Study On The Mechanism Of Cyclophilin A Regulates Thioredoxin Interaction Protein Ubiquitination Promoting Myocardial Ischemia Reperfusion Injury

Objective:The mechanism of myocardial ischemia reperfusion injury（MIRI）has not been fully elucidated.Previous studies have found that oxidative stress and inflammation play a key role in the damage of cardiac function after myocardial reperfusion therapy in patients with myocardial infarction.Cyclophilin A（CypA）is one of the pathogenic proteins mediating inflammation-induced cardiovascular dysfunction.Thioredoxin interacting protein（TXNIP）participates in the pathological process of various diseases as a pro-oxidant.The relationship between CypA and TXNIP has not been fully elaborated,whether CypA regulates the function of TXNIP to aggravate oxidative stress in MIRI remains unclear.The study is aim to investigate the potential mechanism between CypA and TXNIP during myocardial ischemia reperfusion.To analyze the significance of CypA as a biomarker of coronary heart disease,and to provide new ideas for the treatment of myocardial ischemia reperfusion injury.Methods:Part I clinical study,Patients diagnosed with coronary atherosclerotic heart disease by coronary angiography were selected as the coronary heart disease group（CHD）,and health check-up subjects during the same period were selected as the control group.A total of 74subjects were included in each group:1)Clinical characteristics of the two groups were analyzed;2)The serum levels of CypA,TXNIP and IL-1βin CHD group and control group were detected by ELISA;3)The CHD group was divided into single-vessel disease group,double-vessel disease group and three-vessel disease group according to the number of coronary artery lesions.The differences in serum CypA,TXNIP and IL-1βlevels among each group were analyzed;4)According to the Gensini score,the patients were divided into mild lesion group（Gensini score<30 points）,moderate lesion group（Gensini score 30-60 points）and severe lesion group（Gensini score≥60 points）.The difference of serum CypA,TXNIP and IL-1βlevels among different Gensini score groups and the comparison of echocardiographic results among different Gensini score groups were analyzed;5)Pearson test was used to analyze the correlation between Gensini score and serum CypA,TXNIP and IL-1βlevels in CHD group;6)Logistic regression analysis was used to analyze the risk factors of coronary artery lesions in patients with coronary heart disease in this study.The experimental data were statistically analyzed by SPSS 21.0software,and P<0.05 was considered statistically significant.Part II cellular level:1)H₂O₂-induced oxidative stress model in H9c2 cardiomyocytes was established;2)H9c2cardiomyocytes were divided into four groups:Control group,H₂O₂group,NC-si RNA+H₂O₂group,and PPIA-si RNA+H₂O₂group.The activities of SOD,ROS generation,and MDA levels in H9c2 cardiomyocytes were detected by commercial kits.The m RNA expression levels of TNF-α,IL-6,IL-1βand TXNIP were detected by q PCR.Western blot was used to detect the protein expression of Cleaved Caspase-3,Cleaved PARP1,Bax,Bcl-2,CypA and TXNIP.Cell apoptosis was detected by flow cytometry;3)293T/WT and 293T/CypA-were transfected with TXNIP-myc overexpression plasmid,and CHX was used to inhibit the synthesis of new protein to observe the effect of endogenous CypA on TXNIP protein stability.293T/WT and 293T/CypA-were transfected with TXNIP-myc,Ub-HA,K48-HA and K63-HA overexpression plasmids to observe the effect of endogenous CypA on TXNIP ubiquitination;4)293T cells were transfected with TXNIP-myc overexpression plasmid,and the E3 ubiquitin ligases that might interact with TXNIP were identified by mass spectrometry.293T cells were transfected with TXNIP-myc and E3 ligase overexpression plasmids,and Co-IP was used to detect the protein interaction between TXNIP and its E3 ligase.293T cells were transfected with TXNIP-myc,K48-HA overexpression plasmids and ITCH-si RNA to knockdown AIP4 protein expression.CHX was used to inhibit protein synthesis.Cell lysates were collected at different time points,and western blot was used to detect TXNIP protein expression to observe the effect of AIP4 on TXNIP stability.Co-IP was used to detect the effect of AIP4 on the ubiquitination of TXNIP K48 line;5)293T/WT and293T/CypA-were transfected with TXNIP-myc,AIP4-flag and K48-HA overexpression plasmids,and the expression of TXNIP,AIP4 and CypA proteins was detected by western blot to observe the effect of endogenous CypA on the stability of TXNIP.Co-IP was used to detect the effect of endogenous CypA on AIP4-mediated K48-linked TXNIP ubiquitination.293T cells were transfected with AIP4-flag,TXNIP-myc and CypA-myc overexpression plasmids to investigate the mechanism of CypA on the interaction between TXNIP and E3 ligase AIP4 protein.Part III animal level:C57 mice were divided into three groups:sham group,wild type（WT）group and CypA gene knockout（Ppia-/-,KO）group;1)HE staining was used to observe the pathomorphological changes of heart tissues;2)Cardiac function was measured by echocardiography and hemodynamics;3)The changes of myocardial injury markers（CK-MB,c Tn I,Mb）in each group were detected by automatic biochemical analyzer;4)ELISA was used to detect the levels of serum inflammatory cytokines（IL-1β,IL-6,TNF-α）;5)TUNEL staining was used to detect cardiomyocyte apoptosis in each group;6)Immunohistochemistry was used to observe the expression of Cleaved Caspase 3 and Bax;7)Western blotting was used to detect the expression of Cleaved Caspase 3,Cleaved PARP1,Bax,Bcl-2 and CypA in myocardial tissue of mice in each group;8)q PCR was used to detect the m RNA expression of inflammatory factors（IL-1β,IL-6,TNF-α）in each group;9)The changes of oxidative stress indicators（ROS,MDA,SOD）of cardiomyocytes in each group were detected by kits.Results:1)The serum levels of CypA,TXNIP and IL-1βwere significantly higher in CHD group than in control group（P<0.05）.Serum concentrations of CypA,TXNIP and IL-1βincreased with the number of coronary artery lesions.The concentrations of CypA,TXNIP and IL-1βin serum were increased with the increase of Gensini score.The serum levels of CypA,TXNIP and IL-1βwere positively correlated with Gensini score in CHD group.Serum CypA and IL-1βmay a risk factors for CHD;2)The expression of CypA was increased in H₂O₂-induced oxidative stress injury in H9c2cardiomyocytes.CypA promoted H₂O₂-induced oxidative stress injury,the secretion of inflammatory factors（TNF-α,IL-6,IL-1β）and cardiomyocyte apoptosis in H9c2cardiomyocytes.Endogenous CypA enhanced TXNIP stability and inhibited the K48-linked ubiquitination degradation of TXNIP.TXNIP interacted with E3 ubiquitin ligase AIP4,but not with SMURF2.TXNIP is dependent on K48-linked ubiquitination mediated by E3 ubiquitin ligase AIP4.CypA inhibited AIP4-induced TXNIP K48-linked ubiquitination by inhibiting the interaction of AIP4 with TXNIP;3)In vivo myocardial ischemia-reperfusion injury model was established.Compared with WT mice,Ppia-/-mice showed significant improvements in cardiac function,myocardial histopathological changes,myocardial enzyme release,oxidative stress levels,serum cytokine concentrations and gene expression levels.Besides,the degree of myocardial injury caused by apoptosis was milder（P<0.05）.Conclusions:1)The levels of serum CypA,TXNIP and IL-1βwere increased in patients with coronary heart disease.Serum CypA is a risk factor for coronary heart disease and may be a serum biomarker for coronary heart disease;2)Intracellular CypA aggravates oxidative stress and inflammatory injury of cardiomyocytes via inhibiting E3 ligase AIP4-mediated TXNIP ubiquitination to promotes the accumulation of TXNIP;3)In the mouse model of myocardial I/R,knocking out CypA expression can alleviate myocardial I/R injury and improve cardiac function.