| BackgroundLiver fibrosis refers to a pathological state in which the injury part of liver is replaced by scars.Liver fibrosis can cause liver function damage,and even develop into cirrhosis,leading to liver failure.Chronic liver diseases accounts for cirrhosis,such as non-alcoholic fatty liver disease(NAFLD)etc.Liver possesses a remarkable regeneration function,it can rebuild the original structure and function after tissue damage without leaving scars.Therefore,the liver recovers from various injuries.When the liver suffers from chronic damage,such as various chronic liver diseases,its regenerative capacity is usually impaired,and it is not enough to fulfill self-repair.Therefore,another mechanism(wound healing)began to participate in the repair of chronic injuries.In this case,hepatic stellate cells will be activated and transdifferentiate into myofibroblast-like cells,resulting in increased secretion of extracellular matrix protein(ECM),and excessive deposition of ECM protein can cause tissue fibrosis.Promoting liver regeneration is believed to alleviate liver fibrosis.In the partial hepatectomy(PH)model for studying liver regeneration,the liver can recover its mass and function even 70%of liver is removed,which is considered as a consequence of unaffected liver regeneration capacity.The activation of ID1-WNT2/HGF pathway is also believed to inhibite liver fibrosis by promoting hepatocytes regeneration.Bear gall is a traditional Chinese medicine in China.It is used for syndromes such as fever,epilepsy,and eclampsia.Ursodeoxycholic acid(UDCA)is one of the main active ingredients of bear gall,and it is also a hydrolysate of tauroursodeoxycholic acid.Previous studies have shown that UDCA can improve the biochemical indicators,delay histological progress,and improve survival in primary biliary cholangitis patients.However,the mechanism of UDCA has not yet been fully elucidated.ObjectiveThrough the use of bile duct ligation(BDL)model,PH model,Id1 knockdown model and in vitro cell model,this research explores the relationship between Id1,liver regeneration and UDCA’s anti-hepatic fibrosis effect,and explore the effect of UDCA on the treatment of liver fibrosis.Mechanism.MethodsFirst,we use blood biochemistry indicators,histological staining,type I collagen expression,F4/80 expression,hepatic stellate cell activation genes(Acta2,Coll1a1,Loxl2)and Id1 gene expression,α-SMA expression and so on to evaluate the protective effect of UDCA on BDL-induced liver fibrosis.Second,we use blood biochemistry indicators to assess UDCA’s protection on liver hepatectomy.With use cell cycle assay,TUNEL assay,Ki67 stain,cell cycle-related gene expression,we evaluate the effects of UDCA on liver regeneration.We detect the expression of ID1,WNT2,HGF,phosphorylated c-MET and phosphorylated GSK-3β proteins to explore the activation of ID1-WNT2/HGF pathway by UDCA.Third,the cell viability experiment was used to determine the optimal concentration of UDCA to promote the proliferation of HepG2 cells.The effects of UDCA on the expression of ID1 gene and protein at this concentration are then detected by qPCR and Western blot.Using cellular thermal shift assay and molecular docking experiments,we study the interaction between UDCA and ID1 protein.At last,serum ALT,AST,ALP,TBil levels,Masson staining,F4/80 expression,hepatic stellate cell activation genes(Acta2,Coll1a1,Loxl2)expression,α-SMA expression are used to explore the effect of Id1 knockdown on anti-fibrosis effects of UDCA.Then,cell cycle assay,Ki67 expression,TUNEL assay,cell cycle-related gene expression,ID1,WNT2,HGF,and phosphorylation of c-MET and GSK-3β are used to explore the effects of Id1 knockdown on UDCA’s promotion of liver regeneration.Results1.UDCA improves the liver to body weight ratio of the BDL model,reduce the levels of ALT,AST,TBil and ALP,improve liver histology,relieve inflammation,and reduce the expression of hepatic stellate cell activation genes(Acta2,Coll1a1,Loxl2).In addition,UDCA significantly increases the expression of Idl gene in BDL liver.2.UDCA reduces the levels of ALT and AST in the PH model,promotes hepatocytes in the PH model to enter the cell cycle,increases the expression of Ki67,inhibits hepatocyte apoptosis,increases the expression of cyclin genes,inhibits the gene expression of cell cycle inhibitors.UDCA promotes the expression of Id1,Wnt2,Hgf genes and proteins in the PH model,and promotes phosphorylations of c-MET and GSK-3β.3.UDCA promote the proliferation of HepG2 cells,increase the expression of ID1 gene and protein.UDCA can also enhance the thermal stability of ID1.Molecular docking experiments predicted the interaction model between UDCA and ID1.4.UDCA improves the liver coefficient of BDL mice in knockdown control group,reduces serum ALT,AST,TBil and ALP levels,reduces F4/80 expression,inhibits hepatic stellate cell activation genes(Acta2,Call1a1,Loxl2)expressions,inhibits α-SMA expression.Id1 knockdown inhibits the improving effect of UDCA on fibrosis.UDCA promotes liver regeneration by promoting cell cycle entry,increasing Ki67 expression,inhibiting hepatocyte apoptosis,increaseing cyclin gene expression,inhibiting cell cycle inhibitor genes expression,promoting ID1,WNT2,HGF,and phosphorylation c-MET and phosphorylated GSK-3β protein expression.ConclusionOur Research indicates:1.UDCA has significant anti-liver fibrosis effect in BDL mice model;2.UDCA can significantly enhance liver regeneration in PH mice,and play a role by activating the ID1-WNT2/HGF signaling pathway;3.Through thermal stability experiments,it is found that UDCA can enhance the thermal stability of ID1,suggesting that UDCA may interact with ID1;4.Through the Id1 knockdown model,confirm that UDCA can fight liver fibrosis in the BDL model by promoting liver regeneration,and its molecular mechanism is to activate the ID1-WNT2/HGF signaling pathway.In summary,our study found that the new mechanism of UDCA is to promote liver regeneration by activating the ID1-WNT2/HGF signaling pathway,and to combat liver fibrosis caused by BDL. |
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