| Biliary atresia(BA)is a severe occlusive cholangiopathy in early infancy.Progressive inflammation and liver fibrosis are prominent features of BA.In the absence of Kasai surgery(hepatojejunostomy),the progression of BA liver fibrosis is quite astonishing,and cirrhosis can form within a few weeks after birth.After Kasai surgery,obstruction can be effectively alleviated and bile flow can be restored.However,progressive inflammation and fibrosis of the internal and external bile ducts still exist,and nearly 70%of children still require liver transplantation surgery.Therefore,slowing down the progression of liver fibrosis after BA surgery is particularly important.Traditional Chinese medicine has unique advantages in preventing and treating liver fibrosis after BA surgery.Our research group has conducted a series of clinical and basic studies on the prevention and treatment of BA with modified Yinchen Sini decoction in the early stage.On the basis of previous research,this study conducted a network pharmacology study on the relationship between modified Yinchen Sini decoction and liver fibrosis,summarizing and analyzing its related targets and pathways,in order to provide new ideas for clinical and basic research.At the same time,based on literature research and the findings of this network pharmacology study,starting from the Treg/Th17 balance,a young rat model of BA liver fibrosis was constructed using the method of bile duct ligation.Modified Yinchen Sini decoction was used as an intervention drug to further explore the therapeutic targets of this formula for BA liver fibrosis.Objective1.Based on the network pharmacology of traditional Chinese medicine compound formula,this study explores the targeted relationship between modified Yinchen Sini decoction and liver fibrosis.By utilizing the genetic correlation between drugs and clinical diseases,the possible targets and pathways of the drug components in modified Yinchen Sini decoction on liver fibrosis are analyzed,providing a basis for clinical medication and scientific research.2.To observe the therapeutic effect of modified Yinchen Sini decoction on young rats with BA liver fibrosis after biliary ligation,and to study its effects on the symptoms and related indicators of BA liver fibrosis in young mice,and to explore the potential target of modified Yinchen Sini decoction on BA liver fibrosis from Treg/Th17 balance.Methods1.Network Pharmacological Study on Modified Yinchen Sini Decoction and Liver FibrosisThrough TCMSP database and GEO database screening,and using Perl language and R language to collate,the common target genes of each drug of modified Yinchen Sini decoction and liver fibrosis were obtained,and GO functional analysis and KEGG pathway enrichment analysis were carried out;Constructing a regulatory network and KEGG relationship network for traditional Chinese medicine prescriptions using Cytoscape software;Build a PPI protein interaction network through the STRING database and obtain core genes using R language.Based on the literature research in this article,analyze the possible immunological gene targets and pathways of modified Yinchen Sini decoction in the intervention of liver fibrosis.2.Experimental research(1)Construction of BA liver fibrosis model in young mice using bile duct ligation methodSPF grade male SD rat pups(24)were randomly divided into a sham operation group and a model group(n=12).Biliary duct ligation method was used to ligate the common bile duct in the model group of young rats,while the sham operation group of young rats underwent abdominal wall incision and suturing,but the common bile duct was not ligated.Observe the changes in skin color,stool color,and survival status of two groups of young mice;On the 16th day after surgery,two groups of young rats were anesthetized with isoflurane and underwent abdominal wall incision to observe the changes in the liver,gallbladder,and spleen of the two groups of young rats.Blood was taken from the abdominal aorta and blood biochemical tests were performed to detect liver function indicators such as ALT,AST,and TBIL;After blood collection,euthanize the young mice,weigh the liver,calculate the liver index of two groups of young mice,and perform HE staining,Masson staining,and immunohistochemical detection on the liver tissue of both groups of young mice,observing the changes of histopathology,the degree of fibrosis and the expression levels of collagen Ⅰ,α-SMA and TGF-β1.Analyze each indicator to determine whether the mold is successful.(2)Intervention of modified Yinchen Sini decoction on BA liver fibrosis young rat modelSPF grade male SD rat pups(48)were randomly divided into 6 groups:sham surgery group,model group,ursodeoxycholic acid group,high-dose group of modified Yinchen Sini decoction,medium dose group of modified Yinchen Sini decoction,and low-dose group of modified Yinchen Sini decoction(n=8).The sham operated group of young rats underwent abdominal wall incision and suturing,but the common bile duct was not ligated.The remaining groups of young rats underwent common bile duct ligation.Starting from the 5th day after surgery,gavage was performed according to the administration methods of each group,gavage was administered once a day for 10 consecutive days.On the 11th day of administration,the body weight of each group of young mice was recorded.Blood was taken from the abdominal aorta of the mice anesthetized with isoflurane,and separating the serum.After blood was taken,the young mice were euthanized and liver tissue was taken,and liver mass was recorded.Calculate liver index(liver index=liver mass/body weight X 100%);Perform blood biochemical tests to detect liver function indicators such as serum ALT,AST,TBIL in each group of young mice;HE staining and Masson staining were performed on the liver tissues of each group;Detection the expression of collagen Ⅰ、α-SMA、TGF-β 1 in liver tissue using immunohistochemistry;Using flow cytometry to detect the content of Treg cells and Th17 cells in whole blood;Detection the expression of IL-10、TGF-β1、IL-6 and IL-17 in serum using ELISA method;Detection the expression level of Foxp3 mRNA and ROR γ t mRNA in liver tissue using RT-qPCR.Results1.Network Pharmacological Study on Modified Yinchen Sini Decoction and Liver Fibrosis(1)Through screening and sorting,18 common core genes related to liver fibrosis were identified,including BAX,VEGFA,CCND1,FOS,NFKBIA,POR,ODC1,HMOX1,CAV1,MYC,VCAM1,HSPB1,IFNGR1,COL3A1,CXCL2,PPARA,CTSD,ERBB3.(2)Through screening and sorting,a total of 8 effective ingredients were obtained for the effect of modified Yinchen Sini decoction on liver fibrosis,among which Yinchen has 6 active ingredients,namely isorhamnetin,Yinchen flavonoids,Yinchen artemisinin flavonoids,Yinchen artemisinin A,quercetin,β-Sitosterol;Fuzi,Chuanxiong,and Ganjiang each have one active ingredient,which is shifangfeng su,bayberry ketone,βSitosterol.The molecule of β-sitosterol is a common active ingredient of Artemisia and Ginger.Among the active ingredients,quercetin has the highest number of gene targets for liver fibrosis.(3)The enrichment of 1736 biological functions,133 molecular functions,and 95 cell components was obtained through GO enrichment analysis.In terms of biological and molecular functions,it is mainly reflected in the response to a decrease in O2 levels and the binding of DNA binding transcription factors;In terms of cell composition,target genes are mainly enriched in membrane rafts,early endosomes,membrane microregions,and extracellular matrix containing collagen.(4)Through KEGG enrichment analysis,it was found that there are multiple pathways related to liver fibrosis and immunity in the pathway of liver fibrosis induced by modified Yinchen Sini decoction,including the MAPK signaling pathways,NF-κB signaling pathway,JAK-STAT signaling pathway,T cell receptor signaling pathway,Thl and Th2 cell differentiation pathway,Th17 cell differentiation pathway,IL-17 signaling pathway,etc;Among the enriched genes in these pathways,three core genes,FOS,NFKBIA and IFNGR1,were found to be jointly associated,indicating that the intervention of modified Yinchen Sini decoction in liver fibrosis may have immune related regulatory mechanisms.2.Experimental research(1)Construction of BA liver fibrosis model in young mice using bile duct ligation methodAll young rats in the sham operation group survived.A total of 9/12(75%)young rats in the model group survived.Compared with the sham operated group,the model group had sparse and dull body hair,swollen abdomen,and symptoms of bile stasis,manifested as yellow staining of the ears,legs,and tail,as well as dark urine deposition and clay like stools;During laparotomy,hepatosplenomegaly and cystic dilation of the common bile duct were observed,but not in the sham operated group of young mice;Compared with the sham surgery group,the model group showed a significant decrease in body mass(P<0.01)and a significant increase in liver index(P<0.01);Compared with the sham surgery group,there was no significant difference in liver mass between the model group and the control group(P>0.05);Compared with the sham surgery group,the serum levels of ALT,AST and TBIL in the model group were significantly increased(P<0.01);Compared with the sham operated group of young mice,the model group of young mice showed diffuse bile duct hyperplasia between the hepatic tissue portal area and portal area,accompanied by a large amount of collagen fiber hyperplasia,and there was a clear bridging between the portal and portal ducts;Compared with the sham operated group of young mice,a large amount of collagen I protein,TGF-β 1 protein and α-SMA protein aggregates can be observed in the fibrous septa of the portal area of the liver tissue in the model group of young mice.(2)Intervention of modified Yinchen Sini decoction on BA liver fibrosis young rat model①Compared with the sham surgery group,the model group had sparse and matte body hair,swollen abdomen,and accompanied by symptoms of cholestasis,manifested as yellow staining of the ears,legs,and tail;Compared with the model group,the high-dose,medium,and low-dose group of modified Yinchen Sini decoction and ursodeoxycholic acid group improved body hair weight and glossiness,with no significant swelling in the abdomen and reduced yellowing.②Compared with the sham surgery group,the body mass of the model group decreased(P<0.05);Compared with the model group,the high and medium dose groups of modified Yinchen Sini decoction showed a statistically significant increase in body mass(P<0.05),while the low dose group of modified Yinchen Sini decoction and the ursodeoxycholic acid group showed no statistically significant difference in body mass(P>0.05).There was no statistically significant difference in liver quality between groups of young rats(P>0.05).③Compared with the sham surgery group,the liver index of the model group was significantly increased(P<0.01);Compared with the model group,the liver index of the high-dose group of modified Yinchen Sini decoction decreased(P<0.05),while the liver index of the ursodeoxycholic acid group and the middle and low dose groups of modified Yinchen Sini decoction showed no significant difference(P>0.05).④Compared with the sham surgery group,the serum levels of ALT,AST and TBIL in the model group were significantly increased(P<0.01);Compared with the model group,the high-dose group of modified Yinchen Sini decoction showed a statistically significant decrease in serum AST and TBIL levels in young mice(P<0.05 or P<0.01).Although the high-dose group of modified Yinchen Sini decoction showed a decrease in serum ALT levels,the difference was not statistically significant(P>0.05).Although the middle and low dose groups of modified Yinchen Sini decoction and the ursodeoxycholic acid group showed a decrease in ALT,AST and TBIL levels,But the difference was not statistically significant(P>0.05).⑤Compared with the sham surgery group,the model group showed diffuse bile duct hyperplasia between the portal area and the portal area of the liver tissue,accompanied by a large amount of collagen fiber hyperplasia,and there was a clear bridging between the portal and portal ducts;Compared with the model group,the high,medium and low dose group of modified Yinchen Sini decoction and the ursodeoxycholic acid group can to some extent alleviate the proliferation of bile ducts in the portal area of the liver tissue of young rats with BA liver fibrosis and alleviate liver fibrosis.⑥Compared with the sham surgery group,the model group showed a significant increase in collagen Ⅰ,α-SMA,TGF-β 1.The positive surface density values all significantly increased,with statistically significant differences(P<0.01);Compared with the model group,the high-dose group of modified Yinchen Sini decoction had a decrease in the positive surface density value of α-SMA and TGF-β1 in young mice,and the difference was statistically significant(P<0.01 or P<0.05).Although the high dose group of modified Yinchen Sini decoction had a decrease in the positive surface density value of collagen Ⅰ in young mice,the difference was not statistically significant(P>0.05).Although the medium and low dose group of modified Yinchen Sini decoction and the ursodeoxycholic acid group had a decrease in the positive surface density value of collagenⅠ,α-SMA and TGF-β1,the difference was not statistically significant(P>0.05).⑦Compared with the sham surgery group,the serum IL-17 and IL-6 levels in the model group were significantly increased(P<0.01);Compared with the model group,the serum IL-17 levels in the high-dose group of modified Yinchen Sini decoction were significantly reduced,with a statistically significant difference(P<0.01).However,although the levels in the ursodeoxycholic acid group and the middle and low dose group of modified Yinchen Sini decoction were reduced,the difference was not statistically significant(P>0.05);Compared with the model group,the serum IL-6 levels in the high and medium dose group of modified Yinchen Sini decoction were significantly reduced,with a statistically significant difference(P<0.01).However,although the levels in the ursodeoxycholic acid group and the low-dose group of modified Yinchen Sini decoction were reduced,the difference was not statistically significant(P>0.05).⑧Compared with the sham surgery group,the proportion of Th17 cells to CD4-T cells in the model group significantly increased(P<0.01);Compared with the model group,the proportion of Th17 cells to CD4-T cells in the high and medium dose group of modified Yinchen Sini decoction decreased significantly(P<0.01 or P<0.05),while in the low dose group of modified Yinchen Sini decoction and ursodeoxycholic acid group decreased,but the difference was not statistically significant(P>0.05).⑨Compared with the sham surgery group,the expression level of ROR γ t mRNA in the model group significantly increased(P<0.01);Compared with the model group,the expression level of ROR y t mRNA in the high-dose group of modified Yinchen Sini decoction was significantly reduced,and the difference was statistically significant(P<0.01).However,although the ursodeoxycholic acid group and the middle and low dose group of modified Yinchen Sini decoction showed a decrease,the difference was not statistically significant(P>0.05).⑩Compared with the sham surgery group,the model group showed a significant decrease in serum IL-10 levels and TGF-β 1 levels.The level significantly increased,and the differences were statistically significant(P<0.01);Compared with the model group,the serum IL-10 levels in the ursodeoxycholic acid group and the high and medium dose group of modified Yinchen Sini decoction increased with statistical significance(P<0.01 or P<0.05).Although the low dose group of modified Yinchen Sini decoction had an increase in serum IL-10 levels,the difference was not statistically significant(P>0.05);Compared with the model group,the level of the serum TGF-β 1 of the high and medium dose group of modified Yinchen Sini decoction decreased and the difference was statistically significant(P<0.05).Although the level of The serum TGF-β 1 in the ursodeoxycholic acid group and the low-dose group of modified Yinchen Sini decoction decreased,the difference was not statistically significant(P>0.05).⑾Compared with the sham surgery group,the proportion of Treg cells to CD4+T cells in the model group was significantly reduced(P<0.01);Compared with the model group,the proportion of Treg cells to CD4+T cells in the high-dose group of modified Yinchen Sini decoction increased with statistical significance(P<0.05).There was no significant difference in the proportion of Treg cells to CD4-T cells in the ursodeoxycholic acid group and the middle and low dose group of modified Yinchen Sini decoction(P>0.05).⑿Compared with the sham surgery group,the expression of Foxp3 mRNA in the model group was significantly reduced(P<0.01);Compared with the model group,the high-dose group of modified Yinchen Sini decoction significantly increased the expression of Foxp3 mRNA,with a statistically significant difference(P<0.01).Although the expression of Foxp3 mRNA in the ursodeoxycholic acid group and the middle and low dose group of modified Yinchen Sini decoction increased,the difference was not statistically significant(P>0.05).ConclusionThrough network pharmacology research on the relationship between modified Yinchen Sini decoction and liver fibrosis,it was found that modified Yinchen Sini decoction has multiple effective active ingredients that can act on multiple liver fibrosis targets for regulation,manifested at multiple levels such as biological function,molecular function,and cellular composition.The possible mechanism is to regulate liver fibrosis by targeting the enrichment of genes in the liver fibrosis pathway and immune regulation pathway.The use of bile duct ligation can successfully construct a BA liver fibrosis model in young mice,and the intervention with modified Yinchen Sini decoction was found to improve the symptoms and related indicators of BA liver fibrosis in young mice.The mechanism may be through inhibiting the expression of IL-17 and IL-6,inhibiting Th17 cell differentiation,and reducing ROR γ T expression,while inhibiting TGFβ1 expression promotes Treg cell differentiation and increases Foxp3 expression,promoting IL-10 secretion,leading to a steady state of Treg/Th 17 immune regulation,thereby achieving the effect of reducing BA liver fibers. |
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