通腑逐瘀法指导下抵当汤加减可抑制大鼠急性脊髓损伤后胶质瘢痕的形成

Jiajian Didang Tang inhibits glial scar formation in rats with acute spinal cord injury under the guidance of Tongfu Zhuyu therapy

背景:急性脊髓损伤具有较高的致残率和致死率,在急性期进行积极有效的治疗对预防继发性损害具有重要意义。目的:观察通腑逐瘀法指导下抵当汤加减治疗大鼠急性脊髓损伤后水肿和肢体功能恢复的疗效,探讨通腑逐瘀法治疗急性脊髓损伤的作用机制。方法:将60只SD大鼠随机分为假手术组、模型组、抵当汤组、TGN-020组以及抵当汤+TGN-020组,每组12只。假手术组行椎板切除术,不予脊髓打击,其余各组均采用纽约大学打击器制造急性脊髓损伤模型。造模成功后,假手术组和模型组不采取任何治疗措施,抵当汤组术后给予5.04 g/kg抵当汤灌胃;TGN-020组术后给予5 mg/kg TGN-020(水通道蛋白4特异性抑制剂)腹腔注射;抵当汤联合TGN-020组术后采用5 mg/kg TGN-020腹腔注射联合5.04 g/kg抵当汤灌胃处理,1次/d,持续7 d。分别于术后1,3,5,7天评估BBB评分和Reuter评分,术后第7天取材进行脊髓组织大体观察和含水率检测,苏木精-伊红染色观察组织受损程度,免疫荧光检测各组脊髓组织中水通道蛋白4和胶质原纤维酸性蛋白共表达细胞个数,Western Blot法检测各组脊髓组织中水通道蛋白4、胶质原纤维酸性蛋白、硫酸软骨素蛋白聚糖、增殖细胞核抗原蛋白的表达量。结果与结论:(1)行为学评分结果显示,与模型组相比,各用药组BBB评分均显著升高(P<0.05),Reuter评分均显著降低(P<0.05),且各用药组之间无显著差异(P>0.05);(2)脊髓组织含水率结果显示,与模型组相比,各用药组脊髓组织含水率均显著降低(P<0.05),且各用药组之间无显著差异(P>0.05);(3)苏木精-伊红染色结果显示,模型组脊髓组织结构离乱,大量瘢痕组织和脊髓空洞形成;与模型组相比,各用药组可见组织结构较完整,可见少量瘢痕组织增生和脊髓空洞,组织形态得到有效改善;(4)免疫荧光结果显示,与模型组相比,各用药组水通道蛋白4和胶质原纤维酸性蛋白共表达细胞数量较少,亮度较低,细胞肿胀程度减轻(P<0.05),且各用药组之间无显著差异(P>0.05);(5)Western Blot结果显示,与模型组相比,各用药组脊髓组织中水通道蛋白4、胶质原纤维酸性蛋白、硫酸软骨素蛋白聚糖、增殖细胞核抗原蛋白的表达均显著降低(P<0.05),且各用药组之间无显著差异(P>0.05);(6)提示通腑逐瘀法指导下抵当汤加减可促进急性脊髓损伤大鼠肢体功能恢复,其作用机制可能通过抑制水通道蛋白4表达减轻急性脊髓损伤大鼠损伤处水肿,从而有效抑制反应性星形胶质细胞的活化和增生,减少胶质瘢痕的形成。

BACKGROUND:Acute spinal cord injury is a disease with high disability and mortality.An active and effective treatment in the acute phase is of great significance to prevent secondary injury.OBJECTIVE:To investigate the mechanism underlying Tongfu Zhuyu therapy in the treatment of acute spinal cord injury through observing the therapeutic effect of Jiajian Didang Tang on edema and recovery of limb function after acute spinal cord injury in rats.METHODS:A total of 60 Sprague-Dawley rats were randomly divided into sham group,model group,Didang Tang group,2-(nicotinamide)-1,3,4-thiadiazole(TGN-020)group,and Didang Tang combined with TGN-020 group(combined group),with 12 rats in each group.Among them,the sham group only underwent laminectomy without spinal cord shock and the other groups used NYU percussion device to create an acute spinal cord injury model.After modeling,the sham and model groups did not take any treatment measures.The Didang Tang group was given 5.04 g/kg Didang Tang by intragastric administration,the TGN-020 group was given 5 mg/kg TGN-020 by intraperitoneal injection,and the combined group was given TGN-020 by intraperitoneal injection combined with Didang Tang by intragastric administration,once a day for 7 days.Basso,Beattie and Bresnahan score and Reuter score were evaluated on 1,3,5 and 7 days after operation.Spinal cord tissue was taken for general observation and water content measurement at 7 days after operation.The degree of tissue damage was detected by hematoxylin-eosin staining.The number of aquaporin 4/glial fibrillary acidic protein-positive cells in the spinal cord tissue was detected by immunofluorescence assay.The protein expression levels of Aquaporin 4,glial fibrillary acidic protein,chondroitin sulfate proteoglycan,and proliferating cell nuclear antigen protein in spinal cord tissue were detected by western blot.RESULTS AND CONCLUSION:The behavioral scores showed that compared with the model group,the Basso,Beattie and Bresnahan scores were significantly increased in the three administration groups(P<0.05),while the Reuter scores were significantly decreased(P<0.05).Moreover,there was no significant difference among the three administration groups(P>0.05).Compared with the model group,the water content of spinal cord tissue was significantly decreased in the three administration groups(P<0.05),while there was no significant difference among the three administration groups(P>0.05).Hematoxylin-eosin staining results showed that the structure of the model group was disorganized,with a lot of scar tissue and syringomyelia.Compared with the model group,the tissue structure was relatively complete in the three administration group,with a small amount of scar tissue hyperplasia and syringomyelia,and the tissue morphology was effectively improved.Immunofluorescence staining results showed that compared with the model group,the number of aquaporin 4/glial fibrillary acidic protein-positive cells was less in the three administration groups,with lower brightness and reduced degree of cell swelling(P<0.05).However,there was no significant difference among the three administration groups(P>0.05).Western blot results showed that compared with the model group,the expressions of aquaporin 4,glial fibrillary acidic protein,chondroitin sulfate proteoglycan,and proliferating cell nuclear antigen protein in the spinal cord tissue of the three administration groups were significantly decreased(P<0.05).While,there was no significant difference among the three administration groups(P>0.05).To conclude,Jiajian Didang Tang can promote the recovery of limb function in rats with acute spinal cord injury under the guidance of Tongfu Zhuyu therapy.Its mechanism may reduce edema at the injury site by inhibiting the expression of aquaporin 4,so as to effectively inhibit the activation and proliferation of reactive astrocytes and reduce the formation of glial scar.