督脉电针和高压氧对脊髓损伤大鼠脊髓神经元的作用机制探讨

The mechanism of Du meridian electroacupuncture and hyperbaric oxygen on spinal cord neurons in rats with spinal cord injury

目的:探讨督脉电针和高压氧对脊髓损伤大鼠脊髓神经元的作用机制。方法:选择Wistar大鼠共60只,按随机数字表法分为5组,即正常对照组、模型组、督脉电针组、高压氧组、高压氧联合督脉电针组,每组12只。正常对照组只行椎板切除,不造成脊髓损伤;其余组用脊髓横断的方法损伤大鼠胸髓背侧部位,以构建实验模型。高压氧组于造模清醒4 h后,置于高压氧舱内氧疗,持续75 min/次,治疗4次/d。督脉电针组分别于造模清醒4 h后给予干预,频率2 Hz;干预时长为20 min,频率为1次/d。高压氧联合督脉电针组使用上述两种方法相结合,对大鼠进行干预。正常对照组和模型组不予治疗。造模后的第1、3、7天分别对各组进行Basso Beattie Bresnahan(BBB)运动功能评估,记录各组大鼠行为学改变。造模后第7天处死各组大鼠,取大鼠脊髓组织,应用TUNEL法检测凋亡细胞并计算凋亡指数;免疫荧光检测Ras同源基因家族成员A(RhoA)、Rho相关卷曲螺旋蛋白激酶(ROCK)通路变化;同时采用经典的RT-PCR和Western印迹测定大鼠脊髓损伤区轴突生长抑制因子A(Nogo-A)、Nogo-66受体(NgR)mRNA和蛋白的含量。结果:造模后第1、3和7天,督脉电针组、高压氧组和高压氧联合督脉电针组BBB评分均高于模型组(均P<0.05),且高压氧联合督脉电针组评分高于督脉电针组与高压氧组(P<0.05)。TUNEL染色下模型组脊髓灰质区神经元染色明显阳性,提示细胞凋亡较多,高压氧联合督脉电针组损伤部位细胞凋亡显著减少(P<0.05);督脉电针组、高压氧组和联合治疗组RhoA、ROCK蛋白含量、Nogo-A mRNA和蛋白表达量、NgR mRNA和蛋白表达量均低于模型组(F=34.597、39.230、14.783、12.370、21.435、50.435,均P<0.05),且联合组的Nogo-A mRNA和蛋白表达量、NgR mRNA和蛋白表达量均低于督脉电针组和高压氧组(F=13.58、11.253、24.843、41.221,均P<0.05)。结论:督脉电针和高压氧对脊髓损伤大鼠脊髓神经元具有保护作用,其机制可能与RhoA/ROCK通路有关。

Objective:To explore the mechanism of Du meridian electroacupuncture(EA)and hyperbaric oxygen(HBO)treatment on spinal cord neurons in rats with spinal cord injury(SCI).Methods:A total of 60 Wistar rats were divided into five groups by radom number table method:normal control group,model group,Du meridian EA group,HBO group,HBO combined with Du meridian EA group,with 12 animals in each group.The normal control group only underwent laminectomy without spinal cord injury and the SCI rat model was established by transecting the thoracic spinal cord on the dorsal side.At four hours after wakefulness,the rats of HBO group were placed in the hyperbaric oxygen chamber for 75 minutes and 4 times a day.The electroacupuncture intervention was given the rats of the Du meridian EA group for 20 minutes at four hours after wakefulness,with the frequency of 2 Hz and once a day.The HBO combined with Du meridian EA group received the combination treatment of the above two groups to intervene in rats,while the normal control group and model group did not receive any therapy.Basso Beattie Bresnahan(BBB)scores of spinal cord injury were used to assess motor function in each group at the 1st,3rd and 7th day after injury,and the behavioral changes in each group of rats were recorded.On the 7th day after injury,all the rats were sacrificed and their spinal cord tissue was harvested.The number of apoptotic cells were assessed with TUNEL staining and the apoptotic index was calculated.The changes of Ras homolog gene family memberA(RhoA)/Rho associated coiled-coil forming protein kinase(ROCK)pathway were detected by immunofluorescence.RT-PCR and Western blotting were used to detected the expression of the mRNA and protein of Neurite outgrowth inhibitor-A(Nogo-A)and Nogo-66 receptor(NgR)in the injured spinal cord area.Results:On the1st,3rd and 7th day after injury,the BBB scores of the Du meridian EA group,HBO group and combined treatment group were all higher than those of the model group(all P<0.05),and the scores of the combined treatment group were higher than those of the Du meridian EA group and HBO group(P<0.05).TUNEL staining showed a significant increase of apoptotic neurons in the gray matter area of the spinal cord in the model group while in the HBO combined with Du meridian EA group,the apoptosis at the injury site was significantly reduced(P<0.05).The protein contents of the RhoA and ROCK,the levels of the mRNA and protein expression of Nogo-A and NgR in the Du meridian EA group,HBO group and combined treatment group were all lower than those in model group(F=34.597,39.230,14.783,12.370,21.435,50.435,all P<0.05).Furthermore,the levels of the mRNA and protein expression of Nogo-A and NgR in the combined treatment group were lower than those in the Du meridian group and HBO group(F=13.58,11.253,24.843,41.221,all P<0.05).Conclusion:The treatment of the Du meridian EA and HBO have protective effects on spinal cord neurons in rats with SCI,and the mechanism may be related to the RhoA/ROCK pathway.