The Relationship Between IL-22 And Notch Signaling Way In Regulating The Process Of Liver Fibrosis

Hepatic fibrosis is a reversible wound-healing response characterized by the accumulation of extracellular matrix(ECM)to liver injury.In the process of hepatic fibrosis,the key process is the activation and proliferation of hepatic stellate cells(HSCs),which is characterized by the expression of α-smooth muscle actin(α-SMA)and ECM.Therefore,the majority of anti-fibrotic therapies are designed to inhibit the activation,proliferation,or synthetic products of HSCs.Interleukin-22(IL-22)plays an important role in controlling bacterial infection via the upregulation of anti-microbial proteins,and also promotes tissue repair by upregulating a variety of genes expressed in epithelial cells such as hepatocytes.In recent years,more and more studies found that IL-22 ameliorate fibrosis in fibrosis disease such as pulmonary,renal and peritoneal fibrosis.Notch signaling is an ancient cell signaling that regulates cell fate specification,stem cell maintenance,and initiation ofdifferentiation in embryonic and postnatal tissues.More recently,some researches reported that Notch signaling was implicated in human fibrosis diseases,such as pulmonary,renal and peritoneal fibrosis.In order to elucidate the role of IL-22 in hepatic fibrosis,and discuss the relationship between IL-22 and Notch signaling way,and to investigate whether Notch signaling is involved in liver fibrosis by regulating the activation of hepatic stellate cells(HSCs),the research will be divided into the following two parts.Part1:Interventing liver fibrosis in mice with recombinant IL-22,DAPT(inhibitor of Notch signaling way)and IFN-y in vivoObjective:To observe the influence of IL-22 on the state of liver fibrosis and the difference of the expression of Notch signaling pathways related factor in rat liver fibrosis model caused by CCL4 in vivo.Method:A total of 62 mice were treated with 2 mL/kg body weight of 20%CCI4 intraperitoneally for 8 weeks,and then randomly divided into five groups.The mice were given rIL-22,DAPT,rIL-22+DAPT,IFN-γ and IFN-γ+DAPT groups by intraperitoneal injection.All surviving animals were sacrificed on week 10.HE and Masson staining were used to observe the pathological changes of hepatic at six groups.Ishak scoring system was used to assess the degree of liver fibrosis.Immunohistochemical staining was used to detect the expression of α-smooth in the livers of mice.qRT-PCR method was adopted to detect mRNA expressions of Hes-1,Hes-5 and Hey-1.The protein expression ofα-SMA and.Notch3 protein were determined by Western blot.Result:(1)Compared with normal group,HE staining showed liver cells of all the experimental groups gradually edema,degeneration,necrosis,a large number of inflammatory cells infiltration and a large amount of collagen deposition at all the experimental groups,especially in the portal area;Masson staining showed portal area of all the experimental groups had large blue-green collagen deposition.As the intervention of different recombinant protein,the degree of liver fibrosis in all the experimental groups had alleviated,especially in the rIL-22+DAPT group,p<0.05.(2)The results of immunohistochemical staining showed that expression of α-SMA protein were higher than in normal control group,p<0.05,as the intervention of different recombinant protein,the expression of α-SMA protein were down-regulation,especially in the rIL-22+DAPT group,p<0.05.(3)Compared with the control group,the expressions of Hes-1,Hes-5 and Hey-1 at all the experimental groups were down-regulation,especially in the rIL-22+DAPT group,p<0.05.(4)Compared with the control group,the expressions of Notch3 protein at all the experimental groups were down-regulation,especially in the rIL-22+DAPT group,p<0.05.Conclusion:IL-22 ameliorate fibrosis in liver fibrosis;IL-22 and DAPT(the Notch signal pathway inhibitors)can ameliorate the degree of liver fibrosis significantly;IL-22 may play the role in the process of role in liver fibrosis through the Notch signaling pathways.Part2:Interventing the activated hepatic stellate cell with recombinant IL-22,DAPT(inhibitor of Notch signaling way)and IFN-γ in vitroObjective:To observe the influence of IL-22 on the state of the activation of hepatic stellate cells and the difference of the expression of Notch signaling pathways related factor under the different cocultivation status of hepatic stellate cell in vitro.Method:rIL-22,DAPT and IFN-γ of different concentrations were cocultured with hepatic stellate cell-T6(HSC-T6)for 24 h,36h and 48h.CCK8 was used to detect the effects of rIL-22,DAPT and IFN-γ on HSC-T6 proliferation.Flow cytometer was used to detect the effects of rIL-22,DAPT and IFN-γ on HSC-T6 apoptosis.qRT-PCR method was adopted to detect mRNA expressions of Hes-1,Hes-5 and Hey-1.The protein expression ofα-SMA and Notch3 protein were determined by Western-blot.Result:(1)After culture with TGF-β1,immunofluorescence results showed α-SMA(+)and more than 95%of activated HSC positive.Cultured with different concentrations of recombinant protein,cell proliferation of HSC-T6 at IL-22 and DAPT groups was no statistically significant difference compared with the control group by CCK8 detection,p>0.05;and cell proliferation of HSC-T6 at IFN-γ group and IFN-γ+DAPT group was decreased obviously as the concentration of IFN-γ and DAPT rised,the difference was statistically significant,p<0.05,compared with the control group.(2)Compared with the control group,HSC-T6 apoptosis were increased apparently as the concentration of recombinant protein rised,especially in the rIL-22+DAPT group(p<0.05).(3)The expression of α-SMA,compared with the control group,were decreased obviously as the concentration of recombinant protein rised,especially in the rIL-22+DAPT group(p<0.05).(4)Compared with control group,the expressions of Hes-1,Hes-5 and Hey-1 mRNA were down-regulation significantly as the concentration of recombinant protein rised,with optimal concentration effect when the relative expression quantity reduced to a minimum,p<0.05.Compared between different groups,the expressions of Hes-1,Hes-5 and Hey-1 mRNA were down-regulation significantly in the rIL-22 group,p<0.05.(5)Compared with control group,the expressions of Notch3 protein were down-regulation significantly as the concentration of recombinant protein rised,with optimal concentration effect when the relative expression quantity reduced to a minimum,p<0.05.Compared between different groups,the expressions of Notch3 protein were down-regulation significantly in the rIL-22 group,p<0.05.Conclusion:IL-22 can depress the activation of HSC-T6 and Notch signaling pathway may play a role together in the process of hepatic fibrosis.