消肿止痛合剂通过TLR4信号通路对大鼠糖尿病神经痛的作用及机制OA

Objective:To study the effect and mechanism of Xiaozhong Zhitong(消肿止痛)Mixture in the treat-ment 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,Xi-aozhong Zhitong Mixture(0.9 g/kg),Toll-like receptor 4(TLR4)inhibitor(TAK-242,3 mg/kg),and combined treat-ment(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)stai-ning 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 quan-tified 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 lo-cal breakage and sparse and uneven distribution of nerve fibers,widened myelin sheaths with segmental demyelination,lo-cal vascular dilation and lymphocyte infiltration,decreased MWT and PWL at different time points(P<0.05),up-regula-ted expression levels of NF-κB,MyD88,and TLR4 at both protein and mRNA levels(P<0.01),increased positive ex-pression 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,in-creased MWT and PWL(P<0.05),down-regulated mRNA levels of Nfκb,Myd88,and Tlr4(P<0.01),decreased posi-tive 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-regula-ted 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 Zhi-tong 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.