Paeonol Pretreatment Attenuates Hypoxia-induced Myocardial Ischemia/reperfusion Injury By A BRCA1-dependent Antioxidant Pathway

Objective: The incidence and mortality of ischemic heart disease is extremely high,and it is the "number one killer" that threatens human life and health.Once a patient has a myocardial infarction,it is currently recognized that the most effective method is to take revascularization(stent implantation,bypass,drug thrombolysis,etc.)as soon as possible.However,recanalization will also cause myocardial and vascular damage,leading to serious complications such as arrhythmia,so myocardial ischemia/reperfusion(I/R)injury is also attracting more and more attention,Clarifying the mechanisms of I/R injury,exploring effective I/R damage protection methods have become an urgent problem to be solved.During myocardial ischemia/reperfusion injury(I/R),the production of large amounts of oxygen free radicals in the ischemic area is one of the important factors of I/R.Due to the imbalance of oxygen radical generation and scavenging caused by I/R,a large amount of oxygen free radicals such as superoxide anion and hydrogen peroxide are accumulated,which has various toxic effects on cardiomyocytes.The inflammatory response caused by myocardial ischemia/reperfusion releases inflammatory factors such as TNF-?,IL-1,etc.These factors cause secondary damage to tissues in the infarcted area and accelerate apoptosis.Paeonol is the main ingredient of traditional Chinese medicine peony bark.Paeonol is easily absorbed,metabolized quickly and safe for humans.Modern pharmacological studies have shown that paeonol has significant anti-oxidation and free radical scavenging ability,and can also reduce inflammation.Paeonol is widely used in the prevention and treatment of various diseases.In this study,the ischemia/reperfusion(I/R)model was established to study the protective effect of paeonol on ischemiareperfusion injury of rat cardiomyocyte cell H9C2,and to explore its mechanism.The expression of BRCA1 gene was determined by Q-PCR and Western blotting;si RNA was used to block BRCA1 expression in H9C2 cells;The gene silencing efficiency of BRCA1 si RNA was evaluated by Q-PCR and Western blotting.The total antioxidant capacity(T-AOC),LDH activity and CK-MB activity of each group were detected by down-regulation of BRCA1 by si RNA.GST m RNA was detected by Q-PCR.expression;Method : Pretreatment of H9C2 cells with paeonol(10u M)and establishment of myocardial I / R injury model by hypoxia/reoxygenation in culture medium;The cells were divided into three groups: hypoxia/reoxygenation + paeonol group(H/R+Paeonol group),normal culture group(control group),and hypoxia/reoxygenation group(H/R group).The total antioxidant capacity(T-AOC),LDH activity and CK-MB activity of each group were evaluated using a commercial kit.The level of ROS was checked by flow cytometry.The concentrations of L-6,tumor necrosis factor alpha(TNF-?)and IL-1? were measured using an ELISA kit.Result:Paeonol pretreatment significantly reduced the activity of CK-MB and LDH in the medium(p<0.05),indicating that paeonol can significantly reduce H9C2 cell injury induced by hypoxia/reoxygenation;The ROS level in the H/R group was more than twice that in the control group(p<0.05),and the H/R+ paeonol pretreatment group significantly reduced the level of ROS in the cells(p<0.05);The levels of SOD and TAOC in H/R+ paeonol pretreatment group were significantly higher than those in H/R group(p<0.05),suggesting that paeonol significantly enhanced the activity of T-AOC and SOD in hypoxic/reoxygenated H9C2 cells.The expression of BRCA1 in H/R+paeonol pretreatment group was significantly increased(p<0.05),and paeonol could up-regulate the expression of BRCA1 in cells,indicating that paeonol regulate the expression of BRCA1 at the transcriptional level;si RNA was used to block the expression of BRCA1 in H9C2 cells.The BRCA1 m RNA level in the si RNA blocking group was significantly lower than that in the control group,which indicated that BRCA1 si RNA could successfully reduce the BRCA1 expression in cells(p<0.05);BRCA1 si RNA treatment can eliminate the enhancement of GST m RNA expression and T-AOC by paeonol(p<0.05);The activity of CK-MB and LDH in paeonol pretreated BRCA1 si RNA H9C2 cells was significantly lower than that in controlsi RNA group under hypoxia/reoxygenation conditions(p<0.05).The results confirmed that the down-regulation of BRCA1 expression significantly reduced the protective ability of paeonol to hypoxia/reoxygenation-induced H9C2 cells.Conclusion: Paeonol significantly increases the activity of free radical scavenging enzymes and reduces the production of reactive oxygen species(ROS).Paeonol enhances cellular antioxidant capacity by promoting BRCA1 expression.Paeonol will be a potential drug against myocardial ischemia and reperfusion(I / R)damage.