麦粒灸对神经根型颈椎病大鼠脊髓背角Beclin-1/GRP78表达的影响

Effect of wheat-grain moxibustion on the expression of Beclin-1/GRP78 in spinal dorsal horn in rats with cervical spondylotic radiculopathy

目的:观察麦粒灸“大椎”穴对神经根型颈椎病(CSR)大鼠脊髓背角自噬效应蛋白Beclin-1、内质网应激标志蛋白葡萄糖调节蛋白-78(GRP78)表达的影响,探讨麦粒灸治疗CSR的可能镇痛机制。方法:将48只SD大鼠随机分为假手术组、模型组、麦粒灸组、麦粒灸+3-MA组,每组12只。采用椎管插线法制备CSR模型。造模后第3天,模型组予腹腔注射1mL0.9%氯化钠溶液;麦粒灸组在模型组基础上予麦粒灸“大椎”穴(6壮/次);麦粒灸+3-MA组予腹腔注射3-甲基腺嘌呤(3-MA)溶液,并麦粒灸“大椎”穴(6壮/次)。3组均每天1次,连续干预7 d。于干预前、连续干预7 d后观察各组大鼠步态评分,测量机械痛阈值;干预后,采用实时荧光定量PCR法及免疫组化法检测脊髓背角Beclin-1 mRNA及蛋白表达,Western blot法检测脊髓背角GRP78蛋白表达,电镜观察脊髓背角细胞自噬小体及超微结构。结果:干预后,与假手术组比较,模型组大鼠步态评分升高、机械痛阈值降低(P<0.01),脊髓背角GRP78蛋白表达升高(P<0.01);与模型组及麦粒灸+3-MA组比较,麦粒灸组大鼠步态评分降低、机械痛阈值升高(P<0.01),脊髓背角GRP78蛋白表达降低、Beclin-1mRNA及蛋白表达升高(P<0.01)。电镜下观察麦粒灸组大鼠脊髓背角细胞超微结构未见明显损坏,其结构基本接近正常,且较其他3组的自噬小体数量增多。结论:麦粒灸“大椎”穴对CSR大鼠具有镇痛作用,其机制可能在于适度上调Beclin-1表达增强细胞自噬,减轻内质网应激。

Objective To observe the effect of wheat-grain moxibustion at "Dazhui"(GV 14) on the expressions of Beclin-1 and GRP78 in spinal dorsal horn in rats with cervical spondylotic radiculopathy(CSR), and to explore the possible analgesic mechanism of wheat-grain moxibustion for CSR. Methods A total of 48 SD rats were randomly divided into a sham operation group, a model group, a wheat-grain moxibustion group and a wheat-grain moxibustion+3-MA group, 12 rats in each group. The CSR model was prepared by spinal cord insertion method. Three days after modeling, the rats in the model group were intraperitoneally injected with 1 mL of 0.9% sodium chloride solution;the rats in the wheat-grain moxibustion group were treated with wheat-grain moxibustion at "Dazhui"(GV 14, 6 cones per time) on the basis of the model group;the rats in the wheat-grain moxibustion+3-MA group were intraperitoneally injected with 3-MA solution and wheat-grain moxibustion at "Dazhui"(GV 14, 6 cones per time). The three groups were intervened for 7 days, once a day. The gait score and mechanical pain threshold were observed before treatment and 7 days into treatment;after the treatment, the expressions of mRNA and protein of Beclin-1 in spinal dorsal horn were detected by real-time fluorescence quantitative PCR and immunohistochemistry;the expression of GRP78 protein in spinal dorsal horn was detected by Western blot method;the autophagosomes and ultrastructure in spinal dorsal horn neurons were observed by electron microscope. Results After the treatment, compared with the sham operation group, in the model group, the gait score was increased and the mechanical pain threshold was decreased(P<0.01), and the expression of GRP78 protein in spinal dorsal horn was increased(P<0.01). Compared with the model group and the wheat-grain moxibustion+3-MA group, in the wheat-grain moxibustion group, the gait score was decreased and mechanical pain threshold was increased(P<0.01), and the expression of GRP78 protein in spinal dorsal horn was decreased, and the expressions of mRNA and protein of Beclin-1 were increased(P<0.01). Under electron microscope, the ultrastructure of spinal dorsal horn neurons in the wheat-grain moxibustion group was not significantly damaged, and its structure was basically close to normal, and the number of autophagosomes was more than the other three groups. Conclusion Wheat-grain moxibustion at "Dazhui"(GV 14) has analgesic effect on CSR rats. The mechanism may be related to moderately up-regulate the expression of Beclin-1, enhance autophagy and reduce endoplasmic reticulum stress.