[关键词]
[摘要]
【目的】观察电针夹脊穴对腰椎间盘退变大鼠的治疗作用及机制。 【方法】将30只SD大鼠随机分为假手术组、模型组与电针组,每组10只。模型组与电针组大鼠采用纤维环穿刺法构建腰椎间盘退变模型,假手术组仅分离椎间盘,不做其他处理。造模成功后,电针组于L4、L5双侧夹脊穴电针治疗,假手术组与模型组不给予处理。干预结束后,采用电子Von Frey测痛仪检测机械缩足阈值 (PMWT) ,苏木素-伊红 (HE) 染色法观察大鼠腰椎间盘结构变化,酶联免疫吸附分析 (ELISA) 检测腰椎间盘组织上清液白细胞介素1β (IL-1β) 、肿瘤坏死因子α (TNF-α) 水平,流式细胞仪检测髓核细胞周期比例,实时定量 聚合酶链反应 (RT-PCR) 法检测腰椎间盘髓核组织Fas相关死亡域蛋白 (FADD) 、天冬氨酸特异性半胱氨酸蛋白酶8 (Caspase-8) 、B细胞淋巴瘤/白血病2 (Bcl-2) 相关X蛋白 (Bax) 、Bcl-2 mRNA表达水平,Western Blot法检测椎间盘髓核组织FADD、Caspase-8、Bax、Bcl-2蛋白表达水平。 【结果】模型组大鼠腰椎间盘整体结构异常,可见到明显退变;电针组大鼠腰椎间盘组织退变程度较模型组明显改善。与假手术组比较,模型组大鼠PWMT降低,IL-1β、TNF-α水平升高,髓核细胞G0/G1期比例升高,G2/M期细胞比例降低,FADD、Caspase-8、Bax mRNA及蛋白表达水平升高,Bcl-2 mRNA及蛋白表达水平降低,差异均有统计学意义 (P<0.05) ;与模型组比较,电针组大鼠PWMT升高,IL-1β、TNF-α水平降低,髓核细胞G0/G1期细胞比例降低,G2/M期细胞比例升高,FADD、Caspase-8、Bax mRNA及蛋白表达水平降低,Bcl-2 mRNA及蛋白水平升高,差异均有统计学意义 (P<0.05) 。 【结论】电针夹脊穴可通过调控FADD/Caspase-8信号通路抑制细胞凋亡来缓解炎症反应、调控 髓核细胞周期,改善腰椎间盘结构变化,从而延缓大鼠腰椎间盘退变。
[Key word]
[Abstract]
Objective To observe the therapeutic effect and mechanism of electroacupuncture at Jiaji(EX-B2)points for lumbar disc degeneration in rats. Methods Thirty SD rats were randomly divided into the sham-operation group,model group and electroacupuncture group,with 10 rats in each group. The rats in the model group and electroacupuncture group were constructed a lumbar disc degeneration model by annulus fibrosus puncture method,and the sham-operation group was only given separation of intervertebral discs without other treatments. After successful modeling,the electroacupuncture group was treated with electroacupuncture at the L4 and L5 bilateral Jiaji points. No treatment was given to the sham-operation group and the model group. At the end of the intervention,the paw withdrawal mechanical threshold (PMWT) was detected by electronic Von Frey filaments,the changes of the structure of lumbar disc in rats were observed by hematoxylin-eosin (HE) staining,the levels of interleukin 1β (IL-1β) and tumor necrosis factor α(TNF- α) in the supernatant of lumbar intervertebral disc tissues were detected by enzyme-linked immunosorbent assay (ELISA),and the ratio of nucleus pulposus cell cycle was detected by flow cytometry,real-time quantitative polymerase chain reaction (RT-PCR) method was used to detect the mRNA expression levels of Fas-associated death domain protein (FADD),cysteinyl aspartate specific protease 8 (Caspase-8) ,B-cell lymphoma/leukemia 2 (Bcl-2) -associated X protein (Bax) and Bcl-2 in the nucleus pulposus of lumbar intervertebral disc,the protein expression levels of FADD,Caspase-8,Bax,and Bcl-2 in the nucleus pulposus of lumbar intervertebral disc were detected by Western Blot. Results The overall structure of the intervertebral disc in rats of the model group was abnormal and obvious degeneration was seen;the degeneration of intervertebral disc tissue in the electroacupuncture group was significantly improved compared with that of the model group. Compared with the sham-operation group,the model group showed lower PWMT,and the higher levels of IL-1β and TNF- α,the increased proportion of G0/G1 nucleus pulposus cells and decreased proportion of G2/M nucleus pulposus cells ,and the increased mRNA and protein expression levels of FADD,Caspase-8 and Bax and the decreased mRNA and protein levels of Bcl-2,the differences being statistically significant (P<0.05) . Compared with the model group,the electroacupuncture group showed higher PWMT,the lower levels of IL-1β and TNF-α,the decreased proportion of G0/G1 nucleus pulposus cells and the increased proportion of G2/M nucleus pulposus cells,and the decreased mRNA and protein expression levels of FADD, Caspase-8 and Bax and the increased mRNA and protein levels of Bcl-2,the differences being statistically significant(P<0.05) . Conclusion Electroacupuncture at the Jiaji points can alleviate inflammatory reaction,regulate nucleus pulposus cell cycle to improve the structural changes of lumbar disc through regulating the FADD/Caspase-8 signaling pathway to inhibit apoptosis,thereby slowing down rat lumbar disc degeneration.
[中图分类号]
R245.31
[基金项目]
海南省自然科学基金青年基金项目 (编号:821QN1007);海南省临床医学中心建设项目 (琼卫医函[2021]276 号)