瑞香素对坐骨神经慢性压迫性损伤诱导大鼠神经病理性疼痛的作用及其机制

Effects of daphnetin on neuropathic pain in rats induced by chronic constriction injury of sciatic nerve and its mechanism

目的:探讨瑞香素(DAPH)对坐骨神经慢性压迫性损伤(CCI)诱导大鼠神经病理性疼痛(NP)的作用及其机制。方法:将60只SD大鼠随机分为假手术组、模型组、DAPH低剂量组(DAPH-L组)、DAPH高剂量组(DAPH-H组),每组15只。除假手术组外,其它各组均采用CCI诱导大鼠NP,假手术组手术操作同模型组但不结扎坐骨神经。分别于术前、术后测定大鼠机械缩足反射阈值(MWT)、热缩足反射潜伏期(TWL)。酶联免疫吸附试验法(ELISA)检测大鼠脊髓组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)水平,免疫荧光染色检测大鼠脊髓中胶质原纤维酸性蛋白(GFAP)、小钙离子接头蛋白1(Iba-1)的荧光表达量,Western blotting法检测大鼠背根神经节(DRG)中硫氧还蛋白相互作用蛋白(TXNIP)、Nod样受体蛋白3(NLRP3)、凋亡相关斑点样蛋白(ASC)和含半胱氨酸的天冬氨酸蛋白水解酶-1(Caspase-1)蛋白表达水平。结果:与假手术组比较,模型组术后MWT和TWL降低,脊髓组织TNF-α、IL-1β和IL-6水平升高,GFAP和Iba-1荧光强度增强,DRG中TXNIP、NLRP3、ASC和Caspase-1蛋白表达水平升高(均P<0.05)。与模型组比较,DAPH-L组和DAPH-H组术后MWT和TWL升高,脊髓组织TNF-α、IL-1β和IL-6水平降低,GFAP和Iba-1荧光强度减弱,DRG中TXNIP、NLRP3、ASC和Caspase-1蛋白表达水平降低(均P<0.05),且DAPH-H组上述指标变化较DAPH-L组更为显著(P<0.05)。结论:DAPH通过阻断星形胶质细胞和小胶质细胞活化抑制CCI诱导的炎症反应,进而减轻NP,其机制可能与下调TXNIP、NLRP3表达有关。

Objective:To investigate the effects of daphnetin(DAPH)on neuropathic pain(NP)in rats induced by chronic constriction injury(CCI)of sciatic nerve and its mechanism of action.Methods:Sixty rats were randomly divided into sham-operated group,model group,DAPH low-dose group(DAPH-L group),and DAPH high-dose group(DAPH-H group),with 15 rats in each group.Except for the sham-operated group,NP were induced using CCI for rats in all other groups,and the surgical operation of the sham-operated group was the same as that of the model group but without ligation of the sciatic nerve.The mechanical threshold threshold(MWT)and thermal withdrawal latency(TWL)were measured before and after surgery.The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)were measured by enzyme-linked immunosorbent assay(ELISA),and the fluorescence expression of glial fibrillary acidic protein(GFAP)and ionized calciumbinding adapter molecule 1(IBA1)was measured by immunofluorescence staining in the spinal cord of rats.Western blotting was performed to detect the expression levels of thioredoxin-interacting protein(TXNIP),Nod-like receptor protein 3(NLRP3),apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC)and Caspase-1 in dorsal root ganglion(DRG)of the rats.Results:Compared with the shamoperated group,the model group showed decreased MWT and TWL,increased levels of TNF-α,IL-1βand IL-6in spinal cord tissues,enhanced fluorescence intensity of GFAP and Iba-1,and increased expression levels of TXNIP,NLRP3,ASC and Caspase-1 proteins in DRG after surgery(all P<0.05).Compared with the model group,the DAPH-L and DAPH-H groups showed increased MWT and TWL,decreased TNF-α,IL-1βand IL-6levels in spinal cord tissues,decreased GFAP and Iba-1 fluorescence intensity,and decreased TXNIP,NLRP3,ASC and Caspase-1 protein expression levels in DRG(all P<0.05),and the changes of the above indexes were more significant in the DAPH-H group than in the DAPH-L group(P<0.05).Conclusions:DAPH inhibits CCIinduced inflammatory response and thus attenuates NP by blocking astrocyte and microglia activation,and its mechanism may be related to downregulation of TXNIP and NLRP3 expression.