Study On The Mechanism Of Autophagy Mediated By Mammalian Target Of Rapamycin On Immunosuppression Of Sepsis

Background:Sepsis is a life-threatening organ dysfunction caused by the host’s disordered response to infection.Late immunosuppression predominates in sepsis,leading to repeated opportunistic infections and high mortality.Autophagy can participate in immune regulation and maintain immune system homeostasis.Minocycline and 3BDO can induce and inhibit autophagy through the m TOR pathway.In this paper,we studied the effect of autophagy regulation on immunosuppression of sepsis through m TOR pathway.Objective: To investigate the secretion of inflammatory cytokines in human mononuclear/macrophage cell line(THP-1 cells)after multiple lipopolysaccharide(LPS)stimulation.The administration of minocycline or 3BDO in the immunosuppressive stage affects the level of autophagy,and the effect of autophagy on cytokine release and mononuclear/macrophage proliferation in the immunosuppressive stage of sepsis is observed,and the possible mechanism of action is discussed,so as to provide a new strategy for improving the prognosis of immunosuppressive patients with sepsis clinically.Research methods: THP-1 cells were stimulated by LPS several times to simulate the inflammatory cell model of severe clinical infection.Cytokine concentrations at different time points were measured by ELISA,and cell proliferation levels were measured by WST-1 cell proliferation assay,so as to clarify the immune status of THP-1 cells.In the immunosuppressive stage,m TOR inhibitor Minocycline and m TOR activator 3BDO were respectively used to intervene,and Western blot was used to detect the expressions of NF-κB,LC3 and p-m TOR at different times,so as to evaluate the influence of drug intervention on autophagy level.Results: Multiple LPS attacks up-regulated the expression of NF-κB in a time-dependent pattern,and stimulated the release of TNF-α and IL-8 from THP-1 cells.TNF-α/IL-10 decreased slightly in THP-1 cells around day 4 after multiple LPS hits.LPS up-regulated the expression levels of p-m TOR and LC3-Ⅱ/LC3-Ⅰ and induced autophagy,but the autophagy level did not increase significantly with time.Minocycline inhibited cytokine release from NF-κB signaling pathway in dose-dependent mode,and the low-dose multiple administration mode inhibited TNF-α and IL-8 release from THP-1 cells more strongly.Minocycline inhibited pm TOR expression,up-regulated LC3-Ⅱ/LC3-Ⅰ,induced autophagy and inhibited THP-1 cell release of inflammatory cytokines,but its ability to up-regulate autophagy did not increase in a dose-dependent manner.LPS stimulated cell proliferation of THP-1 cells,Minocycline downregulated m TOR to inhibit immune cell proliferation,and 3BDO activated m TOR to promote immune cell proliferation.Conclusion: Regulation of autophagy through m TOR pathway can affect immune status of sepsis,and induction of autophagy in late sepsis provides a new direction for immunosuppressive therapy of sepsis.