不同缺血时间再灌注损伤对大鼠骨骼肌的影响

Effect of reperfusion injury following different ischemic duration on skeletal muscle in rats

目的探讨不同缺血时间再灌注损伤对大鼠骨骼肌的影响。方法选取35只雄性Wistar大鼠,采用单侧夹闭股动脉和压力绷带施压的方法构建下肢骨骼肌缺血再灌注损伤(IRI)模型。根据不同缺血时间分为2 h缺血24 h再灌注(I2R24组)、2.5 h缺血24 h再灌注(I2.5R24组)、3 h缺血24 h再灌注(I3R24组)、4 h缺血24 h再灌注(I4R24组)、假手术组,每组7只。在再灌注终点,收集腓肠肌组织和血浆进行分析。采用湿重/干重比值(W/D)评估组织水肿情况;3-(4,5-二甲基噻唑-2)-2,5二苯基四氮唑溴盐(MTT)检测组织活力;HE染色观察组织病理学变化;免疫荧光染色检测补体C1q和C3b/c沉积、凝血组织因子(TF)表达和纤维蛋白原(FN)沉积、缓激肽受体1(BR1)和BR2表达、内皮血管细胞黏附分子-1(VCAM-1)和E选择素表达、炎症纤维介素蛋白-2(FGL-2)和髓过氧化物酶(MPO)表达;ELISA法检测血浆干扰素-γ(IFN-γ)、白细胞介素-7(IL-7)、IL-18、巨噬细胞炎症蛋白-1α(MIP-1α)、单核细胞趋化蛋白-1(MCP-1)水平。结果延长缺血时间再灌注,组织水肿逐渐加重,I2R24组、I2.5R24组、I3R24组、I4R24组W/D分别为5.3±0.2、6.1±0.3、6.9±0.2、7.6±0.3,高于假手术组的4.5±0.1(P均<0.01)。组织活力逐渐降低,I2R24组、I2.5R24组、I3R24组、I4R24组分别为(62.4±3.5)%、(45.3±3.3)%、(35.4±3.4)%、(27.1±5.9)%,低于假手术组的(93.8±7.2)%(P均<0.01)。病理组织损伤逐渐加重,最重为I4R24组,有严重肌细胞损伤、间质水肿和大量炎性细胞浸润,余依次为I3R24组、I2.5R24组、I2R24组,假手术组肌细胞结构完整、排列整齐。免疫荧光染色提示C1q、C3b/c、FN、BR1、VCAM-1、E选择素、FGL-2水平逐渐升高,由低到高依次为假手术组、I2R24组、I2.5R24组、I3R24组、I4R24组。MPO阳性细胞数/高倍镜(×200)细胞总数的大体比例逐渐升高,从高到低依次为I4R24组、I3R24组、I2.5R24组、I2R24组、假手术组。而TF和BR2表达在各组间无明显改变。血浆IFN-γ、IL-7、IL-18、MIP-1α、MCP-1浓度随缺血时间延长均逐渐升高(P均<0.01),从低到高依次为假手术组、I2R24组、I2.5R24组、I3R24组、I4R24组(P均<0.01)。结论延长缺血时间再灌注增加补体、凝血、激肽、内皮细胞激活及炎症因子释放,从而加重大鼠骨骼肌组织损伤。

Objective To investigate effect of reperfusion injury following different ischemic duration on skeletal muscle in rats.Methods A model of ischemia/reperfusion injury(IRI)was established by unilateral clamping femoral artery and additional application of tourniquet in skeletal muscle of hind limbs in 35 male Wsitar rats.According to different ischemia time,the animals were assigned to 2-hour ischemia and 24-hour reperfusion(I2R24 group),2.5-hour ischemia and 24-hour reperfusion(I2.5R24 group),3-hour ischemia and 24-hour reperfusion(I3R24 group),4-hour ischemia and 24-hour reperfusion(I4R24 group)and sham group,with 7 rats per group.At the end of reperfusion,gastrocnemious tissues and plasma samples were collected and analyzed.The ratio of wet∶dry weight(W/D)was used to measure muscle edema.The assay of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)was conducted to evaluate muscle viability.HE staining was executed to observe histopathological changes.Immunofluorescence staining was performed to assess the levels of C1q,C3b/c,tissue factor(TF),fibrinogen(FN),bradykinin receptor 1(BR1),BR2,vascular cell adhesion molecule-1(VCAM-1),E-selectin,fibrinogen-like protein-2(FGL-2)and myeloperoxidase(MPO)in muscle tissues.ELISA method was used to determine the concentrations of interferonγ(IFN-γ),interleukin7(IL-7),IL-18,macrophage inflammatory1α(MIP-1α)and monocyte chemotactic protein 1(MCP-1)in plasma.Results With prolongation of ischemia time and subsequent reperfusion,tissue edema became severe gradually.The ratio of W/D was 5.3±0.2,6.1±0.3,6.9±0.2,7.6±0.3 in I2R24,I2.5R24,I3R24 and I4R24 groups,higher than that in sham group(4.5±0.1)(all P<0.01).Muscle viability got decreased gradually.Muscle viability was(62.4±3.5)%,(45.3±3.3)%,(35.4±3.4)%,(27.1±5.9)%in I2R24,I2.5R24,I3R24 and I4R24 groups,lower than that in sham group[(93.8±7.2)%](all P<0.01).Histopathological changes became aggravated gradually.The most severe group was I4R24 group,with the most severe myocyte injury,interstitial edema and extensive inflammatory infiltration,followed by I3R24,I2.5R24 and I2R24 groups in order.There was normal structure integrity and neatly arranged myocyte in sham group.Meanwhile,levels of C1q,C3b,FN,BR1,VCAM-1,E-selectin and FGL-2 got increased gradually.The highest levels for these factors were seen in I4R24 group,followed by I3R24 group,I2.5R24 group,I2R24 group and sham group in order.The rough ratio of the number of positive MPO cells/total cell number under high lens(×200)were increased gradually,with the highest level in I4R24 group,followed by I3R24 group,I2.5R24 group,I2R24 group and sham group in order.However,expression of TF and BR2 were not altered significantly among the groups.Plasma levels of INF-γ,IL-7,IL-18,MIP-1αand MCP-1 elevated gradually with prolongation of ischemia time(all P<0.01).The sequence was the sham group,I2R24 group,I2.5R24 group,I3R24 group and I4R24 groups for levels of these factors from low to high(all P<0.01).Conclusion Reperfusion after prolongation of ischemia duration can increase the activation of complement,coagulation,kinin and endothelial cells as well as the release of inflammatory factors,and thus aggravate the degree of skeletal muscle tissue injury.