Effect Of Lipid-induced Kupffer Cell/Macrophages M1/M2 Polarization On Inflammation And Lipid Metabolism In Hepatocytes

Background and Aims: Intrahepatic inflammatory-immune dysregulation and lipid metabolism disorder play an important role in the development of nonalcoholic fatty liver disease(NAFLD).Kupffer cell is the key cell in the regulation of liver immunity,and its unique polarization in NAFLD is attracting more and more attention.It was known that Kupffer cell could regulate a variety of hepatic non parenchymal cells and parenchyma cells,and affected cell differentiation,metabolism,apoptosis etc.Nevertheless,it is still lacking deep understanding on the exact influence of different polarized Kupffer cell on liver parenchyma cells.Our study aims to explore the influence of lipid-induced Kupffer cell/macrophages M1/M2 polarization on inflammation and lipid metabolism in hepatocytes through establishing the Kupffer cell/macrophage and hepatocyte indirect co-culture system in vitro.Methods: Kupffer cell and hepatocyte were isolated by in situ perfusion of the liver with collagenase.Kupffer cells/macrophages were treated with different kinds of fatty acids(saturated fatty acids(SFAs),monounsaturated fatty acids(MUFAs)and polyunsaturated fatty acids(PUFAs)),or treated with PPAR? agonists/antagonists to regulate PPAR? expression meanwhile.Cell culture supernatants were collected to prepare conditioned medium(CM).M1/M2 phenotype markers were detected by Real-time PCR.Hepatocytes were treated with different fatty acids or different CM.Real-time PCR and Western Blot were used to detect NLRP3 inflammasome related mRNA and protein expression,ELISA to detect the secretion of inflammatory cytokines,Caspase-1 colorimetric assay kit to detect Caspase-1 activity.Lipid synthesis and decomposition related mRNA and protein expression in hepatocytes were detected,lipid deposition and lipid content in hepatocytes were detected by oil red O staining and TG,T-CHO quantitative assay kit.Results:(1)SFA-palmitic acid(PA)and MUFA-oleic acid(OA)polarized Kupffer cells/macrophages to an M1 predominant M1/M2 mixed phenotype,while PUFA-docosahexaenoic acid(DHA)polarized Kupffer cells/macrophages to an M2 phenotype.PPAR? agonist shifted lipid-induced M1 macrophages polarization to M2 polarization,while PPAR? antagonist decreased lipid-induced M2 macrophages polarization.(2)PA increased mRNA and protein expression of NLRP3 inflammasome in hepatocytes,and also enhanced NLRP3 activity and secretion of proflammatory cytokines.OA led to increased mRNA and protein expression of NLRP3 inflammasome,and also increased IL-1? secretion,but had no significant effect on its activity.DHA had no significant effect on NLRP3 inflammasome in hepatocytes.CM-PA could further promote the expression and activity of NLRP3 inflammasome as well as pro-inflammatory cytokines secretion,while CM-DHA down-regulated them.(3)PA increased lipid synthesis related mRNA and protein expression,and also promoted lipid deposition in hepatocytes.OA primarily raised lipid synthesis related mRNA and protein expression,but also promoted lipid decomposition.DHA increased lipid decomposition related mRNA and protein expression.CM-PA and CM-OA could further increase lipid synthesis related mRNA and protein expression,and also enhanced lipid deposition in hepatocytes.Conversely,CM-DHA could further increase lipid decomposition related mRNA and protein expression in hepatocytes,and reduced lipid deposition.(4)PPAR? agonist could down-regulate the high expression of lipid synthesis related mRNA and protein induced by CM-PA;while PPAR? antagonist inhibited CM-DHA induced high expression of mRNA and protein related to lipid decomposition.Conclusions:(1)Different kinds of fatty acids had different effect on Kupffer cells/macrophages polarization.(2)PA directly increased NLRP3 inflammasome expression and activity in hepatocytes.M1 macrophages induced by PA could further enhance its expression and activity.While M2 macrophages induced by DHA significantly down-regulated its expression and activity.(3)Lipid-induced M1 macrophages could further strengthen lipid synthesis in hepatocytes,and inhibit lipid decomposition.While DHA-induced M2 macrophages could further enhance lipid decomposition,and inhibit lipid synthesis.(4)PPAR? agonist made lipid-induced macrophage polarization shifting to M2 type,and therefore decreased lipid synthesis in hepatocytes.PPAR? antagonist had the opposite effect.