Inhibitory Effects And Molecular Mechanism Of Ginkgolide A On The LPS-Mediated Inflammation

Inflammation is a crucial and complex pathological process which results from tissue injury or infection.Acute and chronic inflammatory state can lead to serious illnesses such asatherosclerosis,asthma,rheumatoid arthritis,acute liver injury,diabetes and cancer.Therefore,inhibition of the inflammatory response can effectively suppressthe occurrence and progression ofinflammation-related diseases.Interleukin(interleukin-6,IL-6)plays important roles during inflammation and is involved in pathogenesis of different inflammatory diseases.Hence,in the previous study we took advantage of IL-6 gene promoter as a firefly luciferasereporter screening system to screenGinkgolide A(GA)out from over 300 natural compounds,which suggested that GA could have the potential to inhibit the inflammatory response.Therefore,in this study,we aim to further explore anti-inflammatory activitiesof GAand elucidate its working mechanism as well.Macrophages are major players in the inflammatory responses and Lipopolysaccharide(LPS)can potently activate macrophages and induce various pro-inflammatory mediators.Here,we used the LPS-activated macrophagesas an inflammation model in vitro.Firstly,GA was showed tohave no influence on the cell viability of macrophages of three origins,mouse peritoneal macrophages,mouse macrophage cell line RAW264.7 and human monocytes differentiated macrophages(dTHP-1)at the concentrations(0-40μg/mL)by MTT assay,whichexluded the interference ofcell toxic effect to its anti-inflammatory activities.Then,We showed that GA could suppress the expression of the pro-inflammatory mediators(cyclooxygenase-2(COX-2)and nitric oxide(NO))and the pro-inflammatory cytokines(tumor necrosis factor(TNF)-α,IL-6 and IL-1β)in LPS-induced three types of macrophagesby using Western Blot,RT-qPCR(quantitative Real-Time RT-PCR)and ELISA assay(enzyme-linked immunosorbent assay).We also found that GA can improve the phagocytosis of LPS-reduced mouse peritoneal macrophages to accelerate the regression of inflammation.Finally,we have shown that GA canimprovethe survival of mice with septic shockin vivo.Consistently,GA could inhibit the release ofTNF-α,IL-6 and IL-1βin LPS-induced in mice.In addition,wefurther investigated the mechanisms underlyingthe anti-inflammatory activities of GA.First of all,GA appeared to involve inhibition of NF-κB activation by blocking LPS-stimulated IκB(inhibitor of NF-κB)degradation and translocation of NF-κB p65;Secondly,LPS induced MAPKs activation(p38 mitogen-activated protein kinase,extracellular signal-regulated kinase(ERK)and c-Jun N-terminal kinase(JNK)),and GAsignificantly suppressed the phosphorylation of P38 and ERK to weaken inflammatory response;And lastly,GA could obviously enhance activation of AMPK(AMP-activated protein kinase)in LPS-stimulated macrophages,as well as in the immune cell of LPS-induced BALB/c mice,the activation of AMPK could regulate the activation of MAPKsand NF-κB pathways.These results suggest that GA plays an anti-inflammatory role partially via downregulating NF-κB,MAPK(ERK and P38)signaling pathways and up-regulation of AMPK pathway.In summary,GA inhibits LPS-induced inflammatory responses in three types of macrophages and BALB/c mice by mediating multiple signaling pathways.Therefore,the natural compound GA may develop as a potential drug for inflammatory diseases.