Curculigoside Attenuates Myocardial Ischemia-reperfusion Injury By Inhibiting The Opening Of The Mitochondrial Permeability Transition Pore

Objective:The best therapeutic intervention on AMI is the timely revascularization to restore blood flow.Although early reperfusion is effective in decreasing the mortality,recovering blood flow into previously ischemic myocardium tissue causes second wave of injuries,which is referred to as myocardial ischemia reperfusion injury?MIRI?.Mitochondrial permeability transition pore?MPTP?,which is located in the inner membrane,is the critical effectorin the pathway of cell apoptosis.After ischemia,reperfusion may cause reactive oxygen species?ROS?generation,Ca²⁺inflx et al,which may prompt the open of MPTP.The opening of MPTP,which is known as mitochondrial permeability transition and decrease of inner mitochondrial membrane potential???m?,is the key event of the mitochondrial apoptosis pathway.Therefore,MPTP is regard as a crucial target for inhibiting MIRI.Curculigoside extracted from Curculigoorchioides Gaertn is a phenolicglycoside which have various kinds of biological activities such as antioxidant,anti-inflammatory and anti-tumor effects.But whether curculigoside has cardioprotective effect on I/R injury remains unclear.Therefore in this study,our aim was to test the cardio protection provided by curculigoside against I/R or H/R injury through experiments in vivo and in vitro and further investigate whether the underlying mechanism was the inhibition of MPTP opening.The results of this research reveal that pretreatment with curculigoside may decreases myocardium or H9c2 cell apoptosisafter I/R or H/R injury by inhibiting the open of MPTP.Method:1.The medium was exchanged every 2–3 days.The cells were exposed to experimental procedures?received experimental treatment?when the cell confluence level reached 80–90%.After pretreatment with curculigoside at the concentration of 5,10 and15?mol/Lor the same volume of 1‰DMSO separately for 8h,the culture medium of all groups was replaced with glucose-free and FBS-free Earle's medium except control group,and then cells were incubated in a tri-gaschamberwith 95%?v/v?N₂,5%?v/v?CO₂and 5%O₂ at 37?C for 12 h to induce hypoxia injury.After hypoxia,Earle's medium was replaced with normal medium and the cells were subsequent placed into a incubator with5%CO2 at 37?C for 1 h to re-oxygenate the samples.The concentration of 10?mol/L was received above treatment again for investigating the effective mechanism of curculigoside based on the results of the experiment.2.The rats were kept in good hygienic and well-ventilated condition with temperature of22±2°C,relative humidity of 60±5%and a cycle of 12-h light/dark.All rats were ensured to adapting the environment before the experiments and fed with standard food and water.Prior to hearts isolation,the curculigoside-treated groups received treatment of curculigoside at the dose of 5mg/kg?10mg/kg and 15 mg/kg once/day by intraperitoneal injection for 7 days and the solvent group received the same volume of 1‰DMSO without curculigoside.Following anesthetization induced by intraperitoneal injection at a dose of 30 mg/kg using pentobarbital sodium,the rat hearts were taken out quickly and the aortas were used to connect the Langendorff system.After stability reached by perfusing for 15min,global ischemia was induced by stopping perfusion for 30?minutes followed reperfusion for 60 min to mimic MIRI.According to the results of the experiment,the dose of 10 mg/kg was choosed to repeat above treatment to further explore the mechanism of action.3.The cell viability determination of H9c2 was performed by CCK-8 assay with the Cell Counting Kit-8.4.The activity of lactate dehydrogenase?LDH?was determined to evaluate the cytoprotective effects of curculigoside.5.The survival rate of H9c2 was determined by typan blue staining.6.The apoptosis rate of H9c2 cells was evaluated using the Annexin V-Fluorescein isothiocyanate?FITC?/Propidium Iodide?PI?kit.7.The change of??m was measured by Rhodamine 123staining.8.The total RNA was isolated from H9c2 cells.Real time PCR was used to evaluate mRNA expression level of factors from the downstream of mitochondrial apoptotic pathway.9.Protein was extracted from H9c2 cells.Western blot was used to evaluate protein expression level of factors from the downstream of mitochondrial apoptotic pathway.10.The measurement of mPTP opening was determined with Ca²⁺-induced method.11.The total RNA was isolated from H9c2 cells.Real time PCR was used to evaluate mRNA expression level of factors related to MPTP opening.12.Protein was extracted from H9c2 cells.Western blot was used to evaluate protein expression level of factors related to MPTP opening.13.Through connecting the Langendorff system,heart rates and coronary flow were measured to evaluated heart function of Wistar rats.14.Myocardial morphologic characteristic was determined by hematoxylin-eosin taining.15.TTC staining was performed to quantify the infarct size of myocardial tissue.16.TUNEL staining was carried out to assess the myocardium apoptosis activity by the In Situ Cell Death Detection kit.17.The total RNA was isolated from myocardial tissue.Real time PCR was performed to assess mRNA expression level of factors from the downstream of mitochondrial apoptotic pathway.18.Protein was extracted from myocardial tissue.Western blot was used to assess protein expression level of factors from the downstream of mitochondrial apoptotic pathway.19.The total RNA was isolated from myocardial tissue.Real time PCR was performed to assess mRNA expression level of factors related to MPTP opening.20.Protein was extracted from myocardial tissue.Western blot was used to assess protein expression level of factors related to MPTP opening.21.Statistical anslysisi:The experimental results were presented as mean values±standard deviation.One-way analysis of variance was firstly used to compare among?three groups and the least significant difference test was used to conduct multiple comparison analysis.Statistically signifiant differences were accepted at P-value of<0.05.All data were statistically analyzed with SPSS version 20.0.Results:1.After H/R injury,the cell viabilityof H9c2 cells was significantly decreased whereas curculigoside pretreatment restored the cell viability better than H/R group and vehicle group.The protective effects of Cur 10?M group and Cur 15?M group were better than Cur 5?M group.LDH assay indicated that LDH activity in the H/R group was markly higher than those in the control group,and this was significantly ameliorated by curculigoside treatment.LDH activity in Cur 10?M group and Cur 15?M group was lower compared to Cur 5?M group.After H/R injury,the cell survival rate of H9c2 cells was significantly decreased whereas curculigoside pretreatment significantly increased cell survival rate.The protective effects of Cur 10?M group and Cur 15?M group were better than Cur 5?M group.The experimental results show that the above three concentrations of curculigoside have different degrees of protective effect on H9c2cardiomyocytes,and the protective effect of 10?M and 15?M is equivalent,and both are stronger than that of 5?M,so 10?M concentration is selected as the next mechanism research.2.The pretreatment with curculigoside increased the heart rate and coronary flow significantly compared with the I/R group,and heart rate and coronary flow was more at the dose of 10mg/kg and 15mg/kg than 5mg/kg.The pretreatment with curculigoside decreased the infarct size significantly compared with the I/R group,and infarct size was fewer at the dose of 10 mg/kg and 15mg/kg than 5mg/kg.The pathologic changes,including inflammatory cell infiltrate,myocardial cells edema and et al,were fewer in the curculigoside groups and of all the curculigoside groups the dose of10mg/kg and 15mg/kg caused the fewest changes.The experimental results show that the above three doses of curculigoside have different degrees of protective effects on the myocardium of Wistar rats,and the protective effects of 10mg/kg and 15mg/kg are the same,and both of them are stronger than 5mg/kg.Therefore,10mg/kg of curculigoside is selected as the next step of mechanism research.3.Flow cytometric detection indicated that exposure of H9c2 cells to H/R injury significantly increased cellular apoptosis compared to the untreated cells in the control group.Pretreatment with curculigoside significantly inhibited H9c2 cell apoptosis after H/Rcompared with the H/R group,and this change caused by curculigoside can be significantly reversed by the use of ATR.The curculigoside pretreatment effectively prevented the decrease of??m and preserved high??m,and the H/R groups showed the loss of??m and low??m,and this change caused by curculigoside can be significantly reversed by the use of ATR.4.Curculigoside pretreatment significantly attenuated the increased mRNA expression of cytochrome C?APAF-1?caspase 9 and caspase 3 in vitro,and this change caused by curculigoside can be significantly reversed by the use of ATR.Pretreatment with curculigoside before H/R injury signifiantly attenuated the increased protein expression of cytochrome C?APAF-1?cleaved-caspase 9 and cleaved-caspase 3 compared to the H/R group,and this change caused by curculigoside can be significantly reversed by the use of ATR.5.The results revealed that pretreatment with curculigoside significantly decreased the sensitivity of MPTP opening to Ca²⁺compared to the I/R group,indicating that curculigoside inhibited MPTP opening after H/R injury.The value of min/max A540 in the curculigoside group was higher than in the H/R group.The above changes caused by curculigoside can be significantly reversed by the use of ATR.6.H/R injury significantly up-regulated the mRNA expression of Bax,down-regulated the mRNA expression of Bcl-2 and decreased the ratio of Bcl-2/Bax compared to the control group.Curculigoside treatment significantly attenuated the changes of the mRNA expression and increased the ratio of Bcl-2/Bax compared to the H/R group in vitro,and these changes caused by curculigoside can be significantly reversed by the use of ATR.Pretreatment with curculigoside significantly attenuated the increased protein expression of Bax,the decreased protein expression of Bcl-2 and the decreased the ratio of Bcl-2/Bax in vitro,and these changes caused by curculigoside can be significantly reversed by the use of ATR.7.The results of TUNEL assay revealed that myocardial apoptosis after I/R injury was significantly ameliorated following pretreatment with curculigoside.This change caused by curculigoside can be significantly reversed by the use of ATR.8.Curculigoside pretreatment significantly attenuated the increased mRNA expression of cytochrome C?APAF-1?caspase 9 and caspase 3 in vivo,and this change caused by curculigoside can be significantly reversed by the use of ATR.Pretreatment with curculigoside before I/R injury signifiantly attenuated the increased protein expression of cytochrome C?APAF-1?cleaved-caspase 9 and cleaved-caspase 3 compared to the I/R group,and this change can be significantly reversed by the use of ATR.9.I/R injury significantly up-regulated the mRNA expression of Bax,down-regulated the mRNA expression of Bcl-2 and decreased the ratio of Bcl-2/Bax compared to the control group.Curculigoside treatment significantly attenuated the changes of the mRNA expression and increased the ratio of Bcl-2/Bax compared to the I/R group in vivo,and these changes caused by curculigoside can be significantly reversed by the use of ATR.Pretreatment with curculigoside significantly attenuated the increased protein expression of Bax,the decreased protein expression of Bcl-2 and the decreased the ratio of Bcl-2/Bax compared to the I/R group in vivo,and these changes caused by curculigoside can be significantly reversed by the use of ATR.Conclusion:1.We demonstrated that curculigoside protected against H/R or I/R injury.2.The protection may be attributable to the alleviation of mitochondrial apoptosis by Bcl-2 expressed upward,Bax expressed downward and inhibiting MPTP opening.