The Role Of Macrophage Polarization And Hepatic Stellat E Cell Activation In Progression Of Hepatic Inflammation And Fibrosis In NAFL/NASH

Background and Aims: Non-alcoholic fatty liver disease(NAFLD)is a non-alcoholic-induced clinical pathological syndrome of liver abnormal lipid deposition.In the spectrum of NAFLD,non-alcoholic steatohepatitis(NASH)is the most critical pathological stage in the progression of NAFL to NASH.Intrahepatic macrophages polarization and hepatic stellate cells(HSCs)activation play an important regulatory role in this progression.In this study,animal models and cell experiments were conducted to observe the changes of intrahepatic macrophages polarization and HSCs activation in NAFL and NASH and its correlation with intrahepatic inflammation and fibrosis,and to explore the correlation between PPAR-?,intrahepatic macrophages polarization and HSCs activation in the progress of NASH,as well as its effect on intrahepatic inflammation and fibrosis,and to study the effects of PPAR-? on LPS combined with fatty acid-induced macrophages polarization changes and fibroblast activation by regulating its expression.Methods: C57BL/6 mice were fed with either high fat(HF)diet or methionine-choline-deficient(MCD)diet to induce non-alcoholic fatty liver(NAFL)or non-alcoholic steatohepatitis(NASH)model respectively. Some NASH model mice were given gavage of PPAR-? agonist rosiglitazone for 4 weeks.Liver steatosis,inflammation and fibrosis were determined by histopathologic examination of liver tissues through HE staining,Masson staining and Sirius red staining.Macrophages polarization phenotype,HSCs activation and their cell counts were determined by immunohistochemistry examination.Expressions of m RNA and protein of PPAR-? gene as well as hepatic inflammation and fibrosis related genes were determined by Real-time PCR and Western Blot.Serum levels of inflammatory cytokines were measured by CBA.RAW264.7 macrophages or NIH/3T3 fibroblasts were incubated with fatty acids(either saturated fatty acids(SFAs)or polyunsaturated fatty acids(PUFAs))or fatty acids with LPS as well as pre-treated with PPAR-? agonist or antagonist.Expressions of m RNA of macrophages polarization phenotype and HSCs activation related genes were determined by Real-time PCR.Results:(1)Macrophages in liver were significantly increased with predominant M1 phenotype polarization in both NAFL and NASH.Activated HSCs in liver were significantly increased as well.Both cells were increased more significantly in NASH,and Monocyte-derived macrophages were significantly increased in NASH.(2)In the progression of NASH,with the exacerbation of hepatic inflammation and fibrosis,macrophages in liver were gradually increased with predominant M1 phenotype polarization.Monocyte-derived macrophages were gradually increased.Activated HSCs were gradually increased as well.Expressions of PPAR-? gene m RNA and protein in liver tissues were gradually decreased.(3)PPAR-? agonist rosiglitazone intragastric administration inhibited hepatic inflammation,fibrosis and steatosis.Macrophages in liver were significantly decreased.M1 phenotype polarized macrophages were decreased,while M2 polarized phenotype macrophages were increased.Monocyte-derived macrophages were decreased.Activated HSCs were significantly decreased as well.Meanwhile,expressions of PPAR-? gene m RNA and protein in liver tissues were increased.(4)Saturated fatty acid PA polarized macrophages to a M1 predominant phenotype,polyunsaturated fatty acid DHA polarized macrophages to a M2 predominant phenotype,while fatty acids with LPS polarized macrophages to a M1 predominant M1/M2 mixed phenotype.PPAR-? agonist enhanced fatty acids and fatty acids with LPS induced M1 phenotype polarization and weakened M2 phenotype polarization.While PPAR-? antagonist proved an opposite effect.All kinds of fatty acids and fatty acids with LPS promoted the activation of fibroblasts,while PPAR-? agonist reduced this activation.Conclusions:(1)M1 predominant macrophages polarization and HSCs activation initiated in NAFL and exacerbated in NASH.(2)Changes of macrophages polarization phenotype and HSCs activation were closely correlated with hepatic inflammation and fibrosis and the expression change of PPAR-?.(3)PPAR-? agonist relieved liver inflammation and fibrosis by inhibiting M1 polarized phenotype macrophages and HSCs activation.(4)Different kinds of fatty acids or fatty acids with LPS had different effects on macrophages polarization and fibroblasts activation.Regulating the expression of PPAR-? could change macrophages polarization and fibroblasts activation.