The Regulation Of Angiotensin (1-7) And Angiotensin ? On Non-alcoholic Fatty Liver Disease Via Oxidative Stress And Autophagy

Background:Non-alcoholic fatty liver disease(NAFLD)is a clinical syndrome which refers to the exception of alcohol and other clear liver damaging factors and characterized as diffusing macrovesicular steatosis of hepatocytes,including simple steatosis,non-alcoholic steatohepatitis(NASH)and cirrhosis.The development of NAFLD is closely related to metabolic syndrome,including diabetes mellitus type 2,central obesity,insulin resistance,hyperlipidemia and hypertension.The epidemiology research demonstrated that there are about 1.46 billion obese adults worldwide,and about 40-90%of them are suffering from NAFLD.Now the morbidity of this disease is still increasing year by year,which seriously affects the health level and increases the global medical financial burden.Nowadays,the most important pathogenesis of NAFLD is "the two-hit hypothesis":the first hit leads to lipid accumulation in hepatocytes and the second hit leads to oxidative stress and lipid preoxidation.Oxidative stress is defined as the over-production of reactive oxygen species(ROS)and its metabolies,which results in the dysfunction of fatty acid oxidation and causes lipid preoxidation,finally causes inflammation,necrosis and fibrosis in liver.As a result,in order to inhibit the development of NAFLD,we should inhibit hepatocellular oxidative stress and lipid preoxidation.The renin-angiotensin system(RAS)exists in vascular wall,heart,nerve center,kidney and adrenal gland,participating in the regulation of target organs.The most important member of angiotensin family is Angiotensin II(Ang II),which is formed by the cleavage of Angiotensin ?(Ang 1)by angiotensin converting enzyme(ACE).Ang ? can promote vasoconstriction,fibrosis and inflammation.Recent research indicated that Ang ? plays an important role in pro-oxidation,pro-inflammation and fibrosis in liver,it can promote the development of NAFLD by inducing the production of ROS in hepatocytes,which increases the expression of the inflammatory cytokines(such as TNF-aand CRP),and leads to the peroxidation of lipid,inflammation,fibrosis and apoptosis in liver.In recent years,the research about Angiotensin(1-7)(Ang(1-7))is becoming more and more popular.Ang(1-7)is produced by the cleavage of Ang ? by ACE2.It antagonizes the effects of Ang ? by acting on Mas receptors of target organs and plays a role in vasodilatation,anti-fibrosis and anti-oxidation.However,whether Ang(1-7)can inhibit the development of NAFLD by eliminating the oxidative stress in liver is rarely reported.As described above,oxidative stress leads to the increase of cellular ROS.ROS includes superoxide anion(O2-),hydroxyl(OH")and hydrogen peroxide(H2O2)which mainly derives from mitochondrial dysfunction and activation of NADPH oxidase(NOX)family.In addition,mitochondria undertakes crucial physical function in cells,maintains cellular homeostasis and survival,participates in the production and transfer of energy(especially ATP),regulates cellular dynamic equilibrium of redox and calcium and takes part in fatty acid oxidation and cellular apoptosis.Mitochondrial dysfunction mainly represents as the damage of mitochondrial respiratory chain which leads to the over production of mitochondrial ROS,therefore damages mitochondrial DNA(mtDNA)or causes the mutation of mtDNA,and leads to the depolarization of mitochondrial membrane,decrease of mitochondrial membrane potential and inhibition of mitochondrial ATP production,finally affects normal function in cells.On the other hand,NOX is defined as a multimeric transmembrane enzyme complex which uses NADPH as an electron donor to transform molecular oxygen into superoxide anion(O2-)and hydrogen peroxide(H202).The mammalian NOX family includes NOX1,NOX2,NOX 3,NOX4,NOX5,DUOX1 and DUOX2.NOX4 is constitutively active and produces ROS without other regulatory cellular binding partners.Recent researches indicated that NOX4 plays a crucial role when hepatocytes are infected by hepatitis B virus(HBV)and suffer from oxidative stress.What's more,in the process of liver fibrosis,NOX4 can transform fibroblasts into myofibroblasts by acting TGF-?1/Smads signaling.However,in the progress of NAFLD,whether Ang ? can stimulate hepatocellular oxidative stress by inducing mitochondrial dysfunction and activating NOX4,and whether Ang(1-7)can eliminate ROS,decrease lipid peroxidation and inhibit the process of NAFLD by improving mitochondrial function and inhibiting NOX4 is still unknown,we will discuss those questions mentioned above in this research.Moreover,autophagy,which is the self-protection mechanism of cells against injury,it maintains cellular homeostasis and provides energy for cells by packaging and degrading protein or cell organs which were damaged.ROS produced by NOX and damaged mitochondria can activate autophagy.On the process of autophagy,microtubule-associated protein 1 light chain 3(LC3I)combines phosphatidylethanolamine and becomes activated form LC3?.LC3? locates on the inner and outer membrane of autophagosomes as a recognition site for chaperones like p62/SQSTM1(p62)to send their cargo to autophagosomes,and then autophagosomes combine with lysosomes and become autolysosomes,which can degrade the engulfed cargo.Recent studies indicated that Ang ? can induce autophagy by oxidative stress in the process of myocardial remodeling and Ang(1-7)inhibits autophagy by anti-oxidation.But it is still unclear how Ang ? and Ang(1-7)regulate autophagy in NAFLD;we will discuss it in our research.Method:In vitro,we use AML 12 mice normal hepatocytes to perform our experiments.We set experimental groups mentions below:(1)control?Ang ?(10-7mol/L)?Ang(1-7)(10-7 mol/L)?Ang ?+Ang(1-7)?Ang ?+Ang(1-7)+A779(Mas receptor antagonist,10-7 mol/L);(2)control?palmitic acid(PA,0.25mmol/L)?Ang(1-7)?PA+Ang(1-7)?PA+Ang(1-7)+A779;(3)control?PA?Ang ?+PA?Ang(1-7)?PA+Ang(1-7)?Ang ?+PA+Ang(1-7);(4)control?DPI(NOX inhibitor?10mmol/L)?Ang ??PA?Ang ?+DPI?PA+DPI;(5)control?NAC(ROS scavenger,5?mol/L)?Ang ??PA?Ang ?+NAC?PA+NAC;(6)control?mito-TEMPO(mitochondrial ROS inhibitor,10?mol/L)?Ang??PA?Ang ?+mito-TEMPO?PA+mito-TEMPO;(7)control?rapamycin(autophagic inducer,100nmol/L)?Ang ??PA?Ang ?+rapamycin?PA+rapamycin;(8)control?3-Methyladenine(autophagic inhibitor,PI3K inhibitor,3-MA,10mmol/L)?Ang2?PA?Ang?+3-MA?PA+3-MA;(9)control?bafilomycin Al(autophagic inhibitor,H+-ATPase inhibitor,10nmol/L)?Ang ??PA?Ang II+bafilomycin A1?PA+bafilomycin A1)?The mitochondrial ROS content was measured by flow cytometry after hepatocytes were loaded with MitoSOX probe;mitochondrial membrane potential were detected by confocal microscope after hepatocytes were loaded with JC-1 probe;hepatocellular ATP content was measured by ATP detection assay;normal mtDNA expression was measured by qRT-PCR;hepatocellular H2O2 concentration was measured by H2O2 detection assay;fluorescence spectrophotometer was used to detect the expression of ROS;the location of NOX4 and mitochondria was observed by confocal microscope;western blot was used to detect the expression of NOX4,LC3 and p62;the formation of autophagosomes and autolysosomes was observed by confocal microscope after hepatocytes were transfected by mRFP-GFP-LC3 adenovirus;the accumulation of hepatocytes was observed after cells were stained by Oil Red O.In vivo studies,32 male C57 BL/6J mice of 6-8 weeks old were randomly divided into 4 groups,each group containing 8 mice:(1)methionine choline-supplemented(MCS)diet group;(2)methionine choline-deficient(MCD)diet group;(3)MCD diet+normal saline(NS)group(with continuous intraperitoneal infusion of normal saline by osmotic mini-pumps at a rate of 25pg/kg/h);(4)MCD diet+Ang(1-7)group(with continuous intraperitoneal infusion of Ang(1-7)by osmotic mini-pumps at a rate of 25?g/kg/h).After 6 weeks of molding,mice were anaesthetized with 3%pentobarbital,then the eyeballs were extracted and blood samples were collected,the serum was separated and transaminase(ALT and AST)level was measured;histological changes of livers were observed after liver tissues were stained with haematoxylin and eosin(H&E);the level of lipid accumulation was detected by Oil Red O staining;the expression and location of NOX4 and LC3 was observed by immunohistochemistry;western blot was used to measure the expression of NOX4,LC3 and p62 in liver tissues.Statistic analysis:All data are represented as mean ± stand error(x ±S,E.M.)and were analyzed using SPSS 20.0(?).Significant differences between groups were evaluated by one-way ANOVA or Student's t test.P<0.05 was considered as statistically significant.Results:In vitro:1.Ang ? and PA result in mitochondrial dysfunction,while Ang(1-7)improves mitochondrial function.Ang ? and PA increase the production of mitochondrial ROS,inhibit ATP formation,decrease mitochondrial membrane potential and reduce the expression of normal mtDNA.However,Ang(1-7)inhibits the production of mitochondrial ROS,increases ATP formation,improves mitochondrial membrane potential and increases the expression of normal mtDNA when A779 antagonizes the effect of Ang(1-7).2.Ang ? and PA stimulate the expression of NOX4 while Ang(1-7)inhibits the expression of NOX4.Ang ? and PA increase the expression of NOX4 and concentration of H2O2.Ang(1-7)lowers the expression of NOX4 and concentration of H2O2.A779 antagonizes the effect of Ang(1-7).3.Ang ? and PA induce hepatocellular autophagy while Ang(1-7)inhibits autophagy.Ang ? and PA increase the expression of LC3II,reduce the expression of p62 and induce the formation of autophagosomes and autolysosomes(especially autolysosomes).However,Ang(1-7)reduces the expression of LC3?,increases the expression of p62 and decreases the formation of autophagosomes and autolysosomes.4.Ang ? and PA induce hepatocellular autophagy through triggering ROS production.DPI(NOX inhibitor),NAC(cellular ROS scavenger)and mito-TEMPO(mitochondrial ROS inhibitor)inhibit autophagy induced by Ang ? and PA.Rapamycin,the autophagic inducer,can inhibit the production of ROS,and increase the formation of ATP based on Ang ? or PA treatment.3-MA and bafilomycin A1(autophagic inhibitors)can lead to increase of ROS production and inhibit ATP formation based on Ang ? or PA treatment.5.Ang ? and PA can increase hepatocellular lipid accumulation,while Ang(1-7)inhibits lipid accumulation.When PA acts on hepatocytes alone,we can see obvious lipid droplet disposition in hepatocytes,and Ang II+PA aggravates lipid accumulation observed by Oil Red O staining.Ang(1-7)reduces lipid disposition.In vivo results:1.Ang(1-7)reduces MCD diet induced liver oxidative stress and autophagy.Western blot and immunohistochemistry show that NOX4,LC3 and p62 expression in liver tissues of MCD diet group and MCD+NS group up-regulates compared with MCS diet group,while expression of those proteins mentioned above in liver tissues of MCD+Ang(1-7)group obviously down-regulates compared with MCD diet group and MCD+NS group.2.Ang(1-7)attenuates NAFLD induced by MCD diet.As shown by H&E staining and Oil Red O staining,the liver structure of mice in MCS diet group is normal,without macrovesicular fatty degeneration and inflammatory cells infiltration.The livers of mice in MCD diet group and MCD+NS group show obvious fatty droplets disposition and inflammatory cells infiltration,the concentration of serum transaminase ALT and AST up-regulates compared with MCS diet group.The level of fatty degeneration and inflammatory cells infiltration of mice in MCD+Ang(1-7)group significantly decreases compared with MCD diet group and MCD+NS group,and the concentration of serum transaminase also decreases.Conclusion:1.Ang(1-7)inhibits NOX4 stimulation and mitochondrial dysfunction which induced by Ang ? and PA,thus reduces hepatocellular oxidative stress and attenuates lipid accumulation induced by Ang ? and PA.2.Ang ? and PA induce when Ang(1-7)inhibits hepatocellular autophagy,and this procedure is closely related to cellular oxidative stress.3.Ang(1-7)inhibits oxidative stress and autophagy induced by MCD diet in mice liver,and attenuates mice NAFLD induced by MCD diet.