| Chronic liver inflammation is a global health burden,which gradually develop hepatic fibrosis,cirrhosis,liver cancer,eventually leading to death.Liver fibrosis is the pathological condition of the liver resulting from sustained wound healing in response to chronic liver injury.Multiple factors could lead to such injury,including genetic(the accumulation of misfolded alpha1-anntitrypsin),cholestatic(sclerosing cholangitis),metabolic(non-alcoholic fatty liver disease and non-alcoholic steatohepatitis),drug induced(par-acetamolintoxication and alcohol)and viral diseases(hepatitis B and C).Hepatic fibrosis can eventually progress toward cirrhosis,which is characterized by the loss of endothelial fenestrations,excessive scar formation in the space of Disse,and the presence of vascularized fibrotic septa.These distortions of liver architecture and subsequent cellular homeostasis lead to impaired organ function,ascites,encephalopathy,variceal hemorrhage,portal hypertension and the development of hepatocellular carcinoma.Hepatic fibrosis contains complex signaling networks regulating pathways,leading to the excessive deposition of extracellular matrix(ECM).In recent years,studies have demonstrated that activation of hepatic stellate cells(HSCs)plays a crucial role in the liver fibrosis.When a variety of pathogenic factors damage the liver cells,they release the active oxygen species and inflammatory mediators.As a result,large numbers of inflammatory cells accumulate and HSCs are activated.The quiescent fat storing cells are transformed into myofibroblast cells(MFCs)when HSCs activated characterized by their expression of abundant intracellular filaments like ?-SMA,secretion of ECM including fibronectin(FN)and collagen type I,III and IV,resulting in fibrosis.Transforming growth factor ?1(TGF-?1)is an important member among the TGF-? family,which plays a major role in the occurrence of liver fibrosis.After HSCs activation,they produce TGF-?1 which is a strong pro-fibrotic effect molecule,through autocrine and paracrine pathway regulation.HSCs produce ECM,mainly through TGF-? / SMAD signaling pathway.TGF-?1 initiates intracellular signaling by binding to the TGF-? receptor II(T?RII).Then,TGF-?1 activates the TGF-? receptor type I(T?RI)kinase,resulting in phosphorylation of SMAD2 and SMAD3.Subsequently,the activated SMAD2 and SMAD3 form oligomeric complexes with SMAD4.These oligomeric complexes translocate into the nucleus,where they regulate the transcription of target genes and activate HSCs.Simultaneously,SMAD7 is overexpressed,which constitutes a negative feedback regulation loop to maintain the balance of HSCs in stationary state and active state during the TGF-?1 signal pathway.MicroRNA can inhibit the translation of its target gene by binding with 3?-UTR of target mRNA via complementary base pairing,and hence regulate the expression of the genes in post-transcriptional level.miR-29 and miR-19 b are downregulated in HSCs and liver cirrhosis,which overexpression medi-ated by TGF-? pathway can reduce I collagen and ?-SMA expression to inhibit liver fibrosis.SB-525334 has been characterized as a potent and selective inhibitor of the TGF-?1 receptor,activin receptor-like kinase(ALK5).SB-525334 can inhibit TGF-?1 receptor mediated chronic kidney injury.However,the mechanism of SB-525334 on liver fibrosis is unclear,the study intends to observe the expression of type I collagen,miR-29 and miR-19 b in LX-2 cell line when treated with SB-525334 to clarify its effect on liver fibrosis.Objective: Aimming to assess the SB-525334 in the prevention and treatment of hepatic fibrosis application value,hepatic stellate cell line LX-2 were treated with SB-525334 and observed the expression of type I collagen,miR-29 and miR-19 b.Methods:1 LX-2 cells were divided into four groups,control group,TGF-?1 group,SB-525334 group and TGF-?1 + SB-525334 group.2 MTT assay was used to evaluated TGF-?1 and SB-525334 optimum drug concentration,optimum time,proliferation rate and inhibition rate.3 After administered drugs,the growth state of cells in each group was observed under inverted microscope.4 Reverse transcription-quantitative polymerase chain reaction(RTqPCR)was used to determine the expression of type I collagen mRNA,miR-29a,miR-29b,miR-29c and miR-19b in LX-2 cells of each group.5 The protein expression of type I collagen in each group was further evaluated by western blot method.Results: 1 MTT assay indicated that SB-525334 inhibited cell proliferation,the proliferation of LX-2 cells was in a concentration-dependent manner at first increased and then decreased(P < 0.05)in TGF-?1 group compared with the control,the survival rate up to 132% with concentration of 10ng/ml.The proliferation in a concentration-dependent manner showing gradually decreased(P < 0.05)in SB-525334 group compared with the control,the inhibition rate up to 38.4% with concentration of 10umol/l.Both drugs were no significant difference in the cell proliferation rate and inhibition rate of different time(P > 0.05).2 Under inverted microscope was investigated the effect of SB-525334 to LX-2 cells,cells morphology were observed at different time points in each group.We could find that the number of cells and volume increases,cells protrusion extend,cytoplasm increase,the nucleus become larger and cells grow slowly with time in TGF-?1 group compared with the control.While in the SB-525334 group,we found the number of cells reducing,cell size smaller,cytoplasm concentrating and nucleus rounding,with time,cells apotosis compaired with the control group.In the TGF-?1 + SB-525334 group,we found the number of cells reducing,cell size smaller,cytoplasm concentrating and nucleus rounding,apotosis after 24 h compared with the control group.3 RT-qPCR showed that SB-525334 inhibits the expression of type I collagen mRNA,which TGF-?1 + SB-525334 group of type I collagen mRNA expression levels(0.60 ± 0.37)lower than that TGF-?1 group(2.44 ± 0.31)(P < 0.05).TGF-?1 + SB-525334 group of type I collagen mRNA expression levels(0.60 ± 0.37)compared with SB-525334 group(0.55 ± 0.36)was no significant difference(P > 0.05).4 Western blot showed that SB525334 could inhibit the expression of type I collagen,which the expression of type I collagen(82.80 ± 4.39)significantly increased in the TGF-?1 group than the control group(17.89 ± 4.27)(P < 0.01)and type I collagen(62.62 ± 3.72)expression in TGF-?1 + SB-525334 group was lower than the TGF-?1 group(82.80 ± 4.39)(P < 0.05).5 RT-qPCR showed that SB-525334 inhibited the expression of miR-29 a,which the relative expression of miR-29 a in SB-525334 group(0.38 ± 0.06)was lower than control group(P < 0.05),the relative expression of miR-29 a in TGF-?1 group(0.82 ± 0.07)and TGF-?1 + SB-525334 group(0.64 ± 0.08)had no statistically significance(P > 0.05)with the control group,meanwhile,there was no significant difference between the two comparison(P > 0.05).6 RT-qPCR showed that SB-525334 can down-regulate the expression of miR-29 b,which the relative expression of miR-29 b in TGF-?1 group(0.77 ± 0.05),SB-525334 group(0.44 ± 0.04)and TGF-?1 + SB-525334 group(0.61 ± 0.06)were all lower than control group(P < 0.05),the relative expression of miR-29 b in TGF-?1 + SB-525334 group(0.61 ± 0.06)reduced compared with TGF-?1 group(0.77 ± 0.05)(P < 0.05)and increased compared with SB-525334 group(0.44 ± 0.04)(P < 0.05).7 RT-qPCR showed that SB-525334 could down-regulate expression of miR-19 b,which the relative expression of miR-19 b in TGF-?1 group(0.62 ± 0.07),SB-525334 group(0.28 ± 0.06)TGF-?1 + SB-525334 group(0.64 ± 0.20)were all significantly lower than the control group(P < 0.01),the relative expression of miR-19 b in TGF-?1 + SB-525334 group increased compared with SB-525334 group(P < 0.05)and no significant difference compared with TGF-?1 groups(P > 0.05).8 RT-qPCR showed that SB-525334 inhibitated the expression of miR-29 c,which the relative expression of miR-29 c in SB-525334 group(0.43 ± 0.06)was lower than control group(P < 0.05),while the relative expression of miR-29 c in TGF-?1 group(0.80 ± 0.14)and TGF-?1 + SB-525334 group(0.72 ± 0.13)had no statistically significance(P > 0.05)with the control group,meanwhile,there had no significant difference between the two comparison(P > 0.05).Conclusion:1 SB-525334 can inhibited TGF-?1-induced increases in type I collagen expression in HSCs.2 SB-525334 can down-regulate the expression of miR-29 b,miR-19 b through TGF-?1 mediated in HSCs.SB-525334 can inhibite the expression of miR-29a,miR-29b. |
PDF Full Text Request