基于MiRNA-146a调控TRL4信号通路探讨推拿对慢性神经病理性疼痛大鼠镇痛机制研究

The pathogenesis of chronic neuropathic pain (CNP) is complicated, and the mechanism of its occurrence and transmission is still not clarified. Recent studies have found that miRNA-146a and TLR4 are involved in the transmission of NP, but whether these two factors are independent or correlative is not clear. Our previous study indicated that massage can affect the ERK downstream signaling pathways of TLR4 so as to reduce the transmition of CNP. According to the study mentioned above we put forward the hypothesis that massage may affect the activation of TLR4 and its downstream signaling pathway by influencing the expression of miRNA-146a, thereby inhibiting the transmission of CNP. In order to verify the hypothesis, we plan to establish a rat model of SNL and observate the heat pain thresholds and mechanical pain thresholds of rats after massage to definite analgesic effect of massage; and then use PCR, Western blot and immunohistochemistry to check the miRNA-146a expression of the rats after massage and the activation of TLR4 in DRG of rats by up-regulating or down regulating the expression of miRNA-146a. And take the results to compare with the activation of TLR4 in DRG of rats after massage, finally illuminate the molecular mechanism of analgesic effect of massage by affecting the expression of miR-146a thereby put the influence on the activation of TLR4 signaling pathway.

慢性神经病理性疼痛（CNP）病机错综复杂，其发生和传递机制不明，近期研究发现miRNA-146a和TLR4均参与了CNP的传递，但二者是独立存在还是相互关联尚不清楚。我们的前期研究发现推拿可以通过影响TLR4的下游信号通路ERK从而达到减少CNP传递，据此我们提出假说：推拿是通过影响miRNA-146a表达，进而影响TLR4及其下游信号通路的活化，从而达到抑制CNP传递。为验证假说，我们建立SNL大鼠模型，观察推拿后大鼠热痛阈和机械痛阈以明确推拿的镇痛效果；再运用PCR、Western blot、免疫组化等技术检测推拿后大鼠miRNA-146a表达量，以及检测上调或下调miRNA-146a表达后大鼠DRG内TLR4的活化情况，并将该结果与推拿后大鼠DRG内TLR4的活化情况进行比较，以期阐明推拿通过影响miR-146a的表达进而影响TLR4信号通路的活化，从而起到镇痛作用的分子机制。

背景：推拿治疗神经病理性疼痛疗效显著，研究其起效机制具有重要意义。方法：对56只雌性大鼠随机分为空白对照组、假手术组、模型组、假推拿组、枢经推拿组、MiRNA-146a过表达组和阴性对照组（NC组），每组各8只。MiRNA-146a过表达组和阴性对照组大鼠鞘内置管成功后与模型组、假推拿组、枢经推拿组一起建立SNL大鼠模型，假手术组暴露脊神经不予结扎，空白对照组不作任何处理。建模成功后MiRNA-146a过表达组鞘内给予10μl 可以靶向上调MiRNA-146a表达水平的慢病毒；NC组给予等剂量的阴性对照试剂。枢经推拿组采用按摩推拿手法模拟仪每天推拿一次，假推拿组大鼠每天采用布袋束缚并轻轻抚摸大鼠双后肢，连续干预14d。空白对照组、假手术组和模型组不进行干预，观察14d。在造模前及造模后的1，3，7，14d检测各组大鼠的热痛阈值和机械痛阈值。于造模后第14天检测空白对照组、假手术组、模型组、假推拿组、枢经推拿组大鼠的MiRNA-146a、TLR4、IRAK1、TRAF6、TNF-α和IL-6表达水平，检测MiRNA-146a过表达组和NC组MiRNA-146a、IRAK1、TRAF6表达水平。结果：大鼠机械痛阈值和热痛阈值变化：枢经推拿组大鼠与模型组、假推拿组大鼠之间差异有统计学意义（P＜0.05），QPCR结果：模型组、假推拿组TLR4、IRAK1、TRAF6表达水平高于枢经推拿组（P＜0.05）。ELISA结果：模型组、假推拿组TNF-α和IL-6表达水平高于枢经推拿组（P＜0.05）。结论：上调MiRNA-146a的表达进而下调TLR4信号通路的活化情况，减少炎症因子的释放，可能是枢经推拿治疗神经病理性疼痛的起效机制之一。