基于TLR4/NF-κB信号通路研究右美托咪定对脑卒中后中枢痛大鼠的镇痛机制

Based on TLR4/NF-κB signaling pathway study the pain mechanism of Dexmedetomidine on central pain in rats after stroke

目的观察腹腔注射右美托咪定通过TLR4/NF-κB信号通路对脑卒中后中枢痛(CPSP)大鼠的镇痛作用。方法90只SD大鼠随机选取30只为假手术组,剩余60只进行丘脑腹后外侧核(VPL)注射Ⅳ型胶原酶造模,造模后3 d测定热缩足反射潜伏期(TWL),采用随机数字表法将TWL降低的大鼠分为模型组和干预组,每组30只。干预组连续7 d腹腔注射右美托咪定50μg/kg,模型组注射等量的生理盐水。于造模前1 d(T_(0))、造模后10 d(T_(1))、造模后17 d(T_(2))、造模后31 d(T_(3)),测定大鼠TWL;在T_(3)时采用Western blot检测VPL中蛋白表达;免疫荧光观察VPL小胶质细胞及神经元的变化;酶联免疫吸附试验检测VPL中炎症介质的释放水平。结果模型组、干预组T_(1)~T_(3)时TWL低于T_(0)时,T_(2)~T_(3)时TWL低于T_(1)时,T_(3)时TWL低于T_(2)时(P<0.05);模型组T_(1)~T_(3)时TWL值低于假手术组,干预组T_(1)~T_(3)时TWL高于假模型组(P<0.05)。与假手术组比较,模型组VPL中TLR4及pNF-κB的表达升高(P<0.05);与模型组比较,干预组VPL中TLR4及pNF-κB的表达降低(P<0.05);三组VPL中NF-κB表达差异无统计学意义(P>0.05)。与假手术组比较,模型组VPL神经元数量均减少(P<0.05);与模型组比较,干预组VPL神经元数量差异无统计学意义(P>0.05)。模型组VPL中Iba-1及肿瘤坏死因子-α(TNF-α)、白介素(IL)-1β和IL-6水平较假手术升高(P<0.05),干预组VPL中Iba-1及炎症因子TNF-α、IL-1β和IL-6水平较模型组降低(P<0.05)。结论右美托咪定可能通过TLR4/NF-κB信号通路抑制小胶质细胞增殖活化及神经炎症进展治疗CPSP。

Objective To observe the analgesic effect of intraperitoneal injection of Dexmedetomidine on central pain(CPSP)after stroke in rats through TLR4/NF-κB signaling pathway.Methods Thirty SD rats were selected randomly from 90 SD rats for sham operation group,and the remaining 60 rats were modeled by injecting typeⅣcollagenase into ventralis posterolateral nucleus(VPL).The thermal withdrawal latency(TWL)was measured three days after modeling.The rats with reduced TWL were divided into model group and intervention group by random number table method,with 30 rats in each group.The intervention group was intraperitoneal injected Dexmedetomidine 50μg/kg for seven consecutive days and the model group was injected with the same amount of narmal saline.TWL values of rats were measured one day before modeling(T_(0)),10 d after modeling(T_(1)),17 d after modeling(T_(2)),and 31 d after modeling(T_(3)).Western blot was used to detect protein expression in VPL at T_(3).The changes of VPL microglia and neurons were observed by immunofluorescence.The release of inflammatory mediators in VPL was detected by enzyme-linked immunosorbent assay.Results In model group and intervention group,TWL at T_(1)-T_(3) was lower than T_(0),TWL at T_(2)-T_(3) was lower than T_(1),and TWL at T_(3) was lower than T_(2)(P<0.05).The TWL at T_(1)-T_(3) in the model group was lower than that in the sham operation group,and the TWL at T_(1)-T_(3) in the intervention group was higher than that in the sham operation group(P<0.05).Compared with sham operation group,the expression of TLR4 and pNF-κB in VPL of model group was significantly increased(P<0.05).Compared with model group,the expression of TLR4 and pNF-κB in VPL of intervention group was decreased(P<0.05).There was no significant difference in NF-κB expression in VPL among the three groups(P>0.05).Compared with sham operation group,the number of VPL neurons in model group decreased(P<0.05);compared with model group,there was no significant difference in the number of VPL neurons in the intervention group(P>0.05).The levels of IbA-1,tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,and IL-6 in VPL of model group were increased compared with that of sham operation group(P<0.05),while the levels of IBA-1 and inflammatory factors TNF-α,IL-1β,and IL-6 in VPL of intervention group were decreased compared with that in model group(P<0.05).Conclusion Dexmedetomidine treat the CPSP may inhibit the proliferation and activation of microglial cells and the progression of neuroinflammation through the TLR4/NF-κB signaling pathway.