The Role And Mechanism Of FGF21 In Reducing The Expression Of ALCAT1 And Inhibiting The Apoptosis Of Heart And Liver Cells In Myocardial Infarction Mice By Aerobic Exercise

Background:Myocardial infarction leads to myocardial ischemia and anoxia,reactive oxygen species(ROS)burst,abnormal Ca²⁺regulation,endoplasmic reticulum stress(ERs)and mitochondrial damage,which leads to cardiomyocyte apoptosis and cardiac pathological remodeling.After myocardial infarction,reticulin in the central area of liver collapses and proliferates,resulting in cardiogenic liver damage.It is of great significance to seek scientific and effective methods to prevent cardiovascular disease and its organs.Fibroblast growth factor 21(FGF21)as a response factor of liver-derived movement,plays a protective role in a variety of liver diseases.ALCAT1 locates in endoplasmic reticulum and plays an important role in oxidative stress and mitochondrial biological function.Aerobic exercise can significantly increase the expression of FGF21,decrease the expression of ALCAT1 and ROS,and inhibit the apoptosis of myocardial cells.But can it improve the intracellular calcium abnormality by activating PI3K/Akt/SERCA2a signaling pathway,and inhibit the apoptosis of ERs and IRE1?/JNK signaling pathway?At present,there is no experimental evidence.Exercise significantly increased the level of FGF21 in the liver,but whether FGF21 participates in aerobic exercise to improve the cardiogenic liver damage and its mechanism are not reported.Objective:To investigate the role and mechanism of FGF21 in the reduction of ALCAT1 expression and the inhibition of apoptosis of cardiomyocyte and hepatocytes by aerobic exercise in post-MI.Methods:In vivo animal experiment:two months old wild-type(WT)C57 mice were randomly divided into sham operation group(S),myocardial infarction group(MI),myocardial infarction+aerobic exercise group(ME),10 mice in each group.Two months old ALCAT1-/-C57 mice were randomly divided into sham operation group(KS),myocardial infarction group(KMI),myocardial infarction+aerobic exercise group(KME),8 mice in each group.The model of myocardial infarction was established by ligation of left anterior descending coronary artery.The S group and KS group only thread without ligation.The cardiac function was detected by echocardiography one week after operation,and then aerobic exercise was performed 6 weeks later.After the end of the next day,echocardiography was used to assess the heart function.Blood was taken from the orbit and the head was severed.In vitro cell experiments:H9C2 cells were treated with rhFGF21 protein(rhFGF21,75ng/ml/15h),AMPK agonist(AICAR,1Mm/15h),FGFR1 receptor blocker(PD166866,10 ?M/lh,H₂O₂(100 ?mol/L/4h).H9C2 cells were divided into 7 groups:H9C2 control group,H₂O₂ group,H₂O₂+AICAR group,H₂O₂+rhFGF21 group,H₂O₂+rhFGF21+AICAR group,H₂O₂+PD166866 group,H₂O₂+PD166866+rhFGF21 group.Test indexes:RT-qPCR was used to detect the expression of alcatl mRNA,Western blotting was used to detect the expression of FGF21,FGFR1,ALCAT1,CHOP,GRP78,ATF6,pIRE1?/IRE1?,pJNK/JNK,Caspase3,Bax,Bcl-2,pPI3K/PI3K,pAkt/Akt,SERCA2a,PINK1,P62,LC3 ?/? protein,DHE was used to detect the ROS level of cells.Masson staining was used to detect the collagen fiber,CCK-8 was used to detect the cell activity,TUNEL was used to detect the cell activity apoptosis,Mitotracker,EF%,FS%,LVIDd and LVIDs were detected by echocardiography.Results:(1)Aerobic exercise significantly increased the expression of FGF21 and FGFR1(p<0.01),decreased the ratio of pIRE1?/IRE1?,pJNK/JNK,apoptotic proteins such as CHOP,GRP78,Caspase3 and Bax(p<0.05,p<0.01),reduced the number of TUNEL positive particles(p<0.01).The results showed that aerobic exercise can significantly inhibit the ERs and IRE1?/JNK signaling pathway and reduce the apoptosis of cardiomyocytes by activating the expression of FGF21 and FGFR1 in the marginal area of myocardial infarction.(2)Aerobic exercise significantly increased the expression of pPI3K/PI3K,pAkt/Akt ratio and SERCA2a protein in the infarct margin(p<0.01).The results showed that aerobic exercise significantly activated the FGF21/FGFR1/PI3K/Akt/SERCA2a signal pathway in the infarct margin and corrected the abnormal calcium regulation.(3)Aerobic exercise significantly reduced the expression level of alcat1 mRNA and ALCAT1 protein(p<0.01),decreased the content of ROS and MDA(p<0.01),increased the activity of T-SOD and T-AOC antioxidant enzyme(p<0.05,p<0.01),increased the expression of PINK1 and LC3?/? protein(p<0.05,p<0.01),and decreased the expression of P62 protein(p<0.01).The results showed that aerobic exercise reduced the expression of ALCAT1,oxidative stress and mitochondrial autophagy abnormality.(4)Knockout of ALCAT1 significantly increased the endogenous expression of FGF21 in the infarct margin(p<0,01).but did not affect the effect of exercise intervention.The results showed that the expression of FGF21 was closely related to that of ALCAT1.(5)Knockout of ALCAT1 significantly reduced myocardial oxidative stress,improved mitochondrial autophagy abnormality,decreased the ratio of Bax/Bcl-2 and the number of TUNEL positive granules(p<0.01).Aerobic exercise intervention reduced oxidative stress,improved the quality mitochondrial autophagy abnormality,reduce the number of TUNEL positive particles ALC ATI.The results showed that ALCAT1 played an important role in reducing myocardial oxidative stress,improving mitochondrial autophagy abnormality and inhibiting cardiomyocyte apoptosis.(6)Aerobic exercise significantly reduced the percentage of myocardial collagen volume(p<0.01),LVIDd and LVIDs(p<0.01),increased FS%and EF%(p<0.01),and improved cardiac function.Knockout of ALC AT1 significantly inhibited the over proliferation of myocardial collagen fibers(p<0.01).Aerobic exercise intervention further reduced the percentage of myocardial collagen volume,reduced the LVIDd and LVIDs(p<0.01),increased FS%and EF%(p<0.01),improved cardiac function.The results showed that ALC AT1 played an important role in improving the cardiac function of MI by aerobic exercise.(7)AICAR or rhFGF21 significantly reduced the number of TUNEL positive granules(p<0.01),Bax/Bcl-2 ratio,Caspase 3,CHOP,GRP78 and apoptotic protein expression of H9C2 cells induced by H₂O₂(p<0.01,p<0.05),and decreased the ratio of pIRE1?/IRE1? and pJNK/JNK(p<0.01);FGFR1 receptor inhibitor(PD166866),significantly increased the number of TUNEL positive granules,Bax/Bcl-2 ratio,Caspase 3,CHOP,GRP78 and apoptotic protein expression of H9C2 cells(p<0.01).The expression increased the ratio of pIRE1?/IRE1? and pJNK/JNK(p<0.01).The results showed that simulated exercise or rhFGF21 significantly inhibited ERs and IRE1?/JNK apoptosis signaling pathway induced by H₂O₂(p<0.01),and inhibited H9C2 cell apoptosis induced by H₂O₂,and FGF21 could be inhibited by PD166866.(8)AICAR or rhFGF21 significantly increased T-SOD activity of H9C2 cells induced by H₂O₂(p<0.01),decreased ROS,MDA level and ALCAT1,PINK1,P62,LC3 II/I protein expression(p<0.01).The results showed that exercise or rhFGF21 significantly reduced oxidative stress and ALCAT1 expression,improved mitochondrial autophagy abnormality,inhibitied cardiomyocyte apoptosis.(9)After MI,ALT,ALP,AST and FGF21 were significantly increased(p<0.01,p<0.05),and the levels of ALT,ALP and AST were decreased by aerobic exercise intervention(p<0.01).The results showed that aerobic exercise significantly improved myocardial infarction induced liver inj ury.(10)In post-MI,the expression of ATF6,CHOP,GRP78 and IRE1? protein in liver increased significantly(p<0.05,p<0.01),and the number of TUNEL positive particles increased(p<0.01);the number of TUNEL positive granules in liver and the expression of CHOP,GRP78,ATF6 and IRE1? protein in liver decreased significantly(p<0.01,p<0.05)after aerobic exercise intervention.The results showed that aerobic exercise significantly inhibited ERs and hepatocyte apoptosis induced by MI.(11)After MI,the expression of ALCAT1 were significantly increased(p<0.01),PINK1,Parkin and P62 were significantly decreased(p<0.05,p<0.01),LC3II/I were significantly increased(p<0.01).After aerobic exercise intervention,the expression of ALCAT1,LC3 ?/?(p<0.01,p<0.05)were significantly decreased,PINK1?Parkin and P62 were significantly increased(p<0.05,p<0.01).The results showed that after myocardial infarction,the expression of ALCAT1 in liver was significantly increased,the level of mitochondrial autophagy was changed,and aerobic exercise intervention significantly reduced the expression of ALCAT1 in liver and improved mitochondrial autophagy abnormality.Conclusion:(1)Aerobic exercise activated PI3K/Akt/SERCA2a pathway,inhibited ERs and IRE1?/JNK apoptotic signal pathway,reduced ALCAT1 expression,alleviated oxidative stress,improved mitochondrial autophagy abnormality,inhibited myocardial apoptosis,and improved cardiac function by stimulating the expression of FGF21 and FGFR1 in the infarct border area.(2)Exogenous AICAR or rhFGF21 significantly inhibited the apoptosis of H9C2 cells induced by H₂O₂,the apoptosis signaling pathway of ERs and IRE1?/JNK,the expression of ALCAT1,alleviated oxidative stress,improved mitochondrial autophagy abnormality.The protective effect of FGF21 was inhibited by PD 166866.(3)Aerobic exercise significantly stimulated the expression of FGF21,decreased the expression of ALCAT1 and ERs,improve mitochondrial autophagy abnormality,inhibited hepatocyte apoptosis,and improved the liver injury induced by myocardial infarction.In conclusion,aerobic exercise can stimulate the expression of FGF21 and FGFR1 in the heart and liver,activate the myocardial PI3K/Akt/SERCA2a pathway,correct the abnormal calcium regulation,inhibit the apoptosis signal pathway of the heart,liver ERs and myocardial cell IRE1?/JNK,reduce the expression of ALCAT1 in the heart and liver,alleviate oxidative stress,improve mitochondrial autophagy abnormality,inhibit the apoptosis of cardiomyocytes and hepatocytes,improvement of myocardial infarction and liver injury caused by myocardial infarction.