The Molecular Mechanism Of Deubiquitinating Enzyme USP7 Regulating Myocardial Ischemia-reperfusion Injury Through Keap1

【Background】Myocardial ischemia/reperfusion injury(MIRI)injury refers to a pathological process in which the myocardial tissue injury becomes more serious after the ischemic and hypoxic myocardium returns to normal perfusion.The oxidative stress response plays a very important role in the occurrence and development of myocardial ischemiareperfusion injury,and the production of excessive reactive oxygen species(ROS)will cause more serious damage to the myocardium.Studies have shown that Keap1 is one of the important regulatory factors for reducing myocardial injury and improving cardiac function after myocardial ischemia reperfusion injury(MIRI).In our previous studies of ischemia-reperfusion heart tissue models,we found that the expression of ubiquitin-specific protease 7 was significantly increased,and Keap1(Kelch-like ECHrelated protein 1)plays an important role in oxidative stress damage and ROS generation,but Its regulatory mechanism for reperfusion injury after ischemia is not yet clear.The purpose of this study is to explore how USP7 mediates the expression of Keap1 pathway to regulate the production of ROS and the investigation and study of ischemia-reperfusion mechanisms【Methods】1.The mouse myocardial ischemia-reperfusion injury model was established by ligating the left anterior descending coronary artery(LAD)to loosen the thread structure after ischemia.2.P5091 was injected into animals to inhibit the activity of USP7,and si RNA myocardial injection interfered with the expression of USP7 to study the expression of apoptosis-related proteins in mouse hearts.3.TUNEL technology was used to detect the condition and results of cardiac tissue apoptosis.4.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of cardiac injury factors.5.TTC technology to detect the area of mouse heart infarction.6.Ultrasound to Observe the Changes of Heart Function in Mice after Operation.7.Using co-ip technology to study the interaction between USP7 and Keap1.【Results】1.The expression of USP7 increased in myocardial infarction tissue: 1 hour and6 hours after the establishment of the acute myocardial infarction model group in mice,the heart tissue was taken out and the USP7 protein expression was detected by Western blot.The results showed that the expression of USP7 in the model group was significantly increased.2.The expression of USP7 increased in myocardial ischemia-reperfusion injury:After the acute myocardial ischemia-reperfusion model was established in mice,the heart tissue was taken out to detect the expression of USP7 protein by Western blot,RT-q PCR,and immunohistochemistry.The results showed that the expression of USP7 in the model group was obviously height.3.Inhibition of USP7 can reduce the expression of myocardial apoptotic protein:mice were injected with P5091 intraperitoneally and adeno-associated virus into the heart to construct a myocardial ischemia-reperfusion model.Western blot was used to detect the expression of myocardial USP7 and pro-apoptotic protein BAX the amount.The results showed that the expression of USP7 and BAX protein in heart tissues that inhibited USP7 was significantly reduced.4.Inhibition of USP7 can reduce the apoptosis of cardiac tissue: mice are injected with P5091 intraperitoneally and adenovirus-associated virus into the heart to construct a myocardial ischemia-reperfusion model.TUNEL method is used to detect the apoptosis of cardiomyocytes.The results showed that the amount of apoptotic cells in the heart tissue was significantly reduced after USP7 was inhibited.5.Inhibition of USP7 can improve cardiac function: mice are injected intraperitoneally with P5091 and adenovirus-associated virus into the heart to construct a myocardial ischemia-reperfusion model.VEVO2100 ultrasound Doppler is used on the 3rd,14 th,and 21 st days after the model is successfully established.Changes in mouse heart function,the results showed that the changes in mouse heart function after USP7 was inhibited improved over time.6.Inhibition of USP7 can reduce the infarct size and fibrosis caused by myocardial injury: mice were injected with P5091 into the abdominal cavity to construct a myocardial ischemia-reperfusion model,and the EVAN-TTC double staining method and Masson staining were used to detect the infarct size and fibrosis degree of the mouse.ELISA method detects the content of CTNT(troponin T)and CKMB(creatine kinase isoenzyme)in serum.The results showed that the area of infarction and fibrosis of the mouse heart was significantly reduced after USP7 was inhibited,and the levels of serum CTNT and CKMB were significantly reduced.7.USP7 interacts with Keap1 and can promote the degradation of Keap1: Mice were intraperitoneally injected with P5091 and adenovirus-associated virus into the heart to construct a myocardial ischemia-reperfusion model.After the operation,CO-IP and Western blot were used to detect the protein expression levels of USP7 and Keap1,and Western blot was used to detect ubiquitin.(Ub)and Keap1 protein expression level,the results showed that the inhibition of USP7 increased the ubiquitin level of Keap1 and accelerated its degradation.8.Inhibition of USP7 can reduce the content of ROS: mice are injected with P5091 into the intraperitoneal cavity to construct a myocardial ischemia-reperfusion model.After the operation,the heart tissue is prepared into a single-cell suspension.Flow cytometry is used to detect the content of ROS.The results show that the heart after USP7 is inhibited The ROS content of the tissue is significantly decreased【Conclusion】Inhibition of deubiquitinating enzyme USP7 can promote the degradation of Keap1 and reduce the damage caused by myocardial ischemia and reperfusion.