中药药理与临床

目的:研究消肿止痛合剂治疗糖尿病神经痛(diabetic neuropathic pain, DNP)的作用与机制。方法:将40只雄性SD大鼠以链脲佐菌素(STZ)和高糖高脂饲料建立大鼠DNP模型后，随机分为模型对照组、消肿止痛合剂0.9 g/kg组、Toll样受体4(TLR4)抑制剂TAK-242 3 mg/kg组、TAK-242 3 mg/kg+消肿止痛合剂0.9 g/kg组，另设正常对照组，每组10只，各组给予相应药物或生理盐水，1次/d,连续14 d。测后足机械性缩足反射阈值(MWT)、测定热刺激缩足反射潜伏期(PWL); ELISA法检测大鼠坐骨神经组织中肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、IL-1β含量；HE染色观察大鼠坐骨神经组织病理形态改变；Real-time PCR法检测大鼠坐骨神经组织中Tlr4、Nfkb、Myd88 mRNA表达；Western blot法检测大鼠坐骨神经组织中TLR4、核因子κB(NF-κB)及髓样分化因子88(MyD88)蛋白表达；免疫组化法(IHC)检测大鼠坐骨神经组织中NOD样受体蛋白3(NLRP3)、环氧合酶-2(COX-2)、诱导型一氧化氮合酶(INOS)蛋白阳性表达。结果:与正常对照组比较，模型对照组小鼠坐骨神经纤维局部断裂，分布稀疏不均，髓鞘增宽，有节段性脱髓鞘现象，局部可见血管扩展及淋巴细胞浸润，不同时间节点的MWT、PWL明显降低(P<0.05),坐骨神经组织Nfkb、Myd88及Tlr4 mRNA和蛋白表达显著上调(P<0.01),COX-2、INOS及NLRP3蛋白表达和阳性面积均显著升高(P<0.01),TNF-α、IL-6及IL-1β含量显著升高(P<0.01);与模型对照组比较，消肿止痛合剂0.9 g/kg组、TAK-242 3 mg/kg组、TAK-242 3 mg/kg+消肿止痛合剂0.9 g/kg组坐骨神经损伤明显改善，神经纤维排列相对紧密整齐，未见明显淋巴细胞浸润；MWT、PWL均显著升高(P<0.05),坐骨神经组织Nfkb、Myd88及Tlr4 mRNA表达显著下调(P<0.01),COX-2、INOS及NLRP3蛋白表达和阳性面积明显降低(P<0.05),TNF-α、IL-6及IL-1β含量明显降低(P<0.05),消肿止痛合剂0.9 g/kg组和TAK-242 3 mg/kg+消肿止痛合剂0.9 g/kg组大鼠坐骨神经组织中TLR4、NF-κB及MyD88蛋白表达显著下调(P<0.01),TAK-242 3 mg/kg组大鼠坐骨神经组织中仅TLR4蛋白表达明显下调(P<0.05)。结论:消肿止痛合剂可改善DNP大鼠坐骨神经损伤，降低炎症反应与氧化应激反应，缓解DNP疼痛；消肿止痛合剂联合TLR4抑制剂在改善DNP大鼠坐骨神经损伤、降低炎症反应、缓解DNP疼痛方面效果更显著。

Objective:To study the effect and mechanism of Xiaozhong Zhitong(消肿止痛) Mixture in the treatment of diabetic neuropathic pain(DNP). Methods:Forty male Sprague-Dawley(SD) rats were modeled for DNP via streptozotocin(STZ) and a high-fat high-sugar diet. The modeled rats were randomly assigned into model control, Xiaozhong Zhitong Mixture(0.9 g/kg),Toll-like receptor 4(TLR4) inhibitor(TAK-242,3 mg/kg),and combined treatment(TAK-242 at 3 mg/kg + Xiaozhong Zhitong Mixture at 0.9 g/kg) groups(n=10). Another 10 rats were selected as the normal control group. Rats in each group were administrated with corresponding drugs or normal saline once per day for 14 consecutive days. The mechanical withdrawal threshold(MWT) and paw withdrawal latency(PWL) were measured. Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and interleukin-1β(IL-1β) in the sciatic nerve tissue. Hematoxylin-eosin(HE) staining was performed to examine the histopathological changes in the sciatic nerve tissue. The protein and mRNA levels of TLR4,nuclear factor-κB(NF-κB),and myeloid differentiation factor 88(MyD88) in the sciatic nerve tissue were quantified by Western blot and real-time PCR,respectively. Immunohistochemistry(IHC) was adopted to assess the positive expression of NOD-like receptor protein 3(NLRP3),cyclooxygenase-2(COX-2),and inducible nitric oxide synthase(iNOS) in the sciatic nerve tissue. Results:Compared with the normal control group, the model control group showed local breakage and sparse and uneven distribution of nerve fibers, widened myelin sheaths with segmental demyelination, local vascular dilation and lymphocyte infiltration, decreased MWT and PWL at different time points(P<0.05),up-regulated expression levels of NF-κB,MyD88,and TLR4 at both protein and mRNA levels(P<0.01),increased positive expression of COX-2,INOS,and NLRP3(P<0.01),and raised levels of TNF-α,IL-6,and IL-1β(P<0.01). Compared with the model control group, the Xiaozhong Zhitong Mixture, TLR4 inhibitor(TAK-242),and combined treatment groups showed alleviated sciatic nerve damage, neatly and tightly arranged nerve fibers, no obvious lymphocyte infiltration, increased MWT and PWL(P<0.05),down-regulated mRNA levels of Nfκb,Myd88,and Tlr4(P<0.01),decreased positive expression of COX-2,INOS,and NLRP3(P<0.05),and lowered levels of TNF-α,IL-6,and IL-1β(P<0.05). Compared with model control group, the Xiaozhong Zhitong Mixture and combined treatment groups showed down-regulated protein levels of TLR4,NF-κB,and MyD88(P<0.01),and the TLR4 inhibitor(TAK-242) group showed only down-regulated protein level of TLR4 in the sciatic nerve tissue(P<0.05). Overall, compared with the model control group, the combined treatment group showed the most significant changes in all indicators. Conclusion:Xiaozhong Zhitong Mixture can ameliorate the sciatic nerve injury, reduce inflammation and oxidative stress, and relieve the DNP pain in the rat model of DNP. Xiaozhong Zhitong Mixture combined with TLR4 inhibitor is more effective in ameliorating the sciatic nerve injury, reducing inflammation, and relieving DNP pain.