Adiponectin Alleviates The Role Of Lps-induced Macrophage Inflammatory Injury By Regulating Autophagy Level And Its Mechanism

Objective:Existing large amount of epidemiological evidence confirmed the cardiovascular protection effect of adiponectin(APN),including myocardial protection,angiogenesis promotion,anti-inflammatory and anti-atherosclerosis(AS).In this study,the inflammatory injury model of macrophages induced by lipopolysaccharide LPS was established to explore whether APN exerts an anti-as effect by regulating the autophagy level of macrophages,thus providing a new idea for clinical anti-as treatment.Methods:1.In part I,after in vitro cultured RAW264.7 macrophages were induced by LPS,Cell Counting Kit(cck-8)was used to detect the survival rate of cells,and Western blot(WB)was used to detect the autophagy level of cells at different action points.Experimental group:(1)control group,cells were cultured in normal condition without LPS induction.The cells were induced by LPS(100ng/ml)for 6h.(3)cells were induced by LPS(100ng/ml)for 12h;The cells were induced by LPS(100ng/ml)for 24 h.2.In the second part of the experiment,the obvious time point of autophagy was selected in combination with the results of the first part of the experiment.WB method was used to observe the effect of APN on the autophagy level of macrophages induced by LPS.The contents of inflammatory factors(IL-1β、IL-6 and TNF-α)were measured by ELISA.Experimental group:(1)control group,the same as the first part of the experiment;The cells were induced by LPS(100ng/ml)for 12 h.(3)cells were inducedby LPS(100ng/ml)for 12 h,and APN(0.5ug/ml)was added for 24 h.(4)cells were induced by LPS(100ng/ml)for 12 h,and APN(1.0ug/ml)was added for 24 h.(5)cells were induced by LPS(100ng/ml)for 12 h,and APN(2.0ug/ml)was added for 24 h.3.In the third part,the mechanism of adiponectin regulating the autophagy level of macrophages induced by LPS was studied.Experimental group:(1)control group,the same as the first part of the experiment;The cells were induced by LPS(100ng/ml)for12h.(3)cells were induced by LPS(100ng/ml)for 12 h,and APN(1.0ug/ml)was added for 24 h.(4)cells were induced by LPS(100ng/ml)for 12 h.After the addition of Akt inhibitor LY294002,APN(1.0ug/ml)was added to continue the culture for 24 h.Expression levels of microtubule light chain protein 3(LC3),phosphorylated Akt(p-)and total Akt were determined by Western blot.Results:1.CCk-8 results showed that the survival rate of macrophages increased gradually with the increase of LPS concentration at a low concentration of LPS(0-100 ng /ml),and decreased gradually at a high concentration of LPS(100-1000 ng /ml).The cell viability was optimal at a certain level,and LPS(100ng /ml).2.WB results show that the macrophages by LPS(100 ng/ml)induced by different time points(6,12,24 h),autophagy proteins in cells LC3-Ⅱ expression level higher than the control group obviously,the time dependence of the LPS(100 ng/ml)inducing autophagy highest when 12 h,according to the first part of the experimental results,the second and the third part of the experiment select LPS(100 ng/ml,12 h)induced macrophage.3.Macrophages by LPS(100 ng/ml,12 h)after induction of inflammatory lesions,compared with blank control group,LPS induced macrophage can make inflammatory factor(IL-1β、IL-6 and TNF-α)content significantly increased(P < 0.01),and different concentration of APN post-processing all can significantly reduce the IL-1β、IL-6 and TNF-α level(P < 0.01),and with the increase of concentration of APN,difference,the more obvious;At the same time the WB results show that the model of APN treatment group,the LPS group can significantly reduce LC3-Ⅱ autophagy protein expression level,the difference was statistically significant(P < 0.01).4.APN treatment group compared with model group LC3-Ⅱ levels and p-Akt expression quantity increased significantly(p < 0.01),after joining Akt inhibitors LY294002,LC3-Ⅱ expression levels(p < 0.01),p-Akt expression quantity decreased obviously(p < 0.01).Conclusion:1.LPS can reduce the survival rate of macrophages in a dose-dependent manner;2.Lps-induced macrophages can increase the inflammatory level of cells,and APN post-treatment can reduce the inflammatory damage of macrophages.3.Macrophages induced by LPS can increase the level of autophagy,and APN post-treatment can inhibit excessive autophagy.The possible signal pathway is PI3K/Akt/mTOR,which plays an anti-as role.