Effection On H₂O₂-Injuried L6 Skeletal Myoblast By Preconditioning Of Diazoxide

Objectives: Ischemic heart disease (IHD) is one of the diseases which severely impacts on human life.Recent treatment methods include drug therapy, interventional, surgical and other treatment.Although these methods delayed the development of disease in degree, but could not reverse myocardial necrosis and stop the progress of heart failure. Cell transplantation provided a new method for the reconstruction and function recovery of heart failure. Some studies showed stem cell transplantation could promote new blood vessels and myocardial cells to regenerate, improve myocardial blood supply, and enhance cardiac function. In many studies about cell transplantation, skeletal myoblasts have been paid close attention by the scientists because of the probability in extensive clinical application. Skeletal myoblasts lie between the skeletal muscle sarcolemma and basement membrane. They are the precursor cells of skeletal muscle cells. As donor cells,skeletal myoblasts have many desirable properties, such as high proliferative potential, high tolerance to ischemia, autologous transplantation, avoiding immune rejection reaction, gene carrier and so on. Skeletal myoblasts become to be the main stem cells for transplantation because of these advantages.But poor survival rate of SkMs in the host myocardium is a vital problem.A significantly high percentage of SkMs apoptosis after transplantation greatly influnced the effection of transplantation therapy.Oxidase and antioxidant enzymes in the balance is broken after acute myocardial infarction. ROS (reactive oxygen species, ROS) in the infarct area and non-infarct area was increased, and the oxidative stress persist. ROS is an important factor that induced transplanted cells apoptosis in myocardial infarction. In this study, L6 skeletal myoblasts were induced to be injuryed by hydrogen peroxide in vitro and oxidative stress model was established. Then the protective effects and mechanisms of diazoxide (DZ) on L6 skeletal myoblast following the damage of hydrogen peroxide(H₂O₂) were studied. We want to find a effective way that could improve the survival rate of transplanted cells and enhance the effection of transplantation therapy. Methods:1 Effects on L6 Skeletal Myoblast by H₂O₂ injuring with different concentration and time: Rat L6 skeletal myoblast (L6SkM) were cultured in vitro. They were divided into normal control group and five oxidative groups which were injured by H₂O₂ with different concentration (0.10,0.50,1.00, 1.20,1.50mmol/L,respectively12hours,24hours,36hours),the levels of MTT (OD) of cells in each group were measured.We want to find the appropriate concentration and treatment time of H₂O₂ and establish L6SkM oxidate stress damage model.2 Effects of diazoxide preconditioning with different concentration on L6 SkM: Rat L6 skeletal myoblast (L6SkM) were cultured in vitro. They were divided into normal control group and H₂O₂ group and precondition groups which were treated by DZ with different concentration(50,100,200,400umol/L,30minites).The level(sOD)of cells in each group were measured by MTT. We want to find the appropriate concentration of DZ and establish DZ protect model.3 Effects of DZ preconditioning on L6 SkM following oxidative stress injure: Rat L6 skeletal myoblast (L6SkM) were cultured in vitro. They were divided into normal control group and H₂O₂ group and DZ preconditioning groups.Cell activity was measured in each group by MTT assay. Lactate dehydrogenase (LDH) levels and LDH release rate in each group were detected to show the degree of cell membrane damage. The changing of cells morphology was observed by LM and EM.Apoptosis ratio and cell cycle of L6SkM was detected by FCM. Immunocytochemistry was used to show Cytochrome C, PCNA, Bcl-2, Bax protein expression. The quantity of Bcl-2 and Bax protein expression was studied by western-blot assay. Results:1 Effects on L6 Skeletal Myoblast by H₂O₂ injuring with different concentration and time: MTT results indicated that the cell vitality was reduced with the increase of the H₂O₂ concentration and the extention of time. It showed concentration and time-dependent.H₂O₂ concentration which caused the cell vitality to decrease by 55% was selected to establish L6SkM damage model.2 Effects of diazoxide preconditioning with different concentration on L6 SkM: MTT results indicated that the cell vitality was raised with the increase of he DZ concentration.100umol/L,200umol/L,400umol/LDZ group compared with the H₂O₂ group, the average optical density value increased significantly. There was no difference between 200umol/L and 400umol/L DZ.So 200umol/L DZ group was selected to establish experimental model of protection.3 Effects of DZ preconditioning on L6 SkM following oxidative stress injury:3.1 MTT assay showed that, cell viability in the H₂O₂ group SkM was reduced significantly compared with the normal control SkM .Cell viability in the DZ group was increased significantly compared with the H₂O₂ group. There was no significant difference in cell viabilities between DZ group and normal control.3.2 LDH assay showed that LDH leakage in the H₂O₂ group was significant increased compared with the normal control. LDH leakage in the DZ group was significant reduced compared with the H₂O₂ group, but it was still higher than the normal control.3.3 L6SkM morphological changes. In the H₂O₂ group, some cells shrink to become round. Nucleous showed shrinkage and nuclear chromatin was dense. SEM showed that some cells shrink and more spherical protrusions and fold appeared on the cell surface.TEM showed nuclear heterochromatin margination and gradually form a crescent-shaped. Even more nucleous broke to be fragment. L6SkM morphology was improved significantly by DZ pretreatment before H₂O₂-stimulation.3.4 FCM revealed DZ preconditioning could reduce the apoptosis rate of H₂O₂-induced L6SkM and increased proliferation index.3.5 Immunocytochemistry displayed, compared with normal control, for H₂O₂ group L6SkM, PCNA and Bcl-2 protein expressions were decreased and Bax protein expression was increased significantly. Compared with H₂O₂ group, for DZ group L6SkM,PCNA and Bcl-2 protein expressions were increased and Bax protein expression was decreased significantly.For PCNA, Bcl-2 and Bax protein expression,there was no significant difference between DZ group and normal control.Cytochrome C immunoreactivity was significantly increased in some H₂O₂ group L6SkM and showed diffuse distribution in cytoplasm.In DZ group and normal control, brown punctate staining was present in the cytoplasm which coresponded well with the location of mitochondria.3.6 Western-blot reavealed, compared with normal control, Bcl-2 protein expression was decreased and Bax protein expression was significantly increased in H₂O₂ group.Compared with H₂O₂ group, Bcl-2 protein expression was increased and Bax protein expression were significantly decreased in DZ group. Compared with normal control, Bcl-2 protein expression increased in DZ group but still significant lower than the normal control. There was no significant difference for Bax protein expression between the two groups.Conclusion:1 L6SkM could be injuried by H₂O₂, which showed the concentration and time-dependent. 24 hours and 1.00mmol/L were a better condition that was suitable to study the protective effects on L6SkM following oxidative stress in vitro.2 DZ exerted protective effect on H₂O₂ damaging L6SkM. DZ could weak the oxidative stress injure by protecting the cell membrane and mitochondrial membrane integrity, preventing cell morphology to change, promoting proliferation and inhibiting apoptosis.