Effects of acupuncture at Houxi (SI3) and Huantiao (GB30) on NF-κB/iNOS/NO pathway in lumbar disc herniation model rats

Abstract

Objective

To observe the effects of acupuncture at Houxi (SI3) and Huantiao (GB30) on the expression levels of nuclear factor kappa B (NF-κB), inducible nitric oxide synthase (iNOS), and nitric oxide (NO) of NF-κB inflammatory signaling pathway in L₅ spinal nerve root of lumbar disc herniation (LDH) model rats and explore the mechanism of acupuncture in LDH treatment.

Methods

Forty specific-pathogen-free healthy male Sprague-Dawley rats were randomly divided into a sham operation group, a model group, acupuncture group 1, and acupuncture group 2, with 10 rats in each group. The non-compression nucleus protrusion model was made by puncturing L₄–L₅ spinous process space and injecting autologous nucleus suspension. Acupuncture at bilateral Shenshu (BL23), Dachangshu (BL25), and Weizhong (BL40) was carried out in acupuncture group 1, and acupuncture at bilateral Houxi (SI3) and Huantiao (GB30) in acupuncture group 2. All rats were treated with balanced reinforcing and reducing needling manipulations, and the needles were retained for 30 min/time with one episode of needling manipulation every 10 min, once a day, 14 times in total. The threshold value of paw withdrawal pain was measured by a thermal stimulation pain instrument; the serum NF-κB, iNOS, and NO levels were measured by enzyme-linked immunosorbent assay. The pathomorphological changes of spinal nerve roots were observed by hematoxylin-eosin (HE) staining; quantitative reverse transcription polymerase chain reaction was used to detect iNOS mRNA expression in spinal nerve roots; the NF-κB and iNOS protein expression in spinal nerve roots was detected by the immunofluorescence method.

Results

Compared with the sham operation group, the threshold of paw withdrawal pain in the model group was decreased, and the expression levels of serum NF-κB, iNOS, and NO were increased; HE staining showed many degenerated and dissolved Schwann cells in spinal nerve roots with vacuolar changes; meanwhile, the expression levels of NF-κB and iNOS proteins, and the iNOS mRNA in spinal nerve roots were increased. Compared with the model group, the paw withdrawal pain thresholds in acupuncture group 1 and acupuncture group 2 were increased, and the increase in acupuncture group 2 was greater (P<0.05); the expression levels of serum NF-κB, iNOS, and NO in acupuncture group 1 and acupuncture group 2 were decreased, especially in acupuncture group 2 (P<0.01); the vacuolar changes of spinal nerve roots, and the degeneration and lysis of Schwann cells in acupuncture group 1 and acupuncture group 2 were decreased, which were more obvious in acupuncture group 2; the NF-κB and iNOS protein expression and the iNOS mRNA expression levels in spinal nerve roots of acupuncture group 1 and acupuncture group 2 were decreased, especially in acupuncture group 2 (P<0.01).

Conclusion

Acupuncture at Houxi (SI3) and Huantiao (GB30) can improve the morphology of spinal nerve roots, inhibit the NF-κB and iNOS protein expression levels in spinal nerve roots and the serum NO level, and relieve the pain caused by inflammation of spinal nerve roots, which may be one of the mechanisms of acupuncture in LDH treatment.

摘要

目的

观察针刺后溪和环跳对腰椎间盘突出症(LDH)模型大鼠L₅脊神经根核因子κB(NF-κB)炎性信号通路中 NF-κB、诱导型一氧化氮合酶(iNOS)及一氧化氮(NO)表达的影响, 探讨针刺治疗LDH的作用机制。

方法

将40只健康无特定病原体级雄性Sprague-Dawley大鼠按随机数字表法分为假手术组、模型组、针刺1组和针刺2组, 每组10只。通 过穿刺L₄–L₅棘突间隙注射自体髓核混悬液制成非压迫髓核突出模型。针刺1组针刺双侧肾俞、大肠俞和委中; 针刺2 组针刺双侧后溪和环跳。均采用平补平泻手法, 每次留针30 min, 每隔10 min行针1次, 每日1次, 共治疗14次。用热刺 痛仪进行缩爪痛阈值测定; 酶联免疫吸附法测定血清NF-κB、iNOS及NO水平; 苏木素-伊红(HE)染色法观察脊神经根的 病理形态学改变; 定量反转录聚合酶链式反应法检测脊神经根iNOS mRNA表达; 免疫荧光法检测脊神经根NF-κB及 iNOS蛋白表达。

结果

与假手术组比较, 模型组的缩爪痛阈值降低; 血清NF-κB、iNOS及NO表达水平升高; HE染色观察 发现脊神经根内施万细胞大量变性溶解, 并出现空泡样改变; 同时脊神经根NF-κB、iNOS蛋白表达和iNOS mRNA表达 均升高。与模型组比较, 针刺1组和针刺2组的缩爪痛阈值升高, 针刺2组升高幅度更大(P<0.05); 针刺1组和针刺2组的 血清NF-κB、iNOS及NO表达水平降低, 针刺2组降低更显著(P<0.01); 针刺1组和针刺2组的脊神经根空泡样改变和施万 细胞变性溶解减少, 且针刺2组改变更明显; 针刺1组和针刺2组的脊神经根NF-κB及iNOS蛋白表达和iNOS mRNA表达 均降低, 针刺2组降低更明显(P<0.01)。

结论

针刺后溪和环跳可改善LDH模型大鼠的脊神经根形态, 抑制脊神经根中 NF-κB、iNOS蛋白表达和血清NO水平, 缓解脊神经根炎症引发的疼痛, 这可能是针刺治疗LDH的机制之一。