Intervention In The Effect Of Senescent Alveolar Epithelial Cell Succinylation On Pulmonary Fibrosis

Background: Idiopathic pulmonary fibrosis(IPF)occurs primarily in patients over60 years old.Alveolar epithelial cells(AECs)senescence is an important factor in initiating and accelerating IPF.AECs are strongly associated with mitochondrial dysfunction in aging.Mitochondrial dysfunction leads to AECs senescence through a variety of pathways,such as impeded energy synthesis,increased free radical release,antioxidant defense system disorders,and mitochondrial DNA damage.Hypersuccinylation that occurs in mitochondria is an important mechanism responsible for these dysfunctions.Succinylation occur from lysine residues and can be hydrolyzed by sirtuin 5(SIRT5)in the sirtuins family to maintain the stability of mitochondrial function.Objective:SIRT5-mediated lysine desuccinylation is the molecular mechanism that regulates pulmonary fibrosis induced by AECs senescence.Methods: Western blotting and immunofluorescence were used to detect the level of succinylation,the expression of mitochondrial functional protein and SIRT5 in the lung tissue of IPF patients.The cellular senescence model of AECs induced by bleomycin(BLM)was established.The constructed overexpression plasmid pc DNA-SIRT5 intervened in the BLM-induced AECs senescence model,and the effect of western blotting was verified.β-galactosidase staining to observe the aging changes of AECs;Western blotting,immunofluorescence detection of mitochondrial functional proteins,AECs aging marker expression;Mitochondrial cell membrane potential JC-1 and red fluorescent probe detect changes in mitochondrial membrane potential in AECs.Results: In clinical studies,it was found that many proteins in IPF lung tissue were regulated by succinylation,PGC-1α,TOMM20 and SIRT5 was down-regulated;In the aging of AECs in IPF lung tissue,the expression of PGC-1α and TOMM20 was upregulated,and the expression of SIRT5 was down-regulated;In order to verify this clinical phenomenon,the verification of basic experiments was further opened.Firstly,after BLM stimulation of AECs for 72 h,the overall level of succinylation increased compared with the control group,while AECs senescence was accompanied by upregulation of PGC-1αand TOMM20 expression and downregulation of SIRT5 expression.Plasmid pc DNASIRT5 intervened in senescent AECs,and western blotting verified that SIRT5 was successfully transfected into mouse AECs.After plasmid pc DNA-SIRT5 removed the hypersuccinylation modification of AECs,the expression of senescence marker P21 was down-regulated,and the expression of mitochondrial functional proteins PGC-1α and TOMM20 was down-regulated,which also inhibited the increase of mitochondrial membrane potential after AECs senescence.Conclusions: Mitochondrial dysfunction and hypersuccinylation occurred in the lung tissue of IPF patients;SIRT5 as the intervention target can prevent AECs senescence by maintaining the stability of mitochondrial function.