杜仲腰痛丸对腰椎间盘突出症慢性下肢痛大鼠海马BDNF-TrkB-CREB信号通路的影响

Effects of Duzhong Yaotong Pills on BDNF-TrkB-CREB signal pathway in hippocampus of lumbar disc herniation chronic lower limb pain rats

目的:探讨杜仲腰痛丸对腰椎间盘突出症(L D H)慢性下肢痛大鼠海马脑源性神经营养因子(BDNF)-络氨酸激酶受体B(TrkB)-环磷腺苷效应元件结合蛋白(CREB)信号通路的影响。方法:72只SD雄性大鼠随机分为正常组、模型组、模型治疗组,每组24只。模型组、模型治疗组采用自体髓核移植法进行造模,正常组与模型组给予相同剂量0.9%氯化钠溶液灌胃;模型治疗组给予杜仲腰痛丸药液灌胃,灌胃剂量为0.26 g/kg,2次/d。用疼痛行为学(机械痛阈值和热痛阈值)评价造模是否成功,造模成功后,各组大鼠在给药前1天、给药后14、28d取大鼠海马切片,利用免疫荧光技术观察BDNF、TrkB和CREB蛋白在海马神经元细胞内的分布情况、实时荧光定量PCR技术观察海马神经元中BDNF、TrkB、CREB mRNA表达水平、蛋白免疫印迹考察BDNF、TrkB和CREB蛋白在各组大鼠海马神经元中的表达水平。结果:与正常组比较,模型组大鼠海马神经元中BDNF蛋白及其特异性受体TrkB和CREB蛋白表达及mRNA水平在给药前1天(造模后14 d)显著受到抑制(P<0.05,P<0.01);杜仲腰痛丸干预之后,与模型组比较,模型治疗组海马中BDNF、TrkB、CREB蛋白及mRNA表达和分布在给药后14、28 d显著增加(P<0.05,P<0.01)。结论:杜仲腰痛丸通过上调大鼠海马神经元BDNF、TrkB和CREB蛋白及mRNA的表达水平,治疗LDH慢性下肢痛,因此,其治疗LDH的效应机制可能与激活海马神经元BDNF-TrkB-CREB信号通路相关。

Objective: To explore the effect of Duzhong Yaotong Pills on BDNF-TrkB-CREB signal pathway in hippocampus of rats with chronic lower limb pain caused by lumbar disc herniation(LDH). Methods: Seventy-two male SD rats were randomly divided into normal group, model group and model treatment group, with 24 rats in each group. The model group and the model treatment group were modeled by autologous marrow nucleus transplantation, while the normal group and the model group were given the same dose of 0.9% sodium chloride solution by gavage. The treatment group was given Duzhong Yaotong Pills by gavage, with the dosage of 0.26 g/kg twice a day. Pain behavior(mechanical pain threshold and thermal pain threshold) was used to evaluate whether the model was successful. After the model was successful, hippocampal slices of rats in each group were taken 1 day before and 14 and 28 days after drug administration. The distribution of BDNF, TrkB and CREB proteins in hippocampal neurons was observed by immunofluorescence technique, and the expression levels and proteins of BDNF, TrkB and CREB mRNA in hippocampus were observed by real-time fluorescence quantitative PCR(RT-PCR). Results:Compared with the normal group, the expression and mRNA level of BDNF protein and its specific receptors TrkB and CREB protein in the hippocampus of the model group were significantly inhibited 1 day before administration(14 days after modeling)(P<0.05, P<0.01). Compared with the model group, the expression and distribution of BDNF, TrkB, CREB protein and mRNA in the hippocampus of the model treatment group were significantly increased on 14th and 28th day after administration of Duzhong Yaotong Pills(P<0.05, P<0.01). Conclusion: Duzhong Yaotong Pills can treat LDH chronic lower limb pain by up-regulating the expression levels of BDNF, TrkB and CREB protein and mRNA in hippocampus of rats. Therefore, its therapeutic mechanism of LDH may be related to activating BDNF-TrkB-CREB signal pathway in hippocampus.