| Objective: Acute myocardial infarction(AMI)is one of the major fatal and disabling diseases in the world.Myocardial injury is related to the duration of ischemia.Therefore,the treatment principles of AMI are to minimize the duration of ischemia and timely restore blood flow in the ischemic area.However,clinically,Myocardial edema,cell membrane rupture,mitochondrial swelling,excessive contraction of myocardium and other secondary myocardial injuries were observed during reperfusion,a p Henomenon known as Myocardial ischemia reperfusion injury(MIRI).MIRI can cause severe myocardial injury and dysfunction,and how to reduce myocardial ischemia-reperfusion injury is a great clinical challenge.The mechanism of MIRI is complex,among which oxidative stress is considered as one of its core mechanisms.A large number of experimental studies have shown that oxidative stress is closely related to the dysfunction of mitochondrial mass and inhibits the activation of oxidative stress,to improve the quality and function of mitochondria is a therapeutic target for MIRI.Mitochondrial quality control is a dynamic and complex process,which maintains the stability of mitochondrial structure and function through mitochondrial biosynthesis,mitochondrial dynamics and mitochondrial autop Hagy.Among them,mitochondrial dynamics includes mitochondrial fusion and division.When stimulated by ischemia and hypoxia injury,the structure and function of the mitochondria can be restored through certain degree of fusion in the case of mild injury,while the damaged mitochondria can be separated through division and cleared through autop Hagy in the case of severe injury,so as to ensure mitochondrial homeostasis.Maintain normal structural and functional integrity.Siraitia grosvenorii is a perennial vine plant in the cucurbitaceae family.Mogroside V(MV)is the main active component isolated from the fruit of the cucurbitane triterpenoid saponins.In recent years,several p Harmacological activities of MV have been reported,such as anti-inflammatory,lowering blood sugar,lowering blood lipid,enhancing immunity,anti-aging and anti-carcinogenic effects.In addition,several studies have reported that MV is a powerful antioxidant with significant antioxidant,free radical scavenging abilities.MV has been reported to reduce oxidative stress in the liver of obese mice induced by a high fat diet.In RAW264.7 cells,MV decreased the level of reactive oxygen species(ROS)induced by LPS and alleviated oxidative stress damage.These studies have all confirmed that MV can inhibit oxidative stress and protect mitochondria,but the specific mechanism involved has not been further explored,and the role of MV in MIRI is still unclear.In this study,the protective effect of MV on MIRI was observed by constructing H9c2 cell oxygen-glucose deprivation reperfusion model and Wistar rat heart reperfusion model in vitro,and its possible molecular mechanism was revealed to provide theoretical support for further use of MV in clinical prevention and treatment of MIRI.Research Methods: 1.To clarify the protective effect of MV on MIRI(1)To construct an OGD/R model of cardiomyocytes: H9c2 cells were cultured at37℃ with DMEM high glucose complete medium and incubated at 5%C02 until the density reached 80-90%.H9c2 cells were replaced with MEM sugar-free(containing NEAA)base medium and transferred to a three-gas incubator(94% N2,5%CO2and %1O2;37℃)for 6 hours,resulting in cell hypoxia.During reperfusion,the cells were continued to be cultured with DMEM high-sugar complete medium for 3 h in a5%C02 incubator at 37℃.(2)To observe the protective effect of MV treatment on OGD/R cardiomyocytes: 12 hours before oxygen and glucose deprivation,gradient concentration MV(0,10,15,20,25,and 30 μM)was administered.Cell viability was detected by CCK-8 method,and myocardial enzyme(LDH and CK-MB)release was detected by colorimetry.(3)I/R animal model was constructed: healthy male Wistar rats were weighed and then anesthetized by intraperitoneal injection of isopentobarbital(50mg/kg).The completely anesthetized Wistar rats were fixed on the killing table,the heart was separated after thorax was opened with surgical scissors,the blood vessels were cut quickly,the heart was taken out and placed in the pre-cooled heparinized KH solution,the heart was gently squeezed to squeeze out the residual blood congestion in the coronary artery,and the excess fat and connective tissue around the heart and coronary artery were pruned with scissors,so that the aorta was fully exposed.The trimmed rat heart was gently lifted with tweezers,and the aorta was inserted into the perfusion tube in the Langendorff device.After the heart beat was observed to be stable,the connection between the aorta and the perfusion tube was fixed with surgical line,and KH electrolytic solution was injected at 37℃ and 75 mm Hg at constant pressure.About 15 minutes later,cardiac perfusion reached a steady state,and perfusion was completely stopped for 30 minutes,and then re-perfusion was performed for 90 minutes to induce cardiac ischemia-reperfusion injury models in isolated rats.(4)To observe the effect of different concentrations of MV on MIRI in rats: Wistar rats were randomly divided into 6 groups with 3 rats in each group.The storage solution of momorrhodoside V was diluted to the required concentration in each group with1 ml PBS solution,and the rats were injected intraperitoneally once a day for 1 week before cardiac withdrawal.Cardiac function(heart rate,left ventricular diastolic pressure LVDP and±dp/dt)was dynamically recorded by electrop Hysiological recorder,myocardial infarction area was measured by TTC staining,myocardial enzyme release was measured by colorimetry,myocardial cell apoptosis was measured by TUNEL staining,and myocardial ultrastructural damage was observed by transmission electron microscopy.2.To explore the regulatory effect and mechanism of MV on mitochondrial quality control in H9c2 cells after OGD/R injury(1)To explore the effect of MV treatment on mitochondrial energy metabolism function: Mitochondrial oxygen consumption was determined by oxygen electrode method,and the activity of mitochondrial respiratory chain complex I,II,III,IV,V and ATP levels were determined by colorimetry.(2)To investigate the influence of MV treatment on mitochondrial membrane structure: JC-1 fluorescent probe was used to detect changes in mitochondrial membrane potential,and calcium ion induction was used to detect MPTP opening.(3)To explore the effect of MV in the treatment of oxidative stress injury: ROS fluorescence probe-DHE was used to detect cell reactive oxygen species,and Mitosox staining was used to detect mitochondrial reactive oxygen species,so as to clarify the effect of MV on oxidative stress damage.(4)To investigate the effects of MV treatment on mitochondrial biosynthesis,mitochondrial fusion,division and mitochondrial autop Hagy flow: mt DNA was determined to clarify mitochondrial biosynthesis,and mitochondrial specific localization fluorescent probe(HBAD Mito dsred)combined with double fluorescent LC3 cell autop Hagy adenovirus was used to accurately and real-time track the dynamic process of mitochondrial autop Hagy.Mitochondrial fusion and division were detected by immunofluorescence TOM20.Western blot was used to detect the expression changes of autop Hagy signature proteins LC3,Parkin,PINK1,MFN1,MFN2 and DRP1,and to clarify the effect of MV on mitochondrial autop Hagy.(5)By using Label-free quantitative proteomics and bioinformatics,the downstream signaling pathway of MV improving mitochondrial damage was explored,and the protective effect of MV on ischemia-reperfusion injury was verified by the recovery experiment.Results: 1.The cell viability was determined by CCK8 method,the myocardial enzyme(CK-MB,LDH)levels were determined by colorimetry,and the protective effect of MV on OGD/R of H9c2 myocardial cells and the optimal intervention concentration(25μm)were determined.2.MV preconditioning can improve the cardiac function of ischemia reperfusion rats,reduce the myocardial infarction area of rats,reduce the release of myocardial CK-MB and LDH,inhibit myocardial apoptosis,reduce mitochondrial damage,and maintain the stability of mitochondrial structure,and the MV-50mg/kg group has the most significant therapeutic effect.3.MV can improve the energy metabolism of mitochondria damaged by OGD/R in H9c2 cells,inhibit the loss of mitochondrial membrane potential,inhibit the opening of mitochondrial permeability conversion pore,inhibit the generation of ROS in cardiomyocytes and mitochondria,promote mitochondrial biosynthesis,promote mitochondrial autop Hagy and mitochondrial fusion,inhibit mitochondrial division,and maintain the stability of mitochondrial structure and function.4.MV treatment promoted the increase of Sirt1 expression.After transfection of Sir T1-specific small interfering RNA into H9c2 cells and intraperitoneal injection of EX-527 S-enantiomer(Sirt1 inhibitor(EX-527))into Wistar rats,the effect of MV on improving myocardial ischemia reperfusion injury was found to be weakened.Conclusion: 1.Mogroside V can alleviate myocardial ischemia-reperfusion injury.2.MV can ameliorate the dysfunction of mitochondria induced by myocardial ischemia-reperfusion injury.3.MV can maintain the stability of mitochondrial structure and function by promoting mitochondrial biosynthesis,inhibiting mitosis,and promoting fusion and autop Hagy.4.MV attenuates myocardial ischemia-reperfusion injury by up-regulating SIRT1 and regulating mitochondrial quality control... |
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