The Protective Effects And Mechanisms Of Aloin On Myocardial Ischemia Reperfusion Injury

Background:Acute myocardial infarction(AMI)is one of the leading causes of death worldwide.As the population ages,the incidence of AMI will continue to increase.Percutaneous coronary intervention and intravenous thrombolysis have significantly reduced mortality in AMI patients,but the reperfusion process can further exacerbate the myocardial injury,known as myocardial ischemia-reperfusion injury(MI/RI).MI/RI can lead to complications such as arrhythmias,cardiac arrest,and myocardial stunning,affecting the size of myocardial infarction.Therefore,prevention and treatment of MI/RI are critical to improving the prognosis of patients with AMI.The mechanisms of MI/RI are complex and mainly involve pathophysiological processes such as oxidative stress,inflammation,and apoptosis.Aloin has biological activities such as anti-oxidative stress,anti-inflammatory response,and antitumor.Aloin is fulfills pleiotropic protective functions in several disease models including myocardial injury and hepatic injury.Nevertheless,the potential of aloin in MI/RI remains elusive.Objective:This paper aims to explore the role of aloin in MI/RI and its possible mechanisms by constructing a cardiomyocyte-simulated ischemia/reperfusion injury(SI/RI)model and a rat MI/RI model.Methods:1.Protective effects of aloin on cardiomyocyte SI/RI model and its mechanism.The cardiomyocyte SI/RI model was established by pretreatment with aloin for 6 h,hypoxia for 4 h,and reoxygenation for 8 h.The different concentrations(0-100 μM)of alointreated normal cardiomyocytes and cardiomyocyte SI/RI models respectively.The cell viability was determined by the MTT method,and the optimum concentration of aloin was selected according to the results.The cells were divided into three groups:①control group;②SI/R group;③SI/R+Alo 50 μM group.Apoptosis was detected by flow cytometry in cardiomyocytes of each group;To determine the release of lactate dehydrogenase(LDH),the activity of superoxide dismutase(SOD),the content of reactive oxygen species(ROS),malondialdehyde(MDA),interleukin-6(IL-6),interleukin-1 β(IL-1β)and tumor necrosis factor-α(TNF-α),and the protein expression of Bcl-2,Bax,Nrf2,and HO-1 by Western blotting.The levels of TNF-α,IL-6,IL-1β,Nrf2,and HO-1 mRNA were measured by realtime fluorescence quantitative polymerase chain reaction(RT-qPCR).Small interference siRNA was used to silence the expression of Nrf2,and to further explore the protective effect of aloin on the cardiomyocyte SI/RI model by regulating the Nrf2/HO-1 signal pathway.The experimental groups were:①control group;②SI/R group;③SI/R+Alo group④SI/R+Alo+si-con group ⑤SI/R+Alo+si-Nrf2 group.The cell viability,LDH release,apoptosis,SOD activity,MDA content,ROS content,IL-6,IL-1β,and TNF-α content of cardiomyocytes were measured.2.To further verify the protective effect and mechanism of aloin in rat MI/RI model.Female Wistar rats were randomly divided into sham group,model group,Alo 20 mg/kg group and Alo 40 mg/kg group,and Alo+ML385 group(Alo 40 mg/kg,ML385 30 mg/kg).Rats were given aloin or the same volume of PBS respectively,once a day for 7 days.ML385 was injected intraperitoneally 1 hour before intragastric administration,once a day for 7 days.On the day of operation,1 hour after the last administration,three sites of the left anterior descending coronary artery in Wistar rats were ligated with the operation line,ischemia time 30min,and reperfusion time of 4 hours.The rat MI/RI model was established and the protective effect of aloin on MI/RI was observed.The latent period of arrhythmia,the duration of arrhythmia,and the potential difference of the ST segment were measured.The contents of CK-MB and LDH in serum,SOD activity,and MDA content in myocardial tissue were measured.Myocardial infarct area(IA),myocardial risk area(AAR),and myocardial protective area(PA)were determined by triphenyl tetrazolium chloride(TTC)and Evans blue double staining.The morphology of myocardial tissue was observed by HE staining,the apoptosis of cardiomyocytes was detected by TUNEL staining,and the expression levels of Nrf2 and HO-1 protein in myocardial tissue were detected by Western blot.The positive expression of HO-1 protein in myocardial tissue was detected by immunohistochemistry(IHC),and the levels of Nrf2 and HO-1mRNA in myocardial tissue were detected by RTqPCR technique.Results:1.Aloin was not significantly toxic to cardiomyocytes.Aloin has no obvious toxicity to cardiomyocytes.In the cardiomyocyte SI/RI model,50 μM aloin could significantly improve the cell survival rate,and 50 μM aloin was selected as the best concentration.2.In the cardiomyocyte SI/RI model,aloin pretreatment increased the viability of cardiomyocytes in a dose-dependent manner and silencing Nrf2 inhibited the effect of aloin on cardiomyocytes viability.3.In the cardiomyocyte SI/RI model,aloin pretreatment could reduce apoptosis,decrease the expression of Bax protein and increase the expression of Bcl-2 protein.Silencing Nrf2 inhibited the anti-apoptotic effect of aloin.4.In the cardiomyocyte SI/RI model,aloin pretreatment increased SOD activity,and significantly decreased ROS production,LDH release,and MDA content.Silencing Nrf2 significantly inhibited the effect of aloin on antioxidant stress.5.In the cardiomyocyte SI/RI model,aloin pretreatment significantly decreased the contents of TNF-α,IL-6,IL-1β,and mRNA,and inhibited the anti-inflammatory effect of aloin after silencing Nrf2.6.Aloin preconditioning significantly upregulated the expression of Nrf2 and HO-1 in the cardiomyocyte SI/RI model.7.In the rat MI/RI model,the ST potential difference increased,and the myocardial infarction area measured by TTC and Evans Blue double staining increased significantly.The contents of CK-MB and LDH in serum increased,which indicated that the rat MI/RI model was successfully constructed.Aloin pretreatment can reduce the area of myocardial infarction,increase the area of myocardial protection,and reduce the content of serum CKMB and LDH.ML385 treatment inhibited the myocardial protective effect of aloin in rat MI/RI model.8.In the rat MI/RI model,aloin pretreatment could increase myocardial SOD activity,reduce ROS production and reduce MDA content.Inhibitory effect of ML385 on antioxidative stress of aloin in rat MI/RI model.9.In the rat MI/RI model,TUNEL staining showed that the apoptosis rate was significantly increased,and aloin could reduce the apoptosis rate.ML385 inhibited the antiapoptotic effect of aloin in rat MI/RI model.10.In the rat MI/RI model,aloin pretreatment could up-regulate the expression of Nrf2 and HO-1,while ML385 could inhibit the up-regulation of Nrf2 and HO-1 expression by aloin.Conclusions:1.Aloin ameliorates myocardial ischemia reperfusion injury by inhibiting apoptosis,resisting oxidative stress and attenuating inflammatory response.2.Aloin ameliorates myocardial ischemia reperfusion injury by activating the Nrf2/HO1 signaling pathway to inhibit apoptosis,resist oxidative stress,and attenuate the inflammatory response.