白果内酯对大鼠骨骼肌细胞缺血再灌注损伤的作用

Role of bilobalide in ischemia reperfusion injury of skeletal muscle cells in rats

目的探讨白果内酯对大鼠骨骼肌细胞缺血再灌注损伤(IRI)的作用。方法建立大鼠IRI模型:利用缺氧孵箱培养细胞4 h后,在常规孵箱继续培养4 h,按照随机数字表法分为对照组、缺血再灌注组和白果内酯组。对照组常规孵箱培养L6大鼠骨骼肌细胞;缺血再灌注组利用缺氧孵箱和常规孵箱交替培养L6大鼠骨骼肌细胞;白果内酯组在缺氧孵箱培养L6大鼠骨骼肌细胞4 h,更换为含有20μmol/L白果内酯的培养基,继而放入常规孵箱继续培养48 h。CCK-8法检测细胞活性,利用分光光度计检测各组上清液中乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)、黄嘌呤氧化酶(XOD)、丙二醛(MDA)水平和细胞内Ca^2+水平。结果与缺血再灌注组[(62.2±6.8)%]相比,白果内酯组细胞活性[(86.5±5.6)%]增强(P<0.05);与缺血再灌注组[(265.3±35.2)U/mg]相比,白果内酯组LDH水平[(125.4±16.9 U/mg)]下降(P<0.05);与缺血再灌注组[(68.6±10.7)U/mg]相比,白果内酯组SOD水平[(98.5±8.4)U/mg]上升(P<0.05);与缺血再灌注组[(224.7±65.7)U/mg]相比,白果内酯组XOD水平[(105.9±58.9)U/mg]下降(P<0.05);与缺血再灌注组[(198.2±35.7)U/mg]相比,白果内酯组MDA水平[(100.4±25.3)U/mg]下降(P<0.05);与缺血再灌注组[(2.5±0.3)]相比,白果内酯组Ca^2+水平[(1.6±0.3)]降低(P<0.05);但各指标均未恢复至对照组水平(P<0.05)。结论白果内酯能通过抑制氧化应激反应和钙超载发生,减轻IRI。

Objective To examine the effects of bilobalide on ischemia reperfusion injury(IRI)of skeletal muscle cells in rats.Methods IRI of skeletal muscle cells in L6 rats was stimulated by culturing skeletal muscle cells in a hypoxia incubator for 4 hours,which were further cultured in a conventional incubator for 4 hours.The induced rat myoblasts were randomly divided into control group,ischemia reperfusion group and bilobalide group.Rat myoblasts in control group were cultured in the conventional incubator,and cells in ischemia reperfusion group were cultured in hypoxia incubator and conventional incubator alternatively to simulate IRI of skeletal muscle cells.After 4 hours of incubation in hypoxic incubator,L6 rat myoblasts in bilobalide group were replaced with medium containing 20μmol/L bilobalide,and then put into conventional incubator for further culture.CCK-8 assay was used to test cell viability 48 hours later.Related test kits were used to examine the levels of lactate dehydrogenase(LDH),superoxide dismutase(SOD),xanthine oxidase(XOD),malondialdehyde(MDA)and intracellular calcium.Results Compared with ischemia reperfusion group[(62.2±6.8)%],the viability of myoblasts was increased in bilobalide group[(86.5±5.6)%](P<0.05).Compared with ischemia reperfusion group[(265.3±35.2)U/mg],the level of LDH in myoblasts was decreased in bilobalide group[(125.4±16.9)U/mg](P<0.05).Compared with ischemia reperfusion group[(68.6±10.7)U/mg],the level of SOD in myoblasts was increased in bilobalide group[(98.5±8.4)U/mg](P<0.05).Compared with ischemia reperfusion group[(224.7±65.8)U/mg],the level of XOD in myoblasts was decreased in bilobalide group[(105.9±58.9)U/mg](P<0.05).Compared with ischemia reperfusion group[(198.2±35.7)U/mg],the level of MDA in myoblasts was decreased in bilobalide group[(100.4±25.3)U/mg](P<0.05).Compared with ischemia reperfusion group(2.5±0.3),the intracellular Ca^2+ concentration in cell supernatant was decreased in bilobalide group(1.6±0.3)(P<0.05).But all the indexes tested above did not return to the level in control group(P<0.05).Conclusion Bilobalide can protect skeletal muscle cells from IRI by inhibiting oxidative stress reaction and calcium overload.