| ObjectiveCholestasis is the main manifestation of cholestatic liver diseases,which has a risk of progression to end-stage liver disease.Gardenia Fructus is a kind of traditional Chinese medicine with liver clearing and choleretic efficacy,which is often used to treat hepatobiliary dampness and heat.Gardenia extract(GE,total iridoid glycoside extract of Gardeniae Fructus)ameliorates cholestasis,while the underlying mechanism is unclear.To study the pharmacodynamic effects of GE on acute and chronic cholestatic liver injury by using α-naphthylisothiocyanate(ANIT)-induced acute and chronic cholestatic liver injury model,and to clarify the mechanism of Gardenia extract in treating acute and chronic cholestatic liver injury through bile acids(BAs)argeted metabolomics and liver transcriptomic,providing a scientific basis for the clinical application and new drug development of Gardenia Fructus.Method1.Methodology for simultaneous quantitative analysis of 31 bile acids in biological samples by UPLC-MS/MSIn this study,the simultaneous quantitative analysis of 31 BAs in biological samples from 9 sites in the bile acid enterohepatic circulation was established by using UPLC-MS/MS quantitative analysis system.Methodological studies included selectivity,residue,precision,lower limit of quantification(LLoQ),standard curve,stability,matrix effect,accuracy and recovery.2.Study on the improvement effect and mechanism of Gardenia extract on acute cholestatic liver injuryForty SPF male Sprague-Dawley rats were random allocation to five groups:control,model,OCA,GE low-dosage(GE-L),and GE high-dosage(GE-H)(n=8 rats per group).Rats in the OCA,GE-L and GE-H groups were administered 2mg·kg-1 OCA,21mg·kg-1 or 42mg·kg-1 GE,respectively,once daily for five consecutive days via intragastric gavage,whereas those in the control and model group received(10mL·kg-1)drinking water.After 1h administration on the third day,the control group was administered(5 ml/kg)sunflower oil,and the remaining groups were administered ANIT 40 mg·kg-1 by intragastric gavage for modeling.Feces and urine were collected 12 hours before the last administration.After 1 hour of the last drug administration,1%pentobarbital sodium was used for anesthesia.Blood was collected from the abdominal aorta and serum was collected by centrifuging(15 min,3,500 rpm,4℃)the blood samples.Livers were collected,intestinal(duodenal,jejunal,ileal,and cecal)contents were collected for analysis of BAs.Some liver,ileum and colon tissues were collected for qRT-PCR and stored at-80℃ freezer for further use.Part of the liver was fixed in formalin for pathological observation.ALP,GGT,TBA,TBIL,and DBIL concentrations in rat serum were measured using a biochemistry analyzer.The histopathology of liver tissues was observed by HE staining.The levels of BAs in serum,liver,urine,duodenum,jejunum,ileum,cecum and feces were determined by UPLC-MS/MS.RNA-seq of livers was performed using Illumina BeadChips ArrayTM(n=3,control/model/OCA/GE-H),and the RNA-seq results were verified by qRT-PCR.3.Study on the improvement effect and mechanism of Gardenia extract on chronic cholestatic liver injurySixty-five SPF male Sprague-Dawley rats were random allocation to six groups:control,model,OCA,Yinzhihuang(YZH),GE low-dosage(GE-L),and GE highdosage(GE-H)(n=10-11 rats per group).Except for the control group,rats in other groups were administered 40mg·kg-1 ANIT by intragastric gavage three times a week for modeling chronic cholestatic liver injury.From the 3w,rats in the OCA,YZH,GE-L and GE-H groups were administered 2mg·kg-1 OCA,6.35m·kg-1 YZH,21 mg·kg-1 or 42mg·kg-1 GE,respectively,once daily via intragastric gavage,whereas those in the control and model group received(10mL.kg-1)drinking water.The blood,liver,ileum,and colon were taken in batches at 4w and 6w after administration.Serum biochemical indicators,pathological examination,and BAs analysis were as above.snRNA-seq of livers(administration for 6w)was performed using 10x Genomics ChromiumTM(n=3,control/model/GE-H),and the snRNA-seq results were verified by qRT-PCR.Result1.Methodology for simultaneous quantitative analysis of 31 bile acids in biological samples by UPLC-MS/MSA high-throughput and sensitive targeted metabolomics assay for BAs was successfully developed based on UPLC-MS/MS,allowing the simultaneous quantification of 31 BAs in biological samples from nine sites including serum,liver,urine,feces,and intestinal contents(duodenum,jejunum,ileum,cecum,colon).The established method exhibited good results in selectivity,residual amount,LLoD,accuracy,standard curve,matrix effects,precision,stability and recovery,which met the bioanalysis guidelines stated in the Chinese Pharmacopoeia(2020 edition).The developed analytical method of BAs analysis solved the separation of nature similar and conformers in the determination of BAs,the peak shapes did not interfere with each other and good separation.2.Study on the improvement effect and mechanism of Gardenia extract on acute cholestatic liver injury(1)Study on the pharmacodynamic of Gardenia extract on ANIT-induced acute cholestatic liver injuryCompared with the control group,the serum levels of TBA,TBIL,DBIL,ALP and GGT were significantly increased in the model group(p<0.05).Compared with model group,serum TBA,TBIL,and DBIL levels were significantly decreased in the GE-H group(p<0.05),but not in the OCA and GE-L groups.In addition,ALP level in the GE-H group showed a tendency to decrease compared with model group,and the decrease was greater than that in the OCA group.Histopathological examinations showed that the liver tissue of the model group had marked bile duct hyperplasia(7/8 rats scored≥ 3),and inflammatory cells infiltration in the portal area were observed(5/8 rats scored≥ 3)compared with the control group.Compared with the model group,the infiltration of inflammatory cells and bile duct hyperplasia in the GE-L and GE-H groups were significantly reduced,especially in improving the bile duct hyperplasia.The effect of GE-H group was more obvious,with 2/8 of the liver morphology of rats basically normal.The ameliorative effect of the OCA group was similar to that of the GE-H group.These results suggested that GE attenuates ANIT-induced acute cholestatic liver injury in a dose-dependent manner.(2)The mechanism of Gardenia extract on ameliorating acute cholestatic liver injuryThe content and proportion of total bile acid(TBA),primary bile acid(PBA),secondary bile acid(SBA),unconjugated bile acid(UnconBA),taurine conjugated bile acid(TaurineBA),glycine conjugated bile acid(GlycineBA)and conjugated bile acid(ConBA)in various parts of the bile acid enterohepatic circulation significantly changed,especially the changes of TaurineBA and PBA in the liver,serum,urine and feces.Compared with the control group,the levels of TBA,PBA,and TaurineBA in the liver,serum,and urine of the model group were significantly increased(p<0.01),and the proportion of TaurineBA increased by 55.98%→94.99%,17.13%→88.87%,and 8.87%→77.51%,respectively.The TBA and PBA in the feces of the model group were significantly reduced(p<0.01).Compared with the control group,the levels of TBA,PBA and TaurineBA in the liver of GE-H and OCA groups were obviously increased.The TBA,PBA,and TaurineBA in serum and urine were significantly decreased in GE-H group.The TBA and PBA in feces of GE-H groupwere significantly increased(p<0.05).The differential BAs were identified based on statistical significance in unit analysis(p<0.05)and multivariate analysis(VIP>1)analyses were used to identify the differential BAs between the control and model group,PBA(Tβ-MCA,TCA and GCA)were the differential BAs in rat liver.The differential BAs in serum and urine were basically similar to those in liver,while the differential BAs in feces were β-MCA and DCA.Compared with the control group,The level of TCA and Tβ-MCA in the liver,serum and urine of the model group was significantly increased(p<0.05),β-MCA and DCA in feces significantly decreased(p<0.05).The TCA in serum and urine of GE-H group was significantly decreased,and β-MCA and DCA in feces significantly increased(p<0.05).The results of liver RNA-seq differentially expressed genes(DEGs)and their pathways showed that the DEGs in the model group,GE-H,and OCA groups were significantly enriched in the PBA biosynthesis and bile secretion pathways(padj<0.05).Compared with the model group,some genes involved in PBA biosynthesis and bile secretion(such as Cyp8b1,Cyp27a1,Fxr,Oatp1,Ntcp and Ugt1a1)were upregulated after treatment with GE and OCA.The RNA-seq results were verified by qRT-PCR.Compared with the control group,the expression of Cyp8b1,Fxr,Shp,Oatp1 and NtcP mRNA in the model group was significantly decreased(p<0.001).The expression of Cyp8b1,Fxr and Oatp1 mRNA in GE-H group was significantly upregulated compared with the model group(p<0.05).In summary,GE could inhibit PBA biosynthesis in liver by upregulating the expression of Cyp8b1 mRNA,thus reducing BAs level,and upregulate the expression of Fxr and Oatp1 mRNA,promote bile acid excretion through the liver-intestine-fecal pathway,and inhibit the PBA in the liver entry into the blood through alternative ways,thereby reducing liver cholestasis,and improving the bile acid enterohepatic circulation.3.Study on the improvement effect and mechanism of Gardenia extract on chronic cholestatic liver injury(1)Study on the pharmacodynamic of Gardeniae extract on ANIT-induced chronic cholestatic liver injuryAfter repeated stimulation with 40mg·kg-1 ANIT by gavage three times a week,biochemical and liver pathological examinations at 6w and 8w demonstrated that the model of chronic cholestatic liver injury was successfully established.The levels of serum TBIL,DBIL,ALP,and GGT in model group showed a continuous increase(p<0.05).The hepatic pathology showed that there was obvious bile duct hyperplasia,inflammatory cell infiltration in and around the bile duct and lamellar fibrosis.GE was administered from 2w after modeling,and serum levels of TBIL,DBIL,ALP,and GGT at 4w and 6w after dosing were not statistically significantly different compared with the model group.Due to the formation of grayish blue pigments in the body after repeated and prolonged administration of geniposide,it may interfere with the determination of the above indicators,which may be the reason why there is no significant change in biochemical indicators.Therefore,the improvement of pathology was taken as the direct basis in this study.In the control group,there was no obvious abnormality in the morphology of hepatocytes,cells arranged in order,hepatic cord and hepatic sinuses demarcated clearly,occasional mild inflammatory cell infiltration in the portal vein area,and no pathological changes such as bile duct hyperplasia.After 6w and 8w of modeling,severe bile duct hyperplasia could be seen in the portal duct area,with diffuse hyperplasia extending into the hepatic lobule,and obvious inflammatory cell infiltration could be seen around the proliferative bile duct,and the lesions were aggravated with time.After administration of GE,pathological damage did not improve significantly after 4w of administration,but the degree of bile duct hyperplasia was significantly reduced and the scope of lesion was reduced after 6w of prolonged administration in the low-dose group.In the GE-H group,4w and 6w administration could significantly improved the proliferation of bile duct and narrow the scope of lesions.The ameliorative effect of the YZH group was similar to that of the GE-H group.Masson staining results were basically consistent with HE staining.The results showed that GE had an obvious ameliorativcholestasise effect on the pathology of chronic cholestatic liver injury.(2)The mechanism of Gardenia extract on improving chronic liver injuryAfter 6w of modeling,ConBA,TaurineBA,PBA,TBA and UnconBA levels in the liver and serum in the model group were significantly increased(p<0.05),while the other types of BAs showed an increasing trend.SBA was significantly decreased in duodenum(p<0.05).After 4w of treatment with GE,OCA and YZH,compared with the model group,the BAs levels in liver and serum of OCA,YZH and GE groups did not change significantly,while the levels of various types of BAs in duodenum showed an uptrend.After 8w of modeling,PBA in the liver,and ConBA,GlycineBA and TaurineBA in the liver and serum were significantly increased in the model group(p<0.05).Compared with the model group,the various types of BAs levels in the liver and serum of GE,OCA and YZH groups were decreased,and the improvement effect of GE-H group was stronger than that of GE-L group.After 8w of modeling,the differential BAs between the control and model group was mainly PBA(Tβ-MCA,TCA,TCDCA and GCA).The differential BAs in serum were basically similar to these in liver.Compared with the control group,Tβ-MCA,TCA,TCDCA and GCA levels significantly increased(p<0.01).At the same period,the levels of Tβ-MCA,TCA,TCDCA,and GCA were obviously decreased in the GE and OCA group,and the TCDCA level was significantly decreased in GE-H group(p<0.01).Through liver tissue mononuclear transcriptome sequencing(snRNA-seq)analysis,we further elucidate the mechanism of GE improving chronic cholestatic liver disease by affecting bile acid metabolism and cholangiocyte proliferation.The KEGG pathway enrichment analysis of liver DEGs showed that pharmacodynamic related genes in the GE-H group were significantly enriched(padj<0.05)in PBA biosynthesis and bile secretion pathways.Compared with the model group,GE could upregulate the expression of some genes involved in PBA biosynthesis and bile secretion pathway(such as Cyp8b1,Cyp27a1,Fxr,Bsep,Ntcp,Shp,Mrp2,Oatp1,and Asbt),and downregulate the expression of some genes(such as Cyp7a1,Slc27a5,and Baat).The results of snRNA-seq was validated by qRT-PCR.Compared with the control group,Cyp7a1 and Mrp3 mRNA were significantly upregulated,Sult2a1 and Oatp1 mRNA were significantly downregulated in the model group(p<0.05).After administration of GE and YZH,the expression of Cyp7a1 and Bacs mRNA in rat liver were obviously downregulated,Osta mRNA was significantly downregulated(p<0.05).In summary,GE could mainly affect the synthesis of bile acid in the liver by inhibiting the expression of the BAs synthesis rate-limiting enzyme Cyp7a1 mRNA,and regulate various transporters related to bile acid transport,promote the excretion of bile acid,and reduce the level of BAs in serum and liver,resulting in improving the metabolism of BAs in rats with chronic bile accumulation liver injury and alleviating liver damage caused by bile stasis.According to the analysis of seurat clustering results,the cells were defined by differentially expressed marker genes.The liver cells of the control,model and GE-H group all defined 8 kinds of cell clusters:hepatocytes,hepatic stellate,inflammatory macrophages/monocytes,Kuffer cells,endothelial cells,γδ T cells,mature B cells and cholangiocytes.Compared with the control group,the number of cholangiocytes in the model group significantly increased(control vs model:0.05%vs 14.90%),while the number of hepatocytes significantly decreased(66.90%vs 50%).After treatment with GE-H,the number of cholangiocytes significantly decreased(GE-H vs model:9.40%vs 14.90%),while the number of hepatocytes increased(55.50%vs 50%).Then the cholangiocytes were further divided into three cell clusters:hepatocyte-like,cholangiocyte-like,and hepatic progenitor cells.The number of hepatic progenitor cells and hepatocyte-like cells in the model group obviously increased compared with the control group.The number of hepatocyte-like cells and hepatic progenitor cells obviously decreased in GE-H group.It is suggested that some hepatocytes may be differentiated into cholangiocytes,and GE may interfere with this differentiation.In order to verify this conclusion,pseudotime analysis was performed on hepatocytes and cholangiocytes.The results showed that hepatocytes and cholangiocytes in the control group were in two different states,and some hepatocytes in the model group appeared in the state of cholangiocytes,indicating that some hepatocytes were differentiated into cholangiocytes after long-term cholestasis of the liver.The two kinds of cells in the GE-H group were in a similar state to the model group,but the number of hepatocytes in the bile duct cell state was smaller than that in the model group.Cholangiocytes transformed from hepatocytes,which belong to the hepatobiliary biphenotypic cells,possess both the biliary synthetic and secretory properties of hepatocytes and the ability of cholangiocytes to activate proliferation in response to inflammatory stimuli,and play an important role in the development of chronic cholestatic liver injury conditions.The results suggested that GE may attenuate the progression of chronic liver injury by inhibiting of liver cell differentiation into bile duct cells,inhibiting hepatobiliary biphenotypic cell hyperplasia and abnormal bile secretion,and thus alleviating bile duct proliferation and inflammation.Conclusion1.GE ameliorated acute and chronic cholestatic liver injury by alleviating liver injury and inflammatory infiltration,and inhibiting bile duct and fibrous tissue proliferation,and its effects may be related to improving BAs homeostasis in rats.2.GE could inhibit the synthesis of BAs(especially primary TaurineBAs,such as TCA and Tβ-MCA)in the liver by upregulating the expression of Cyp8b1,and promote BAs excretion via the duodenal-faecal pathway by upregulating the expression of Fxr,and Oatp1,inhibiting PBA entry into the blood via the alternative pathways,thereby reducing the levels of BAs in liver and producing anti acute cholestatic liver injury.3.On the one hand,The results suggested that GE may attenuate the progression of chronic liver injury by inhibiting of liver cell differentiation into bile duct cells,inhibiting hepatobiliary biphenotypic cell hyperplasia and abnormal bile secretion,and thus alleviating bile duct proliferation and inflammation.On the other hand,GE could inhibit the synthesis of BAs(especially TCDCA,GCA,Tβ-MCA and TCA)in the liver bydowgulating the expression of BAs synthesis rate limiting enzyme Cyp7a1 mRNA,and promote BAs excretion via the duodenal-faecal pathway by regulating the expression of nuclear receptors and multiple transporters involved in the transport of BAs,inhibiting PBA entry into the blood via the alternative pathways,thereby reducing the levels of BAs in liver and producing choronic cholestatic liver injury.The BAs analysis method established in this study can simultaneously quantitatively analyze the BAs levels in various parts of the bile acid enterohepatic circulation,and evaluate the impact of drugs on the bile acid enterohepatic circulation.Differential bile acids TCA,Tβ-MCA and GCA in serum can not only be used as biomarkers for the diagnosis of cholestatic liver injury,but also can judge whether drugs produce liver protection. |
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