天麻素通过调节PI3K/AKT信号通路减轻大鼠坐骨神经离断吻合后的损伤

Gastrodin alleviate nerve injury in rats of anastomosis of transected sciatic nerve via regulating PI3K/AKT signaling pathway

目的观察天麻素(Gas)对坐骨神经离断吻合模型大鼠神经功能修复的影响,并初步探讨其作用机制。方法32只雄性SD大鼠随机分为对照组(Control)、模型组(Model)、天麻素组(Gas)和甲钴胺组(Cob)。除Control组外,均距梨状肌下缘1 cm处锐性离断左侧坐骨神经,随即使用9-0显微外科缝合线进行端端吻合以建立坐骨神经离断吻合模型。Gas组每天灌胃给予Gas(15 mg/kg)1次,Cob组每天灌胃给予Cob(0.15 mg/kg)1次,Control组和Model组每天灌胃给予等体积双蒸水。60 d后,采用坐骨神经指数(SFI)观察坐骨神经功能恢复情况;计算腓肠肌湿重恢复率;HE与Masson染色观察坐骨神经远端有髓神经纤维的形态学变化,计数完整有髓神经纤维数;HE染色观察腓肠肌的形态学变化,统计腓肠肌横截面积;Western blot检测腓肠肌中PI3K、AKT和mTOR蛋白水平;药物亲和反应的靶点稳定性(Darts)实验检测Gas与PI3K p110 gamma的相互作用。结果与Control组相比,Model组SFI明显降低(P<0.01),腓肠肌萎缩严重,湿重显著降低(P<0.01);与Model组相比,Gas组和Cob组SFI明显升高(P<0.01,P<0.05),腓肠肌湿重增加;HE与Masson染色显示Gas和Cob可以改善坐骨神经离断所致的神经纤维排列稀疏,连续性差,增加了有髓神经纤维数量;HE染色显示Gas和Cob增加了腓肠肌纤维横截面积(P<0.01);WB结果显示,Gas和Cob增加了腓肠肌中PI3K、AKT和mTOR蛋白水平(P<0.05);药物亲和反应的靶点稳定性(Darts)实验显示,Gas可以与PI3K p110 gamma结合(P<0.01)。结论Gas通过靶向结合PI3K,上调PI3K/AKT/mTOR信号通路,发挥促进大鼠坐骨神经损伤后修复的作用。

Objective To investigate the effect of gastrodin(Gastrodin,Gas)on the nerve function repair of sciatic nerve dissection anastomosis model rats and preliminarily explore its possible mechanism.Methods Thirty-two male SD rats were randomly divided into 4 groups:control group,model group,gas group and mecobalamin group.Except for the control group,transection of the left sciatic nerves conducted 1 cm under the piriformis muscle was performed in all animals,and then end-to-end anastomosis was performed with 9-0 microsurgical suture to establish the sciatic nerve dissection anastomosis model.The gas group was given gas(15 mg/kg)once a day by gavage,the mecobalamin group was given mecobalamin(0.15 mg/kg)once a day by gavage,and the control group and model group were given double distilled water of equal volume every day.After 60 days,the functional recovery was evaluated by sciatic functional index(SFI);Calculation of wet weight recovery rate of gastrocnemius muscle;Morphological changes of the distal myelinated nerve fibers of the sciatic nerve were observed by HE and Masson staining,and the number of intact myelinated nerve fibers was counted;Morphological changes of the gastrocnemius muscle were observed by HE staining,and the cross-sectional area of the gastrocnemius muscle was counted;The expression of PI3K,AKT and mTOR protein in the gastrocnemius muscle was detected by Western blot;Finally,Drug affinity responsive target stability(Darts)assay was performed to detect the interaction of Gas with PI3K p110 gamma.Results Compared with the control group,the model group had significantly lower SFI(P<0.01),severe atrophy of gastrocnemius muscle and lower wet weight(P<0.01).compared with the model group,the Gas and mecobalamin groups had significantly higher SFI(P<0.01,P<0.05)and increased wet weight of gastrocnemius muscle;HE and Masson staining showed that Gas and mecobalamin improved the sparse arrangement and poor continuity of nerve fibers due to sciatic nerve dissection and increased number of myelinated nerve fibers;HE staining showed that Gas and mecobalamin increased the cross-sectional area of gastrocnemius fibers(P<0.01);WB results showed that Gas and mecobalamin increased the protein expression of PI3K,AKT and mTOR in gastrocnemius muscle(P<0.05);In additions,the Drug Affinity Responsive Target Stability(Darts)showed that Gas could bind to PI3K p110 gamma(P<0.01).Conclusion By targeting PI3K/AKT/mTOR signaling pathway,Gas promotes the repairement after sciatic nerve injury in rats.