Protective Effect And Its Mechanism Of Angiotensin Converting Enzyme 2 (ACE2) In Nonalcoholic Fatty Liver Disease (NAFLD)

In recent years,the study of angiotensin converting enzyme 2(ACE2)in the liver has received increasing attention.In this study,rat and rat BRL-3A cells were used as research objects to study the role of ACE2 in nonalcoholic fatty liver disease and its mechanism by making animal models and cell models of nonalcoholic fatty liver disease.The research includes the following three aspects:1.Effects of Negative Reversal of two axes of Renin Angiotensin(RAS)and Liver Injury induced by Non-alcoholic Fatty Liver Disease(NAFLD)in RatsIn this chapter we explored the negative regulation of the two axes of renin angiotensin system(RAS)on liver injury in rat non-alcoholic fatty liver disease(NAFLD).Sixty male SD rats were randomly divided into normal control group,model group and medication group.In addition to the normal control group,the other two groups were fed with high-fat diet.The medication group was additionally given Lovastatin 50 mg/kg·d as a positive control every day.Three weeks later,10 rats were killed in each group,and the rest were fed for another 3 weeks,and all the remaining rats were killed 6 weeks later.The contents of TG,ALT and AST in serum were determined.The liver homogenate was prepared and the activities of·OH,TNOS,SOD and TAOC were measured.The contents of AngⅡ,Ang1-7,IL-1β and inflammatory factors in rat liver tissue were determined using ELISA.The levels of ACE,ACE2,AT1R and MasR in liver tissues were analyzed by Western Blot.HE staining was used to observe the pathological changes of liver.Results:1)Compared with the normal control group,the serum levels of TG,AST and ALT in the model group increased.Liver pathological morphology changed.Oxidative stress and inflammatory factor release increased in liver tissue.The protein expression of ACE,ACE2,MasR AT1R was increased.The content of AngⅡ and Ang1-7 was increased,and the ratio of ACE/ACE2 and AngⅡ/Ang1-7 was significantly lower.2)In the 6-week trial,serum TG,AST and ALT levels increased.Liver pathological morphology changed.Oxidative stress activation and inflammatory factor release increased significantly.The protein expression of ACE,AT1R and the content of AngⅡ and Ang1-7 levels increased.The protein expression of ACE2 and MasR was significantly decreased,and the ratio of ACE/ACE2 and AngⅡ/Ang1-7 were decreased.The drug group improved the damage of oxidative stress inflammation and down-regulated the ACE/AngⅡ/AT1R pathway.Conclusion:The RAS system exists in the liver in the rat model of NAFLD induced by high-fat feeding.Both pathways are activated.The ACE2/Ang1-7/Mas pathway is dominant in the 3-week induced model,and the 6-week induced model.The ACE/AngⅡ/AT1R pathway predominates.The results suggest that endogenous ACE2 produced by the liver may exert a certain anti-injury effect by mediating the activation of the Ang1-7/MasR pathway in the case of exogenous stimuli-induced liver injury.2.Role of angiotensin-converting enzyme 2 in oleic acid-induced injury of non-alcoholic fatty liver cells in BRL-3A cellsIn this chapter,a suitable non-alcoholic fatty liver cell model was established by oleic acid treatment to investigate the anti-injury effect of ACE2/RAS in NAFLD cells at the cellular level.BRL-3A cells were treated with different concentrations of oleic acid(0.025,0.05,0.1,0.2 mmol/L)for 24 h,and the morphology of the cells was observed.NAFLD cells were established by cell viability,intracellular TG,ALT,AST content and oil red staining.Three oleic acid concentrations(0.025,0.1,0.2 mmol/L)were applied to the cells for 24 h for subsequent experiments.ELISA was used to detect inflammatory factors and AngⅡ and Ang1-7 levels.Western Blot analysis of intracellular ACE,ACE2,AT1R,MasR protein levels.RESULTS:1)The in vitro NAFLD model of BRL-3A cells was established by oil red staining and TG content,ALT and AST activity.The oleic acid concentration was 0.1 mmol/L and the culture time was 24 h.2)ACE2 is uniformly expressed on the cell membrane.In the low concentration group(0.025 mmol/L),the expression of ACE2,MasR and the content of Ang1-7 increased.The content of AngⅡ and the expression of AT1R decreased.The ratio of ACE/ACE2 and AngⅡ/Ang1-7 decreased.The medium concentration group(0.1 mmol/L)ACE,AT1R expression was significantly increased,other RAS members did not change significantly.The high concentration group(0.2 mmol/L)the expression of ACE2,MasR and the content of Angl-7 decreased.The content of AngⅡ,the expression of ACE and AT1R,the ratio of ACE/ACE2 and AngⅡ/Ang1-7 were increased.Conclusion:The NAFLD cell model of BRL-3A cells was successfully established.The local RAS system is activated and participates in the occurrence and development of NAFLD.The ACE2-mediated Ang1-7/MasR pathway predominates when low concentration oleic acid-treated cells are less damaged;the high concentration oleic acid-treated cells exacerbate the damage,and the ACE-mediated AngⅡ/AT1R pathway predominates.The results are consistent with the results of in vivo experiments,that is,ACE2 has a protective effect on oleic acid-mediated non-alcoholic fatty liver cell injury.3.Angiotensin-converting enzyme 2 attenuates the mechanism of oleic acid-induced injury in rat liver BRL-3A cellsACE2 is associated with hepatocyte injury induced by NAFLD.It suggests that ACE2 has a protective effect on hepatocyte injury.To further validate the protective effect of ACE2 on hepatocyte lipid accumulation,this chapter uses siRNA to reduce the expression of ACE2 in a cell model and add exogenous ACE2 active protein in the NAFLD cell to verify ACE2 by detecting inflammatory factors and oxidative stress indicators.Analyze the changes in the expression or content of RAS members and the changes in key enzymes or protein expression in the lipid metabolism pathway,reveal the mechanism of ACE2 against NAFLD hepatocytes.Results:1)Adding ACE2 active protein,oxidative stress decreased.Inflammatory factor release decreased.The expression of NO and iNOS decreased.After transfection siRNA,oxidative stress increased.Inflammatory factor release increased.And the expression of NO and iNOS increased.2)After adding ACE2 active protein,TG content and lipid accumulation were alleviated,the expression of ACC and Fas in lipid synthesis was significantly decreased(P<0.05),and the expression levels of CPT-I and FABP were increased.(P<0.01,P<0.05,P>0.05).After transfection siRNA,TG content and lipid accumulation is up-regulated.The expression of ACC and Fas in lipid synthesis was significantly increased(P<0.05).The expression levels of CPT-I and FABP were increased(P>0.05).Conclusion:This experiment successfully reduced the expression of ACE2 protein by siRNA technology.It was confirmed that ACE2 had a certain alleviation effect on oleic acid-induced injury of NAFLD cell model,and ACE2 improved lipid accumulation induced by oleic acid in NAFLD cell model.Its mechanism:ACE2 can regulate the degradation of Ang Ⅱ,increase the formation of Ang1-7,and reduce liver steatosis,oxidative stress and inflammatory damage by acting on NO/iNOS signaling pathway,ACE2/Ang1-7/Mas pathway liver fat accumulation can be partially reduced by regulating lipid.