电针预处理对兔脊髓缺血再灌注损伤后炎性因子的影响

Effect of Electroacupuncture Preconditioning on Inflammatory Factors of Rabbits after Spinal Cord Ischemia-Reperfusion Injury

目的:观察电针预处理对兔脊髓缺血再灌注损伤后脊髓组织病理变化以及白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1受体拮抗剂(IL-1Rα)等炎性细胞因子表达的影响,探讨电针对脊髓缺血再灌注损伤的保护作用机制。方法:24只新西兰大白兔采用随机数字表法分为模型组、假手术组、电针组,每组8只。模型组与电针组兔均采用Zivin造模法进行造模,耳缘静脉注射麻醉后打开腹腔,暴露分离腹主动脉并临时夹闭,使其缺血30 min建立脊髓缺血再灌注损伤兔模型。假手术组模型制备及实验动物处理同模型组,但只暴露分离并不夹闭腹主动脉。在兔脊髓缺血再灌注模型诱导前30 min,电针组采用SDZ-II型电针仪针刺双侧"悬钟""阳陵泉"穴进行干预,时间为30 min。干预12 h后处死全部动物,取兔L2~5段脊髓组织,采用HE染色观察3组脊髓组织病理变化;采用免疫组化法观察IL-1β、IL-6、TNF-α的定位及含量变化;采用酶联免疫吸附法(ELISA)检测造模前及缺血再灌注后4、8、12 h 4个时间点血清中IL-1β、IL-6、TNF-α、IL-1Rα的含量。结果:①HE染色结果:与假手术组比较,模型组脊髓组织中神经元的数目减少,细胞胞核及胞浆颜色均变浅,核膜的边界不清晰,严重者核溶解甚至出现核仁消失,细胞周围存有透亮区,轴突出现扩大、肿胀、消失等不同程度的变性;与模型组比较,电针组神经元的数量增加,大部分神经元的结构较为清晰,核仁、胞核萎缩程度较轻,只有少量出现变形、坏死,细胞的破坏程度较轻,存活神经元的密度增加。②免疫组化法结果:与假手术组比较,模型组兔脊髓组织中IL-1β、IL-6、TNF-α的表达均明显上升(P<0.05);与模型组比较,电针组兔脊髓组织中IL-1β、IL-6、TNF-α的含量明显降低(P<0.05)。③ELISA结果:造模前,3组间比较差异无统计学意义(P>0.05);与造模前比较,3组缺血再灌注后4、8、12 h血清中IL-1β、IL-6、TNF-α、IL-1Rα的表达均有不同程度的升高,差异具有统计学意义(P<0.05);与假手术组同一时间比较,模型组再灌注后4、8、12 h IL-1β、IL-6、TNF-α、IL-1Rα的表达均明显升高(P<0.05);与模型组同一时间点比较,电针组再灌注后4、8、12 h IL-1β、TNF-α的表达均明显降低(P<0.05)。结论:电针预处理可有效减轻脊髓组织病理损伤,对脊髓缺血再损伤具有良好的保护作用,其机制可能与抑制IL-1β、IL-6、TNF-α的表达,促进IL-1Rα的表达有关。

Objective:To observe the effects of electroacupuncture preconditioning on the pathological changes of spinal cord tissue and the expression of inflammatory cytokines such as interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),interleukin-1 receptor antagonist(IL-1Rα)after spinal cord ischemia-reperfusion injury,and to explore the protective mechanism of electroacupuncture on spinal cord ischemia-reperfusion injury in rabbits.Methods:A total of twenty-four New Zealand white rabbits were randomly divided into the sham operation group,the model group and the electroacupuncture group according to the random number table method,with eight cases in each group.The model was made by Zivin method in the model group and the electroacupuncture group,the abdominal cavity was opened after intravenous anesthesia at ear margin,the abdominal aorta was exposed and temporarily clamped,and the rabbit model of spinal cord ischemia-reperfusion injury was established after ischemia for thirty minutes.The model preparation and experimental animal treatment of the sham operation group were the same as those of the model group,however,the dissection was fully exposed without clamping the abdominal aorta.At thirty minutes before the induction of rabbit spinal cord ischemia-reperfusion model,SDZ-II electroacupuncture instrument was used to acupuncture bilateral"Xuanzhong"and"Yanglingquan"acupoints in the electroacupuncture group,intervention for thirty minutes.All rabbits were killed after intervention for twelve hours,and the L2-5 segments of spinal cord tissue were captured.HE staining method was used to observe the pathological changes of spinal cord tissues in the three groups;Immunohistochemical method was used to observe the localization and content changes of IL-1β,IL-6 and TNF-α;Enzyme-linked immunosorbent assay(ELISA)was used to detect the contents of IL-1β,IL-6,TNF-αand IL-1Rαin serum before modeling and 4,8,12 hours after ischemia-reperfusion.Results:①HE staining results:compared with the sham operation group,the number of neurons in spinal cord tissue in the model group decreased,the color of nucleus and cytoplasm of cells became lighter,and the boundary of nuclear membrane was not clear.In severe cases,the nucleus dissolved or even nucleolus disappeared,there were transparent areas around cells,and axons showed different degrees of degeneration such as enlargement,swelling and disappearance;compared with the model group,the number of neurons in the electroacupuncture group increased,the structure of most neurons was clear,the atrophy degree of nucleolus and nucleus was light,only a small amount of cells became deformed and necrosis,the damage degree of cells were lighter,and the density of surviving neurons increased.②Immunohistochemistry results:compared with the sham operation group,the expressions of IL-1β,IL-6 and TNF-αof spinal cord tissue in the model group were significantly increased(P<0.05);compared with the model group,the contents of IL-1β,IL-6 and TNF-αin the spinal cord of rabbits in the electroacupuncture group were significantly decreased(P<0.05).③ELISA results:Before modeling,there was no significant difference in the three groups(P>0.05);compared with before modeling,the expressions of IL-1β,IL-6,TNF-α,IL-1Rαin serum of the three groups increased in different degrees at 4,8 and 12 h after ischemia-reperfusion,with significantly significance(P<0.05);compared with the sham operation group at the same time,the expressions of IL-1β,IL-6,TNF-αand IL-1Rαin the model group increased significantly at 4,8 and 12 h after reperfusion(P<0.05);compared with the model group at the same time,the expression of IL-1βand TNF-αin the electroacupuncture group decreased significantly at 4,8,12 h after reperfusion(P<0.05).Conclusion:Electroacupuncture pretreatment can effectively alleviate the pathological injury of spinal cord and has a good protective effect on spinal cord ischemia-reperfusion injury,which may be related to inhibit the expression of IL-1β,IL-6 and TNF-αand promote the expression of IL-1Rα.