Autophagy Participates In LPS-stimulated Inflammation In Macrophage And Its Mechanisms

Objective:To investigate whether autophagy was involved in lipopolysaccharide (LPS)-stimulated inflammation in macrophage and the underlying mechanisms.Methods:Part I:LPS was used to stimulate and induce the production of proinflammatory cytokines in Raw264.7macrophage. Western blot was applied to examine the expression of TNF-a. LC3puncta formation was monitored by immunohistochemistry. The expression of autophagy-related proteins (LC3, p62and Beclin1) at different time points was determined by western blot analysis. Part II:The expression of autophagy related proteins was determined by western blot analysis. The levels of proinflammatory cytokines such as TNF-a, ICAM-1and NO in cell-free culture supernatant were determined by ELISA and Griess Reagent, respectively. The level of p62mRNA was measured by reverse transcription PCR. p62siRNA was used to knockdown p62, and the production and release of TNF-a was determined by ELISA.Results:Part Ⅰ:The stimulation with LPS markedly increased the levels of TNF-a and autophagy related proteins of LC3, p62and Beclinl in Raw264.7macrophage. When LPS was removed, the expression of autophagy related proteins were maintained but not decreased for up to12hours. Fluorescent immunohistochemistry for LC3showed that there was significant increase of puncta formation following LPS stimulation for12hours. Part Ⅱ:Pretreatment with Rapamycin, an autophagy activator, significantly increased the levels of autophagy related proteins and also enhanced the production of inflammation cytokines including TNF-a, ICAM-1and NO induced by LPS. But, pretreatment with3MA, an autophagy inhibitor, significantly decreased the levels of autophagy related proteins and suppressed the LPS-stimulated expression of inflammation cytokines. Reverse transcript PCR confirmed that the expression of p62mRNA was in paralleled with the change of p62protein level. transfection with effective p62siRNA markedly superssed the expression of TNF-a in response to LPS.Conclusion:LPS may trigger the dysfunction of autophagy and inflammatory responses in macrophage. Autophagy activator, at an early stage, can increase the expression of p62mRNA and inflammatory cytokines in LPS-stimulated macrophage. Conversely, autophagy inhibitor, can decrease the accumulation of p62and inflammatory cytokines. Therefore, the present study presents that p62play an important role in LPS-stimulated inflammation in macrophage.