不同缺血预适应模式对大鼠骨骼肌缺血再灌注损伤的影响

Effect of different ischemia preconditioning modes on ischemia-reperfusion injury of skeletal muscle in rats

目的探讨不同缺血预适应(IPC)模式对大鼠骨骼肌缺血再灌注(IR)损伤的影响。方法选取40只雄性SD大鼠, 采用动脉夹夹闭一侧股动脉的方法构建大鼠骨骼肌IR损伤模型。IPC模式采用夹闭右侧股动脉10 min后松开动脉夹再灌注10 min, 重复3次进行预适应。按随机数字表法将大鼠分为常规IR组、IPC即刻组、IPC24 h组、IPC48 h组和假手术组, 每组8只。常规IR组夹闭右股动脉3 h后松开动脉夹再灌注3 h;IPC即刻组为预适应后即刻行常规IR组相同处理;IPC24 h组为预适应后缝合皮肤, 放回笼中喂养24 h后行常规IR组相同处理;IPC48 h组为预适应后缝合皮肤, 放回笼中喂养48 h后行常规IR组相同处理;假手术组钝性分离右股动脉但不夹闭。在再灌注终点, 收集胫前肌组织、腓肠肌组织和血清;计算胫前肌组织湿重/干重比值(W/D), 评估组织水肿情况;HE染色后观察腓肠肌组织病理学变化情况并对腓肠肌组织损伤程度进行评分;ELISA法检测血清肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和丙二醛(MDA)水平;实时荧光定量PCR(qRT-PCR)检测腓肠肌组织中低氧感受器(EGLN1)、缺氧诱导因子-1α(HIF-1α)的表达情况。结果不同模式IPC后, 组织水肿程度均较常规IR组减轻(P均<0.01), 常规IR组、IPC即刻组、IPC24 h组、IPC48 h组W/D分别为6.05±0.19、5.70±0.12、5.25±0.13、5.50±0.08, 高于假手术组的3.80±0.08(P均<0.01);与IPC即刻组、IPC48 h组比较, IPC24 h组水肿程度最轻(P<0.05或0.01)。假手术组肌纤维排列整齐, 结构清晰, 余各组肌纤维均出现不同程度损伤和炎性浸润。IPC即刻组、IPC24 h组、IPC48 h组的组织损伤程度评分总分分别为(8.15±0.15)分、(6.15±0.38)分、(6.90±0.19)分, 低于常规IR组的(9.60±0.50)分, 高于假手术组的(0.16±0.16)分(P均<0.01);与IPC即刻组比较, IPC24 h组、IPC48 h组组织损伤程度评分总分降低(P<0.05或0.01), 但IPC24 h组与IPC48 h组组织损伤程度评分总分差异无统计学意义(P>0.05)。ELISA法检测显示, 不同模式IPC后, 血清TNF-α、IL-1β、MDA水平较假手术组升高(P均<0.01), 从高到低依次为常规IR组、IPC即刻组、IPC48 h组、IPC24 h组、假手术组;与IPC即刻组、IPC48 h组比较, IPC24 h组IL-1β、TNF-α、MDA水平降低(P均<0.05)。qRT-PCR检测显示, EGLN1 mRNA表达由高到低依次为常规IR组、IPC48 h组、假手术组、IPC即刻组、IPC24 h组;与常规IR组、IPC即刻组、IPC48 h组比较, IPC24 h组EGLN1 mRNA表达下降(P均<0.01)。不同模式IPC后, HIF-1α mRNA表达较假手术组增加(P均<0.01), 由高到低依次为IPC24 h组、IPC即刻组、IPC48 h组、常规IR组、假手术组;与常规IR组、IPC即刻组、IPC48 h组比较, IPC24 h组HIF-1α mRNA表达升高(P<0.05或0.01), IPC即刻组与IPC48 h组差异无统计学意义(P>0.05)。结论不同缺血预适应模式能够通过减轻组织水肿、炎性症状和氧化应激反应等, 减轻大鼠骨骼肌IR损伤, 其中IPC后再灌注24 h模式减轻IR损伤的效果相对最佳;EGLN1、HIF-1α可能参与IPC减轻大鼠骨骼肌IR损伤的过程。

Objective To investigate the effect of different ischemic preconditioning(IPC)modes on ischemia-reperfusion(IR)injury of skeletal muscle in rats.Methods Forty male SD rats were selected to construct the model of IR injury of skeletal muscle by clamping one side of the femoral artery.In the IPC mode,the right femoral artery was clipped for 10 minutes,and reperfusion ensued for 10 minutes after the release of artery clamp.Such preconditioning procedure was repeated 3 times.The rats were divided into conventional IR group,IPC immediate group,IPC24-hour group,IPC48-hour group and sham operation group according to the random number table method,with 8 rats in each group.In conventional IR group,the right femoral artery was clipped for 3 hours and then the artery clamp was released to allow reperfusion for 3 hours.In IPC immediate group,the same treatment as conventional IR group was performed immediately after preconditioning.In IPC24-hour group,the rats received skin suturing after preconditioning and then fed in a cage for 24 hours before the same treatment as conventional IR group.In IPC48-hour group,the rats received skin suturing after preconditioning and then fed in a cage for 48 hours before the same treatment as conventional IR group.In sham operation group,the right femoral artery was bluntly separated but not clipped.At the end of reperfusion,the tibialis anterior muscle tissue,gastrocnemius muscle tissue and serum were collected.The ratio of wet weight to dry weight(W/D)of the tibialis anterior muscle tissue was calculated to evaluate tissue edema.After HE staining,histopathological changes of gastrocnemius muscle were observed and the injury severity of gastrocnemius tissue was scored.Serum levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and malondialdehyde(MDA)were determined by ELISA.Expressions of hypoxia receptor(EGLN1)and hypoxia-inducible factor-1α(HIF-1α)in the gastrocnemius tissue were detected by real-time fluorescence quantitative PCR(qRT-PCR).Results The degree of tissue edema was reduced under different IPC modes when compared with conventional IR group(all P<0.01).The W/D in conventional IR group,IPC immediate group,IPC24-hour group and IPC48-hour group were 6.05±0.19,5.70±0.12,5.25±0.13 and 5.50±0.08,higher than 3.80±0.08 in sham operation group(all P<0.01).IPC24-hour group had the least degree of edema when compared with IPC immediate group and IPC48-hour group(P<0.05 or 0.01).The muscle fibers in sham operation group were neatly arranged with clear structure,and the muscle fibers in other groups showed different degrees of injury and inflammatory infiltration.The total scores for tissue injury severity in IPC immediate group,IPC24-hour group and IPC48-hour group were(8.15±0.15)points,(6.15±0.38)points and(6.90±0.19)points,lower than(9.60±0.50)points in conventional IR group and higher than(0.16±0.16)points in sham operation group(all P<0.01).The total scores for tissue injury severity in IPC24-hour group and IPC48-hour group were not significantly different(P>0.05),but both decreased when compared with IPC immediate group(P<0.05 or 0.01).ELISA showed that the levels of TNF-α,IL-1βand MDA under different IPC modes were higher than those in sham operation group(all P<0.01),and the order from high to low was conventional IR group,IPC immediate group,IPC48-hour group,IPC24-hour group and sham operation group.Levels of IL-1β,TNF-αand MDA in IPC24-hour group were decreased when compared with IPC immediate group and IPC48-hour group(all P<0.05).qRT-PCR showed that the expression of EGLN1 mRNA from high to low was conventional IR group,IPC48-hour group,sham operation group,IPC immediate group and IPC24-hour group and that the expression of EGLN1 mRNA in IPC24-hour group decreased when compared with conventional IR group,IPC immediate group and IPC48-hour group(all P<0.01).The expression of HIF-1αmRNA increased under different IPC modes when compared with sham operation group(all P<0.01),and the order from high to low was IPC24-hour group,IPC immediate group,IPC48-hour group,conventional IR group and sham operation group.The expression of HIF-1αmRNA in IPC24-hour group increased when compared with conventional IR group,IPC immediate group and IPC48-hour group(P<0.05 or 0.01),but there was no significant difference between IPC immediate group and IPC48-hour group(P>0.05).Conclusions Different ischemic preconditioning modes can reduce IR injury of skeletal muscle in rats by reducing tissue edema,inflammatory symptoms and oxidative stress response,among which reperfusion 24 hours after IPC has the best effect on IR injury.EGLN1 and HIF-1αmay be involved in IPC to alleviate IR injury of skeletal muscle in rats.