PM-induced Mice Liver Lipid Metabolism Disorder: Roles Of Hepatic Macrophages Mediated Inflammatory Response

Objective:More and more evidence had linked ambient particulate matter(PM)exposure to the development of hepatic lipid metabolism disorder and steatosis,yet,the underlying mechanism remains to be elucidated.In the present study,a real-ambient PM exposure system and transwell co-culture model were used to investigate the role of hepatic macrophages in the process of PM-induced lipid metabolism disorder and steatosis in mice liver and the mechanism by which PM modulates macrophage inflammation response.Methods:1.Using a real-ambient PM exposure system,we performed whole-body exposure of mice to PM in Shijiazhuang City,Hebei Province.Sixty 7-week-old male C57 mice were randomly divided into control group and exposure group,with 30 mice in each group.Mice were placed in individual ventilated cages for 16 hours per day.The exposure chamber was ventilated with unfiltered air from outdoor,while the control chamber was ventilated with air after high efficiency particulate air filtered.After 42 days of PM exposure,the pathological changes of mice liver were observed by HE staining,and the number of macrophages was observed by F4/80 immunohistochemistry.2.Primary hepatocytes and hepatic macrophages(HMs)were extracted from normal male C57 mice based on a 2-step perfusion method followed by purification on density gradient.HMs/J774 A.1 macrophages were co-cultured with primary hepatocytes in a transwell plate.The macrophages in the upper transwell were exposed to PM and the triglyceride content of hepatocytes in the lower well was detected by oil red O staining and triglyceride detection kit.3.The concentration of IL-1β in cell culture medium and mice liver was detected by enzyme-link immunosorbent assay(ELISA).4.The expression levels of NLRP3 inflammasome,NF-κB and IL-1β related genes in J774 A.1 macrophages were detected by q RT-PCR after 24 and 72 hours of PM exposure.The expression levels of ASC and caspase-1 protein in J774 A.1were detected by Western blotting.Immunofluorescence was used to detect whether NLRP3 protein in macrophages aggregation into multiple puncta after PM exposure.5.NLRP3 inflammasome inhibitor(MCC950)was used to treat macrophages in the co-culture system to construct the NLRP3 inflammasome inhibition model.The experiment was divided into four groups: Con(vehicle),Exp(PM),Con-I(vehicle+MCC950),Exp-I(PM+MCC950).After 72 hours of PM exposure,the triglyceride content of hepatocytes and the concentration of IL-1β in cell culture medium were detected,and the m RNA expression of some transcription factors of lipid metabolism in hepatocytes was detected by q RT-PCR.6.Transmission electron microscope and Lyso Tracker Red was used for monitoring the endo-lysosomal compartment in J774 A.1 cells exposure to PM,and the expression level of Cathepsin B protein was detected by Western blotting.Results:1.Hepatocyte swelling,increased lipid droplets and liver steatosis was observed in the liver of mice after 42 days of PM exposure,along with increased number of activated hepatic macrophages.2.After 72 hours of PM exposure to macrophages in the co-culture model,increased triglyceride accumulation in hepatocytes was revealed by oil red O staining and triglyceride level detection.3.The results of ELISA assay showed that PM exposure increased the level of IL-1β in mouse liver and co-culture model.4.Compared to Con group,the m RNA expression of NLRP3,ASC,caspase-1 and IL-1βincreased significantly in J774 A.1 macrophages after PM exposure.Western blotting results showed that PM exposure for 72 hours induced significantly increased caspase-1 protein level in J774 A.1 macrophages.5.Immunofluorescence results indicated that NLRP3 protein was diffused across the cytosol under basal condition but formed multiple small puncta after PM exposure for 72 hours.6.Inhibition of macrophage NLRP3 inflammasome with MCC950 significantly alleviated hepatocyte triglyceride accumulation and increased IL-1β level caused by PM exposure.7.The expressions of some key genes that control lipid metabolism in hepatocytes were detected by q RT-PCR.Among these genes,PM exposure significantly decreased the m RNA expression level of PPARα in Exp group,and inhibition of macrophage NLRP3 inflammasome activation significantly alleviated this change.8.Transmission electron microscope and lysosomal fluorescence showed that increased number of swollen lysosomes in J774 A.1 cells after PM exposure,and the protein expression level of cathepsin B in J774 A.1 cells increased significantly.Conclusion:1.PM exposure induced hepatic steatosis and hepatic macrophages infiltration in mice model.2.PM exposure induced NLRP3 inflammasome and caspase-1 activation in hepatic macrophages,activated caspase-1 promotes maturation and secretion of IL-1β,which leads to the hepatocytes triglyceride accumulation.