Notch Signaling In Liver Fibrosis Rats And The Research On Halofuginone Intervention

Objective To research the exact interaction between Notch and transforming growth factor(TGF)-? signaling,which the effect on the balance of Th17/Treg in hepatic fibrosis rats;exploring the changes of Notch and TGF-? pathway in hepatic fibrosis rats with halofuginone and the mechanism of its anti-fibrosis.Methods Rats were divided into control(n=12)and model(three groups,n=12 per group)group.The rats in control were iv injected with 300 ?l PBS from tail vain once a week,the rats in model were iv injected with 12.5 mg/kg Con A(in 300 ?l PBS)once a week for 8 consecutive weeks.The biochemical analysis was used to determine ALT,AST and ALB in serum while HE staining was conducted in liver tissue to observe the pathological variations.At 8 wk later,the model group rats were randomly divided into DAPT,TGF-? inhibitor and DMSO group(n=12 per group).We collected blood 3ml via cardiac puncture,and isolated peripheral blood mononuclear cells(PBMCs)from it.These cells were treated with DAPT and TGF-? inhibitor for 24 h,with 0.1% DMSO as a control.The relative m RNA levels of Notch and TGF-? were detected by RT-PCR.Expression of Notch and TGF-? proteins was analyzed by Western blotting.The frequencies of Th17 and Treg cells in PBMCs were measured by flow cytometry(FCM),and the contents of IL-17,IL-23,IL-10 and TGF-?1 in the culture supernatant were measured by ELISA.Rats were randomly divided into model group(n =10),halofuginone group(n=12)and control group(n=12).The mothed of molding,ditto.Halofuginone was given to rats by gavage in halofuginone group while molding and continuous administration for eight weeks.The relative m RNA levels of Notch and TGF-? were detected by RT-PCR.Expression of Notch and TGF-? proteins was analyzed by Western blotting.The immunohistochemical staining was used to test the expression of Notch and TGF-? signaling in liver tissue.Results Compared with the control group,the levels of ALT and AST in model group were increased significantly(P<0.05),while ALB was decreased remarkably(P<0.05).HE staining showed hepatocyte necrosis and false lobules formed.The Notch and TGF-? signaling was activated in liver fibrosis.RT-PCR analysis revealed that the mRNA levels of Notch1,Hes1,Hes5,TGF-?1 and Smad3 were significantly increased in PBMCs of model rats as compared with controls(P<0.05).The relevant protein levels had the same changes(P<0.05).The mRNA and protein levels of Notch and TGF-? signaling were prominent reducing in DAPT and TGF-? inhibitor group compared with DMSO group(P<0.05).The frequency of Th17 and its cytokines(IL-17,IL-23)were lower(P<0.05),while the frequency of Treg and its cytokines(IL-10,TGF-?1)were lower markedly(P<0.05).Furthermore,the ratio of Th17/Treg increased in DAPT group and TGF-? inhibitor group compared with DMSO control group.Compared with the model group,the mRNA and protein levels of Notch1,Hes1,Hes5,TGF-?1 and Smad3 were significantly decreased in halofuginone group(P<0.05),there were no significant differences in control group.Immunohistochemical staining showed the expression of Notch1,Hes1,Hes5,TGF-? and Smad3 were remarkably reducing compared with the model group(P<0.05),there were no remarkable changes in control group.Conclusion Notch and TGF-? signaling plays a role in liver fibrosis.Treatment with the ?-secretase inhibitor DAPT significantly inhibited Notch and TGF-? signaling in PBMCs of liver fibrosis rats,which is similar to the TGF-? inhibitor.Blocking Notch signaling inhibits Treg cells function and reduces the level of TGF-?1,which changes the balance of Th17/Treg cells.Halofuginone may inhibit Notch and TGF-? signaling,and it exerts anti-fibrosis effect by reducing the expression of Smad3.