| Objective:Acute myocardial infarction（AMI）,the most severe type of coronary artery disease,is one of major deadly diseases all over the world.In patients suffering ST-elevation myocardial infarction（STEMI）,minimizing ischemic time by urgent revascularization,either by percuta-neous coronary intervention（PCI）or thrombolytic therapy,has been a successful strategy to reduce mortality.However,restoration of flow kills some myocardial cells by a process known as reperfusion injury.Therefore,how to effectively prevent against myocardial IR injury has gained increased interest from researchers.Sweroside,a secoiridoid glucoside extracted from Swertia pseudochinensis Hara,is reported to possess antioxidant and anti-inflammatory activities.More interestingly,sweroside has been found to prevent myocardial cells against aconitine-induced cardiac toxicity.However,whether sweroside has a protective effect on myocardial ischemia-reperfusion（IR）injury is yet to be elucidated.Pyroptosis,a new form of programmed cell death,commonly starts with the formation of an inflammasome complex containing NLRP3,the adaptor proteins ASC and pro-caspase-1.Pro-caspase-1 is cleaved into its active form by the inflammasome complex,and then,on the one hand,active caspase-1 cleaves GSDMD to facilitate the formation of pore at the cell membrane,eventually leading to cell lysis.On the other hand,the precursors of IL-1βand IL-18 are cleaved by active caspase-1 to produce active IL-1βand IL-18.The inflammatory response is induced when active IL-1βand IL-18 are released out of cells.In the process of MIRI,reactive oxygen species（ROS）large accumulate in tissue and induce NLRP3inflammasome-mediated pyroptosis.Nuclear factor E2-associated factor 2（Nrf2）is a key transcription factor that serves an important role in the regulation of oxidative stress and inflammation.In a physiological state,Nrf2 mainly resides in the cytoplasm by binding to Kelch-like ECH-associated protein 1（Keap1）,which is a physiological inhibitor of Nrf2.However,under an oxidative stress condition,Nrf2 translocates into the nucleus by separating from Keap1,and subsequently activates the transcription of antioxidant genes,such as heme oxygenase（HO）-1,which protects cells against oxidative stress-and inflammation-induced injury.Recent studies have demonstrated that activation of Nrf2 can repress NLR family pyrin domain containing 3（NLRP3）inflammasome-mediated pyroptosis via modulation of ROS.Moreover,as predicted by Molecular Operating Environment（MOE）software,sweroside may interact with Keap1.Hence,we hypothesized that sweroside may regulated ischemia-reperfusion-induced pyroptosis via the Keap1/Nrf2/ROS axis.This study is aimed at investigating the cardioprotective effects of sweroside on ischemia reperfusion injury in rats and identifying its underlying mechanism.Methods:Rat myocardial cell line（H9c2）was pretreated with sweroside for 24h,and then underwent 6h-hypoxia,followed by 3h-reoxygenation.Cell Counting Kit-8assay was used to detect cell viability,creatine kinase-MB（CK-MB）and lactate dehydrogenase（LDH）assays were conducted to detect myocardial injury.ROS content was measured using 2,7-dichlorofuorescin diacetate（DCFH-DA）probe.The malondialdehyde（MDA）content,and the activities of superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）enzymes were measured using MDA,SOD and GSH-Px detection kits.Animal anesthesia was induced via an intraperitoneal injection of sodium pentobarbital（30 mg/kg）.The rat heart was isolated from the thoracic cavity,hung on a Langendorff perfusion device,and perfused with O2-saturated Krebs-Henseleit（K-H）solution at a constant pressure.Myocardial IR was induced by stopping K-H solution perfusion for a period of 30min,followed by reperfusion with K-H solution for 120 min.The fluid-filled latex balloon,which was connected to a pressure sensor,was inserted into the left ventricle to dynamically monitor the alteration of cardiac function using a homodynamic system.Triphenyltetrazolium chloride staining was performed to detect myocardial infarct size.Propidium iodide（PI）staining was used to detect cell membrane integrity.The activity of caspase-1 was detected using a caspase-1 activity assay kit.The concentration of IL-1βin the culture medium were measured using an enzyme linked immunosorbent assay（ELISA）with the Rat IL-1βELISA kit.Nuclear translocation of Nrf2 was detected using immunofluorescence staining.The protein expressions of NLRP3,ASC,IL-1β,cleaved caspase-1,HO-1 and Nrf2 were analyzed using Western blotting.Results:1.The results demonstrated that cell viability was remarkably enhanced（10,25,50and 100μM sweroside vs.HR:0.652±0.009,0.697±0.007,0.732±0.009,0.735±0.011 vs.0.597±0.016）and the release of CK-MB and LDH was significantly decreased by 10,25,50 and 100μM sweroside pretreatment.Moreover,the 25μM sweroside treatment group displayed higher cardioprotection compared with that in the 10μM sweroside treatment group,while the 50μM sweroside treatment group had an improved cardioprotection compared with 25μM sweroside treatment group.However,no additive protective effect was found in the 100μM sweroside treatment group compared with the 50μM sweroside treatment group.Therefore,these results suggested that sweroside pretreatment dose-dependently alleviated HR-induced myocardial injury in vitro,and 50μM was used for subsequent experiments.2.Myocardial infarct size in the sweroside treatment group（25,50 and 100 mg/kg）was significantly decreased compared with that in the IR group（25,50 and 100mg/kg sweroside vs.IR:28.90±2.35%,14.14±2.38%,15.51±2.78%vs.41.14±4.37%）.The control group showed stained brick red region,an indicator of viable tissue.Moreover,the infarct size in the 50 mg/kg sweroside treatment group was small compared with that in the 25 mg/kg sweroside treatment group.However,there was no significant difference in unstained region between the 50 mg/kg sweroside treatment group and the 100 mg/kg sweroside treatment group.In addition,the left ventricular developed pressure（LVDP）and positive/negative first-order derivative of ventricular pressure（±dp/dt）values in the sweroside treatment group（25,50 and 100 mg/kg）were significantly increased compared with those in the IR group,although there was no significant difference in the heart rate.Similar to the result of the infarct size,the LVDP and±dp/dt values in the 50 mg/kg sweroside treatment group were higher compared with those in the 25 mg/kg sweroside treatment group,but these showed no significant difference as compared with the 100 mg/kg sweroside treatment group.Taken together,these results suggested that sweroside decreased myocardial infarct size and ameliorated cardiac function in a dose-dependent manner and that 50 mg/kg sweroside treatment induced the best cardioprotection ex vivo.3.Sweroside pretreatment significantly attenuated the levels of ROS and MDA content that were induced by HR,while it enhanced the activities of SOD and GSH-Px that were repressed by HR.Collectively,these results suggested that sweroside could inhibit HR-induced oxidative stress.4.Sweroside pretreatment markedly prevented against the loss of cell membrane integrity induced by HR,and the percent of pyroptotic cells was reduced by sweroside pretreatment（sweroside vs.HR:14.76±2.58%vs.30.44±2.82%）.Moreover,the activities of caspase-1 and IL-1βin culture medium were significantly decreased by sweroside pretreatment.The expression levels of NLRP3,ASC,IL-1βand cleaved caspase-1 were also decreased by 55%,48%,65%and 49%by sweroside pretreatment,respectively.Altogether,these results indicated that sweroside inhibited HR-induced pyroptosis.5.As predicted by the molecular docking model,it was found that sweroside may interact with Keap1.Furthermore,it was identified that the expression level of Keap1 was significantly decreased by 51%after sweroside treatment.The results demonstrated that the expression level of Nrf2 in the nucleus was nearly enhanced by two fold,while the level of Nrf2 in cytoplasm was reduced by 50%,which supported the hypothesis that sweroside repressed Keap1 and promoted nuclear translocation of Nrf2.6.To determine the regulation of Nrf2 in the inhibition of sweroside on oxidative stress and pyroptosis,we examined whether inhibition of Nrf2 by transfection of si-Nrf2 could abrogate the effect of sweroside on oxidative stress and pyroptosis.It was demonstrated that the suppression of ROS by sweroside were completely rescued by inhibition of Nrf2.However,the repression of NLRP3,ASC,IL-1βand cleaved caspase-1 expression levels by sweroside were only rescued 49%,41%,38%,39%by inhibition of Nrf2,respectively.Taken together,these results suggested that the effect of sweroside on oxidative stress was Nrf2-dependent and its anti-pyroptotic effect was partially Nrf2-dependent.Conclusion:In conclusion,our study demonstrated that sweroside pretreatment could protect against myocardial IR injury by inhibiting of oxidative stress and NLRP3 inflammasome-mediated pyroptosis at least partially via modulation of the Keap1/Nrf2/ROS axis. |
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