Urolithin B Protects Against Myocardial Ischemia Reperfusion Injury Via Autophagy And P62/Keap1/Nrf2 Signaling Pathway

Background: Acute myocardial infarction is one of the most common cardiovascular diseases with high-morbidity and mortality worldwide.Restoring of blood flow to the ischemic myocardium itself per se can induce additional injury which is called myocardial ischemia reperfusion(IR)injury.Urolithin B(UB)is an intestinal metabolite of ellagic acid with various biological activities including anti-cancer,antiinflammatory,anti-oxidative stress and hormone-like effects.Urolithin B was demonstrated to be protective against atherosclerosis and diabetic cardiomyopathy in recent years,but the effect of UB in myocardial IR remains unclear to date.Aim: To investigate the effect and the mechanism of UB in myocardial IR injury.Methods:(1)Myocardial IR injury were established by ligating and releasing left anterior descending of coronary artery in adult male Sprague Dawley rats,different concentrations of UB were intraperitoneal injected.The protein expression of Cleaved Caspase 3 and plasma concentration of UB were evaluated by Western blotting and LCMS/MS respectively.(2)The hemodynamic parameters before and during IR period were recorded by left ventricular intubation.The infarct size,morphology structure,biochemical factors and cell apoptosis of heart were detected after rats were sacrificed.(3)H9c2 cardiomyocyte was subjected to hypoxia reoxygenation(HR)to establish an IR model in vitro.Cell viability and apoptosis were assayed after HR and UB treatment.(4)The level of autophagy and the activation of Akt/m TOR/ULK1 pathway were evaluated by Western blotting and GFP-LC3 adenovirus.(5)Cells were treated with LY294002,an Akt inhibitor,and the activation of Akt/mTOR/ULK1 pathway and cell apoptosis were detected.(6)The contents of Reactive Oxygen Species(ROS)and the activation of Nrf2-ARE pathway were measured by fluorescence probe and western blot respectively.The activation of p62/Keap1/Nrf2 signaling pathway in H9c2 cells was detected by Western blot,Co-immunoprecipitation and immunofluorescence.(7)Cells were treated with or without siNrf2 and the effect of UB on Nrf2 downstream protein expression,ROS contents and cell apoptosis were evaluated.Results:(1)0.7 mg/kg of UB significantly inhibited the expression of Cleaved Caspase 3 in IR heart,and it’s maximal concentration in plasma reached 15.8μM.(2)UB increased the left ventricular end-diastolic pressure(LVSP),decreased left ventricular systolic pressure(LVSP),maximal rate of the increase of left ventricular pressure(+dp/dt max)and maximal rate of the decrease of left ventricular pressure(-dp/dt max).UB inhibited the expression of myocardial injury markers including CK,LDH,MDA,and enhanced the activity of SOD.UB reduced infarct size,morphology disorder and cell apoptosis after IR insult.(3)UB significantly mitigated the H9c2 cells viability decreasing and apoptosis induced by HR.(4)UB treatment significantly inhibited the autophagy induced by IR/HR,and upregulated the expression of p-Akt(S473),pmTOR(S2448)and p-ULK1(S757)in IR hearts and HR cells.(5)LY294002 downregulated Akt/mTOR/ULK1 pathway and abolished UB’s apoptosis inhibition effect.(6)UB reduced the contents of ROS,activated Nrf2 and upregulated the expression of antioxidant including HO-1,NQO1 and GSTP1.HR led to the decrease of p62 binding with Keap1,while UB increased the binding level.(7)Nrf2 interfering reversed the antioxidants upregulation,ROS reduction and apoptosis inhibition effect of UB.Conclusion:(1)UB can protect IR induced myocardial injury.(2)Autophagy inhibition contributes to the protective effect of UB.(3)UB activates Nrf2 at least partially via p62/Keap1/Nrf2 signaling pathway,and Nrf2 is critical for UB’s protective effect.