夹脊电针对脊髓损伤大鼠脊髓病理形态及死亡受体6表达的影响

Effect of Jiaji Electro-Acupuncture on Pathological Morphology of Spinal Cord Tissue and Expression of Death Receptor 6 Protein in Rats with Spinal Cord Injury

目的观察夹脊电针对脊髓损伤(SCI)大鼠脊髓病理形态及死亡受体6表达变化的影响,分析夹脊电针改善SCI的机制。方法健康成年SD雌性大鼠分为假手术组(Sham组)、假手术+电针组(Sham+EA组)、模型组(Model组)、模型+电针组(Model+EA组),每组6只。采用钳夹方式制作SCI模型,给予相应干预方式,各组大鼠一同捆绑,常规饲养,假手术组:仅打开椎板;Sham+EA组:仅打开椎板,给予电针干预;Model组:按手术操作造模。Model+EA组:按手术操作造模,同时给予电针干预,治疗7天。采用运动功能评分(Basso,Beattie&Bresnahan,BBB)对大鼠后肢运动功能进行评分;肉眼及HE染色观察脊髓组织形态和病理变化;采取蛋白免疫印迹(Western blot)检测各组大鼠脊髓组织中死亡受体6(DR6)蛋白及少突胶质细胞标志蛋白CNpase表达差异;采用实时荧光定量PCR(Real-time PCR)检测各组大鼠脊髓组织中DR6 mRNA表达水平差异。结果BBB评分结果示:与Sham组比较,Model组BBB评分显著降低(P<0.05),与Model组比较,Model+EA组BBB评分升高(P<0.05)。肉眼及HE染色观察脊髓形态结果示:Model组脊髓组织损伤段可见大面积淤血现象,组织结构破坏严重;光镜下可见Model组脊髓组织疏松严重,灰白质界限隐约可见,存在部分出血灶,大量炎症浸润,胶质细胞增生,灰质内有神经元残存,核浓缩,体积变小甚至消失,白质内可见大量空泡细胞。Model+EA组脊髓组织损伤段淤血面积及结构破坏程度明显轻于Model组,光镜下出血点减少,淋巴细胞浸润少于Model组,神经细胞出现修复,出现一定量髓鞘再生。Western blot检测脊髓组织DR6表达变化结果示:Model组大鼠脊髓组织DR6蛋白表达显著升高(P<0.05)。经治疗7天后,Model+EA组DR6蛋白表达低于Model组(P<0.05);Model组大鼠脊髓组织CNpase蛋白表达显著降低(P<0.05)。经治疗7天后,Model+EA组CNpase蛋白表达高于Model组(P<0.05);Real-time PCR检测脊髓组织DR6 mRNA表达变化结果示:Model组大鼠脊髓组织DR6 mRNA表达水平显著升高(P<0.05)。经电针治疗7天后,Model+EA组大鼠脊髓组织DR6 mRNA表达低于Model组(P<0.05)。结论夹脊电针可能通过抑制脊髓组织中DR6mRNA及蛋白的表达,促进少突胶质细胞标志蛋白CNpase表达,进而改善损伤的脊髓组织微环境,从而促进髓鞘形成与再生,改善SCI。

Objective The effect of Jiaji electro-acupuncture on the pathological morphology of spinal cord tissue and the expression of death receptor 6 in spinal cord injury(SCI)rats was observed,and the mechanism of Jiaji electroacupuncture on improving SCI was analyzed.Methods Healthy adult female SD rats were divided into Sham operation group(Sham operation group),Sham operation+EA group(Sham operation+EA group),Model group(Model group),and Model+EA group(Model+EA group),with 6 rats in each group.The SCI model was made by clamp method,and the corresponding intervention methods were given.The rats in each group were tied together and fed conventionally.In Sham+EA group,only the lamina was opened and electroacupuncture was given.Model group:The Model was made according to the operation.Model+EA group:The Model was established according to the operation,and electroacupuncture intervention was given at the same time.The treatment lasted for 7 days.BBB score was used to score the hindlimb motor function of rats.The morphology and pathological changes of the spinal cord were observed by eye and HE staining.Western blotting was used to detect the expression of death receptor 6(DR6)protein and oligodendrocyte marker protein CNpase in spinal cord tissues of rats in each group.The mRNA expression of DR6 in spinal cord was detected by real-time PCR.Results The BBB score results showed that compared with Sham group,the BBB score of Model group was significantly decreased(P<0.05),and compared with Model group,the BBB score of Model+EA group was increased(P<0.05).Macroscopic and HE staining results of spinal cord morphology showed that the Model group had a large area of congestion in the injured segment of spinal cord tissue,and the tissue structure was seriously damaged.Under the light microscope,the spinal cord tissue of the Model group was severely loose,the gray matter boundary was faintly visible,there were some bleeding foci,a large number of inflammatory infiltrates,gliosis,residual neurons in the gray matter,nuclei concentrated,smaller or even disappeared,and a large number of vacuolar cells in the white matter.In Model+EA group,the area of congestion and the degree of structural damage in the injured segment of spinal cord tissue were significantly lighter than that in Model group,the bleeding points under light microscope were reduced,the lymphocyte infiltration was less than that in Model group,the nerve cells were repaired,and a certain amount of myelin regeneration occurred.The results of WB detection of DR6 expression in spinal cord showed that the expression of DR6 protein in Model group was significantly increased(P<0.05).After 7 days of treatment,the expression of DR6 protein in Model+EA group was lower than that in Model group(P<0.05).The expression of CNpase protein in the spinal cord of Model group was significantly decreased(P<0.05).After 7 days of treatment,the expression of CNpase protein in Model+EA group was higher than that in Model group(P<0.05).Realtime PCR detection of DR6 mRNA expression in spinal cord showed that the expression level of DR6 mRNA in Model group was significantly increased(P<0.05).After 7 days of EA treatment,DR6 mRNA expression in the spinal cord of Model+EA group was lower than that of Model group(P<0.05).Conclusion Jiaji electro-acupuncture may inhibit the expression of DR6 mRNA and protein in spinal cord tissue,promote the expression of oligodendrocyte marker protein CNpase,and then improve the injured spinal cord tissue microenvironment,thereby promoting myelination and regeneration,and improving SCI.