| BackgroundCardiovascular disease is currently responsible for the death of the primary disease of our people, and in recent years, a large number of research evidence shows that cardiomyocyte apoptosis is an important mechanism for the occurrence and progress of heart failure, dilated cardiomyopathy (DCM), adriamycin-induced cardiomyopathy and viral myocarditis, myocardial infarction, myocardial ischemia and other cardiovascular diseases. Apoptosis is also involved in ventricular remodeling, and is a predictor of long-term prognosis of cardiac function.A variety of stimulus factors can induce apoptosis in cardiac cells, such as hypoxia, hypoxia-reoxygenation, acidosis, oxidative stress, pressure, stress, etc. Among them, Oxygen free radical metabolism disorder is an important precipitating factor in cardiomyocyte apoptosis. In vivo reactive oxygen species can with the role of DNA, proteins and polyunsaturated fatty acids Etc, and cause DNA strand breaks and oxidative damage, protein-protein cross-linking, protein-DNA cross-linking and lipid peroxidation. When reactive oxygen species H2O2 of high concentrations acts on cardiomyocyte, intracellular reactive oxygen species levels can be induced increase rapidly and in vivo the activity of anti-oxidants such as:superoxide dismutase, catalase, glutathione peroxidase enzymes can be induced decrease, so that the structure and function of membrane integrity have been destroyed and cell apoptosis has been caused.Cell apoptosis is an active death process controlled by gene, in which multi-cell organism maintain their autologous tissue and keep the balance between cell proliferation and death. In apoptosis studies, the known signal transduction pathways of apoptosis are:the death factor-mediated apoptosis; ceramide-mediated apoptosis; p53+gene-triggered apoptosis; mitochondrial damage-mediated apoptosis. In those many motivations, free radical produced in body metabolism or extrinsic factor can induce cell apoptosis. Through the above pathway, the results in cell apoptosis make the number of somatic cells reduce, especially those with important functions such as brain cells, myocardial cells, causing the vital organs such as the ventricular muscle a recession in pathological processes. In research of apoptosis in myocardial cells, the two main signal transduction pathways are proposed, namely mitochondrial pathway and the membrane death receptor pathway.It has been confirmed that inhibiting cadiomyocytes apoptosis can protect heart on certain degree. Scientists show great interests on the effect of drugs anti-apoptosis on cardiomyocytes. Ever since a long time ago, China medicine plays an important role in cardiovascular diseases treatment. Scientists are paying attention to the relation between China medicine and cardiomyocytes apoptosis, and have obtained some success. The researches have to be done further, especially on the effective simple component of China medicine.In our laboratory Dangguibuxuetang was isolated and purified by using ultrafiltration membrane technology on the traditional recipe. And preliminary studies have confirmed that the ultra-filtration extract from the mixture of angelica sinensis and hedysarum polybotrys (UFE-AH) has effect of clear oxygen free radicals, anti-lipid peroxidation and anti-apoptosis of myocardial cells. In this study we established the model of myocardial cell apoptosis H2O2-induced in vitro through simulating the process of oxidative damage of myocardial cells in vivo, and observed the effect of UFE-AH on apoptosis induced by oxidative stress in neonatal rat cardiomyocytes in vitro culture. It can identify the mechanism of UFE-AH's anti-apoptosis effect and its value of clinical appliancation.Objective1.To separate and purify traditional Chinese medicine mixture through the use of ultrafiltration membrane technology.2. To study anti-oxidative damage mechanism of UFE-AH.3. To study anti-apoptotic mechanism of UFE-AH.4. To study the effect of UFE-AH on expression of Hsp70 in Cardiomyocyte.5. To provide experimental theoretic foundation for clinical applications of UFE-AH.Methods1. Refined the mixture of angelica sinensis and hedysarum polybotrys (1:5) using ceramic membrane and PAN (Ultra-filtration membrane) of the hollow fiber (cut to stay the molecular weight to measure for 10,0000).2. Primary culture of neonatal rat cardiomyocytes:neonatal rats of 1-3 days were removed, soaked in disinfecting the skin with 75%ethanol, and moved to Clean Bench. Ventricular muscle was removed from chest and digested over and over again using enzymatic digestion. The cell suspension was collected and cardiomyocytes were Separated and Purified with differential adhesion method. Cardiomyocytes were cultivated under the conditions of 37℃,5%CO2, after the concentration of which had been adjusted to per ml of 5×105with DF medium of containing 15%fetal calf serum.Those with good growth state were elected to carry our experiments in the 72h. We detected the survival rate of cardiac cells using trypan blue staining. We identified myocardial cell purity with a-Sarcomericactin immunohistochemistry staining.3. Constructing oxidative stress mode by using peroxide:Myocardial cell apoptosis was induced by 400μmol-L-1 H2O2.There were 5 groups in our research:normal control group; model group(pretreatment myocardial cells with H2O2 for 6h); UFE-AH high-dose intervention group(pretreatment myocardial cells with UFE-AH of 15 g-L"1 for 48h, H); UFE-AH middle-dose intervention group(pretreatment myocardial cells with UFE-AH of 7.5 g-L"1 for 48h, M); UFE-AH low-dose intervention group(pretreatment myocardial cells with UFE-AH of 3.75 g-L-1 for 48h, L). The morphological changes of myocardial cell were observed under inverted phase contrast microscope; The cell viability was detected by MTT chromatometry; Myocardial cell apoptosis was detected by Annexin V-FITC/PI double staining marks; Myocardial cell apoptosis index was detected by flow cytometry in each group.4. The expression of apoptosis-related gene (Hsp70, Caspase-3, Bcl-2 and Bax) were detected by RT-PCR and Western-blotting.5. The changes of enzymes in oxidative stress myocardial cells were detected by biochemical technology, such as SOD, LDH, CK, MDAand MPO.RESULETS1. The UFE-AH (100,000 molecular weight) was found that it did not contain ferulic acid, and polysaccharide concentrations of its mixture closed to single-dose levels by UV spectrophotometer and high performance liquid chromatography (Angelica polysaccharide content of 23.84 g-L"1, HPS content of 27.55 g-L"1, mixture content of 22.08 g-L"1). 2. Observing myocardial cell morphology under inverted phase contrast light microscopy: Normal myocardial cells extended pseudopods with each other and blended them into films. The refractive indexes of nuclears were good. Cells beat with integrity, coherence and regularity (98.2±13.4 times/min). In H2O2 injury group, cells shrinked, nuclears were bleak, and the beats of cells were significantly decreased (20.1±3.3 times/min) and some stopped beating. The pulse amplitude of cells changed of varying intensity and their beating rates had significantly difference, compared with the normal control group(P<0.01).In UFE-AH intervention group of high, medium and low-dose, myocardial pulsating frequencies were 86.9±8.4,71.1±6.7 and 43.7±9.6 times/min, and cell pseudopods were thinner than those in control group, and beat frequency was slightly reduced, but higher than those in group of oxidative damage.3. Detecting the survival rate of myocardial cells by MTT assay:In oxidative injury model group, myocardial cells were severely damaged, cell survival rates were decreased significantly(29.5%) and there were statistically significant compared with normal control group (89.7%, P<0.01). In UFE-AH intervention group the damages of myocardial cells were significantly improved. In high, middle and low-dose group the cell survival rates were 77.6%, 64.4%,43.9, and were significantly increased compared with oxidative injury model group (P< 0.01).4. Biochemistry test:The permeability of cell membrane was increased by H2O2 treatment. So the levels of LDH(274.36±15.16U/L) and CK(201.47±2.71U/L) in the culture medium were increased, and compared with the control group (LDH 138.07±11.27U/L, CK 42.69±1.53U/L), the content of LDH and CK were significant differences (P<0.01). UFE-AH could significantly inhibit the changes of myocardial cell membrane permeability H2O2-induced and reduce the leakage of LDH and CK.The leakage of LDH and CK in high-dose group (LDH 167.22±10.11 U/L, CK 88.14±1.06U/L), middle-dose group (LDH 183.48±9.88U/L, CK 99.66±4.18U/L) and low-dose group (LDH 201.06±18.13U/L, CK 132.54±1.92U/L) were significantly reduced, and there were significantly different compared with the model group (P<0.01).The contents of MDA(3.82±0.64nmol/mL)and MPO(114.00±3.24U/L)were increased and the activity of SOD (58.23±2.45U/mL) was reduced in H2O2 injury group. UFE-AH could significantly reduce the intracellular contents of MDA(H:2.51±0.28nmol/mL; M:2.80±0.32nmol/mL; L:3.11±0.35 nmol/ mL) and MPO(H:59.76±1.84U/L; M:71.52±1.03U/L; L:85.22±4.18U/L), and increase the activity of SOD (H:101.34±2.14U/mL; M:83.36±6.63U/mL; L:71.45±7.11U/mL). The difference was significant between H2O2 injury group and UFE-AH treatment group (P<0.01). And dose-dependent manner, the contents of LDH, CK, MDA and MPO were decreased and the activity of SOD was increased gradually with the concentration of UFE-AH increasing.5. RT-PCR test results:The mRNA of Hsp70, Caspase-3, Bcl-2 and Bax showed low expression in normal control group.In model group, the expression of Hsp70, Caspase-3, and Bax mRNA were increased significantly and the expression of Bcl-2 mRNA was significantly reduced. The difference between model group and normal control group was statistically significant (P< 0.05).6.Western-blotting test results:The expression of Hsp70 and Bax protein were significantly increased and Bcl-2 protein was significantly reduced in myocardial cells after having been treated for 6h by 400μmol·L-1 H2O2. There were significant difference compared with the normal control group (P<0.01). And the protein ratio of Bcl-2/Bax was decreased (P<0.01).However, in myocardial cells after UFE-AH treatment, the expression of Hsp70 protein was increased significantly than those in model group (P<0.05); In myocardial cells of high and middle dose group, the expression of Bcl-2 protein was increased significantly (P<0.05), Bax was markedly reduced (P<0.01), and the ratio of Bcl-2/Bax was increased significantly (P<0.01); There was statistical significance. But in low-dose group, the expression of Bcl-2 and Bax protein had not changed significantly (P>0.05).Conclusions1. Oxidative stress is a major reason of inducing cardiomyocyte apoptosis.2.UFE-AH has an effect of anti anti-oxidative stress of myocardialcells induced by H2O2, It can reduce the decline of cell viability and inhibit myocardial cell apoptosis.3.The activation of Bax and Caspase-3 plays an important role in myocardial cell apoptosis, and the increases of Bcl-2 and Hsp70 expression have an anti-myocardial cell apoptosis effect.4. UFE-AH may play a role in the treatment of cardiovascular disease by inhibiting apoptosis of myocardial cells and preventing oxidative damage of myocardial cells. |
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