活血通络方调控Rho/ROCK通路对脑卒中大鼠肢体运动功能恢复的影响

Effect of Huoxue Tongluo Recipe on the Recovery of Limb Motor Function in Stroke Rats by Regulating Rho/ROCK Pathway

目的:探讨活血通络方对脑卒中大鼠前肢运动功能的影响,及对肉瘤同源基因A(RhoA)/Rho相关螺旋卷曲蛋白激酶(ROCK)通路的调控作用,初步探讨其影响肢体运动功能恢复的机制。方法:取Wistar大鼠,用中动脉线栓阻塞法复制大鼠脑卒中模型,并随机分为假手术组、模型组、活血通络方低剂量组(8.68 g/kg)、高剂量组(34.72 g/kg)、RhoA激动剂(LPA,5 nmol/kg)组、活血通络方+LPA组(活血通络方34.72 g/kg+LPA 5 nmol/kg),每组20只。各组于术后1周开始给药,术后5周后,足失误试验及走横木试验评价大鼠肢体运动功能恢复状况;生物素化葡聚糖胺(BDA)法染色结合生长相关蛋白(GAP-43)免疫荧光法检测颈髓纤维束走行变化;蛋白免疫印迹法(Western Blot)检测颈髓组织RhoA、ROCK2、髓鞘相关阻断因子(Nogo-A)、髓鞘相关糖蛋白(MAG)等蛋白表达。结果:与假手术组相比,模型组大鼠足失误比率升高,走横木评分降低,大鼠出现肢体运动功能障碍。术后5周给药结束后,模型组大鼠肢体运动功能较术后1周有所恢复,损伤侧与未损伤侧颈髓组织BDA、GAP-43阳性表达及未损伤侧颈髓组织RhoA、ROCK2、Nogo-A、MAG蛋白表达降低(P<0.05)。活血通络方低剂量组、高剂量组大鼠肢体运动功能恢复好于模型组,未损伤侧颈髓组织BDA、GAP-43阳性表达及RhoA、ROCK2、Nogo-A、MAG蛋白表达较模型组进一步降低,损伤侧颈髓组织BDA、GAP-43阳性表达较模型组进一步升高(P<0.05)。LPA组大鼠肢体运动功能恢复最差,上述指标变化与模型组相反(P<0.05)。活血通络方+LPA组肢体运动功能恢复低于活血通络方高剂量组(P<0.05)。结论:活血通络方可能通过抑制脑卒中未损伤侧皮质脊髓束RhoA/ROCK2通路活化,促进皮质脊髓束异常路径重塑,发挥促进肢体运动功能恢复的作用。

Objective:To investigate the effect of Huoxue Tongluo Recipe on the motor function of the forelimbs in stroke rats,and the Ras homolog gene family member A(RhoA)/Rho-associated coiled-coil containing kinase(ROCK)pathway.Methods:Wistar rats were randomly divided into sham operation group,model group,Huoxue Tongluo Recipe low-(8.68 g/kg),high-(34.72 g/kg)dose groups,LPA group(RhoA agonist,5 nmol/kg),Huoxue Tongluo Recipe+LPA(Huoxue Tongluo Recipe 34.72 g/kg+LPA 5 nmol/kg)group,with twenty rats in each group.The rat stroke model was replicated by the middle artery thread occlusion method.The rats in each group were given drugs one week after the operation,and 5 weeks after the operation,the foot error test and the walking cross-wood test were used to evaluate the recovery of the rat′s limb motor function.Biotinylated dextran amine(BDA)staining combined with growth-associated protein(GAP-43)immunofluorescence method was used to detect cervical spinal cord fiber bundles.Western Blot was used to detect the expression of RhoA,ROCK2,myelin production inhibitors-myelin-associated blocking factor(Nogo-A),and myelin-associated glycoprotein(MAG)in cervical spinal cord tissue.Results:Compared with sham operation group,the foot error rate of rats in model group was increased,the walking crossbar score was decreased,and the rats developed limb motor dysfunction.Five weeks after operation(the end of the administration),the motor function of the limbs of the rats in model group was recovered compared with that one week after the operation,the positive expression of BDA and GAP-43 in the injured and uninjured cervical spinal cord tissue,and the expression of RhoA,ROCK2,Nogo-A,and MAG proteins in the uninjured cervical spinal cord tissue were decreased(P<0.05).The motor function of rats in Huoxue Tongluo Recipe low-and high-dose groups was recovered better than that of the model group,and the positive expressions of BDA and GAP-43 and the expression of RhoA,ROCK2,Nogo-A,and MAG proteins in the uninjured cervical spinal cord tissue were further lower compared with those of model group,the positive expressions of BDA and GAP-43 in the injured cervical spinal cord tissue were further increased compared with those of model group(P<0.05).The recovery of limb motor function of rats in LPA group was the worst,and the changes in the above indexes were opposite to those in model group(P<0.05).The recovery of limb motor function in Huoxue Tongluo Recipe+LPA group was lower than that of Huoxue Tongluo Recipe high-dose group,and the changes in above indexes were opposite to those of the Huoxue Tongluo Recipe high-dose group(P<0.05).Conclusion:Huoxue Tongluo Recipe may inhibit the activation of the RhoA/ROCK2 pathway of the corticospinal tract on the uninjured side of stroke,promote the remodeling of the abnormal path of the corticospinal tract,and the recovery of limb motor function.