| Objectives To investigate whether N-acetyl-seryl-aspartyl-lysyl-proline（N-acetyl-seryl-aspartyl-lysyl-proline,Ac-SDKP）affects the FAS/FASL apoptosis pathway of human alveolar typeⅡepithelial cells（A549 cell line）through the regulation of heat shock protein27（heat shock protein 27,HSP27）.Methods The A549 cell line was cultured in vitro for 6 h,12 h,24 h and 48 h.A549 cell line was pretreated with Ac-SDKP(1×10-8 mol/L)and J2（2μM）2 h before induction.A549 cell line was induced by 5 ng/ml transforming growth factor-beta 1（transforming growth factor-beta 1,TGF-β1）.The experiment was divided into four groups:control group,TGF-β1 induction group,Ac-SDKP intervention group,J2（HSP27 inhibitor）intervention group.The morphological changes of cells were observed by inverted phase contrast microscope and HE staining;the expression of HSP27,FAS,FASL,Caspase-3 and Caspase-8 was detected by immunocytochemical staining;the protein expression of HSP27,FAS,FASL,Caspase-3 and Caspase-8 was detected by Western blotting（Western blot）;the expression of HSPB1 and FAS m RNA was detected by real-time fluorescence quantitative PCR（real time fluorescence quantitative PCR,q PCR）;the localization expression of HSP27 and FAS was detected by immunofluorescence staining;the apoptosis of cells was detected by flow cytometry.Results 1 The results of inverted phase contrast microscope and HE staining showed that compared with the control group,the morphology of A549 cells induced by TGF-β1 for 24h and 48 h changed more obviously from polygonal or epithelioid to long fusiform,and the number of cells increased;compared with the TGF-β1 induction group,the change of cells to long fusiform in Ac-SDKP and J2 intervention group was not obvious.2 The results of immunocytochemical staining showed:in 6 h and 12 h groups,compared with the control group,the brown-yellow expression of HSP27,FAS,FASL cytoplasm and the cytoplasmic nucleus of Caspase-3 and Caspase-8 in TGF-β1 induction group were deeper than those in control group;compared with TGF-β1 induction group,the brown-yellow expression of HSP27,FAS,FASL cytoplasm became lighter in Ac-SDKP and J2 intervention group,and the brown-yellow expression of Caspase-3 and Caspase-8 in cytoplasm and nucleus became lighter.In 24 h and 48 h groups,compared with the control group,the brown-yellow expression of HSP27 cytoplasm in TGF-β1 induction group was deeper,and the cytoplasmic brown-yellow expression of FAS,FASL,Caspase-3 and Caspase-8 became lighter;compared with TGF-β1 induction group,the brown-yellow expression of HSP27cytoplasm was lighter,FAS and FASL cytoplasmic brown-yellow expression was deeper,and Caspase-3 and Caspase-8 cytoplasmic nucleus expression was deeper in Ac-SDKP and J2 intervention group.3 The results of Western blot showed that the protein expressions of HSP27,FAS,FASL,Caspase-3 and Caspase-8 in TGF-β1 induction group were significantly higher than those in control group at 6 h and 12 h groups,the difference was statistically significant（P<0.05）;the protein expressions of HSP27,FAS,FASL,Caspase-3 and Caspase-8 in Ac-SDKP and J2 intervention group were significantly lower than those in TGF-β1 induction group,the difference was statistically significant（P<0.05）.In 24 h and 48 h groups,compared with the control group,the expression of HSP27 protein in TGF-β1 induction group increased,while the expression of FAS,FASL,Caspase-3 and Caspase-8 protein decreased,the difference was statistically significant（P<0.05）;compared with TGF-β1 induction group,the expression of HSP27 protein in Ac-SDKP and J2 intervention group decreased,while the expression of FAS,FASL,Caspase-3 and Caspase-8 increased,the difference was statistically significant（P<0.05）.There was no significant difference between Ac-SDKP intervention group and J2 intervention group（P>0.05）.4 The results of real-time fluorescence quantitative PCR showed that compared with the control group,the expression of HSPB1 and FAS m RNA in TGF-β1 induction group was up-regulated at 6 h and 12 h groups;while the expression of HSPB1 and FAS m RNA in Ac-SDKP and J2intervention group was down-regulated compared with TGF-β1 induction group,the difference was statistically significant（P<0.05）.In 24 h and 48 h groups,compared with the control group,the expression of HSPB1 m RNA was up-regulated and the expression of FAS m RNA was down-regulated in TGF-β1 induction group;while HSPB1 m RNA expression was down-regulated and FAS m RNA expression was up-regulated in Ac-SDKP and J2 intervention group compared with TGF-β1 induction group,the difference was statistically significant（P<0.05）.5 The results of immunofluorescence staining showed that in 6 h and 12 h groups,the fluorescence intensity of HSP27（red fluorescence）and FAS（green fluorescence）in TGF-β1 induction group was higher than that in control group;while the fluorescence intensity of HSP27（red fluorescence）and FAS（green fluorescence）in Ac-SDKP and J2 intervention group was lower than that in TGF-β1 induction group.In24 h and 48 h groups,compared with the control group,the fluorescence intensity of HSP27（red fluorescence）increased and FAS（green fluorescence）decreased in TGF-β1induction group;while HSP27（red fluorescence）fluorescence intensity decreased and FAS（green fluorescence）fluorescence intensity increased in Ac-SDKP and J2 intervention group compared with TGF-β1 induction group.6 The results of flow cytometry showed that the percentage of apoptosis in TGF-β1 induction group increased in 6 h and 12 h groups compared with the control group;compared with TGF-β1 induction group,the percentage of apoptosis in Ac-SDKP and J2 intervention group decreased,The difference was statistically significant（P<0.05）.In 24 h and 48 h groups,the percentage of apoptosis of induced by TGF-β1 decreased compared with the control group;compared with TGF-β1induction group,the percentage of apoptosis in Ac-SDKP and J2 intervention group increased,The difference was statistically significant（P<0.05）.Conclusions Ac-SDKP may promote the apoptosis of A549 cells by regulating the expression of HSP27 and activating FAS/FASL apoptosis pathway,which is similar to that of J2（HSP27 inhibitor）.Figure 22;Table 13;Reference 91... |
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