The Effect And Mechanism Of GS Rb1 And IA On Ischemia And Hypoxia Myocardial Cells

Objective1.By establishing the model of ischemia and hypoxia in neonatal rat cardiomyocytes, to observe the changes of injury and apoptosis in the myocardial cells when evironment changes to ischemia and hypoxia model.2. To acquaintant the changes of myocardial cells energy metabolism under ischemia and hypoxia evironment, and observes the cure effect of ischemia and hypoxia myocardial cell of the mainly effective components of ginseng and Ilex pubescens-ginsenoside Rbl and Ilexonin A.3.preliminarily study the energy metabolism access of ginsenoside Rbl and Ilexonin A intervent ischemia and hypoxia myocardial cells, and provide a theoretical basis for the prevention and treatment of heart failure by supplementing Qi and activating blood.Methods1.Establishing the ischemia and hypoxia neonatal rat cardiomyocytes model:Selected neonatal SD rat myocardial tissue and digested into myocardial cells in single cell suspension, normal cultured 72h then used hypoxia device respectively on myocardial cells of ischemia for 30min, lh,2h,3h,4h,6h, 12h,24h modeling, CCK-8 cell viability method detected with survival rate, inverted microscope and electron microscope observated cell injury situation, to fullfill establishment of myocardial ischemia and hypoxia in cell model.2. Experiments of the effect of GS Rbl and IA on hypoxia neonatal rat cardiomyocytes:Normal cultured myocardial cells of neonatal rats for 48h then divided into 11 groups:blank control group, model group, trimetazidine group, ginsenoside Rbl high dose group, ginsenoside Rbl low dose group, Ilexonin A high dose group, Ilexonin A low dose group, ginsenoside Rbl high dose bombined Ilexonin A high dose group, ginsenoside Rbl high dose combined Ilexonin A low dose group, ginsenoside Rbl low dose combined Ilexonin A high dose group, ginsenoside Rbl low dose combined Ilexonin A low dose group. Normal group and model group continuly cultrued with complete culture solution in culture box, the drug group were replaced with the corresponding concentration of medicine liquid solution to continue to cultivate 24h. The model group and each drug group were treated with hypoxia and ischemia method for 6h by co culture when cultured 72h, and the normal group were cultured in normal enviorment for 6h. cells were collected for detection. The LDH and MDA levels were detected by the colorimetric method, the cell damage was observed by electron microscope, the apoptosis rate was detected by TUNEL method, and the mPTP condition was detected by flow cytometry.3. Experiments of the metabolism mechanism of GS Rbl and IA on hypoxia neonatal rat cardiomyocytes:The cells were divided into 2 groups:normal group, model group. Using the aforementioned test method on cultured myocardial cells to culture, modeling and drug intervention, use western blot detecting energy substrate metabolism-related protein AMPK, PGC-la, PPARa, GLUT4, CPT-1, ACADM and mitochondria generate relevant protein Tfam, NRF2 protein expression levels. The cells were divided into 10 groups, model group, trimetazidine group, ginsenoside Rbl high dose group, ginsenoside Rb1 low dose group, Iexonin A high dose group, Iexonin A low dose group, ginsenoside Rbl high dose combined Iexonin A high dose group, ginsenoside Rbl high dose combined Iexonin A low dose group, ginsenoside Rbl low dose combined Iexonin A high dose group, ginsenoside Rbl low dose combined Iexonin A low dose group. Western blot detecting the use of energy metabolism-related protein expression. HPLC assayed AMP, ADP, ATP content.Results1. Establishing the ischemia and hypoxia neonatal rat cardiomyocytes model:Confirmed the myocardial cells fulfill the standard of ischemia and hypoxia model by oxygen indicator. The cell activity was detected by CCK8 kit and light microscope and electron microscope observation of myocardial ischemia and hypoxia in cells at various time node morphological changes, select the 6h model for hypoxia time. Model group and the normal group, the myocardial cells compared with obvious cell injury and morphological change, pump power was significantly decreased, cell viability decreased significantly, appear obvious failure of myocardial cells, cardiomyocytes subjected to ischemia animal model was established.2. Experiments of the effect of GS Rbl and IA on hypoxia neonatal rat cardiomyocytes:2.1. Observation by optical microscope:The myocardial cells were grouped after inervention, optical microscope observed that hypoxia and ischemia model group of myocardial cells showed obvious cell injury, myocardial pump power decreased, pulsation frequency slower than control group. Trimetazidine group compared with the IH group, the myocardial cell viability was significantly enhanced and cell morphology were not significantly affected by the hyposia and ischemia enviorment. The GS Rbl and IA sighnificantly improved the cell pump power and protected the cell morphology when compared with the model group, the GS Rbl high dose combined IA high dose group had the most sighnificantly effect of protecting myocardial cells, and dosage and cell protective effect showed a dose effect relationship.2.2. Observation by electron microscope:compared the the cell morphology, organelles and mitochondrial mropholygy among the drug intervented groups and IH group, discovered that the GS Rbl and IA has protecting effect of reducing myocardial cells damage in ischemia and hyposia enviorment, the mitochondrial functions retained, besides, the GS Rbl high dose combined IA high dose group had the similar extent as Trimetazidine group. Through electron microsope test, the results confirmed that GS Rbl and IA had the dose effect relationship among monotherapy and GS Rbl combined IA, the GS Rbl high dose group and IA high dose group had the similar extent on IH myocardial cells.2.3. Cell viability assay:The cell viability was sighnificantly reduced in IH group which compared with control group by CCK8 assay, compared to IH group, the drug intervention groups has a increased effect on cell viability which GS Rbl comined groups had better effect than monotrerapy groups. GS Rbl high dose combined IA high dose group had the most sighnificantly action which obviously improved the myocardial cells damage in IH enviorment.2.4. LDH, FFA, MDA detection:Compared with the control group, LDH, FFA and MDA were significantly increased in IH group, hypoxia ischemia cell injury model was built. The drug intervention groups compared with the model group, LDH, FFA and MDA were obviously reduced and had a dose effect relationship. The drug intervention groups sighnificantly reduced the LDH, FFA, MDA and had a dose effect relationship, GS Rbl high dose combined IA high dose group had the greatest action among drugs intervention groups (P<0.01)2.5. MPTP assay:By staining the cytoplasm and mitochondrion with calcein AM, cobalt chloride and calcein AM would react as fluorescence quenching in cytoplasm to examing the MPTP and mitochondrial functional state in IH enviorment. Expression of fluorescence in the model group declined compared to control group and trimetazidine group had the similar expression of fluorescence with GS Rbl high dose combined IA high dose group. Compared to IH group, the effect of modulating MPTP on GS Rbl and IA intervented groups were sighnificantly improved which had the dose effect relationship.2.6.Cell apoptosis:TUNEL method was used to detect the apoptosis rate, compared to control group, apoptosis rate in IH group was sighnificantly increased which comfirmed that the IH enviorment stimulated the apoptosis program in myocardial cells. Compared to IH group, the apoptosis rate decreased among trimetazidinethe group, GS Rbl high dose group, GS Rbl low dose group, IA high dose group, GS Rbl high dose combined IA high dose group, GS Rbl high dose combined IA low dose group, GS Rbl low dose combined IA high dose group, which showed that all drug inervation can obviously inhibit the cell apoptosis. Experiments of the metabolism mechanism of GS Rbl and IA on hypoxia neonatal rat cardiomyocytes:3.1. expression of myocardial metabolism proteins."Compared to control group, expression of AMPK、PPARa、PGC-la、CPT-1、ACADM、NRF2、Tfam proteins declined varying degrees in IH group, expresstion of GLUT4 protein remained the same. After drug intervention, compared with other groups, trimetazidine group and GS Rbl high dose combined IA high dose group had much higher expression and GS Rbl high dose combined IA high dose group had the highest expression of AMPK protein (P<0.01). In this experiment we didn’t find the interventions had any sighnificant improvement effect on the expresstion of PPARa. Compared to IH group, GS Rbl high dose group, GS Rbl low dose group can obviously increase the expression of PGC-la protein in IH myocardial cells (P<0.01). GS Rbl high dose combined IA high dose group, GS Rbl high dose combined IA low dose group can sighnificantly improve the expression of PGC-la(P<0.01).GS Rbl high dose group, GS Rbl low dose group, GS Rbl high dose combined IA high dose group, GS Rbl high dose combined IA low dose group, GS Rbl low dose combined IA high dose group, GS Rbl low dose combined IA low dose group had the effect of increasing the expresstion of PGC-la protein and the effect related with drugs concertration. Trimetazidine can sighnificantly declines the expresstion of CPT-1 protein in IH myocardial cells(P<0.01). Compared to IH group, the expression of CPT-1 protein in GS Rbl high dose group, IA high dose group, GS Rbl high dose combined IA high dose group and GS Rb1 low dose combined IA high dose group declined(P<0.01).GS Rbl and IA didn’t show the improvement of FFA metabolism functions in this experiment. Except GS Rbl low dose combined IA high dose group each drug intervented groups have no effect on ACADM protein. The expression of GLUT4 in trimetazidine group sighnificantly rised when compared with IH group which confirmed that the mechanism of trimetazidine was improving the function of glucose metabolism. Compared to IH group, GS Rbl high dose group, GS Rbl low dose group, IA high dose group, GS Rbl high dose combined IA high dose group, GS Rbl high dose combined IA low dose group had the improvement effect on GLUT4 protein, and GS Rbl high dose combined IA high dose group had the most sighnificant action among these groups. Compared to IH group, all drug intervention groups could rise the expression of NRF-2 protein (P<0.01). GS Rbl low dose combined IA high dose group, GS Rbl high dose combined IA high dose group, GS Rbl high dose combined IA low dose group, GS Rbl high dose group, GS Rbl low dose group and IA high dose group had the similar action of increasing the expression of NRF-2 protein, GS Rbl high dose combined IA high dose group had the most sighnificant action among these groups. Compared to IH group, each drug intervented group could rise the expression of Tfam protein and GS Rbl high dose combined IA high dose group had the most sighnificant action(P<0.01). 3.2.3.2. creation of High-energy phosphate compounds:Compared to control group, the content of AMP increased in IH group, ADP and ATP content sighnificantly declined(P<0.01). Compared to IH group, each drug intervented groups declined the content of AMP and ADP in varying degrees(P<0.01). The content of ATP increased in trimetazidine group, GS Rbl high dose group, GS Rbl low dose group, GS Rbl high dose combined IA high dose group, GS Rbl high dose combined IA low dose group, GS Rbl low dose combined IA high dose group(P<0.01) but not in IA high dose group(P>0.05).Conclusion1. The myocardial ischemia and hypoxia model was established with the condition of ischemia and hypoxia condition for 6h as the model. Ginsenoside Rbl and I lexonin A on ischemia hypoxia myocardial cell morphology integrity, reduce apoptosis rate, reduce lipid deposition, alleviating lipid oxidative damage, reduction of mitochondrial permeability transition pore opening protection of mitochondrial function and have a significant role. Group of ginsenoside Rbl in the protective effect on myocardial cells is better than that of ilexonin A.2. AMPK-PGC-la pathway may be the key pathway to regulate the energy metabolism of myocardial cells in ischemia and hypoxia condition. Ginsenoside Rbl and ilexonin A can activate the AMPK-PGC-la pathway, stimulate the metabolism of glucose and the formation of mitochondria. Ginsenoside Rb1 can reduce the content of AMP, promote the formation of ADP and ATP, and improve the energy metabolism of myocardial cells after ischemia and hypoxia. Mao Dongqing a can reduce the content of AMP, promote the generation of ATP, the ADP generation has no obvious effect.3. ginsenoside Rb1 and ilexonin A high dose combination in ischemia hypoxia myocardial cell morphology integrity, reduce apoptosis rate, reduce lipid deposition, alleviating lipid oxidative damage, reduction of mitochondrial permeability transition pore opening, protection of mitochondrial function. The possible mechanism is through the interaction of the two drugs on AMPK-PGC-la energy metabolism pathway, promote glucose metabolism, inhibit lipid peroxidation and promote adenylate synthesis.