麦粒灸对神经根型颈椎病大鼠细胞自噬与内质网应激的影响

Effects of wheat-grain moxibustion on autophagy and endoplasmic reticulum stress in rats with cervical spondylotic radiculopathy

目的:观察麦粒灸对神经根型颈椎病(CSR)模型大鼠脊髓及神经根组织细胞自噬及内质网应激凋亡因子表达的影响,探讨麦粒灸缓解CSR神经病理性疼痛的可能作用机制。方法:将48只SPF级SD大鼠随机分为假手术组、模型组、麦粒灸组和麦粒灸+3-甲基腺嘌呤(3MA)组,每组12只。模型组、麦粒灸组和麦粒灸+3MA组大鼠采用椎管插线法制备CSR模型。造模成功后第3天,麦粒灸组于“大椎”行麦粒灸干预,每日1次,每次6壮;麦粒灸+3MA组先予3MA溶液腹腔注射(2.5 mg/kg),后进行与麦粒灸组相同的麦粒灸干预。均每日1次,连续干预7 d。分别于干预前后观察各组大鼠步态障碍评分并测量外周神经机械性痛阈;干预后采用Western blot法检测大鼠脊髓及神经根组织内质网应激凋亡因子C/EBP同源蛋白(CHOP)、半胱氨酸天冬氨酸蛋白酶-12(Caspase-12)及自噬底物P62蛋白表达,透射电镜观察大鼠脊髓及神经根组织自噬小体及细胞超微结构。结果:造模后(干预前),与假手术组比较,模型组、麦粒灸组、麦粒灸+3MA组大鼠步态障碍评分升高(P<0.05),外周神经机械性痛阈降低(P<0.05)。干预后,与假手术组比较,模型组大鼠步态障碍评分升高(P<0.05),外周神经机械性痛阈降低(P<0.05);与模型组比较,麦粒灸组、麦粒灸+3MA组大鼠步态障碍评分降低(P<0.05),外周神经机械性痛阈升高(P<0.05);与麦粒灸+3MA组比较,麦粒灸组大鼠步态障碍评分降低(P<0.05),外周神经机械性痛阈升高(P<0.05)。与假手术组比较,模型组大鼠脊髓及神经根组织CHOP、Caspase-12蛋白表达升高(P<0.05);与模型组及麦粒灸+3MA组比较,麦粒灸组大鼠脊髓及神经根组织CHOP、Caspase-12、P62蛋白表达降低(P<0.05)。麦粒灸组大鼠脊髓及神经根组织自噬小体数量较模型组及麦粒灸组+3MA组增多,且细胞超微结构清晰完整,与假手术组相近。结论:麦粒灸“大椎”可有效缓解CSR模型大鼠神经病理性疼痛,其镇痛机制可能与促进自噬,抑制内质网应激,减少内质网应激介导的细胞凋亡,修复受损神经有关。

Objective To observe the effects of wheat-grain moxibustion on autophagy and endoplasmic reticulum stress(ERS) in the spinal cord and nerve root tissues of rats with cervical spondylotic radiculopathy(CSR), and to explore the potential mechanisms by which wheat-grain moxibustion alleviates neuropathic pain in CSR. Methods Forty-eight SPF-grade SD rats were randomly divided into a sham operation group, a model group, a wheat-grain moxibustion group, and a wheat-grain moxibustion + 3-methyladenine(3MA) group, with 12 rats in each group. The CSR model was established in the model group, the wheat-grain moxibustion group, and the wheat-grain moxibustion + 3MA group using the spinal canal insertion method. From the third day after successful modeling, the wheat-grain moxibustion group received wheat-grain moxibustion at "Dazhui"(GV 14), once daily, six cones each session. The wheat-grain moxibustion + 3MA group received intraperitoneal injection of 3MA(2.5 mg/kg), followed by the same wheat-grain moxibustion intervention as the wheat-grain moxibustion group. Interventions were performed once daily for seven consecutive days. The gait disturbance scores and peripheral nerve mechanical pain thresholds were observed before and after the intervention. Western blot was used to detect the expression of ERS apoptosis factors C/EBP-homologous protein(CHOP) and Caspase-12, as well as autophagy substrate P62 in spinal cord and nerve root tissues. Transmission electron microscopy was used to observe autophagosomes and cellular ultrastructure in spinal cord and nerve root tissues. Results After modeling, compared with the sham operation group, gait disturbance scores were increased(P<0.05) and peripheral nerve mechanical pain thresholds were decreased(P<0.05) in the model group, the wheat-grain moxibustion group, and the wheat-grain moxibustion + 3MA group. After intervention, compared with the sham operation group, gait disturbance scores were increased(P<0.05) and peripheral nerve mechanical pain thresholds were decreased(P<0.05) in the model group;compared with the model group, gait disturbance score was decreased(P<0.05) and peripheral nerve mechanical pain threshold was increased(P<0.05) in the wheat-grain moxibustion and the wheat-grain moxibustion + 3MA group;compared with the wheat-grain moxibustion + 3MA group, gait disturbance score was decreased(P<0.05) and peripheral nerve mechanical pain threshold was increased(P<0.05) in the wheat-grain moxibustion group. Compared with the sham operation group, the expression of CHOP and Caspase-12 proteins in spinal cord and nerve root tissues was increased in the model group(P<0.05);compared with the model group and the wheat-grain moxibustion + 3MA group, the expression of CHOP, Caspase-12, and P62 proteins in spinal cord and nerve root tissues was decreased in the wheat-grain moxibustion group(P<0.05). Compared with the model group and the wheat-grain moxibustion + 3MA group, the number of autophagosomes in spinal cord and nerve root tissues was increased in the wheat-grain moxibustion group, and the cellular ultrastructure was clear and intact, similar to the sham operation group. Conclusion Wheat-grain moxibustion at "Dazhui"(GV 14) could effectively alleviate neuropathic pain in CSR model rats. The analgesic mechanism may be related to promoting autophagy, inhibiting ERS, reducing ERS-mediated apoptosis, and repairing damaged nerves.