The Mechanism And Effection On H₂O₂-Injuried L6Skeletal Myoblast By Preconditioning Of Diazoxide

Objectives: In our country the incidence of ischemic heart diseaseincreased year by year, and it has become the one of the main causes ofhuman death. Nowadays the treatment of ischemic heart disease has madegreat progress by cell transplantation, but the treatment effect is restricted byvarious factors. One of the important factors of stem cells transplantationwhich influenced the treatment effect was the low survival rate. The studiesshowed that a70-80%cells transplaned into damage myocardium were deadwithin3days. Improving the survival rate of transplanted cells can be donefrom two aspects, One is to enhance their tolerance ability of the transplantedcells in bad environment; The second is to improve transplantingmicroenvironment. Previously, L6skeletal myoblasts were induced to beinjuryed by hydrogen peroxide in vitro and oxidative stress model wasestablished. Then the protective effects and mechanisms of diazoxide （DZ） onL6skeletal myoblast following the damage of hydrogen peroxide（H₂O₂） werestudied. Preliminary observations showed that it could improve the cellsurvival rate and reduce the oxidative stress damage by promotingproliferation and inhibiting apoptosis. However, the specific mechanism isunclear. The activation of PI3K/Akt pathway is very important to cell survival.In this study, L6skeletal myoblasts were induced to be injuryed by hydrogenperoxide in vitro and oxidative stress model was established. Then theprotective effects and mechanisms of diazoxide （DZ） on L6skeletal myoblastfollowing the damage by hydrogen peroxide（H₂O₂） were further studied. Therelationship between protection and PI3K/Akt signal channel was discussed.Methods:1Effects of H₂O₂on L6Skeletal Myoblast at different concentration andtime: L6skeletal myoblasts （L6SkM） of Rat were cultured in vitro. They were divided into normal control group and another five groups which wereinjured by H₂O₂with different concentration (0.20,0.40,0.60,0.80mmol/L,respectively（12hours,24hours）, MTT was used to measure cellsurvival.We want to find the appropriate concentration and treatment time ofH₂O₂and establish L6SkM oxidate stress-damaged model.2Effects of LY294002preconditioning with different concentration onL6SkM: Rat L6skeletal myoblasts （L6SkM） were cultured in vitro. Theywere divided into normal control group and H₂O₂group（0.40mmol/L，24hours）and DZ group （pretreated with200μmol/L DZ for30mins before24hours of0.40mmol/L H₂O₂treatment）. Precondition groups which were treated by DZwith concentration200μmol/L about30minites and LY294002withconcentration25μmol/L,50μmol/L about30minites. The levels（OD）of cellsin each group were measured by MTT. We want to find the appropriateconcentration of LY and establish LY inhibitor model.3Effects of DZ preconditioning on L6SkM damage induced by H₂O₂:Rat L6skeletal myoblast （L6SkM） were cultured in vitro. They were dividedinto normal control group and H₂O₂group and DZ group and LY groups.Cellactivity was measured in each group by MTT assay. Cell morphology changewas observed by HE staining. Mitochondrial membrane potential changes wasdetected by Laser confocal microscope which was stained by JC-1.Cellapoptosis rate was detected by AnnexinV-FITC&PI in each group.Immunocytochemistry was used to show caspase-3protein expression. Thequantity of p-Akt and caspase-3and caspase-9protein expression was studiedby western-blot assay.Results:1Effects on L6Skeletal Myoblast by H₂O₂with different concentration andtime: MTT results indicated that the cell vitality was reduced with the increaseof the H₂O₂concentration and the extention of time. H₂O₂concentrationwhich caused the cell vitality to decrease by50%was selected to establishL6SkM damage model.2Effects of LY294002preconditioning with different concentration on L6 SkM: MTT results indicated that the cell vitality of the H₂O₂group wasdeclined compared with the normal group. The cell vitality of the DZ groupwas obvious increased compared with the H₂O₂group. The cell vitality of theLY group was suppressed which was no obvious difference between the H₂O₂group.3The cell apoptosis rate revealed by FCM: Apoptosis rate of the H₂O₂groupwas significantly increased compared with the normal group.Compared withthe H₂O₂group, the apoptosis rate of the DZ group was decreased. Theapoptosis rate of the LY group was increased but still be lower compaired withthe H₂O₂group.4Fluorescent changes of mitochondrial membrane potential were detectivedby Laser confocal microscope: Normal cells have high membrane potential ofmitochondrion, but they depolarizatied when cell apoptosis. Normal cellsshowed a strong red fluorescence and green light relatively weak.When cellapoptosis, the green fluorescence enhanced but red light is abate.We use thechang in green/red fluorescence intensity to show the chang of membranepotential. Compared with the normal group, green/red fluorescence intensityratio increased in H₂O₂group, and mitochondral membrane potentialdecreased. The green/red fluorescence intensity ratio is decreased and close tothe normal group in DZ group, it can maintain a higer membrane potential.The green/red fluorescence intensity ratio is increased in LY group and closeto H₂O₂group, illustrating the membrane potential depolarizatied.5Immunocytochemistry results showed: Compared with normal control,caspase-3protein expression in H₂O₂group was increased significantly.Compared with H₂O₂group, caspase-3protein expression was decreasedsignificantly in DZ group L6SkM. Caspase-3protein expression wasincreased in LY group compared with DZ group,but still was less than H₂O₂group.6Western-blot reavealed6.1Compared with normal control, P-Akt protein expression was significantlydecreased in H₂O₂group. Compared with H₂O₂group, P-Akt protein expression was significantly increased in DZ group. Compared with DZ group,P-Akt protein expression was decreased in LY group, but there was nodifference between H₂O₂group and LY group.6.2Compared with normal control, caspase-3,9protein expression wassignificantly increased in H₂O₂group. Compared with H₂O₂group,caspase-3,9protein expression was significantly decreased in DZ group.Compared with DZ group, caspase-3,9protein expression was increased in LYgroup,however still lower than the H₂O₂group.Conclusions:1DZ can improve cell morphology, increase the cell survival rate,improve the level of mitochondrial membrane potential. So DZ exertedprotective effect on H₂O₂-damaged L6SkM2DZ pretreatment could promote H₂O₂-damaged L6SkM survival andinhibit apoptosis by activating the PI3K/Akt cell signaling pathway andreducing caspase-3,9protein expression.