NLRP3/GSDMD/IL-1β通路在发育期大鼠癫痫后抑郁行为中的作用及其干预方法

Effect of NLRP3/GSDMD/IL-1β pathway in post epileptic depressive behavior of developmentalrats and its possible intervention approaches

目的探索NLRP3/GSDMD/IL-1β通路在发育期大鼠癫痫后抑郁行为中的作用及其可能的干预方法。方法60只21日龄SD雄鼠随机分为对照组、癫痫持续状态组(SE组)、癫痫持续状态+地塞米松(DEX)组(SE+DEX组),每组20只。SE组及SE+DEX组均采用腹腔注射氯化锂-匹罗卡品诱发大鼠SE,建立颞叶癫痫(TLE)模型,SE+DEX组在终止癫痫发作后腹腔注射地塞米松3 mg/(kg·d),连续7 d,SE组及对照组大鼠在相同时点以相同给药方式给予等量生理盐水。采用蔗糖偏爱实验(SPT)、旷场实验(OPT)及强迫游泳实验(FST)检测大鼠癫痫后抑郁行为(n=8);Western blotting及RT-qPCR分别测定大鼠海马组织中NLRP3、GSDMD蛋白及mRNA的表达水平(n=3);ELISA法检测大鼠海马组织中白细胞介素(IL)-1β的表达水平(n=3)。结果与对照组比较,SE组蔗糖消耗率降低(68.50%±8.65%vs.87.13%±3.31%),在旷场中央区停留时间减少[(1.25±0.55)s vs.(4.73±2.57)s],总运动距离减少[(7.34±1.48)cm vs.(11.01±1.94)cm],在水中静止不动时间增加[(53.74±22.54)s vs.(27.18±11.86)s];与SE组比较,SE+DEX组蔗糖消耗率增高(78.8%±2.80%vs.68.50%±8.65%),在中央区停留时间延长[(4.00±2.35)s vs.(1.25±0.55)s],在水中静止不动时间缩短[(22.85±7.85)s vs.(53.74±22.54)s],差异均有统计学意义(P<0.05)。SE组的NLRP3、GSDMD蛋白和mRNA表达水平以及IL-1β表达水平均明显高于对照组(P<0.05);而SE+DEX组NLRP3的mRNA表达水平低于SE组(1.40±0.66 vs.2.70±0.22,P<0.05),蛋白表达水平两组比较无明显差异,SE+DEX组GSDMD蛋白及mRNA表达水平均低于SE组(0.38±0.11 vs.0.72±0.03,1.14±0.44 vs.1.80±0.08),IL-1β表达水平也低于SE组[(299.44±119.35)pg/ml vs.(571.37±18.48)pg/ml],差异均有统计学意义(P<0.05)。结论NLRP3/GSDMD/IL-1β通路可能参与了癫痫伴发抑郁的发生发展;地塞米松短疗程干预可能通过下调GSDMD/IL-1β的表达而改善发育期大鼠的癫痫后抑郁行为。

Objective To investigate the effect of NLRP3/GSDMD/IL-1βpathway in post epileptic depressive behavior of developmental rats and its possible intervention approaches.Methods Sixty SD male rats aged 21 days were randomly divided into negative Control group,status epilepticus(SE)group,and SE+Dexamethasone(SE+DEX)group(20 each).The temporal lobe epilepsy(TLE)model was established by intraperitoneal injection of Lithium chloride-Pilocarpine(Licl-Pilocarpine)in SE group and SE+DEX group,the SE+DEX group was intraperitoneally injected with DEX[3 mg/(kg·d)]after the convulsion ceased for 7 consecutive days. Rats in SE group and Control group were given the same amount of normal saline at the same time point and bythe same way. The post epileptic depressive behavior of rats was evaluated by sucrose partiality test (SPT), open field test (OPT) andforced swimming test (FST, n=8). Western blotting and RT-qPCR were used to detect the expression levels of protein and mRNAof NLRP3 and GSDMD in the hippocampus (n=3), and the expression level of IL-1β in the hippocampus was detected by ELISA(n=3). Results Compared with control group, the sucrose consumption rate decreased (68.50%±8.65% vs. 87.13%±3.31%),the time spent in the central area of the open field was reduced [(1.25±0.55) s vs. (4.73±2.57) s], the total movement distancedecreased [(7.34±1.48) cm vs. (11.01±1.94) cm], while the immobility time (IMT) in water increased [(53.74±22.54) svs. (27.18±11.86) s] in SE group;Compared with SE group, the sucrose consumption rate increased (78.8%±2.80% vs.68.50%±8.65%), the time spent in the central area of the open field was prolonged [(4.00±2.35) s vs. (1.25±0.55) s], whilethe IMT was reduced [(22.85±7.85) s vs. (53.74±22.54) s] in SE+DEX group. All the differences were statistically significant(P<0.05). The expression levels of protein and mRNA of NLRP3 and GSDMD, as well as the IL-1β content, were significantlyhigher in SE group than in Control group (P<0.05);While the expression level of mRNA of NLRP3 was lower in SE+DEX groupthan in SE group (1.40±0.66 vs. 2.70±0.22, P<0.05), but no obviously difference between the two groups on the expression levelof NLRP3 protein, The expression levels of GSDMD protein and mRNA were lower in SE+DEX group than in SE group (0.38±0.11vs. 0.72±0.03, 1.14±0.44 vs. 1.80±0.08), the IL-1β content was also lower than that in SE group [(299.44±119.35) pg/ml vs.(571.37±18.48) pg/ml] with statistically significant difference (P<0.05). Conclusions NLRP3/GSDMD/IL-1β may be involvedin the occurrence and development of epilepsy with depression. Short course intervention with dexamethasone may improveepileptic related depressive behavior in developmental rats by down-regulating GSDMD/IL-1β expression.