六味地黄丸通过TXNIP/NLRP3信号通路对SAMP8小鼠的神经保护作用

Neuroprotective Effect of Liuwei Dihuang Pills(六味地黄丸)on SAMP8 Mice through TXNIP/NLRP3 Signaling Pathway

目的:探讨六味地黄丸通过调控硫氧还蛋白相互作用蛋白(Thioredoxin-interacting protein, TXNIP)/NLR家族Pyrin结构域3(NOD-like receptor, pyrin domain containing 3,NLRP3)炎症通路对快速老化小鼠8(Senescence accelerated mouse/prone 8,SAMP8)的神经保护作用。方法:采用随机数字表法将实验小鼠分为模型对照组、盐酸多奈哌齐0.75 mg/kg组和六味地黄丸0.68、1.35、2.7 g/kg组,用同月龄抗快速老化小鼠(SAMR1)作为正常对照组,连续灌胃给药2个月;采用Morris水迷宫行为学检测小鼠学习记忆能力;通过HE染色和透射电镜观察小鼠脑神经细胞形态及线粒体超微结构;Western Blot法检测小鼠脑组织海马与皮质中TXNIP、NLRP3、半胱氨酸天冬氨酸蛋白水解酶-1(Caspase-1)和白细胞介素-1β(interleulin-1β,IL-1β)蛋白的表达。结果:与正常对照组比较,模型对照组小鼠的逃避潜伏期显著延长,目标象限停留时间与穿越平台次数显著减少(P<0.01),模型对照组SAMP8小鼠海马和皮质内HE染色神经元数量减少,核固缩,电镜结果显示神经元胞质中线粒体数量较少,线粒体嵴减少、断裂,同时,皮质海马中TXNIP、NLRP3、Caspase-1、IL-1β蛋白的表达显著上调(P<0.01);与模型对照组比较,给药各组SAMP8小鼠逃避潜伏期均明显缩短,目标象限停留时间与穿越平台次数明显增多(P<0.05或P<0.01);SAMP8小鼠脑内神经元损伤程度均减轻,可见胞质中线粒体数量增多,线粒体嵴较好融合,给药各组的皮质海马中TXNIP、NLRP3、Caspase-1、IL-1β蛋白的表达明显上调(P<0.05或P<0.01)。结论:六味地黄丸可能通过调控TXNIP/NLRP3炎症信号轴,下调Caspase-1、IL-1β的表达,抑制炎症,保护神经元,从而改善SAMP8小鼠的学习记忆能力。

Objective:To investigate the neuroprotective effect of Liuwei Dihuang Pills(LWDHP)(六味地黄丸)on senescence-accelerated mouse prone 8(SAMP8)mice by regulating thioredoxin-interacting protein(TXNIP)/NOD-like receptor family pyrin domain containing 3(NLRP3)inflammatory pathway.Methods:The experimental mice were randomly divided into a model group,a positive control group(donepezil hydrochloride,0.747 mg/kg),and high-,medium-,and low-dose LWDHP groups(2.7,1.35,and 0.675 g/kg)according to the random number table method.SAMR1 mice aging the same month were assigned to the control group.Mice were administered orally for two months.The learning and memory ability of mice in each group was measured by the Morris water maze(MWM)behavioral test.The morphology of cerebral nerve cells and mitochondrial ultrastructure in each group were observed by HE staining and transmission electron microscopy(TEM).The protein expression of TXNIP,NLRP3,cysteinyl aspartate-specific protease 1(Caspase-1),and interleukin(IL)-1βin the cortex and hippocampus in the mouse brain was detected by Western blot.Results:Compared with the control group,the model group showed prolonged escape latency(P<0.01)and reduced residence time in the target quadrant and the number of crossing the platform(P<0.01).As revealed by HE staining,the model group showed reduced neurons and nuclear pyknosis in the hippocampus and cortex of SAMP8 mice.TEM results showed reduced mitochondria in the cytoplasm of neurons and reduced and broken mitochondrial cristae.At the same time,the protein expression levels of TXNIP,NLRP3,Caspase-1,and IL-1βincreased(P<0.O1).Compared with the model group,the groups with drug intervention showed shortened escape latency of SAMP8 mice(P<0.05 or P<0.01),increased residence time in the target quadrant and the number of crossing the platform(P<0.05 or P<0.01),relieved damage degree of neurons in the brain,partially increased mitochondria in the cytoplasm,fused mitochondrial cristae,and decreased protein expression levels of TXNIP,NLRP3,Caspase-1,and IL-1β(P<0.05 or P<O.O1).Conclusion:LWDHP may improve the learning and memory ability of SAMP8 mice by regulating TXNIP/NLRP3 inflammatory signaling axis,reducing the activity of Caspase-1 and the content of IL-1β,and inhibiting inflammation to protect neurons.