The Role Of Erbin Protein In Autophagy Induced By Muramyl Dipeptide In Macrophages

Object To investigate the relationship between Erbin protein and autophagy in murine acyl dipeptide（MDP）-induced inflammatory reaction of mouse peritoneal macrophages（RAW264.7）,and provide a new way for the clinical treatment of sepsis.Methods RT-PCR was used to detect the mRNA expression of tumor necrosis factor alpha（TNF-α）and interleukin-6（IL-6）in RAW264.7 cells stimulated by 10μg/ml MDP for 6 hours.The activation of Nuclear factor kappa B p65（NF-κB）was detected by immunofluorescence.Transmission electron microscope was used to observe the formation of autophagosome in each group after RAW264.7 cells were stimulated by10μg/ml MDP for 6 hours.Western-blot technique was used to detect the expression of autophagy-related proteins in RAW264.7 cells stimulated by 10μg/ml MDP for 6hours.Results After 10μg/ml MDP stimulated RAW264.7 cells and Erbin^-/-RAW264.7 cells for 6h,the mRNA levels of TNF-αand IL-6 were significantly increased,the difference was statistically significant（P<0.05）,and compared to RAW264.7+MDP group,the mRNAs of TNF-αand IL-6 in the Erbin^-/-RAW264.7+MDP group were significantly increased,the difference was statistically significant（P<0.05）.The immunofluorescence results indicated that NF-κB p65 activation was enhanced after RAW264.7 cells were stimulated by MDP for 6 hours,and NF-κB p65 activation was enhanced in the Erbin^-/-RAW264.7+MDP group compared with the RAW264.7+MDP group.Under the electron microscope we find the autophagosome formation increased after RAW264.7 cells were stimulated by 10μg/ml MDP for 6h,which was statistically significant（P<0.05）,and compared to RAW264.7+MDP group,the formation of autophagosomes decreased in the Erbin^-/-RAW264.7+MDP group,and the differencewas statistically significant（P<0.05）.The expressionof autophagy-associated proteins LC3,p62,Atg5,Atg7 and Beclin1increased after MDP stimulation of RAW264.7 cells for 6 h,the difference was statistically significant（P<0.05）,and compared to RAW264.7+MDP group,the expressions of autophagy-related proteins LC3,p62,Atg5,Atg7 and Beclin1 decreased in Erbin^-/-RAW264.7+MDP group,and the difference was statistically significant（P<0.05）.Conclusions Erbin can promote MDP-induced autophagy in macrophages.The mechanism may be through the formation of Atg5-Atg12-Atg16 complex,affect the conversion of LC3-I to LC3-II under the influence of Atg7,and finally realize the promotion of Erbin in autophagy in macrophages induced by MDP.