| Background and objective:Inflammatory reaction is involved in the development of various diseases,including autoimmune disorders,diabetes,cardiovascular diseases and cancer.The recruitment and activation of immune cells are important for inflammatory process.Macrophages,as the first defending line of external stimulus,are key players in innate immunity.Many chemokines are secreted by activated macrophages,and the abnormal expression and dysfunction of chemokines are closely associated with inflammation-related diseases,namely autoimmune disorders,infectious disease,diabetes and cancer.The two major chemokine sub-families are CC chemokines and CXC chemokines,which play crucial role in regulating the migration of inflammatory cells and immune cells.There is now accumulating evidence that an AMPK activator metformin,the first line of anti-diabetic drug in clinic,can alter inflammation independently of its effect on glycemic control,also suppress NF-κB signaling pathway and inhibit the production of inflammatory cytokines by activating AMPK.However,the effects of AMPK on chemokines and the mechanisms involved are still unknown.Here,we aims to study the effects of metformin on lipopolysaccharide induced chemokines CCL2,CXCL10,CXCL11 and the mechanisms involved.Methods:1.Detect the effect of lipopolysaccharide(LPS)and metformin,respectively,on the expression of chemokines.In order to determine the appropriate drug dosage and incubation time,RAW264.7 macrophage was incubated with LPS of different concentration or different time points,the production of CXCL10,CXCL11 and the phosphorylation level of AMPK were detected by Western blot;The cells were incubated with metformin at various time point,and then the m RNA expression of CCL2,CXCL10,CXCL11 was determined by q RT-PCR.2.Detect the effect of metformin on LPS induced chemokines expression.The groups are: control,metformin,LPS,L+M.RAW264.7 macrophages at about 70% confluent were given normal medium and the medium containing corresponding drugs for the indicated time,then the m RNA expression of CCL2,CXCL10,CXCL11 was determined by q RT-PCR.3.Detect the effect of the treatment of AMPK inhibitor Compound C(CC)and metformin on LPS induced chemokines.The groups are: control,metformin(M),LPS(L),L+M,L+M+CC.RAW264.7 macrophages at about 70% confluence were given normal medium and the medium containing corresponding drugs for the indicated time,then the m RNA expression of CCL2,CXCL10,CXCL11 was determined by q RT-PCR.4.Detect the effect of the treatment of AMPK inhibitor Compound C and AICAR(AI)on LPS induced chemokines.The groups are: control,metformin,LPS,L+AI,L+AI+CC.RAW264.7 macrophages at about 70% confluence were given normal medium and the medium containing corresponding drugs for the indicated time,then the m RNA expression of CCL2,CXCL10,CXCL11 was determined by q RT-PCR.5.Detect the effect of metformin on AMPK/NF-κB signaling pathway.The phosphorylation levels of AMPK,ACC,NF-κB p65 and IκB were detected by Western blot.Results:1.The production of CXCL10 and CXCL11 was induced by LPS in a dosage and time-dependent manner.2.The m RNAs of CCL2,CXCL10 and CXCL11 were downregulated by metformin in a time-dependent manner.3.Metformin treatment inhibited the m RNAs of CCL2,CXCL10 and CXCL11 induced by LPS,an event that was abrogated by Compound C.4.AICAR treatment suppressed the m RNAs of CCL2,CXCL10 and CXCL11 induced by LPS,but Compound C could not reverse the effect of AICAR.5.Metformin significantly activated AMPK and prevented the phosphorylation of IκB and p65(Ser536)instead of p65(Ser276).Conclusion:Metformin treatment inhibits the expression of CCL2,CXCL10 and CXCL11,and the action of metformin is most likely mediated by AMPK/NF-κB signaling pathway. |
