with the same amount of normal saline for a total of four weeks. Before and after modeling， mice in each group were subjected to open field tests for determining their activities and mental states. Such general conditions as body mass， food intake， fur， and urine and stool of mice in each group were observed and recorded weekly for judging the damp-heat syndrome. After the intervention， the sampled liver and gallbladder tissues of mice in each group were stained with hematoxylin-eosin （HE） staining， and the serum γ-glutamyltransferase （GGT）， alkaline phosphatase （ALP）， and total bilirubin （TBIL） were determined. The total cholesterol （TC） and total bile acid （TBA） contents in bile were measured by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expression levels of FXR， FGF15， FGFR4， and cholesterol 7α-hydroxylase gene （CYP7A1） were assayed by real-time fluorescence quantitative polynucleotide chain reaction （Real-time PCR） and Western blot. ResultCompared with the blank group， the model group exhibited enlarged gallbladder， brown turbid bile with flocculent precipitation visible to the naked eye， obvious damp-heat syndrome， lipoid degeneration in the liver tissue， rough and thickened gallbladder wall， elevated ALP， GGT， and TBIL in serum （P<0.01） and TC in bile （P<0.01）， reduced TBA （P<0.01）， up-regulated FXR， FGF15， and FGFR4 mRNA and protein expression in ileum （P<0.05， P<0.01）， and down-regulated CYP7A1 mRNA and protein expression （P<0.01）. Compared with the model group， the two medication groups displayed improved bile turbidity， and the bile in the modified Da Chaihutang group became clearer. After intervention， the damp-heat syndrome of mice in the modified Da Chaihutang group was significantly alleviated. The liver and gallbladder lesions of mice in the two medication groups were significantly relieved， manifested as reduced serum ALP， GGT， and TBIL （P<0.01）. The reduction in ALP and TBIL of the modified Da Chaihutang group was more significant （P<0.01）. The TC contents in the bile of mice from the two medication groups were significantly lowered， whereas the TBA contents were elevated （P<0.01）， with more significant changes present in the modified Da Chaihutang group （P<0.01）. The mRNA and protein expression levels of FXR， FGF15， and FGFR4 in the modified Da Chaihutang group were down-regulated （P<0.05， P<0.01）， while the mRNA and protein expression levels of CYP7A1 rose （P<0.05）， except that the elevation in FGF15 and FGFR4 protein expression and reduction in CYP7A1 protein expression were not significant. The mRNA and protein expression levels of FXR， FGF15， and FGFR4 in the ursodeoxycholic acid group all decreased， among which the reduction in FXR was remarkable （P<0.05）， and the mRNA and protein expression levels of CYP7A1 were significantly up-regulated （P<0.05）. ConclusionModified Da Chaihutang significantly improves the stone， liver function， bile composition， abnormal cholesterol-bile acid metabolism， and damp-heat syndrome in the model mice of CS differentiated into damp-heat syndrome， which may be related to its regulation of key factors FXR， FGF15， FGFR4， and CYP7A1 mRNA and protein expression in the cholesterol-bile acid metabolism pathway.

The Role of Bile Acid Receptors in Non-alcoholic Fatty Liver Disease (NAFLD) / 中国生物化学与分子生物学报

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease and its incidence is increasing year by year worldwide. Insulin resistance and derangement of lipid metabolism, accompanied by activation of the pro-inflammatory response and fibrosis, play an important role in its occurrence and development. However, the understanding of NAFLD is still very poor, and there is still a lack of effective drugs in clinical practice. Researchers are trying to explore the etiology of NAFLD and a new breakthrough in its treatment. Bile acid is one of the many metabolites synthesized in the liver. In addition to helping fat digestion and absorption, bile acid also acts as a signal molecule to activate the bile acid receptor, which is an important transcriptional regulatory factor and plays an important role in maintaining normal physiological metabolism of the body. More and more evidence shows that the function of the bile acid receptor is closely related to the occurrence of NAFLD, and the study of its related roles and functions can provide new insights and drug therapeutic targets for the treatment of NAFLD. Bile acid receptors include nuclear receptors, such as farnesoid X receptor (FXR), pregnane X receptor (PXR), etc., and cell surface receptors, such as transmembrane G protein-coupled receptor 5 (TGR5), sphingosine-1-phosphate receptor 2 (S1PR2) and M3 muscarinic receptor. In this review, we summarize the research progress on the role of bile acid receptors in the pathogenesis of NAFLD by regulating bile acid homeostasis, lipid and glucose metabolism, energy metabolism, liver inflammation and fibrosis, and we further elaborated on the current status of bile acid receptor agonists in the treatment of NAFLD, in order to have a more comprehensive understanding of the pathogenesis of NAFLD and find a more effective way of treatment.

Research on Therapeutic Mechanism of Canhuang Tablets on Jaundiced Rats Induced by ANIT / 中国实验方剂学杂志

Objective:To explore the therapeutic mechanism of Canhuang tablets on the mRNA and protein expression of farnesoid X receptor (FXR), uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) and multidrug resistance associated protein 2 (MRP2) in the liver of jaundiced rats induced by α-naphthalene isothiocyanate (ANIT). Method:The rats were divided into normal group, model group, Canhuang tablets (CHP) group and ursodeoxycholic acid tablets (UDCA) group. The jaundice model was reproduced by ANIT. After the intervention of the corresponding drugs, the contents of total bilirubin (TBIL), total bile acid (TBA), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in serum and the liver histopathology were examined to evaluate the therapeutic effect of CHP. The relative mRNA and protein expressions of FXR, UGT1A1 and MRP2 in rat liver tissues were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Result:CHP can significantly reduce the increase of TBIL, TBA, ALT, AST and ALP caused by ANIT in rat serum, and inhibit the liver pathological changes, which showed that the removing jaundice effect of CHP was better than UDCA. Compared with the normal group, ANIT significantly inhibited the mRNA levels of FXR, UGT1A1 and MRP2 in rat liver tissues after modeling (P<0.01). Compared with the model group, CHP and UDCA significantly increased the mRNA levels of target genes of each protein after intervention (P<0.01), and CHP was superior to UDCA in improving the mRNA level of bilirubin metabolizing enzyme UGT1A1 (P<0.01). In the aspect of affecting protein expression, compared with the normal group, ANIT modeling significantly increased the expression of FXR in rats (P<0.05). CHP intervention showed a tendency to promote the expression of FXR, while UDCA did not, but there was no significant difference between them. In the aspects of promoting bilirubin metabolism and bile excretion, the expressions of UGT1A1 and MRP2 were significantly decreased by ANIT modeling (P<0.01), while the expressions of UGT1A1 and MRP2 proteins were significantly increased after treatment of CHP (P<0.01). CHP was superior to UDCA in increasing the expression of bilirubin and bile acid efflux protein MRP2 (P<0.01). Conclusion:The jaundice abating mechanism of CHP is related to activating FXR mRNA expression in liver, promoting the mRNA and protein expression of bilirubin metabolizing enzyme UGT1A1 and bile acid transporter MRP2, improving liver metabolism of free bilirubin and promoting bile acid excretion from the liver, and alleviating cholestatic liver injury.

Alterations in bile acid levels in metabolic diseases and related treatment strategies / 上海交通大学学报（医学版）

Bile acid as a signaling molecule can specifically bind to bile acid receptors (such as farnesoid X receptor and G-protein-coupled bile acid receptor) to mediate a series of biological regulation reactions. In recent years, it has been found that bile acids are widely involved in glucose metabolism, lipid metabolism and energy metabolism. The development of metabolic diseases is usually accompanied by the changes of bile acid profiles and receptors, and thus bile acids may be applied as potential biomarkers for clinical diagnosis, prediction, and evaluation of therapeutic effects. This article reviews the relationship between bile acids and metabolic diseases, and the treatment of metabolic diseases based on the regulation of bile acid metabolism.

FXR expression is associated with the prognosis and pathological staging of patients with pancreatic cancer / 基础医学与临床

Objective To explore the correlation among farnesoid X receptor (FXR), clinical stage and survival time of patients with pancreatic cancer. Methods The total protein and mRNA were extracted from cultured 8 pan-creatic cancer cell lines,and the expression level of FXR in pancreatic cancer cells was detected by Western bolt and real-time PCR. We collected 5 cases of normal pancreatic tissue and 50 cases of pancreatic cancer tissues,and used immunohistochemistry method to detect FRX expression in normal pancreatic tissue and pancreatic cancer. Ac-cording to the different expression level of FXR,these 50 patients were divided into low expression group and high expression group, the correlation of clinical data and FRX expression level was analyzed. Furthermore, Kaplan-Meier and log-rank analysis of prognostic factors was assessed in a multivariable analysis using a Cox proportional hazards model. Results FXR was differently expressed in 8 pancreatic cancer cell lines and pancreatic cancer tis-sues. FXR was closely related to the pathological G stage of pancreatic cancer(P＜0.05). FXR and pathological G stage were significantly correlated with the patients' survival time. The survival time of the patients with high FXR expression was significantly longer than that of patients with low FXR expression (P＜0.05). Conclusions The expression of FXR is closely related to the pathological G stage in patients with pancreatic cancer. Both FXR expression and pathological G stage are independent prognostic factors in patients with pancreatic cancer.