复灌Ⅰ号注射液对大鼠肢体缺血-再灌注损伤骨骼肌细胞凋亡的影响研究

The Influence of Fuguan Injection Ⅰ in Apoptosis of Rat Skeletal Muscle Cells Suffering from Ischemia-Reperfusion Injury

目的:探讨复灌Ⅰ号注射液对大鼠肢体缺血-再灌注骨骼肌细胞凋亡的影响。方法:复制SD大鼠后肢缺血-再灌注模型。对照组动物不给药物;IR组于造成缺血前12h、造成缺血时和再灌注时于腹腔内各注射生理盐水1ml;Ⅰ-IR组于造成缺血前12h、缺血时和再灌注时腹腔各注射复灌Ⅰ号注射液1ml。各组大鼠于相应时间点切取约1000mg右侧腓肠肌中部肌肉进行固定、脱水、包埋后切片,采用SABC法进行免疫组化染色,经Motic I mage advanced3.1图像分析系统测定Bax、Bcl-2蛋白灰度值,然后对Bax、Bcl-2免疫组化阳性反应产物进行定量分析。结果:(1)Bax免疫组化结果示:对照组骨骼肌组织中未见有阳性细胞表达,在再灌注损伤8h内,IR组与Ⅰ-IR组各时间点灰度值随时间的延长而逐渐下降,除IR-2组和Ⅰ-IR-2组与对照组比较差异无统计学意义(P>0.05),其余各组与对照组比,其差异均有统计学意义(P<0.01);但Ⅰ-IR各组在相应时间点上其灰度值均较IR组为高,除2h时比较差异无统计学意义(P>0.05)外,其余各时间点比较差异均有统计学意义(P<0.05);(2)Bcl-2免疫组化结果示:对照组未见阳性细胞表达,再灌注损伤8h内,2h时表达最为明显,其后随时间的延长其表达逐渐减弱,灰度值逐渐升高,与对照组相比,其差异有统计学意义(P<0.01)。与IR组相比,Ⅰ-IR组各时间点的阳性表达均稍高,除2h外,其他各时间点差异均有统计学意义(P<0.01);(3)Bcl-2/Bax结果示:再灌注8h内,随时间的延长,其比值逐渐增加,Ⅰ-IR各组在相同时间点上较IR各组为低,除2h时间点外,其他时间点其差异均有统计学意义(P<0.05),说明复灌Ⅰ号注射液在缺血-再灌注损伤中有抗骨骼肌细胞凋亡的作用。结论:复灌Ⅰ号注射液可以降低Bax的表达,升高Bcl-2的表达,从而减少骨骼肌细胞凋亡,对肢体缺血-再灌注损伤的骨骼肌起到一定的保护作用。

Objective：To study the influence of Fuguan Injection Ⅰ in the apoptosis of rat skeletal muscle cells suffered limb ischemia-reperfusion injury.Methods：After the rat models of ischemia-reperfusion（IR）injury on the hind limbs were established,they were divided into group IR-2,group IR-4 and group IR-8 according to the respective reperfusion time.The same was done to Fuguan InjectionⅠ treated rats as group I-IR-2,group I-IR-4 and group I-IR-8.At the points of 12 hours before ischemia,right at ischemia and reperfusion time,intraperitoneal injection of 1 ml normal sodium and the same amount of Fuguan Injection Ⅰwas respectively given to the model groups and treatment groups.At each corresponding time point,1000 mg muscle from the middle of the right gastrocnemius was taken for fixation,dehydration,paraffin imbedding,immunohistochemistry staining for measuring the expression of Bax and Bcl-2.Results：（1）Eight hours within reperfusion injury,the grey levels of Bax at each time point decreased with time extension.They were remarkably higher in Fuguan Injection Ⅰ groups than IR groups at each time point except 2 hours（P0.05）.（2）The same results were found in the expression of Bcl-2.（3）The ratio of Bcl-2 and Bax within 8 hours reperfusion injury increased the prolongation of time.There were statistical significant differences between IR groups and I-IR groups（P0.01）,except between the two 2 hour groups（P0.05）.Conclusion：Fuguan Injection Ⅰcan reduce the apoptosis of skeletal muscle cells and protect skeletal muscle suffered ischemia-reperfusion injury,which might be realized by lowering the expression of Bax and increasing the expression of Bcl-2.