Mechanism Study On Anti-inflammatory Effect Of Andrographolide Via Activating AMPK

OBJECTIVE Andrographolide(Andro)is a diterpene lactone compound derived from Andrographis,which has been extensively used as traditional herbal medicine in China,and possess a wide range of biological activities.In order to be closer to the clinical and to the new use of old drugs,this research intends to further explore the anti-inflammatory activity and mechanism of Andro,discover new anti-inflammatory targets,and provide scientific basis for the future development of anti-inflammatory drugs.METHODS The effect of different concentrations of Andro on cell viability was compared by MTT assay.After pretreatment of macrophages with Andro,the inflammatory response was induced by LPS,and the expression levels of nitric oxide(NO)and its related proteins i NOS and COX-2 were detected by Griess assay and Western Blot(WB);the production and secretion of inflammatory cytokines were detected by ELISA and RT-PCR;the protein expression levels of NF-kB,MAPK and upstream signaling pathway proteins were detected by WB;the nuclear transpositions of NF-kB and AP-1 were detected by nucleocytoplasmic separation technique and immunofluorescence assay(IF).The intracellular signal array assay and WB were used to detect the effect of Andro on some specific marker proteins related to inflammation,metabolism,apoptosis,etc in LPS-induced RAW264.7 cells;the effects of Andro AMPK/NF-kB/MAPK signaling pathways were compared with AMPK activator AICAR and inhibitor Compound C.After AMPKa2 gene knockdown using si RNA transfection,the effects of Andro on the production of NO,the protein levels of AMPK/NF-kB/MAPK signaling pathways and the nuclear translocation of translocation factor NF-kB and AP-1 were detected.After establishing DSS-induced acute colitis model in mice,clinical signs of colitis and histology changes of colon were detected by mouse disease activity index(DAI),H&E staining and IHC,proinflammatory cytokine levels in colon and spleen were determined by PCR and ELISA,and protein expression levels of AMPK/NF-kB/MAPK signaling pathways in colon tissue was detected by WB.RESULTS Andro inhibited the secretion of NO and its related proteins i NOS and COX-2 in LPS-induced macrophages without cytotoxicity;decreased IL-6,TNF-a and IL-1b production and m RNA levels;inhibited the activation of NF-kB,MAPK and its upstream signaling pathways;inhibited nuclear translocation of NF-kB and AP-1.Andro inhibited the activation of cleaved Caspase-3 and the phosphorylation of Akt,m TOR,Bad,p70S6 K,PRAS40,S6 RP,Stat1 and Stat3,and also significantly increased LKB1,AMPKa,ACC and GSK3b phosphorylation in LPS-induced macrophages;the effect of Andro on the AMPK pathway was similar to that of the AMPK activator AICAR.After AMPKa2 gene silencing,Andro could not inhibit the production of NO;it had no significant regulatory effect on AMPK/NF-kB/MAPK signaling pathways and the nuclear translocation of NF-kB and AP-1.In the DSS-induced acute colitis model of mice,the high-dose group of Andro compared with DSS group significantly decreased DAI and increased colon length;the epithelial cell defects of the colonic mucosa was not obvious,and its basic morphology similar to the normal group,the number of infiltrating macrophages was also reduced;the production and m RNA levels of IL-6,TNF-a and IL-1b in colon and spleen were reduced;Andro inhibited NF-kB and MAPK pathways and simultaneously activated phosphorylation of the AMPK pathway.CONCLUSION Andro effectively inhibits LPS-induced macrophage inflammatory response,its anti-inflammatory mechanism inhibits NF-kB and MAPK signaling pathways by activating AMPKa2,and reduces inflammation,whereby Andro improves DSS-induced acute colitis in mice.