BRF1 Regulates Inflammation Through Autophagy And MAPK/ERK Signaling Pathway

Objective:To elucidate the function of BRF1 in the regulation of inflammation and autophagy response inmacrophages and the possible mechanism of BRF1 affecting the expression of inflammatory factors.Which provides potential molecular targets for a variety of inflammatory conditions.Simultaneously exploring the function of BRF1 in immune inflammatory diseases in mice.New insight into the mechanistic basis of inflammation and autophagy will provide new perspective of molecular targeted therapies.Methods:The adenovirus Ad-BRF1 and Ad-siBRF1 was constructed and packaged by the Ad-Easy adenovirus packaging system,the expression level of protein and RNA of this gene was detected by Western blotting and real-time quantitative PCR,RAW264.7 cells were infected with Ad-BRF1 and the effect of apoptosis and autophagy was detected.LPS(Lipopolysaccharide)/TNFa/IL1β was used to construct an inflammation model.Inflammatory factors such as IL6(Interleukin 6),NOS2(Nitricoxide synthase 2)and COX2(Cytochrome c oxidase subunit II)and autophagy related protein were determined after inffected by Ad-BRF1 under inflammatory conditions,meanwhile the effect of BRF1 on apoptosis was detected by FCM,both 3-MA(3-Methyladenine)and U0126 inhibitors inhibiting autophagy and MAPK/ERK signaling pathways,respectively,which were used to determine whether both signaling pathway participate in inflammation inhibition.Finally,the model of psoriasis-like dermatitis was established on the back skin of mouse with imiquimod(IMQ)for 6days,and subcutaneous injection of BRF1 adenovirus.And the model of CIA mice was established by injecting with equal ratio of chicken type II collagen and Freund’s complete adjuvant.After 21 days,the animals were boosted and their clinical manifestations were observed.Results:We successfully constructed adenovirus Ad-BRF1 and Ad-si BRF1 through the identification of protein and RNA levels.Western blot and flow cytometric analysis was performed to detect the autophagy and apoptosis,which showed that Ad-BRF1 reduced autophagy and apoptosis compared to Ad-GFP group.while immunofluorescence showed that BRF1 could reduce rapamycin-induced autophagy.The inflammation model of RAW264.7 macrophage was successfully established.The inflammatory factors IL6,NOS2,and COX2 were significantly increased,and the expression of BRF1 was contrary toautophagy.In the presence of inflammation,overexpression of BRF1 can suppress the increase of inflammatory factors,autophagy,and apoptosis,while both 3-MA and U0126 inhibitors inhibit autophagy and MAPK/ERK signaling pathways,3-MA and U0126 could effectively reverse the inhibitory effect of BRF1 on inflammation,which demonstrating that BRF1 inhibits the expression of inflammation factors may through downregulation of autophagy and MAPK/ERK signaling pathway.The dermatitis and CIA models were successfully constructed,Injection of Adenovirus and observation of its effect on tissue structure,autophagy,and inflammation during the occurrence of autoimmune diseases,which providing new therapeutic targets for the study and treatment of subsequent autoimmune diseases.Conclusions:In RAW264.7 cells,overexpression of BRF1 can inhibit autophagy and apoptosis of cells,and can alleviate the occurrence of inflammation,and this function may depend on autophagy and MAPK/ERK signaling pathway.At the same time,the levels of autophagy and Inflammatory factors increase during the course of the immune disease,and BRF1 can alleviate immune diseases.