小续命汤调控NLRP3对CPZ诱导脱髓鞘小鼠的保护机制

Protective mechanism of Xiaoxuming Decoction regulating NLRP3 against cuprizone induced injury in demyelinating mice

目的:探究小续命汤(Xiaoxuming Decoction,XD)对双环己酮草酰二腙(cuprizone,CPZ)诱导的脱髓鞘小鼠的神经炎症影响。方法:C57BL/6雄性小鼠随机分为正常对照(Control)组、CPZ组、XD1组(低剂量)和XD2(高剂量)组。每组10只小鼠,Control组不建模,其他3组每日喂饲0.2%CPZ建模,第28日起XD1和XD2组分别给予30 g·kg^(-1),bid和30 g·kg^(-1),tid XD灌胃,Control组和CPZ组分别给予等量生理盐水灌胃,持续至第42日。通过高架十字迷宫检测小鼠行为学表现,LFB染色和MBP免疫荧光染色观察小鼠胼胝体髓鞘脱失情况,脑组织小胶质细胞IBA1染色,荧光定量PCR和Western blot检测CD11b,HMGB1,TLR4/NLRP3/Caspase-1炎症小体和炎症介质的mRNA和蛋白质表达水平。结果:与CPZ模型组比较,XD治疗组小鼠进入闭臂次数减少和闭臂运动距离缩短,高剂量治疗组效果尤为显著(P<0.05,P<0.01),改善胼胝体髓鞘脱失(P<0.05),降低脑组织中小胶质细胞浸润(P<0.05);减少HMGB1、TLR4和炎症小体NLRP3的mRNA和蛋白质表达(P<0.05),降低细胞因子Caspase-1、IL-1β和IL-18的mRNA(P<0.05),抑制Akt/NF-κB通路活化(P<0.05)。结论:XD可能通过调控NLRP3炎症小体表达,抑制脑组织中小胶质细胞活化和浸润,进而降低CPZ模型神经炎性损伤,发挥神经保护作用。

OBJECTIVE To explore the effect of Xiaoxuming Decoction(XD) on neuroinflammation induced by cuprizone(CPZ) in demyelinating mice.METHODS Forty C57BL/6 male mice were randomized into four groups of control,CPZ,XD1(low-dose) and XD2(high-dose)(n=10 each).Except for Control group,the other three groups received a daily diet of 0.2%CPZ for modeling.From Day 28,XD1 and XD2 groups received gavages of 30 g·kg^(-1),bid and 30 g·kg^(-1),tid XD while Control and CPZ groups equal amounts of physiological saline until Day 42.Murine behaviors were detected by an elevated cross maze.Luxol fast blue(LFB) stain and myelin basic protein(MBP) immunofluorescent stain were utilized for observing the loss of myelin sheath in corpus callosum.IBA1 stain of microglia in brain tissue was employed.mRNA and protein expression levels of CD11b,HMGB1,TLR4/NLRP3/Caspase-1 inflammasomes and inflammatory mediators were detected by fluorescent quantitative polymerase chain reaction(PCR) and Western blot.RESULTS As compared with CPZ model group,XD treatment group lowered the frequency of entering closed arm and distance of closed arm movement in mice,especially in high-dose treatment group(P<0.05,P<0.01),improved the demyelination of corpus callosum(P<0.05) and minimized the infiltration of microglia in brain tissue(P<0.05).XD treatment group down-regulated the mRNA/protein expressions of HMGB1,TLR4 and NLRP3 of inflammasomes(P<0.05),decreased the mRNA of Caspase-1,IL-1β and IL-18(P<0.05) and blunted Akt/NF-κB pathway activation(P<0.05).CONCLUSION XD may play a neuroprotective role through regulating the expression of NLRP3 inflammasome,suppressing the activation and infiltration of microglia in brain tissue and minimizing neuroinflammatory injury in CPZ model.