The Material Basis And Mechanism Of The Protective Effect Of Suxiao Jiuxin Pill On Cardiomyocyte Injury Induced By Glucose Deprivation

Ischemic heart disease is a major disease that seriously affects the quality of life and lives of people,which making up more than 40%deaths of our country.The process of the disease initially results from coronary atherosclerosis,narrowing of the lumen,leading to myocardial insufficiency,and even myocardial infarction.As an effective traditional recipe developed by national expert Professor Zhang Chengui,it firstly combines classical theory of traditional Chinese medicine with modern preparation technology.It features by rapid absorption,fast acting,favorable bioavailability,eutherapeutic effect and convenient to dose.For the reasons above it obtains reliable efficacy for clinical first aid and conventional treatment.However,the chemical material basis and mechanism of Suxiaojiuxin Pill is still not clear.Therefore,this subject will conduct further research on this region to provide more evidence for add-on development.The current research on anti-myocardial ischemia drugs focuses on the initial factors to evaluate the anti-ischemic effect such as dilating blood vessels,thrombolysis,inhibiting platelet aggregation,promoting angiogenesis,and improving blood flow.In the state of acute myocardial infarction caused by thrombosis and blood vessel blockage,the effect of improving blood flow is extremely limited before hospitalization which indicating that the mechanism of protecting cardiac myocytes plays an important role on rescuing from myocardial ischemia.Under ischemic conditions,oxygen supply is insufficient,following after mitochondrial dysfunction,mitochondrial energy metabolism dysfunction and mitochondrial apoptosis,furthermore induced apoptosis of myocardial cell.Therefore,this study will take Suxiaojiuxin Pill as a study subject,conducting a study on its material basis by in vitro cell model and validating its mitochondria protection by the drugs and two main active ingredients ligustilide and senkyunolide I.The mechanism of anti-ischemic myocardial ischemia was studied.The main research content is as follows:(1)UHPLC-QTOF was used to analyze the composition of Suxiaojiuxin Pill,and the absorbed components in blood were identified.HPLC-QQQ was used to quantify the concentration of ferulic acid,ligustilide in the recipe.In addition,determination of the compounds above in rat plasma after intragastric administration of 30 minutes was completed.Results:From the research,15 phthalide compounds were identified,of which 9 compounds were also detected in the blood.The concentration of ferulic acid,ligustilide,senkyunolide I and senkyunolide H were 17.63 ?g/pill(contents accordance with National Pharmacopeia standards),47.52 ?g/pill,35.07 ?g/pill,4.91 ?g/pill.After rats were intragastrically administered with 162 mg/mL Suxiao Jiuxin Pill for 4 mL,serum levels were measured after 30 min.The content of ligustilide,senkyunolide I and senkyunolide H were 64.19 ng/mL and 336.80 ng/mL and 131.65 ng/mL respectively.The above results indicate that ligustilide is the main component of the Suxiao Jiuxin Pill,which is prone to phase I metabolites giving priority to senkyunolide I.(2)Ligustilide and senkyunolide I were the main components enter in blood.Phase I metabolim of ligustilide can be mimic very well in vitro through the electrochemical reaction.By this method we can prepare the metabolites of ligustilide in vitro.Firstly,the phase I metabolism of ligustilide in vivo was simulated and analyzed by electrochemical reaction-mass spectrometry(EC-MS)technology.It was found that the ligustilide oxidizes to generate the senkyunolide I,senkyunolide H and epoxyligustilide,which is consistent with the metabolites in the body,furthermore,optimizing the reaction voltage,electrolyte,and flow rate to get the best reaction condition:ammonium acetate concentration:40 mM,electrolyte solution pH:7.4,flow rate:50 ?L/min,reaction voltage:1.6 V.So a type of prepare reaction cell was selected to prepare and purify the metabolites of 120 mg ligustilide.Finally we obtained the amount of senkyunolide I,senkyunolide H and epoxyligustilide was 5 mg,2 mg,6 mg respectively.Samples for the activity studies will be described later.(3)In addition,the myocardial ischemia environment in vivo is mainly simulated by building an oxygen and glucose deprived cardiomyocyte model.Therefore,in this study,mitochondrial morphology was further observed by transmission electron microscopy.The mitochondrial respiratory chain transmission process and glycolytic level were examined using a seahorse cell energy metabolism analysis system to evaluate the effect of Suxiaojiaoxin Pill and main components ligustilide and senkyunolide I.We clarify the effect of these compounds on how to improve mitochondrial structure and function after myocardial cell injury.The results showed that:? Through TEM observation of mitochondrial morphology,we found the mitochondrial swelling,sparsely populated,incomplete indole,and the structure is fuzzy.The mitochondria were significantly increased,and the swelling was significantly reduced.The results showed that Suxiaojiuxin Pill,ligustilide,and senkyunolide I protected the mitochondrial physiology of the cells under hypoxia conditions.?Through evaluation of mitochondrial function by seahorse cell energy metabolism system,it was found that after administration of Suxiaojiuxin Pill,ligustilide,and senkyunolide I,the mitochondrial basal respiration and mitochondrial maximum respiration capacity increased.Proton leaking level and the amount of generated ATP was increased.The protective effect of ligustilide was better than that of the Suxiaojiuxin Pill.?Through the examination of the glycolytic capacity,it was found that the glycolytic capacity of the cardiac muscle cells was strong after oxygen and glucose was deprived.The positive drugs trimetazidine and senkyunolide I both significantly improved the cell's glycolytic capacity,maximal glycolytic capacity,and maximum glycolytic reserve capacity.The Suxiaojiuxin Pill and ligustilide can effectively improve the glycolytic reserve capacity of cardiomyocytes,but has no significant effect on the maximum glycolysis capacity.?Through the determination of the mitochondria respiration control ratio and mitochondria complex ? and complex ? respiration activity.The results shows that Suxiaojiuxin Pill and senkyunolide I can siginificantly improve the efficiency of mitochondrial oxidative phosphorylation,besides have the protection effect on mitochondria complex ? and complex ?.Therefore,Suxiaojiuxin Pill,ligustilide,and senkyunolide I have a protective effect on mitochondrial damage and mitochondrial dysfunction caused by oxygen and glucose deprivation.(4)Based on previous studies,we found that Suxiaojiuxin Pill,ligustilide,and senkyunolide I can protect the integrity of mitochondrial structure,and mitochondrial function has also been significantly improved.Therefore,this study further explored the protective effect of Suxiaojiuxin Pill,ligustilide,and senkyunolide I on myocardial mitochondrial apoptosis induced by oxygen and glucose deprivation and its mechanism.In this study,we evaluated the effects of extracellular lactate dehydrogenase activity,mitochondrial content,intracellular Ca2+ concentration,mitochondrial membrane potential,reactive oxygen concentration,mitochondrial membrane MPTP channel openness,apoptosis rate,for the purpose of describe the improvement on mitochondrial apoptosis.The protective effect,as well as the detection of pro-apoptotic protein Bax,anti-apoptotic protein Bcl-2,mitochondrial MPTP channel protein VDAC1,mitochondrial and cytoplasmic cytochrome C levels,cysteine aspartate proteolytic enzyme Caspase-3,apoptosis-inducing factor(AIF)was used to elucidate the protective mechanism of Suxiaojiuxin Pill on mitochondrial apoptosis induced by oxygen and glucose deprivation.The results showed that:Suxiaojiuxin Pill can significantly inhibit the release of LDH,increase intracellular mitochondrial content,reduce mitochondrial Ca2+ concentration,maintain mitochondrial membrane potential stability,reduce mitochondrial ROS levels,inhibit mitochondrial apoptosis.In addition it also regulates Bax/Bcl-2 ratio to inhibit the opening of MPTP channels and the release of cytochrome C from mitochondria to the extracellular domain.Moreover,apoptosis was decrease by inhibiting the increase of caspase 3 expression,blocking the caspase pathway of apoptosis and down-regulating the expression of AIF.However,ligustilide and senkyunolide I can significantly inhibit the release of LDH,increase intracellular mitochondrial content,decrease mitochondrial Ca2+ concentration,maintain mitochondrial membrane potential,decrease mitochondrial ROS level and inhibit mitochondrial apoptosis.But it has no significant inhibitory effect on the opening of the MPTP channel,indicating that it does not inhibit mitochondrial apoptosis through the inhibition of MPTP channel opening and exerts cardio-protective effect.Conclusion:A total of 15 phthalide compounds were identified from the Suxiaojiuxin Pill.Among them,9 compounds were absorbed into the blood.The main components of the phthalide were ligustilide and senkyunolide I.Under the conditions of hypoxia conditions,Suxiaojiuxin Pill and its main components ligustilide and senkyunolide I have obvious protective effects on the structure and function of mitochondria,and both can inhibit mitochondrial apoptosis,and ligustilide has a better effect than that of Suxiaojiuxin Pill.In addition,Suxiaojiuxin Pill can inhibit the release of MPTP channel,reduce the release of cytochrome C,block caspase apoptosis pathway,thereby inhibiting cardiac myocyte apoptosis,and play a role in protecting myocardial ischemia.