| Objective:Myocardial ischemia reperfusion(MI/R)injury is a serious clinical problem in the during treatment of ischemic heart disease,and there is no effective treatment.USP15 is a member of the ubiquitin specific protease(USP)family.It is widely expressed in different tissues and regulates a variety of cellular processes,such as cell proliferation,cell invasion,apoptosis,autophagy,cell cycle,genome integrity,transcription regulation,and immune response.The role of USP15 in MI/R injury is unclear.PHD3 is a member of the proline-4-hydroxylase domain(PHD)protein family and has long been considered as a pro-apoptotic protein.PHD3 is highly expressed in the heart and has been associated with a variety of heart diseases.It is predicted that USP15 can bind to PHD3,and it should be further investigated whether the interaction between the two factors can affect MI/R damage.rAj-Tspin is a recombinant peptides containing RGD motif derived from sea cucumber.Whether it plays a protective role in MI/R injury is unclear.This article is aimed(1)to explore the role of USP15 in MI/R injury;(2)to explore whether USP15 affects MI/R injury by regulating PHD3;(3)to explore the protective effect of rAj-Tspin on MI/R injury;(4)to explore whether rAj-Tspin plays a protective role in MI/R injury by regulating USP15/PHD3 pathway.And this study will be helpful to improve the pathogenesis of MI/R injury and provide a new idea for the treatment of MI/R injury.Method:1.The expression of USP15 in MI/R injuryHealthy male SD rats were randomly divided into two groups: sham operation group and model group(MI/R).Rat MI/R injury model was established by ligation of the anterior descending branch of the left coronary artery(LAD),ischemia for 30 min,reperfusion for 2 h.After perfusion,blood was taken from the abdominal aorta and cardiac tissue samples were retained.The ST segment elevation during the modeling process was detected by using ECG.Serum levels of MB-type creatine kinase isoenzyme(CK-MB)and troponin I(CTn I)were determined by ELISA kit.Serum lactate dehydrogenase(LDH)was detected by using the kit.Hematoxylin and Eosin(H&E)staining were used to observe pathological changes of myocardial tissue.USP15 protein expression was detected by using Western Blot.2.Effect of USP15 on MI/R injuryThe H/R injury model of H9C2 cells was replicated by hypoxia for 6 h and reoxygenation for 12 h and transfected with USP15 interference sequence or USP15 overexpression plasmid.The protein levels of USP15,BAX,BCL2,Cleaved Caspase3 were detected by Western Blot.H9C2 cell apoptosis was detected by TUNEL kit.3.USP15 binds to PHD3 and affects its protein expressionH9C2 cells were divided into negative control(Ig G)group and blank group.The whole protein samples were extracted and the interaction between USP15 and PHD3 was verified by immunocoprecipitation method.H9C2 cells were modeled by H/R,transfected with USP15 interference sequence or USP15 overexpression plasmid,and PHD3 protein level was detected by Western Blot.4.USP15 affects the stability and ubiquitination level of PHD3 proteinH9C2 cells were divided into negative control(Ig G)group,blank group,and H/R group.The ubiquitization level of PHD3 was detected by immunoprecipitation method.H9C2 cells were transfected with USP15 interference sequence,and the ubiquitination level of PHD3 was detected by immunoprecipitation method.USP15 interference sequence was transfected into H9C2 cells and MG132 was administered.The protein expression levels of USP15 and PHD3 were detected by Western Blot.H9C2 cells were transfected with USP15 interference sequences,and four time points(0,1,2,4 h)were set.Cycloheximide(CHX)was added at different time points,and the expression level of PHD3 protein was detected by Western Blot.5.USP15 affects MI/R injury by regulating PHD3Rat MI/R injury model was established by ligation of the anterior descending branch of left coronary artery(LAD).H9C2 cells were modeled by H/R and Western Blot was used to detect PHD3 protein expression levels in vitro and in vivo.H9C2 cells were modeled with H/R and transfected with PHD3 interference sequences.BAX,BCL2,Cleaved Caspase3 protein expression levels were detected by Western Blot.H9C2 cells were modeled with H/R,transfected with USP15 overexpression plasmid and transfected with PHD3 interference sequence.BAX,BCL2,Cleaved Caspase3 protein expression levels were detected by Western Blot.6.Protective effect of rAj-Tspin on MI/R injuryH9C2 cells were modeled by H/R and given a certain concentration of rAj-Tspin.Cell viability was determined by CCK8 assay and effective drug concentration was selected.USP15,PHD3,BAX,BCL2,Cleaved Caspase3 protein expression levels were detected by Western Blot after administration of rAj-Tspin at different concentrations.7.rAj-Tspin plays a protective role in MI/R injury by regulating USP15/PHD3 pathwayH9C2 cells were modeled with H/R,transfected with USP15 overexpression plasmid and administered different concentration of rAj-Tspin.BAX,BCL2,Cleaved Caspase3 protein expression levels were detected by Western Blot.Results:1.The expression level of USP15 was increased in MI/R injuryCompared with the sham operation group,the levels of CK-MB,CTn I and LDH in the serum of rats in the MI/R group were significantly increased.H&E staining of myocardial tissue showed structural disorder of myocardial cells in MI/R group.The protein expression level of USP15 in myocardial tissue was significantly up-regulated.2.Effect of USP15 on MI/R injuryCompared with normal group,protein expression levels of USP15,BAX,Cleaved Caspase3 were significantly increased in H/R group,while protein expression level of BCL2 was significantly decreased,indicating increased apoptosis of H9C2 cells after H/R.Apoptosis was significantly improved after USP15 knockdown in vitro.Overexpression of USP15 promoted cell apoptosis and aggravated H/R injury.The TUNEL experiment also showed the same trend.3.USP15 binded to PHD3 and affected its protein expressionThe results of immunoprecipitation showed that USP15 could be combined with PHD3.The protein expression level of PHD3 decreased significantly after USP15 knockdown,but increased significantly after USP15 overexpression.4.USP15 affected the stability and ubiquitination level of PHD3 proteinThe ubiquitization level of PHD3 was reduced in MI/R models.USP15 could deubiquitinate PHD3,inhibit its protein degradation and increase its protein stability.5.USP15 affected MI/R injury by regulating PHD3In vitro and in vivo models,the protein expression level of PHD3 was significantly increased.After PHD3 knockdown in vitro,the apoptosis level was significantly decreased.Compared with the USP15 overexpression group,simultaneous PHD3 knockdown significantly inhibited apoptosis and alleviated MI/R injury,indicating that the effect of USP15 on MI/R was generated by regulating PHD3.6.Protective effect of rAj-Tspin on MI/R injuryTest concentration(0.2 μ M、0.4 μ M、0.8 μ M)rAj-Tspin had obvious protective effects on H/R injury of H9C2 cells in a concentration-dependent manner.Compared with the model group,the protein expression levels of USP15 and PHD3 decreased with the increase of drug concentration after administration of rAj-Tspin.Apoptosis was also significantly improved after administration of rAj-Tspin in a concentration-dependent manner.7.rAj-Tspin played a protective role in MI/R injury by regulating USP15/PHD3pathwayCompared with the USP15 overexpression group,rAj-Tspin significantly reduced the expressions of USP15 and PHD3,inhibited apoptosis,and alleviated the MI/R injury,indicating that the protective effect of rAj-Tspin on MI/R injury was caused by regulating USP15/PHD3.Conclusion:1.USP15 promotes apoptosis and aggravate myocardial ischemia reperfusion(MI/R)injury.Inhibition of USP15 expression can improve myocardial ischemia reperfusion(MI/R)injury.2.USP15 deubiquitinates PHD3,up-regulate the protein expression of PHD3,promotes apoptosis,and then affects MI/R injury.3.rAj-Tspin plays a protective role in MI/R injury by regulating USP15/PHD3. |
