PI3K-Akt-mTOR Pathway Mediated LPS-induced Autophagy Inhibition In Lung Fibroblasts

The lipopolysaccharide(LPS)in the cell walls of gram negative bacilli is the main component of endotoxin,and is also an important pathogenic factor of acute respiratory distress syndrome and pulmonary fibrosis.LPS induces abnormal proliferation and activation of lung fibroblasts and causes collagen deposition in lung tissue,finally resulting in pulmonary fibrosis.The underlying mechanism remains unknown.Recent studies showthat autophagy inhibition in lung fibroblasts is associated with the process of pulmonary fibrosis.However,whether autophagy inhibition in lung fibroblasts exists in LPS-induced pulmonary fibrosis and the related regulatory mechanism still remain to be elucidated.Our previous study discovered that PI3K-Akt pathway mediated LPS-induced fibroproliferation in lung fibrobasts,while recent studies revealed that PI3K-Akt-m TOR pathway is an important regulatory pathway in the process of autophagy.For these reasons,we speculate that the activation of PI3K-Akt-m TOR pathway mediates the LPS-induced autophagy inhibition in lung fibroblasts,which is closely related to LPS-induced pulmonary fibrosis.Purpose In this study,we use LPS-inhibited lung fibroblast autophagy model to investigate the effects of LPS-stimulating mouse lung fibroblasts and the underlying mechanism,to clarify the function of PI3K-Akt-m TOR pathway in it,to further substantiate and enrich the mechanism of LPS-induced pulmonary fibrosis and to lay the foundation for the way of preventing and controling this disease.Methods Primary-cultured mouse lung fibroblasts were treated with LPS.The expression of autophagy-associated proteins LC3,Beclin1 and SQSTM1/p62 were detected by Western Blot or Immunofluorescence to determine the effect of LPS stimulation on autophagy in lung fibroblast.Western Blot were used to clarify the phosphorylation of PI3K-Akt-m TOR signaling pathway after LPS stimulation with or without PI3K-Akt-m TOR inhibitors,Rapamycin and Ly294002.The expression levels of autophagy-associated proteins LC3,SQSTM1/p62 and the activation of PI3K-Akt-m TOR signaling molecules Akt and m TOR were detected by Western Blot or Immunofluorescence.The formation of autophagosomes was observed by Transmission Electron Microscope.Results 1.In primary-cultured mouse lung fibroblasts,LPS stimulation decreased the expression of autophagy-associated protein Beclin1 and the relative expression of LC-II/I.2.LPS stimulation increased the expression of SQSTM1/p62,an autophagy associated protein which is negatively correlated with autophagy level,inhibited the formation of autophagosomes and promoted the phosphorylation of Akt and m TOR in primary-cultured mouse lung fibroblasts.Pretreatment with m TOR inhibitor Rapamycin decreased the high expression of p62 and rescued the formation of autophagosomes.Pretreatment with PI3K-Akt signaling pathway inhibitor Ly294002 suppressed the activation of downstream signaling molecule m TOR,decreased the high expression of p62 and rescued the formation of autophagosomes,reversing the LPS-induced autophagy inhibition in mouse lung fibroblasts.Conclusion Data from current study demonstrated that LPS inhibited autophagy in primary-cultured mouse lung fibroblasts,and the activation of PI3K-Akt-m TOR pathway mediated LPS-induced autophagy inhibition in lung fibroblasts.