| BackgroundThe incidence rate of nonalcoholic fatty liver disease(NAFLD)has increased year by year in China and has caused tremendous burden on our society and economy.Fatty liver tissue is often accompanied by elevated levels of oxidative stress and the formation of inflammation and fibrosis.Some NAFLD can progress to liver fibrosis,cirrhosis and even liver cancer if not pretreated in time.Therefore,in-depth study on the pathogenesis of NAFLD,and to find related therapeutic targets and develop new therapeutic methods have very important social benefits.Liver is the largest digestive gland in the body,in which hepatocytes absorb chylous particles from the small intestine,convert them into VLDL and LDL,transport them outside the liver and provide energy to other organs.However,diet,insulin resistance and other factors can cause a large number of free fatty acids to enter the liver through the portal vein and be absorbed by hepatocytes.Lipid droplets accumulate in hepatocytes,which can induce the increase of oxidative stress,activate the inflammatory pathway in cells,and then lead to the formation of Nash or NAFLD related liver fibrosis.Studies have shown that renin-angiotensin system(RAS)plays an important role in the regulation of cellular oxidative stress.Our group has long been committed to studying the regulation of Ras axis on the level of oxidative stress in liver.It has been confirmed that the increase of oxidative stress level is closely related to the formation of liver fibrosis,and ACE2/ang(1-7)axis in RAS system can negatively regulate the level of oxidative stress and inflammation.However,little is known about the role of RAS system in NAFLD related liver fibrosis,so it is very important to explore the role of RAS system,especially ACE2/ang(1-7)in NAFLD model.NAFLD patients often have the phenomenon of iron overload in the liver.It has been proved that ferrous ion can dramatically increase the level of intracellular oxidative stress through Fenton reaction in a variety of inflammatory disease models,and even induce iron death.However,the significance of iron overload in NAFLD and its regulatory mechanism are not very clear,and studies have shown that reducing intrahepatic iron overload will significantly improve the prognosis of patients with NAFLD.Therefore,it is of great significance to study the relationship between intrahepatic iron overload and NAFLD.AimThe purpose of this study is to investigate the relationship between the two factors1.To explore the relationship between ACE2 and NAFLD related liver fibrosis and its therapeutic prospect.2.Explore how ACE2 regulates oxidative stress and inflammation in NAFLD.3.Objective to explore how ACE2/ang(1-7)axis alleviates lipid accumulation in hepatocytes.Method1.Clinical exploration:Bioinformatics was used to explore the relationship between oxidative stress and the disease severity of clinical NAFLD patients,to explore the potential significance of ACE2 in NAFLD-related liver fibrosis,and to collect clinical samples to verify the expression of ACE2 in clinical NAFLD-related liver fibrosis by immunohistochemical staining,Masson staining and Western blotting.The relationship between iron accumulation and hepatic steatosis was detected by Prussian blue staining.2.In vitro:hepatocytes were stimulated by palmitic acid and oleanic acid to establish a hyperlipidemic model and treated with Ang(1-7).The therapeutic effect of Ang(1-7)was determined by oil red O staining and triglyceride content detection.Transcriptome sequencing was used to explore the specific mechanism of Ang(1-7)in the treatment of cell hyperlipidemia model.The effect of Ang(1-7)on mitochondria was determined by mitoTracker,and the change of cell oxidation level was detected by the content of hydrogen peroxide and malondialdehyde.Western blotting was used to detect the changes of inflammatory body NLRP3 and oxidative stress related protein NOX4 after Ang(1-7)treatment.The activation of NLRP3 inflammatory bodies was detected by immunofluorescence assay.3.In vivo:ACE2 was overexpressed in model animals and wild-type mice were fed with high-fat diet.The mice were weighed and killed before the beginning of the experiment,8 weeks,16 weeks and 24 weeks respectively.The severity of steatosis in mice was detected by HE staining,oil red O staining,triglyceride in liver tissue and plasma;the degree of hepatic fibrosis in mice was detected by Masson staining and α-SMA immunohistochemical staining;the degree of iron accumulation in liver was determined by Prussian blue staining and iron ion content;the degree of liver injury was detected by ALT,AST and ALP.The level of liver inflammation and the expression of oxidative stress-related proteins were detected by immunofluorescence,immunohistochemical staining,qPCR and western blotting.Result1.Clinical exploration:WGCNA analysis of clinical liver steatosis showed that oxidative stress-related protein was positively correlated with the severity of the disease,and ACE2 protein was in this module.Clinical tissue samples showed that gene expression increased in patients with NAFLD liver fibrosis,but the actual protein level showed a significant downward trend and negatively correlated with the accumulation of iron in tissue and the activation of NLRP3 inflammatory bodies.2.In vitro:significant lipid droplets appeared in hepatocytes stimulated by palmitic acid and oleic acid,while Ang(1-7)could alleviate intracellular fat accumulation in hepatocytes.The results of transcriptional sequencing showed that Ang(1-7)upregulated the level of antioxidant stress molecules in hepatocytes,especially the enzymes related to glutathione system.Western blotting and the contents of hydrogen peroxide and malondialdehyde showed that Ang(1-7)down-regulated the expression of oxidative stress-related protein NOX4 and inhibited the accumulation of oxidative stress products,which protected hepatocyte mitochondria from hyperlipidemia,while glutathione system inhibitor Erastin could restore the effect of hyperlipidemia on mitochondrial function.Immunofluorescence assay showed that Ang(1-7)inhibited the activation and assembly of NLRP3 inflammatory bodies.Western blotting showed that the inhibitory effect of Ang(1-7)on inflammatory expression of NLRP3 could also be blocked by Erastin.3.In vivo experiment:histological experiments(HE staining,oil red O staining,Masson staining,α-SMA immunohistochemical staining)showed that the hepatic steatosis and hepatic fibrosis gradually aggravated with the increase of high-fat feeding weeks.Immunohistochemical staining showed that the expression of NLRP3 increased with the increase of high-fat feeding weeks,while Prussian blue staining showed that iron accumulation in the liver of mice gradually increased,which was positively correlated with ACE2(P<0.05).After 24 weeks of high fat feeding,the body weight,liver injury index(ALT,AST,ALP,P),liver fibrosis level(Masson staining,α-SMA immunohistochemical staining)and fat accumulation(oil red O staining and triglyceride detection in plasma and liver tissue)were significantly reduced in ACE2 overexpression mice and wild type mice.QPCR showed that the expressions of inflammatory cytokines CCL2,CCL5,TNF-α,IL-6 and fibrosis related factors COL1A,TGF-β and CTGF in the liver of ACE2 overexpression mice were significantly lower than those of wild high-fat-diet mice.Western blotting and immunohistochemical staining showed that the overexpression of ACE2 promoted the down regulation of NOX4 expression and the up regulation of GPX4 expression.Ly6G and CD11b staining showed that the infiltration of inflammatory cells in overexpressed ACE2 mice was significantly lower than that in wild type mice after high-fat-diet treatment,while qPCR and immunofluorescence staining showed that over-expressed ACE2 significantly inhibited the expression and activation of NLRP3 inflammasome and its related components.At the same time,we found that the expression of NLRP6 in ACE2 overexpressed mice was significantly higher than that in wild type mice regardless of whether they were fed with high-fat-diet or not.ConclusionACE2/Ang(1-7)inhibit the formation of NAFLD related liver fibrosis through up regulating the expression of antioxidant stress molecules such as GPX4 and inhibiting the activation and assembly of inflammasome NLRP3 by down regulating oxidative stress. |
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