Mechanism Of Ac-SDKP Regulating Apoptosis Of Pulmonary Fibroblasts Through HSP27

Objectives To investigate whether N-acetyl-seryl-aspartyl-lysyl-proline(Ac-SDKP)affects the apoptosis of mouse embryonic fibroblasts(MEF)cells and rat lung fibroblasts by regulating the activation of FAS/FASL signal pathway by heat shock protein 27(HSP27).Methods Two kinds of cells,MEF and rat lung fibroblasts,were cultured in vitro and divided into four groups: 1)control group,2)TGF-β1 induction group(TGF-β1),3)AcSDKP intervention group(TGF-β1 + Ac-SDKP),4)J2 intervention group(TGF-β1 + J2).The morphological changes of cells were observed by HE staining.The expression of HSP27,FAS,FASL,Caspase-8 and Caspase-3 was detected by immunocytochemical staining,immunofluorescence staining,immunoblotting(western blot)and real-time fluorescence quantitative PCR.Apoptosis was observed by flow cytometry and in situ fluorescence.Results 1 The results of HE staining showed that compared with the control group,the morphology of MEF cells induced by TGF-β1 changed from polygonal to long fusiform at24 h and 48 h,and the morphology of rat lung fibroblasts induced by TGF-β1 changed significantly to long fusiform at 24 h and 48 h,while the morphological transformation of MEF cells and rat lung fibroblasts in Ac-SDKP and J2 group was not obvious compared with TGF-β1 group.2 The results of immunocytochemical staining showed that compared with the control group,the brown expression of HSP27 in the cytoplasm of MEF cells and rat lung fibroblasts induced by TGF-β1 at 6 h,12 h,24 h and 48 h was deeper,and the brown expression of HSP27 was lighter in 12 h,24 h and 48 h groups treated with AcSDKP and J2 compared with TGF-β1 group.Compared with the control group,the brown expression of FAS,FASL,Caspase-8 and Caspase-3 in MEF cells and rat lung fibroblasts induced by TGF-β1 for 6 h was deeper,while the brown expression of FAS,FASL,Caspase-8 and Caspase-3 in MEF cells and rat lung fibroblasts induced by TGF-β1 for 24 h and 48 h became lighter.Compared with TGF-β1 induced group,the brown expression of FAS,FASL,Caspase-8 and Caspase-3 in cytoplasm was deepened in 12 h,24 h and 48 h groups treated with Ac-SDKP and J2.3 The results of western blot showed that compared with the control group,the expression of HSP27 protein in MEF cells and rat lung fibroblasts induced by TGF-β1 increased at 6 h,12 h,24 h and 48 h,while the expression of HSP27 protein decreased in Ac-SDKP and J2 treated groups at 12 h,24 h and 48 h compared with TGF-β1 group.Compared with the control group,the expression of FAS,FASL,Caspase-8 and Caspase-3 in MEF cells and rat lung fibroblasts induced by TGF-β1 increased for 6 h,while the expression of FAS,FASL,Caspase-8 and Caspase-3decreased in MEF cells and rat lung fibroblasts induced by TGF-β1 for 24 h and 48 h.Compared with TGF-β1 induced group,FAS,FASL,Caspase-8 and Caspase-3 protein expression increased in 12 h,24 h and 48 h groups treated with Ac-SDKP and J2,the differences of the above results were statistically significant.4 The results of real-time quantitative PCR showed that compared with the control group,the mRNA expression of Hspb1 in MEF cells and rat lung fibroblasts induced by TGF-β1 for 48 h was up-regulated,while the mRNA expression of Fas and Fasl was down-regulated.Compared with the TGF-β1 induced group,the mRNA expression of Hspb1 was down-regulated and the mRNA expression of Fas and Fasl was up-regulated in Ac-SDKP and J2 group.5 The results of immunofluorescence staining showed that compared with the control group,the fluorescence of HSP27 red protein in the cytoplasm of MEF cells and rat lung fibroblasts induced by TGF-β1 increased for 24 h and 48 h,and the fluorescence of FAS green protein decreased.Compared with TGF-β1 induced group,the fluorescence of HSP27 red protein in Ac-SDKP and J2 group decreased and the fluorescence of FAS green protein enhanced.6 The results of flow cytometry and in situ fluorescence showed that there was no significant difference in the proportion of apoptosis of MEF cells and rat lung fibroblasts induced by TGF-β1 for 24 h and 48 h compared with the control group,but compared with the TGF-β1 induced group,the apoptosis trend of MEF cells and rat lung fibroblasts in AcSDKP and J2 groups was significantly different.Conclusions The effect of Ac-SDKP is similar to that of J2(HSP27 inhibitors),which can promote the apoptosis of MEF cells and rat lung fibroblasts by inhibiting HSP27 expression and activating FAS/FASL apoptosis pathway.Figure 29;Table 14;Reference 79...