糖络宁调控细胞骨架干预糖尿病周围神经病变脱髓鞘的作用机制

Mechanism of Tangluoning regulating cytoskeleton intervention on demyelination of diabetes peripheral neuropathy

目的 基于PDZ和LIM结构域蛋白7(PDZ and LIM domain protein 7,Pdlim7)和突触极蛋白(synaptopodin-2,Synpo2)调控的丝状肌动蛋白(filamentous actin, F-actin)探讨糖络宁改善糖尿病周围神经病变(diabetic peripheral neuropathy, DPN)脱髓鞘的干预作用及机制。方法 采用腹腔注射链脲佐菌素建立高血糖大鼠模型。将SD大鼠随机分为空白组、模型组、阳性药组(α-硫辛酸,20 mg/kg)和糖络宁组(10.9 g/kg),每组12只。干预12周后,观察透射电镜观察大鼠坐骨神经超微结构并统计髓鞘相对面积和G-ratio;检测鼠尾热痛阈值与坐骨神经神经传导速度,观察各组大鼠神经功能;4D-label free蛋白质组学联合生物信息学分析筛选DPN大鼠坐骨神经的差异性蛋白。采用蛋白免疫印迹法与免疫荧光法检测坐骨神经细胞骨架相关胶质纤维酸性蛋白、微管蛋白、F-actin和Pdlim7、Synpo2的表达。结果 与空白组比较,模型组坐骨神经髓鞘相对面积和G-ratio数值降低(P<0.05),热痛阈值增加(P<0.01),运动和感觉神经传导速度均降低(P<0.01);与模型组比较,糖络宁组髓鞘相对面积和G-ratio数值显著升高(P<0.01),热痛阈值降低(P<0.01),运动和感觉神经传导速度均增加(P<0.01)。蛋白质组学显示,糖络宁干预12周后,DPN大鼠坐骨神经差异蛋白多与细胞骨架、肌动蛋白结合及PDZ结构域和LIM结构域蛋白相关。与空白组比较,模型组坐骨神经中F-actin、Pdlim7和Synpo2表达降低(P<0.05,P<0.01,P<0.01);与模型组比较,糖络宁组F-actin、Pdlim7和Synpo2表达明显升高(P<0.01)。共定位结果表明,与空白组比较,模型组髓鞘上F-actin表达显著降低(P<0.01),与F-actin共定位的Pdlim7表达明显降低(P<0.01);与模型组比较,糖络宁组髓鞘上F-actin表达显著升高(P<0.01),与F-actin共定位的Pdlim7表达明显升高(P<0.01)。结论 糖络宁改善DPN坐骨神经脱髓鞘与调控细胞骨架相关,机制与增加Pdlim7和Synpo2的表达,进而增加细胞骨架蛋白F-actin生成而促进髓鞘生成有关。

Objective To explore the intervention effect and mechanism of Tangluoning in improving demyelination of diabetes peripheral neuropathy(DPN)based on the filamentous actin(F-actin)regulated by PDZ and LIM domain protein 7(Pdlim7)and Synaptopodin-2(Synpo2).Methods The hyperglycemic rat model was established by intraperitoneal injection of streptozotocin(STZ).SD rats were randomly divide into the blank group,the model group,the positive drug group(α-Lipoic acid,20 mg/kg)and the Tangluoning group(10.9 g/kg),each group has 12 mice.After 12 weeks of intervention,the ultrastructure of the sciatic nerve in rats was observed by TEM,and the relative area of myelin sheath and G-ratio were statistically analyzed.Detect the thermal pain threshold of the rat tail and sciatic nerve conduction velocity to observe nerve function.Using 4D label free proteomics combined with bioinformatics analysis to screen for differential proteins in the sciatic nerve of DPN rats.Western blot and immunofluorescence were used to detect the expression of glial fibrillary acidic protein,tubulin,and F-actin related to the cytoskeleton of sciatic nerve and the expression of Pdlim7 and Synpo2.Results Compared with the blank group,the relative area and G-ratio of the sciatic nerve myelin sheath in the model group was decreased(P<0.05),the thermal pain threshold was increased(P<0.01),and motor nerve conduction velocity and sensory nerve conduction velocity was decreased(P<0.01).Compared with the model group,the relative area and G-ratio of myelin sheaths in the Tangluoning group was significantly increased(P<0.01),thermal pain threshold was decreased(P<0.01),and motor nerve conduction velocity and sensory nerve conduction velocity was increased(P<0.01).The results of proteomics showed that after 12 weeks of intervention with Tangluoning,the differential proteins in the sciatic nerve of DPN rats were mostly related to the cytoskeleton,actin binding,as well as PDZ domain and LIM domain proteins.Compared with the blank group,the expression of F-actin,Pdlim7,and Synpo2 in the sciatic nerve of the model group was decreased(P<0.05,P<0.01,P<0.01).Compared with the model group,the Tangluoning group showed significantly increased expression of F-actin,Pdlim7,and Synpo2(P<0.01).Co-localization results showed that compared with the blank group,the expression of F-actin on the myelin sheath of the sciatic nerve in the model group was decreased(P<0.01),and the expression of Pdlim7 co-localized with F-actin was significantly decreased(P<0.01).Compared with the model group,the Tangluoning group showed an significant increase in F-actin expression on the myelin sheath(P<0.01),and the expression of Pdlim7 co-localized with F-actin was significantly increased(P<0.01).Conclusion The effect of Tangluoning on improving demyelination of DPN sciatic nerve is related to regulating the cytoskeleton,and its mechanism is related to increasing the expression of Pdlim7 and Synpo2,thereby increasing the production of cytoskeletal protein F-actin and promoting myelination.