温和灸对神经根型颈椎病大鼠脊髓组织LC3/Bax表达的影响

Effect of mild moxibustion on pain and expression of LC3 and Bax in spinal cord in cervical spondylotic radiculopathy rats

目的:观察温和灸“大椎”对神经根型颈椎病(CSR)大鼠脊髓组织自噬与凋亡因子LC3、Bax蛋白及mRNA的影响,探讨运用温和灸治疗CSR的镇痛作用机制。方法:SD大鼠随机分为空白组、模型组、温和灸组、温和灸+自噬抑制剂3-甲基腺嘌呤(3-MA)组,每组10只。采用渔线压迫颈局部神经根法制备CSR大鼠模型。于造模3 d后,温和灸组取“大椎”进行温和灸10 min治疗并予腹腔注射1 mL 0.9%氯化钠溶液,温和灸+3-MA组予温和灸“大椎”和腹腔注射1 mL 3-MA,模型组仅予腹腔注射1 mL 0.9%氯化钠溶液,均每日1次,连续干预7 d。治疗后进行大鼠步态障碍评分,并采用YLS-3E型痛分析仪测量机械痛阈,免疫组化法检测脊髓组织LC3、Bax的蛋白表达,RT-PCR法检测脊髓组织LC3、Bax mRNA表达水平。结果:与空白组比较,模型组步态障碍评分明显升高(P<0.01),机械痛阈明显降低(P<0.01),脊髓组织LC3蛋白及mRNA表达水平差异无统计学意义(P>0.05),Bax蛋白及mRNA表达水平明显升高(P<0.05)。与模型组比较,温和灸组、温和灸+3-MA组步态障碍评分明显降低(P<0.01,P<0.05),机械痛阈明显升高(P<0.01,P<0.05),脊髓组织LC3蛋白及mRNA表达水平明显升高(P<0.05),Bax蛋白及mRNA表达水平明显降低(P<0.05)。与温和灸+3-MA组比较,温和灸组步态障碍评分明显降低(P<0.05),机械痛阈明显升高(P<0.05),脊髓组织LC3蛋白及mRNA表达水平明显升高(P<0.05),Bax蛋白及mRNA表达水平明显降低(P<0.05)。结论:温和灸“大椎”可有效改善CSR大鼠的神经病理性疼痛,其作用机制可能与上调LC3的自噬水平,从而抑制Bax促凋亡蛋白表达,减少细胞凋亡,修复神经损伤有关。

Objective To observe the effect of mild moxibustion(Moxi)at“Dazhui”(GV14)on neuropathic pain,expression of autophagy and apoptosis factor LC3 and Bax proteins and mRNAs in the spinal cord tissue in rats with cervical spondylotic radiculopathy(CSR),so as to explore its underlying mechanism underlying relief of CSR-induced pain.Methods Forty rats(half male half female)were randomly divided into blank control,model,Moxi,Moxi+autophagy inhibitor 3-methyladenine(3-MA,Moxi+3-MA)groups,with 10 rats in each group.The CSR model was established by loose ligature of the local cervical nerve roots.Three days after modeling,mild Moxi was applied to GV14 for 10 min,once daily for 7 days.Rats of the Moxi+3-MA group received intraperitoneal injection of 3-MA(1 mL,15 mg/kg+saline)before Moxi,once daily for 7 consecutive days.Rats of the model and Moxi groups were also given normal saline(i.p.,1 mL),once daily for 7 days.The gait behavior score(1-3 points)was scaled according to the rats’pain reaction and foot paw contracture produced walking disorder and the mechanical pain threshold(MPT)was detected before and after the treatment.The expression of spinal cord LC3 and Bax proteins and mRNAs were detected by immunohistochemistry and quantitative RT-PCR,respectively.Results Compared with the blank control group,the gait disorder score,and percentage of Bax positive cells and expression of Bax mRNA were significantly increased(P<0.01,P<0.05),and MPT was markedly decreased in the model group(P<0.01).After the treatment,the gait disorder score,percentage of Bax positive cells and Bax mRNA expression were significantly down-regulated(P<0.01,P<0.05),while the MPT and percentage of LC3 positive cells and LC3 mRNA expression were considerably increased(P<0.01,P<0.05)in both Moxi and Moxi+3-MA groups.The therapeutic effects of mild Moxi were remarkably superior to those of Moxi+3-MA in downregulating gait disorder score,Bax positive cell percentage and Bax mRNA expression,and in up-regulating MPT,LC3 positive cell percentage and LC3 mRNA expression(P<0.05),suggesting a reduction of the function of mild Moxi after administration of 3-MA.Conclusion Mild Moxi at GV14 can relieve neuropathic pain in CSR rats,which may be related to its functions in up-regulating LC3 autophagy,thereby inhibiting the expression of Bax pro-apoptotic protein in spinal cord to reduce apoptosis and to repair nerve injury.