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目的探讨雷公藤多甙对异体神经移植后靶肌肉萎缩和细胞凋亡的影响。方法20只雄性Wistar大鼠为供体,体重200～250g,取双侧坐骨神经实验。50只雄性SD大鼠为受体,体重200～250g。将受体大鼠制备右侧坐骨神经10mm缺损模型后,随机分为A、B、C、D、E组(n=10):A、B组为新鲜异体神经移植组,C、D组为异体神经雷公藤多甙预处理后再移植组,E组为自体神经移植组。神经移植术后第2天A、E组予生理盐水,B、D组予雷公藤多甙5.0mg/(kg·d),C组予雷公藤多甙2.5mg/(kg·d),时间均为5周。术后3、6周行大体观察、骨骼肌HE染色观察,采用Image-ProPlusv5.2图像处理系统分析肌纤维直径,透射电镜观察骨骼肌超微结构,免疫组织化学检测Bax、Bcl-2表达,TUNEL法检测骨骼肌细胞凋亡。结果术后大鼠全部存活至实验完成。大体观察发现术后3周各组大鼠胫骨前肌均不同程度萎缩;术后6周A组肌肉萎缩严重,其余各组肌肉萎缩呈好转趋势。A组肌湿重、肌纤维直径及Bcl-2表达均显著低于B、C、D、E组(P<0.01),B、C、D组低于E组(P<0.05),B、C、D组间比较差异无统计学意义(P>0.05);A组细胞凋亡指数和Bax表达显著高于B、C、D、E组(P<0.01),B、C、D组高于E组(P<0.05),B、C、D组间差异无统计学意义(P>0.05)。术后3周,光镜下各组肌纤维变细;透射电镜示肌质网不同程度扩张。术后6周,A组光镜示肌纤维间隙增宽、结缔组织明显增生;透射电镜示肌小节结构消失,肌丝溶解严重和残存“Z”线片段;其余组肌原纤维均排列整齐,明带、暗带和肌小节结构清晰。结论异体神经移植术后予雷公藤多甙能减轻靶肌肉萎缩和细胞凋亡,供体神经经雷公藤多甙预处理能减少异体神经移植术后受体免疫抑制剂用量。
Objective To investigate the effects of Tripterygium wilfordii on target muscle atrophy and apoptosis after allograft nerve transplantation. Methods Twenty male Wistar rats were used as donors, and the body weight was 200-250 g. Bilateral sciatic nerve experiments were performed. Fifty male Sprague-Dawley rats were recipients weighing 200-250 g. After the right sciatic nerve 10mm defect model was prepared in recipient rats, they were randomly divided into groups A, B, C, D, and E (n=10): fresh allograft group A and B were grouped, and allograft C and D were allogeneic. Nerve Tripterygium wilfordii was pretreated before transplantation and E group was autologously transplanted. On the 2nd day after nerve transplantation, rats in group A and group E received normal saline, rats in group B and D received Tripterygium wilfordii 5.0 mg/(kg·d), and rats in group C received 2.5 mg/(kg·d) of tripterygium wilfordii. All are 5 weeks. Gross observation and skeletal muscle HE staining were performed at 3 and 6 weeks after operation. The diameter of muscle fibers was analyzed by Image-ProPlus v5.2 image processing system. Ultrastructure of skeletal muscle was observed by transmission electron microscope, and Bax and Bcl-2 expression was detected by immunohistochemistry. The method detects skeletal muscle cell apoptosis. Results All rats survived until the completion of the experiment. Gross observation showed that the anterior tibial muscles of the rats in each group were atrophied in different degrees at 3 weeks postoperatively. Muscular atrophy was severe in group A at 6 weeks postoperatively. Muscular atrophy showed a trend of improvement in the other groups. Muscle wet weight, muscle fiber diameter, and Bcl-2 expression in group A were significantly lower than those in group B, C, D, and E (P<0.01), while those in group B, C, and D were lower than those in group E (P<0.05). There was no significant difference between group D and group D (P>0.05). The apoptosis index and Bax expression in group A were significantly higher than those in group B, C, D, and E (P<0.01), and higher in group B, C, and D. E group (P <0.05), there was no significant difference between B, C, D group (P> 0.05). At 3 weeks after operation, the muscle fibers of each group became thinner under light microscope; transmission electron microscopy showed that the sarcoplasmic reticulum expanded in different degrees. At 6 weeks after operation, group A showed a widening of the intermuscular space and a clear hyperplasia of connective tissue; transmission electron microscopy showed that the sarcomere structure disappeared and the myofilament dissolved and the remaining “Z” line segment was observed; the rest of the myofibrils were arranged neatly. The clear bands, dark bands, and sarcomere are clear. Conclusion Allograft tripterygium can reduce target muscle atrophy and apoptosis after allogeneic neurotransplantation. Donor nerve preconditioning with tripterygium wilfordii can reduce the dose of receptor immunosuppressive agent after allograft nerve transplantation.