丙泊酚通过调节脊柱GluN2B-p38 MAPK/EPAC1通路减轻慢性收缩损伤引起的神经性疼痛的大鼠模型研究

A rat model study of propofol reducing neuropathic pain caused by chronic contractile injury by modulating the spinal GluN2B-p38 MAPK/EPAC1 pathway

目的探讨丙泊酚减轻慢性收缩损伤(CCI)引起的神经性疼痛的分子作用机制。方法12只大鼠通过结扎坐骨神经构建CCI的模型,然后根据丙泊酚注射剂量分为:模型组(CCI大鼠,皮下注射0.9%氯化钠溶液)、丙泊酚A组(CCI大鼠,皮下注射10 mg/kg丙泊酚)、丙泊酚B组(CCI大鼠,皮下注射20 mg/kg丙泊酚)和丙泊酚C组(CCI大鼠,皮下注射40 mg/kg丙泊酚),每组3只。另取3只大鼠进行假手术,即仅暴露坐骨神经,不进行结扎(假手术组,皮下注射等量0.9%氯化钠溶液)。连续注射14 d后,采用疼痛行为测量法测定各组大鼠疼痛行为变化;采用酶联免疫吸附实验(ELISA)法检测了上述分组大鼠的背根神经节(L_(4)~L_(6))的白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)炎性因子水平;采用蛋白质印迹(Western blotting)法检测了假手术组、模型组和丙泊酚C组的大鼠背根神经节(L_(4)~L_(6))的EPAC1的相对表达水平,以及p-GluN2B,痛觉相关因子p-ERK、p-JNK和p-p38的磷酸化水平。结果与假手术组相比,模型组疼痛行为和痛觉敏感明显加重,大鼠背根神经节(L_(4)~L_(6))的IL-6、IL-1β和TNF-α炎性因子水平显著升高,在给大鼠皮下注射不同剂量的丙泊酚(10、20、40 mg/kg)后,大鼠的疼痛行为和痛觉敏感随着给药丙泊酚剂量的增加显著缓解,差异有统计学意义(P<0.05)。与假手术组相比,模型组和丙泊酚C组EPAC1和4种因子的磷酸化水平均显著上升,差异有统计学意义(P<0.05);与模型组相比,丙泊酚C组EPAC1和4种因子的磷酸化水平均显著降低,差异有统计学意义(P<0.05)。结论丙泊酚镇痛的分子作用机制为降低脊柱EPAC1的表达水平,并降低脊柱GluN2B-p38 MAPK信号通路的磷酸化水平。

Objective To investigate the molecular mechanism of propofol in reducing neuropathic pain caused by chronic contractile injury(CCI).Methods Twelve rats were modeled with CCI by ligating the sciatic nerve,then according to the propofol injection dose,they were divided into:sham operation group(sham operated rats,subcutaneous injection of the same amount of saline),model group(CCI rats,subcutaneous injection of the same amount of saline),propofol A group(CCI rats,subcutaneous injection of 10 mg/kg propofol),Propofol B group(CCI rats,subcutaneous injection of 20 mg/kg propofol)and propofol group C(CCI rats,subcutaneous injection of 40 mg/kg propofol),3 rats in each group.Another 3 rats were taken for sham operation,that is,only the sciatic nerve was exposed without ligation(in the sham operation group,the same amount of 0.9%sodium chloride solution was injected subcutaneously).After 14 days of continuous injection,the pain behavior measurement method was used to measure the pain behavior changes of rats in each group.The inflammatory factor interleukin-6(IL-6),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)levels of the dorsal root ganglia(L_(4)-L_(6))of the rats in the above groups were detected by enzyme-linked immunosorbent assay(ELISA).The EPAC1 and p-GluN2B levels,as well as the pain-related factors,p-ERK,p-JNK and p-p38 of the dorsal root ganglia(L_(4)-L_(6))of the rats in the sham group,the CCI group and the propofol(40)were detected by Western blotting.Results Compared with the sham group,the successfully constructed rat animal model group(CCI group)significantly aggravated the pain behavior and hyperalgesia,and the levels of the inflammatory factor,IL-6,IL-1βand TNF-αin the dorsal root ganglia(L_(4)-L_(6)).After subcutaneous injection of different doses of propofol(10,20 and 40 mg/kg)to CCI rats,the pain behavior and hyperalgesia of those rats were significantly relieved following the increasing dose of propofol.Compared with the Sham group,the phosphorylation levels of EPAC1 and 4 factors in the CCI group and propofol group C group increased significantly,the differencs were statistically significant(P<0.05);compared with the model group,the phosphorylation levels of EPAC1 and 4 factors in propofol group C were significantly reduced,the differencs were statistically significant(P<0.05).Conclusion The molecular mechanism of propofol analgesia is to reduce the expression level of EPAC1 and reduce the phosphorylation levels of the GluN2B-p38 MAPK signaling pathway.