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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 investigate the effect of re-irrigation I injection on apoptosis of rat skeletal muscle cells after limb ischemia-reperfusion. METHODS: SD rat hind limb ischemia-reperfusion model was duplicated. Animals in the control group received no drug; in the IR group, 1 ml normal saline was injected intraperitoneally 12 hours before ischemia, ischemia, and reperfusion; 12 hours before ischemia, ischemia and reperfusion in the I-IR group. When the abdominal cavity was injected with resuscitation I injection 1ml. Each group of rats was cut at the corresponding time point about 1000mg right middle gastrocnemius muscles were fixed, dehydrated, embedded in slices, immunohistochemical staining by SABC method, measured by Bacillus, Bcl-MosisImage advanced3.1 image analysis system 2 protein gray value, and then quantitative analysis of Bax, Bcl-2 immunohistochemical positive reaction products. Results: (1) Bax immunohistochemistry results showed that there was no positive cell expression in the skeletal muscle tissue of the control group. Within 8 hours of reperfusion injury, the gray value at each time point in IR group and I-IR group increased with time. Gradually decreased, except for IR-2 group and I-IR-2 group, there was no significant difference between the control group and the control group (P>0.05). The difference between the other groups and the control group was statistically significant (P<0.01). However, the gray values of each I-IR group at the corresponding time point were higher than that of the IR group, except for 2h, there was no statistically significant difference (P>0.05), the difference was statistically significant at all other time points. (P<0.05); (2) The results of Bcl-2 immunohistochemistry showed that no positive cells were expressed in the control group, and the expression was most pronounced at 8 hours after reperfusion injury, and then gradually decreased with time. The degree value gradually increased compared with the control group, the difference was statistically significant (P<0.01). Compared with IR group, the positive expression of I-IR group was slightly higher than that of IR group. Except for 2 hours, there were significant differences at other time points (P<0.01). (3) Bcl-2/Bax result showed : Within 8 hours of reperfusion, the ratio gradually increased with time, I-IR groups were lower than IR group at the same time point, except for the 2h time point, the difference was statistically significant at other time points ( P<0.05), indicating that re-irrigation I injection has anti-apoptotic effect on skeletal muscle in ischemia-reperfusion injury. Conclusion: Re-irrigation I injection can reduce the expression of Bax and increase the expression of Bcl-2, which can reduce the apoptosis of skeletal muscle cells and protect the skeletal muscle of limb ischemia-reperfusion injury.