APG And Thymoquinone Against Myocardial Ischemia Reperfusion Injury

Background:Ischemic heart disease is a major disease that threatens the health of human beings.Myocardial ischemia reperfusion?IR?injury is not only an important damage type,but also an important reason of postoperative myocardial infarction.Though there exists some medicines which have the cardiovascular protective effect,but they also possess severe adverse reactions.It is urgent to find new medicines and therapeutics for myocardial IR injury.Few side effects were found in traditional Chinese medicine,especially the contributing compounds isolated from them which have low toxicity and high efficiency advantages.APG?apigenin-7-O-?-D-?-6''-p-coumaroyl??is the active ingredient of a traditional Chinese medicine Clematis tangutica.The extracts of this plant is the main component of Yixin Kangtai capsule which is used for the treatment of cardiovascular disease.Previous studies have shown that APG plays a significant role in protecting ischemic brain damage,but whether it has myocardial ischemia reperfusion protective effect remains unknown.If yes,the mechanism is also unclear.Protein kinase c?PKC?is a serine/threonine kinase with a single peptide chain structure and is one of the molecules of multiple signal transductions in myocardial ischemiareperfusion injury.PKC could translocation to mitochondria or initiate the nucleus signal transduction directly or indirectly performance the myocardial protection effect.Studies have shown that PKC?,a subtype of PKC,plays an important role in myocardial ischemic preconditioning against IR injury.Thus,we hypothesized that APG might play a cardio-protective role by activating the PKC? pathway.Nuclear factor-erythroid 2 p45-related factor 2?Nrf2?is a key transcription factor in oxidative stress.In recent years,Nrf2 has been found to protect cardiomyocytes from oxidative stress-induced cell death.Hemeoxygenase-1 is a kind of inducible enzymes,studies have shown that increase the expression of HO-1 in tissues can enhance its antioxidant capacity,and reduce cerebral ischemia reperfusion injury.Some studies have found that several drugs protect brain against cerebral ischemia injury via activation of Nrf2/HO-1 pathway.Whether APG plays a cardio-protective role by activating the PKC? pathway is not clear,and whether the protective act is relate to Nrf2/HO-1 pathway is also unknown.Thymoquinone?TQ?is the main active ingredient from the seed of Nigella sativa Linn,which was traditionally used in the Middle East,the South Asia.Previous studies have shown that thymoquinone has anti-hypertensive,anti-inflammatory,anti-oxidation,anti-tumor and other biological activity.It also could protect liver and kidney from ischemia reperfusion injury,but there is few researches on thymoquinone protect cardiomyocytes from ischemia reperfusion injury.SIRT1 is a histone deacetylase?HDAC?,which can participate in the physiological and biochemical processes such as apoptosis and senescence under stress.In recent years,studies have found that SIRT1 plays an important regulatory role in cardiovascular disease.P53 is a non-histone-acting substrate for SIRT1.Acetylated P53 promotes oxidative stress-induced apoptosis,whereas SIRT1 deacetylates acetylated P53 to alter the cell apoptosis process.We speculate that thymoquinone may regulate the SIRT1 pathway to play a role in myocardial ischemia protection.In conclusion,we suggest that APG and thymoquinone may have a protective effect on myocardial IR injury.In this study,we will clarify the specific mechanism of APG and thymoquinone on myocardial ischemiareperfusion injury by establishing a series of methods such as myocardial ischemia reperfusion model.The aim of this study was to provide a theoretical basis for the development of APG and thymoquinone as new therapeutics for myocardial ischemia reperfusion injury,and to provide theoretical basis for clinical use.Objective:1.To clarify whether APG has anti-myocardial ischemia-reperfusion injury function and its possible protective mechanism.2.To figure out whether thymoquinone has anti-myocardial ischemia-reperfusion injury function and its possible protective mechanism.Methods: Part ?: Study of APG against myocardial IR injury and PKC? signaling pathway in APG myocardial protectionTo observe the effect of different concentration of APG on IR cardiomyocytes and clarify whether APG has protective effect against myocardial IR injury.At the same time,find out the effect of APG on PKC? related molecules in this process.The effect of PKC? siRNA or ?-V1-2 on cardiomyocytes and in vivo cardiac-specific blocking of PKC? pathway was observed,to explore whether APG exerts its cardio-protection through the regulation of PKC? pathway.1.To observe the effect of APG on the IR of H9C2 cardiomyocytes and the effect on PKC? molecules.The experiment was divided into four groups:?1?Control;?2?IR;?3?IR+APG 2?mol/L;?4?IR+APG 4?mol/L.APG pretreatment time was 24 h,followed by simulated ischemia for 45 min,reperfusion for 3 h,and then the cell viability was measured.The release of lactate dehydrogenase?LDH?,malondialdehyde?MDA?and the intracellular Superoxide dismutase?SOD?activity were detected.The expression of PKC?,Caspase-3,Bax and Bcl-2 was detected by Western blot.2.To observe the effect of different concentrations of APG on the IR of primary rat cardiomyocytes and the effect on PKC? signaling pathway.The experiment was divided into 5 groups:?1?Control;?2?IR;?3?IR+APG 2?mol/L;?4?IR+APG 4?mol/L;?5?IR+APG 6?mol/L.Similarly,APG incubated cells for 24 h,simulated ischemia 45 min,reperfusion 3h.The cell viability,LDH release,cell apoptosis rate,and parameters of mitochondrial oxidative stress were measured.Western blot was used to detect the expression of PKC? and related proteins.3.To find out the effect of PKC? siRNA blocking PKC? pathway on anti-myocardial IR injury effect of APG.The experiment was divided into four groups:?1?IR;?2?IR+PKC? siRNA;?3?IR+APG;?4?IR+APG+PKC? siRNA.The changes of mitochondrial oxidative stress,apoptotic pathway molecules and Nrf2/HO-1 pathway were observed.4.To observe the effect of different concentrations of APG in C57BL/6 mice and the effect on PKC? signaling pathway.The experiment was divided into 5 groups:?1?Control;?2?IR;?3?IR+1mg/kgAPG;?4?IR+5mg/kg APG;?5?IR+10mg/kg APG.Male C57BL/6 mice were anesthetized with 2.5% isoflurane.The heart was exited by left thoracic incision,and the left anterior descending coronary artery was ligated for 30 min by suture.At 10 min before reperfusion,mice were injected intraperitoneally with APG?1,5 or 10 mg/kg?for 3 days.The cardiac function of mice was evaluated after the treatment of each group,and the mitochondrial oxidative stress?SOD,H₂O₂,MDA,SDH,COX?were measured.Western blot was used to detect the expression of PKC? and related proteins.5.To observe the effect of PKC? pathway-specific blocker ?-V1-2 on the IR effect of APG in vivo.The experiment is divided into four groups:?1?IR;?2?IR+?-V1-2;?3?IR+APG;?4?IR+APG+?-V1-2.The cardiac function of mice was evaluated after the treatment of each group,and the mitochondrial oxidative stress was measured.The changes of apoptotic pathway molecules and Nrf2/HO-1 pathway were detected by western blot.Part ?: To investigate thymoquinone against myocardial IR injury and whether SIRT1 signaling pathway is involved in cardio-protective effects1.To observe the effects of TQ on normal isolated heart perfusion.After the heart perfusion was stable,the experiment was divided into 4 groups:?1?Control;?2?TQ 2.5?mol/L;?3?TQ 5?mol/L;?4?TQ10?mol/L.KHB perfused for 120 min,the hemodynamic parameters,the LDH release,the cardiac infarct,and the expression of SIRT1 were detected.2.To observe the effect of TQ on isolated hearts.The experiment was divided into 5 groups:?1?Control;?2?IR;?3?TQ 2.5?mol/L+IR;?4?TQ 5?mol/L+IR;?5?TQ 10?mol/L+IR.The isolated hearts were perfused with KHB containing TQ for 5 min and stop perfusion for 45 min,followed byreperfusion for 60 min.The hemodynamic parameters were recorded and the LDH release was measured.The changes of SIRT1 and apoptotic proteins were detected by Western blot.3.To observe the effect of sirtinol and TQ on IR isolated heart.After the heart perfusion was stable,the experiment was divided into 4 groups:?1?IR;?2?sirtinol+IR;?3?TQ+IR;?4?TQ+sirtinol+IR.The isolated hearts were perfused with KHB containing TQ?10?mol/L?and sirtinol?3.75?mol/L?for 5 min and stop perfusion for 45 min,followed reperfusion for 60 min.The hemodynamic parameters were recorded and the LDH release was measured.The changes of SIRT1 and apoptotic proteins were detected by Western blot.4.To observe the effect of TQ on IR in primary rat cardiomyocytes.The experiment was divided into 5 groups:?1?Control;?2?IR;?3?TQ 1?mol/L+IR;?4?TQ 2?mol/L+IR;?5?TQ 4?mol/L+IR.The cells were incubated for 24 h,simulated ischemia for 45 min,and then incubated for 3 h.The cell viability,the release of LDH and cell apoptosis were measured.Western blot was used to detect the expression of SIRT1 and related proteins.5.To observe the effect of SIRT1 siRNA blocking SIRT1 pathway on anti-myocardial IR injury effect of TQ.The experiment was divided into four groups:?1?IR;?2?IR+SIRT1 siRNA;?3?IR+TQ;?4?IR+TQ+SIRT1 siRNA.The cell viability and the changes of mitochondrial oxidative stress were observed.SIRT1 and apoptotic pathway proteins were detected.Results: Part ?: Study of APG against myocardial IR injury and PKC? signaling pathway in APG myocardial protection1.This part firstly explored whether APG has a protective effecton IR injury,while observing the process of PKC? pathway-related molecular changes.H9C2 cells and primary cells have found that APG?2,4,6?mol/L?has a definite protective effect on cells,which shows that APG treatment significantly improves cell viability,improves cell morphology and reduces LDH release in culture medium?vs.IR group,P<0.01?.APG could also increase the activity of SOD,the levels of SDH and COX,and significantly decrease the levels of H₂O₂ and MDA?vs.IR group,P<0.01?.Western blot analysis showed that APG could significantly increase the expression of Bcl2,decrease the expression of Bax and Cleaved Caspase3,decrease the expression of cytoplasmic PKC? protein and increase the expression of mitochondrial PKC? protein?vs.IR group,P<0.01?.The PKC? siRNA was used to weakened the protective effect of APG?vs.APG+IR group,P<0.01?.2.In vivo cardiac studies have yielded consistent results.APG?1,5,10mg/kg?treatment on the heart of the IR has a clear protective effect.APG treatment improved heart function and increased SOD,SDH,COX levels,significantly reduced H₂O₂ and MDA levels?vs.IR group,P<0.01?.Western blot analysis showed that APG could significantly increase the expression of Bcl2,decrease the expression of Bax and Cleaved Caspase3,decrease the expression of cytoplasmic PKC? protein and increase the expression of mitochondrial PKC? protein?vs.IR group,P<0.01?.Further treatment with ?-V1-2 found that ?-V1-2 weakened the protective effect of APG?vs.APG+IR group,P<0.01?.Part ?: To investigate thymoquinone against myocardial IR injury and whether SIRT1 signaling pathway is involved in cardio-protective effects1.Firstly,this part explores the effect of thymoquinone on isolated heart.We found that different concentrations of thymoquinone?2.5,5,10?mol/L?on the IR isolated heart has a significant protective effect.TQ significantly reduced myocardial infarct size and the release of LDH in coronary effluent,increased LVDP?vs.IR group,P<0.01?.The levels of SOD,SDH and COX in mitochondria of IR rats were significantly lower than those in TQ group?P<0.01?.Western blot analysis showed that TQ could significantly increase the expression of SIRT1,Bcl2 and decrease the expression of Bax and Caspase3?vs.IR group,P<0.01?.After treatment with sirtinol,the myocardial protective effect of TQ was weakened?vs.TQ+IR group,P<0.01?.2.The results of myocardium study were consistent.TQ treatment could improve the morphology of damaged cardiomyocytes,the cell viability and decrease the apoptotic rate?vs.IR group,P<0.01?.TQ can also increase the SOD activity,the levels of SDH and COX,increase the expression of Bcl2 and decrease the expression of Bax and Caspase3?vs.IR group,P<0.01?.At the same time,SIRT1 siRNA was found to weakened the protective effect of TQ?vs.TQ + IR group,P <0.01?.Conclusions:1.APG has a clear protective effect that fight against myocardial IR injury.Meanwhile,TQ alleviates the IRinduced mitochondrial oxidative stress injury and apoptosis through the activation of PKC? pathway or activation of Nrf2/HO-1 pathway,and further plays a role in myocardial protection.2.TQ has a definite effect that fight against IR injury and through the SIRT1 pathway to play a role in myocardial protection.