Protective Effects And Mechanisms Of Rosamultin Against H₂O₂-Induced Oxidative Stress Injury On H9c2 Cardiomyocytes

Objectives: Our previous studies have shown that the n-butanol extract of Potentilla anserine L can obviously improve the ischemic reperfusion rat myocardial cell vitality, inhibit rat myocardial cell apoptosis. rosamulin,an active monomers, which was extracted and separated from the n-butanol extract of Potentilla anserine L, which have significant protective effect hypoxia injury of vascular endothelial cell, whether have the myocardial protection effect has not been reported, thus this study selected H2O2 damage model to simulate human myocardial cells ischemia-reperfusion injury, explored the protective effects and mechanisms of rosamultin against H2O2-induced oxidative stress injury on H9c2 cardiomyocytes.Methods: 1 The established progress of model: Through measuring Lactate dehdrogenase(LDH) and MTT, we ascertained the best time and concentration of H2O2 and established the H2O2-induced oxidative stress injury model. 2 The design of the experimental group: The good growth state of H9c2 cardiomyocytes were divided randomly into control group 、 H2O2 –induced injury model group 、 different concentrations of rosamultin intervention group(1′10-11 mol/l, 1′10-12 mol/L and 1′10-13 mol/L) and velapamil group(1′10-12 mol/L). 3 The measurements of experiment 3.1 Using the biochemical method to determine lactate dehydrogenase(LDH) and creatine kinase(CK) activity in culture medium, superoxide dismutase(SOD) activity, the content of malondialdehyde(MDA) catalase(CAT) activity and glutathione oxidase(GSH-Px)activity in the cells to study the protective effects on the cell membrane damage, lipid peroxidation damage and REDOX enzyme damage caused by H2O2. 3.2 Using Hochest/PI staining, DAPI staining to observe the rusamultin influence on the morphology and ultrastructure of cells. 3.3 Using fluorescent tags with Annexin V/PI and using flow cytometry to detected the rosamultin impact on apoptosis rate. 3.4 Using flow cytometry to determine the rosamultin on the role of ROS levels in the cell. 3.5 Immune fluorescent confocal microscopy are used to observe the rosamultin’s effects of intracellular Ca2 +。 3.6 Using Western Blot technology to detect cryab, Pcryab, akt, pakt, bcl-2, bax, cyt c, caspase 9, caspase3 protein expression level. After join the PI3K/AKT pathway blockers LY294002, to observe the cell ablility and pakt,caspase3 protein expression level. To explore the protective mechanism of rosamultin.Results: 1 The established progress of model:with the increasing concentration of hydrogen peroxide, the cell vitality gradually reduced, in 200μmol/l hydrogen peroxide induced to 3h, the cell survival rate steady moderate, suitable for for further research. 2 Through determined by MTT method and LDH to determine the concentration of rosamultin respectively as(1×10-11mol/l、1×10-12mol/l、1×10-13mol/l), the concentration of positive control drug verapamil choice for 1×10-11mol/l. 3 The protective effects of rosamultin against H2O2-induced oxidative stress injury on H9c2 cardiomyocytes 3.1 MTT results showed that compared with normal group, the damage model group cell vitality was obviously lower(P<0.01), giving rosamulitn and verapamil treatment after cell vitality were increased significantly(P<0.01). 3.2 Biochemical indicator: Each rosamultin treatment groups can significantly reduce the release of LDH and CK, reduced the generation of MDA(P<0.01) and improved of SOD(P<0.01), besides each concentration of rosamultin group are able to improve the level of the CAT(P<0.01) and GSH-Px enzymes in the cell(P<0.01). 3.3 Hochest PI staining: Compared with control group, H2O2 oxidative damage group the nuclear chromatin agglutination, nuclear membrane rupture manycells necrotic appeared red fluorescence, and each concentration of rosamultin groups can significantly alter oxidative damage caused by restore the nucleus condensation, reduce the number of dead cells in the red- dye. 3.4 DAPI staining: In each rosamultin treatment group, the shapes and structures change caused by oxidative damage were remarkably reversed. 3.5 Flow cytometry: results showed that rosamultin in various concentration group can significantly decrease the cell apoptosis rate caused by H2O2(P<0.01), reduce the levels of ROS in cells.(P<0.01) 3.6 Confocal laser scanning microscope results showed that rosamultin could reduce the level of intracellular ca2 +. 4 The protective mechanisms of rosamultin against H2O2-induced oxidative stress injury on H9c2 cardiomyocytes 4.1 Western blot: The bcl-2 expression level was significantly decreased induced by H2O2, in contrast, pcryab, pakt, bax, cyt c, caspase3, caspase9 expression levels were significantly increased compared with the control group(P<0.01), and each rosamultin groups can enhanced the expression levels of pcryab, pakt, bcl-2(P<0.01), depressed the levels of bax, Cyt-c, caspase9, csapsae3(P<0.01). after giving Pi3k/akt pathway Specific LY294002 can obviously decreased the cell vitality and the expression level of Pakt(P<0.01), caspase3(P<0.01). 4.2 After rosamultin treatment group was given PI3K/AKT pathway blockers LY294002,compared with the oxidative damage model group, myocardial cell viability was obviously decreased(P<0.05), compared with rosamultin treatment group, the cell vitality level was also decreased(P<0.01). 4.3 westernblot results After given PI3K/AKT pathway blockers LY294002, show that the rosamultin treatment group after joining LY294002 pakt the expression level of the damage model and rosamultin treatment group were significantly lower than it(P<0.01), the expression of caspase 3 levelswas decreased in the oxidative damage model group(P<0.05) the rosamultin treatment group significantly increased(P<0.01).Conclusions:1 Rosamultin could significantly decrease the injury induced by H2O2.2 Rosamultin improved the cells ability of clear oxygen free radicals, reactive oxygen species.3 Rosamultin could restore cells morphology and function, moreover,it also could reduced the apoptosis, inhibiting calcium overload.4 Rosamultin can protect myocardial cells, the role of the mechanism is probably by regulating αB-crystalin protein phosphorylation,the PI3K/AKT signal pathway, improve mitochondrial function, reduce mitochondrial apopotis pathway, thus inhibiting myocardial apoptosis.