Mechanism Of LPS-induced Autophagy Inhibition And Pulmonary Fibrosis In Lung Fibroblasts

Background and PurposeLipopolysaccharide is an important pathogenic factor of sepsis and sepsis-associated pulmonary fibrosis.The in-depth study of the pathogenesis of LPS-induced pulmonary fibrosis has always been an important research direction in the field of severe medicine,but its related mechanism has not yet been elucidated.In this study,LPS induced lung fibroblast autophagy and LPS-induced pulmonary fibrosis in mice was used to study the protective effect and mechanism of Thy-1 deficiency and integrin ?3 up-regulation on autophagy of lung fibroblasts.Methods1.Part ?: Mouse lung fibroblasts were stimulated with LPS.The expression of Thy-1,integrin ?3,autophagy-related proteins LC3 and Beclin-1 was detected by Western Blot.Autophagosomes were detected by transmission electron microscopy.2.Part ?: Inhibitors and siRNA were used to inhibit integrin ?3,siRNA or overexpression of lentivirus to inhibit or overexpress Thy-1,and Western Blot was used to detect the expression of Thy-1,integrin ?3,PI3K-Akt-m TOR signaling pathway and autophagy-related proteins after LPS stimulation.Autophagosomes were detected by transmission electron microscopy.The pulmonary fibrosis model was established by LPS multiple-strike method.HE and Masson staining and immunohistochemical staining of ?-SMA were performed in lung tissue sections.Thy-1,integrin ?3,LC3,P62 and ?-SMA protein expression of lung tissues were detected by Western Blot.Detection of hydroxyproline and type I collagen content in lung tissue.Thy-1 overexpressing adeno-associated virus was performed intratracheally or integrin ?3 inhibitor cilengitide was performed intraperitoneally,following by establishing a pulmonary fibrosis model according to the above method,and comparing the changes of the above indicators.Results1.Part ?: LPS stimulation caused a decrease in Thy-1 expression,an increase in integrin ?3 expression,a decrease in the expression of autophagy-related proteins Beclin-1 and LC3-II/I,and a decrease of autophagosomes.2.Part ?: Vitro experiments confirmed that inhibition of integrin ?3 and overexpression of Thy-1 reversed LPS-induced PI3K-Akt-m TOR pathway activation and autophagy inhibition.In the absence of LPS,inhibition of Thy-1 mimics LPSinduced PI3K-Akt-m TOR pathway activation and autophagy inhibition.Vivo experiments confirmed that LPS multiple strokes can cause pulmonary fibrosis in mice,accompanied by down-regulation of Thy-1,up-regulation of integrin ?3 and inhibition of autophagy in lung tissues.Intratracheal injection of Thy-1 overexpressing AAV or intraperitoneal pre-injection cilengitide can reverse LPSinduced pulmonary fibrosis,down-regulation of Thy-1,up-regulation of integrin ?3 and autophagy inhibition in mice.ConclusionLPS can inhibit autophagy and cause pulmonary fibrosis in lung fibroblasts.LPS activates PI3K-Akt-m TOR pathway by inducing Thy-1 deficiency and up-regulation of integrin ?3,inhibiting autophagy of lung fibroblasts and promoting pulmonary fibrosis.