Effect of hepatocyte fatty degeneration induced by free fatty acid on macrophage polarization / 临床肝胆病杂志

ObjectiveTo investigate the effect of hepatocyte fatty degeneration induced by free fatty acid on macrophage polarization and the possible mechanism. MethodsPrimary hepatocytes of C57BL/6 mice were isolated by in situ collagenase perfusion, and then the hepatocytes were divided into control (NC) group and mixed free fatty acid (FFA) treatment group. A conditioned medium (CM) was prepared for hepatocytes and was used for the intervention of RAW264.7 macrophages. Oil red O staining was used to observe lipid deposition in hepatocytes; real-time PCR was used to measure the mRNA expression of lipid metabolism genes and macrophage M1/M2 polarization markers; ELISA was used to measure the levels of cytokines in supernatant; Western blot was used to measure the expression of proteins involved in the Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) pathway in macrophages. The independent samples t-test was used for comparison between two groups; a one-way analysis of variance was used for comparison between multiple groups, and the Tukey test was used for further comparison between two groups. ResultsCompared with the NC group, the FFA treatment group had the deposition of massive lipid droplets in hepatocytes and significant increases in triglyceride and total cholesterol (t=15.65 and 3.49, both P＜005). Besides, FFA significantly increased the mRNA expression of the lipid synthesis genes SREBP1C and FASN (t=2.89 and 2.82, both P＜0.05) and reduced the mRNA expression of the lipid decomposition genes ACOX1 and CPT1A (t=14.30 and 3.36, both P＜005) in hepatocytes. FFA also induced significant increases in the levels of the inflammatory cytokines interleukin-6 (IL-6), interleukin-1β, and tumor necrosis factor-α (TNF-α) in supernatant (all P＜0.05). Compared with the CM-NC group, the CM-FFA group had significant increases in the mRNA expression of the M1 phenotype markers iNOS2, TNF-α, and IL-6 (all P＜0.05) and a significant reduction in the mRNA expression of the M2 phenotype marker interleukin-10 (P＜0.05). Moreover, Western blot showed that CM-FFA significantly upregulated the protein expression of TLR4, p-NF-κBp65, and p-IκBα in macrophages (t=2.88, 3.69, and 3.54, all P＜0.05). ConclusionFFA-induced hepatocyte fatty degeneration and inflammation can promote M1 macrophage polarization, thereby initiating and triggering the development and progression of nonalcoholic fatty liver disease.

PPARγ Agonists Alleviate the High Fat-induced Oxidative Stress and Inflammatory Response in vitro and in vivo Through Nrf2-mediated Antioxidant Pathway / 胃肠病学

Background: Oxidative stress is important for the development and progress of non-alcoholic fatty liver disease. Recent studies have demonstrated the anti-inflammatory and antioxidant activities of peroxisome proliferator-activated receptor γ (PPARγ) agonists. Aims: To investigate the protective effect of PPARγ agonists (rosiglitazone and GW1929) on high fat-induced hepatocyte oxidative stress injury in vitro and in vivo and the underlying mechanism. Methods: C57BL/6J mice were fed with high-fat diet and administered intragastrically with rosiglitazone 30 mg/kg per day for 4 weeks. The primary mouse hepatocytes were isolated and incubated with mixed free fatty acids (FFA) after GW1929 pretreatment. HE staining and oil red staining were used to evaluate the histopathological changes and lipid deposition in the liver. Serum contents of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) were measured. Reactive oxygen species (ROS) in hepatocytes was detected by DCFH-DA fluorescence probe. Real-time PCR and Western blotting were used to detect the expressions of genes related to oxidative stress and inflammation. Results: High-fat diet induced significant hepatic steatosis and up-regulated gene expressions of inflammatory cytokines. Serum MDA content was significantly increased, and SOD activity and GSH content were significantly decreased. Rosiglitazone intervention could reduce the high fat-induced hepatic steatosis, inflammation and improve the serum indicators of oxidative stress. Primary hepatocytes incubated with FFA produced a large amount of ROS, whereas GW1929 pretreatment could diminish the FFA-induced ROS production, inflammation and increase the antioxidant factors, nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target gene heme oxygenase-1 (HO-1) expression. Conclusions: High-fat environment can induce steatosis, oxidative stress and inflammation in hepatocytes in vitro and in vivo. PPARγ agonists may alleviate the high fat-induced oxidative stress injury and inflammatory response through inhibiting ROS production and activating Nrf2/HO-1 antioxidant pathway.

Roles of Kupffer Cell Polarization and Hepatic Stellate Cell Activation in Hepatic Inflammation and Fibrosis in NAFLD / 胃肠病学

Background:Kupffer cells and hepatic stellate cells(HSC)play important roles in the initiation and progression of hepatic inflammation and fibrosis in chronic liver injury. Aims:To investigate the roles of Kupffer cell polarization and HSC activation in the development of hepatic inflammation and fibrosis in progression of non-alcoholic fatty liver disease (NAFLD). Methods:C57BL/6 mice were fed with high fat(HF)diet and methionine-choline-deficient(MCD)diet to induce experimental non-alcoholic fatty liver(NAFL)and non-alcoholic steatohepatitis(NASH),respectively. Liver steatosis,inflammation and fibrosis were determined by histopathological examination through HE staining and Masson staining. Kupffer cell phenotypes and HSC activation were assessed by immunohistochemistry. Expressions of PPAR-γ as well as inflammation-and fibrosis-related genes were measured by real-time PCR. Results:F4/80-positive Kupffer cells, CD11c-positive M1 polarized macrophages and α-SMA-positive HSC were significantly increased in liver tissue of mice with HF diet-induced NAFL and MCD diet-induced NASH(P<0.05),and was more apparent in MCD diet-induced NASH (P<0.05). Expressions of TNF-α and TGF-β1 mRNA in liver tissue were significantly increased in both groups(P<0.05),whereas expressions of α-SMA and Col1 mRNA were significantly increased only in NASH(P<0.05). Hepatic PPAR-γ mRNA expression was significantly increased in NAFL(P<0.05)but significantly decreased in NASH(P<0.05). Conclusions:Hepatic Kupffer cells exists a sustained M1 polarization in the development of NAFLD. Activation of HSC appears in the early stage of NAFLD,and accelerates in the progression of NAFL to NASH. Polarization of Kupffer cells and activation of HSC initiate and promote hepatic inflammation and fibrosis in NAFLD.