Mechanism Responsible For The Effect Of Hydrogen Sulfide In Improving Pulmonary Fibrosis During Exercise And In Epithelial Cell Senescence Of AT?

Objective:Pulmonary fibrosis is a progressive lung disease caused by a variety of factors.The alveolar type II epithelial cell senescence is closely related to the pathogenesis of pulmonary fibrosis.Exercise has been widely considered as a safe and effective means in disease prevention and rehabilitation.As an important gas signal molecule,H₂S plays an important regulatory role in the occurrence of many diseases,for example,it can improve related diseases by reversing the senescence of human umbilical vein endothelial cells and neurons.Therefore,the purpose of this paper is to first explore whether exercise improve pulmonary fibrosis is related to H₂S,and what is the mechanism?In addition,to investigate whether H₂S can reverse the alveolar epithelial cell senescence and improve pulmonary fibrosis.Methods:1^st part:Through the establishment of BLM-induced pulmonary fibrosis mice,exercise intervention and exogenous H₂S supplementation were carried out in mice,and the collagen deposition in the lung tissue of mice was observed by Masson staining method,the area of fibrosis in the lung tissue was calculated.The contents of hydroxyproline,type I collagen and TGF-?1 in lung tissue were detected by ELISA.The expression of epithelial marker e-cadherin and mesenchymal marker?-sma?were observed by immunofluorescence co-staining.Western Blot analysis was used to detect lrp-6/?-catenin signal pathway related protein expression;Zinc capture method was used to compare endogenous H₂S content in lung tissues of mice.2^nd partFirst,lung tissues were collected at different time points after endotracheal intubation BLM,to observe the changes of cellular senescence markers P53,P21protein expression,and endogenous H₂S content in lung tissues during the formation of pulmonary fibrosis,and whether there was a correlation between them.The changes of lung tissue and the area of pulmonary fibrosis were observed by Masson staining after BLM infusion.Western Blot was used to detect the expressions of P53,P21,CBS and CSE in lung tissues of mice.The content of endogenous H₂S in lung tissues of each group was determined by zinc capture method.Then we tested whether exogenous H2S could reverse the senescence of alveolar epithelial cells in vitro and in vivo.The expressions of P53 and P21 were detected by Western Blot.Senescence of L2 cells was observed by SA-?-gal staining.The expressions of SASP factors MCP-1,Pai1,MMP10 and TGF-?were detected by RT-PCR.CCK8 was used to detect the proliferation of NIH3T3 fibroblasts.Finally,verify whether H₂S promotes P53 degradation through the proteasome pathway.MG132 was added to observe whether the role of H₂S in promoting P53degradation could be reversed by MG132.Results:1^st part1)Exercise training attenuates bleomycin-induced pulmonary fibrosis;2)Exercise training ameliorates bleomycin-induced EMT in lung tissue;3)Exercise training ameliorates bleomycin-induced activation of the LRP6/?-catenin signaling pathway in lung tissue;4)Exercise training restores endogenous H₂S production in lung tissues of bleomycin?treated mice;5)H₂S donor attenuates bleomycin?induced pulmonary fibrosis;6)H₂S donor ameliorates bleomycin?induced EMT in lung tissue;7)H₂S donor ameliorates bleomycin?induced activation of the LRP6/??catenin signalling pathway in lung tissue.2^nd part1)Decreased H₂S production is correlated with higher levels of cell senescence markers p53 and p21 in bleomycin-induced lung fibrosis;2)H₂S donor can improve pulmonary fibrosis by inhibiting the P53/P21 signaling pathway,inhibiting the BLM-induced senescent phenotype of AT? cells and abnormal proliferation of fibroblasts;3)H₂S donor promotes P53 degradation by proteasome pathway.Conclusion:Exercise may improve pulmonary fibrosis by increasing endogenous H₂S content in lung tissue,inhibiting the activation of LRP6/?-catenin signal pathway,and reversing EMT..Meanwhile,H₂S inhibits the activation of P53/P21 signaling pathway,inhibits the BLM-induced senescent phenotype of AT? cells and abnormal proliferation of fibroblasts,thereby improving pulmonary fibrosis.