Research On Anti-inflammatory Molecular Mechanism Of Astragalus Membranaceus Injection By Regulating Autophagy

Objective: Astragalus membranaceus(AM)has played a variety of pharmacological roles in enhancing immunity,regulating metabolism,antioxidation and anticancer in its long history of application.Recent studies show that AM has an obvious anti-inflammatory activity,and its anti-inflammatory mechanism needs further study.Inflammation is the inherent defense mechanism of the body,but excessive and persistent inflammation can cause irreversible damage.Conventional anti-inflammatory drugs treatment can cause immune imbalance,and often accompanied by adverse reactions.AM has double effects on immune regulation and anti-inflammatory and low side effects,which meet the needs of modern anti-inflammatory treatment.AM injection as a standardized AM extract has been used in the clinical treatment of cardiovascular system,kidney and other diseases.In this study,the inflammatory model and macrophage model of mice were established by lipopolysaccharide(LPS)treatment to observe the anti-inflammatory effect of AM injection,and explore the molecular mechanism of AM in the LPS stimulated macrophage model.The results will provide a reference for the application of AM injection in clinical anti-inflammatory treatment,and help to understand the anti-inflammatory mechanism of AM injection.Methods: 1.Mice were injected with different concentrations of lipopolysaccharide by tail vein injection to establish the inflammatory animal model.After different doses of AM injectionwere administrated from the tail vein,the changes of body weight,and the characters of spleen and lung were observed.The levels of IL-1β and IL-6 in the serum of mice were detected by ELISA to evaluate the anti-inflammatory effect of AM injection in vivo experiment.2.To evaluate the anti-inflammatory effect of AM injection in vitro,murine macrophages ANA-1 were treated with different concentrations of lipopolysaccharide and AM injection.The levels of IL-1β and IL-6 in the supernatant of cell culture were detected by ELISA,and the expressions of NLRP3,caspase-1,IL-1β and IL-6 in the cells were detected by Western-blot.3.After treaded with LPS,AM injection and the combo treatment(LPS+AM),the transcriptome of macrophage cells were sequenced.The differential expression genes between groups were analyzed by GO analysis,KEGG Pathway analysis,and STRING analysis to find out the gene change rule and molecular mechanism of LPS induced inflammatory response,as well as the molecular basis and signal pathway information of anti-inflammatory effect of AM injection.These results pointed a direction for further experimental research.4.For the purpose of evaluating the effect of LPS and AM injection on autophagy,the ANA-1 cells were treated with different concentrations of LPS and AM injection for different time.The intracellular autophagy marker proteins LC3 and p62 were labeled by immunofluorescence staining.The puncta of LC3 and the level of p62 were observed by immunofluorescence microscope.The cell image was collected by high-content imaging analysis system and the average fluorescence intensity of p62 was calculated.LPS and LPS combined with the inhibitors of PI3K/Akt/m TOR pathway,LY294002 and Torin 1,were used to treat ANA-1 cells.The type conversion of LC3 and the level of p62 in cells were detected by western-blot method,and the effects of LPS on autophagy and signaling pathways were investigated.For the purposes to estimate the affection of AM injection on autophagy and signaling pathways ANA-1 cells were treated by AM injection,and westernblot method was used to detect the type conversion of LC3,degradation level of p62,and phosphorylation level of AMPK in cells.5.To evaluate the effect of AMPK activation on the anti-inflammatory effect of AM injection,the ANA-1 cells were treated with different concentrations of AMPK activator AICAR and LPS,and the levels of p-AMPK and IL-6 were detected by Western-blot.Moreover,after the expression of AMPK was down regulated by specific si RNA,the levels of AMPK,p-AMPK,p-m TOR,Beclin,IL-6 and the transform of LC3 were observed by Western-blot.6.In order to evaluate the role of autophagy on the anti-inflammatory effect of AM injection,the expression of ATG5 was interfered by si RNA transfection,and the changes of ATG5,LC3,p62 and IL-6 were observed by Western-blot.7.The combining relationship between the several major components in AM injection and AMPK were predicted by STITCH analysis.Different concentrations of formononetin and LPS were used to treat ANA-1 cells.In order to analyze the anti-inflammatory effects of formononetin,the levels of p-AMPK,IL-6,NLRP3,and IL-1 β were detected by western-blot subsequently.Results: 1.In LPS induced inflammatory murine model,AM injection treatment inhibited the weight loss,splenomegaly and pulmonary hemorrhage,and reduced the levels of IL-1β and IL-6 in the serum of mice caused by LPS.In LPS stimulated inflammatory macrophages model,AM injection treatment reduced the levels of IL-1β and IL-6 in the culture supernatant of cells.AM injection also lowered the levels of NLRP3,IL-1 β and IL-6 in LPS induced macrophages and inhibit the activation of caspase-1.2.The results of transcriptome sequencing showed that the inflammation related genes had the most significant differences between LPS group and mock group,including inflammatory cytokines: IL-1,IL-6,TNF,IFN,chemokines and receptor: CCL5,CCR7,acute phase reaction proteins: SAA3,thbs1,etc.GO enrichment analysis showed that most of the differential expression gene(DEG)were distributed in stress,containing 141 genes,followed by 113 genes of immune system program.In DEG of Group mock vs Group LPS,IL-6 was located in the center of interaction between inflammatory molecules.IL-6 were activated by IL-1α,CCL 12,CCL 7,CCR 7 and IL-27.This showed that IL-6 was the downstream inflammatory cytokines.LPS treatment increased significantly the levels of IL1 B,IL6,NOD1 and NOD2 genes which were corresponding to IL-1β,IL-6 and NLRP3 inflammatory protein.This result was consistent with the results in the cell experiment.The GO enrichment of DEG in Group LPS vs Group LPS+AM were defined as post transcriptional regulation,m RNA binding,m RNA stability regulation and m RNA destabilization et al.KEGG Pathway of DEG was enriched in ribosome,PPAR and autophagy signaling pathways.The intersection of DEG in Group mock vs Group LPS,Group LPS vs Group LPS+AM was enriched in autophagy pathway.3.LPS inhibited the type conversion of LC3 and the degradation of p62 in ANA-1 cells in a time and dose-dependent manner.Furthermore,LPS increased the phosphorylation level of Akt and m TOR in ANA-1 cells in a dose-dependent manner.LY294002 and Torin 1,inhibitors of PI3K/Akt/m TOR pathway,reversed the inhibition of LPS on autophagy.Am activated autophagy in ANA-1 cells with or without LPS treatment in a dose-dependent manner.AM injection phosphorylated and activated AMPK in a dose-dependent manner,thus up regulating autophagy.4.AICAR,the activator of AMPK,activated the phosphorylation of AMPK and inhibited the expression of IL-6 in LPS induced macrophages.AMPK si RNA reduced the levels of AMPK and p-AMPK in cells and increased the expression of IL-6.After downregulating the expression of AMPK,the decreasing effect of AM injection on the expression of IL-6 was blocked.5.The activator of autophagy played a similar repressive effect on the LPS-stimulated expression of IL-6 with those shown by AM injection,while the inhibitor of autophagy up regulated the production of IL-6.The deletion of ATG5 which a key gene in the initial stage of autophagy,blocked the anti-inflammatory effect of AM injection.6.STITCH analysis showed that formononetin had indirect binding with AMPK.Formononetin activated AMPK in macrophages in a dose-dependent manner,and reduced the expression of LPS induced inflammatory molecules: NLRP3,IL-1β and IL-6.Conclusion: 1.LPS can cause inflammatory reaction in mice and murine macrophages.AM injection can reduce the LPS induced inflammatory reaction.2.LPS can the change of gene transcription group of ANA-1 cells,and the inflammatory molecules showed the most significant difference.IL-6 and IL-1β are located in the center of the interaction of inflammatory DEGs,and related to most inflammatory genes.IL-6 is located in the downstream of regulation.The differential gene signaling pathways induced by AM injection treatment in LPS-stimulated cells are concentrated in autophagy and PPAR pathways.The intersection of the DEGs change by LPS stimulated and the DEGs changed by LPS+AM treatment were enriched in autophagy signal pathway.3.LPS can inhibit autophagy by activating Akt/m TOR,AM injection can activate autophagy by phosphorylating AMPK,and reverse inhibitory effect of LPS on cell autophagy.4.The phosphorylation of AMPK is involved in the anti-inflammatory effect of AM injection.5.AM injection plays an anti-inflammatory role through AMPK activated autophagy.6.Formononetin,one of the components of AM injection can activate AMPK and play an anti-inflammatory role.