基于Nlrp3/ASC/Caspase-1通路探讨加减薯蓣丸对APP/PS1痴呆小鼠神经炎症的影响

Effect of Modified Shuyuwan on Neuroinflammation in APP/PS1 Dementia Mice Based on Nlrp3/ASC/Caspase-1 Signaling Pathway

目的:观察加减薯蓣丸对淀粉样蛋白前体蛋白/早老素1(APP/PS1)痴呆小鼠认知障碍的治疗效果并探讨其作用机制。方法:将10只野生型小鼠设为正常组,40只APP/PS1小鼠,分为模型组,加减薯蓣丸低、高剂量组(14,64 g·kg^(-1))和多奈哌齐组(1 mg·kg^(-1)),每组10只,正常组、模型组给予等体积生理盐水,连续灌胃35 d。给药结束后,Morris水迷宫检测小鼠记忆和空间探索能力;尼氏(Nissl)染色观察小鼠海马神经元形态;免疫组化检测小鼠海马β淀粉样蛋白1-42(Aβ1-42)沉积;免疫荧光检测海马小胶质细胞(MG)标志物离子钙结合衔接分子1(Iba1)和激活状态标志物诱导型一氧化氮合酶(iNOS)的表达;蛋白免疫印迹法(Western blot)检测小鼠海马NOD样受体3(Nlrp3),接头蛋白指向相关斑点蛋白(ASC),半胱氨酸蛋白酶-1(Caspase-1)通路及白细胞介素-1β(IL-1β)蛋白表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测海马IL-1β,肿瘤坏死因子-α(TNF-α),白细胞介素-18(IL-18)mRNA的表达。结果:与空白组比较,模型组小鼠记忆和空间探索能力明显降低(P<0.05),海马神经元数量减少,Aβ1-42沉积增多,MG激活标记物Iba1,iNOS增加,Nlrp3,ASC,Caspase-1,IL-1β蛋白的表达均明显增高(P<0.05),IL-1β,IL-18,TNF-αmRNA表达均明显增加(P<0.05);与模型组比较,加减薯蓣丸组能够明显改善APP/PS1小鼠的空间探索能力和记忆能力(P<0.05),增加海马神经元数量,减少Aβ1-42沉积,减轻MG的激活,降低Nlrp3,ASC,Caspase-1,IL-1β蛋白的表达(P<0.05),减少炎症因子mRNA的表达(P<0.05)。结论:加减薯蓣丸能够通过抑制Nlrp3/ASC/Caspase-1通路,降低APP/PS1小鼠海马IL-1β等炎症因子的表达,缓解神经炎症,减轻阿尔兹海默症(AD)病理损伤。

Objective: To observe the effect of modified Shuyuwan in amyloid precursor protein/presenilin 1(APP/PS1)dementia mice on cognitive and memory impairment and to explore its mechanism.Method: The 40 APP/PS1 mice were divided into model group(given Physiological saline),low and highdose modified Shuyuwan(14,64 g·kg^(-1))group,and donepezil group(1 mg·kg^(-1))and 10 wild mice were set as the blank control group(given Physiological saline). All of the mice were administered intragastrically for 35 days. The memory and space exploration ability of mice was detected by Morris water maze,the morphology of mouse hippocampal neurons were observed by Nissl staining. The deposition of β amyloid 1-42(Aβ1-42)in mouse hippocampus was detected by immunohistochemistry,and the expression of ionized calcium-binding adapter molecule 1(Iba1),a marker of hippocampal microglia(MG)and Nitric oxide synthase(iNOS),a marker of actived MG, were detected by immunofluorescence. The protein expression of NLR family pyrin domain containing 3(Nlrp3),Apoptosis-associated speck-like protein containing a Caspase-recruitment domain(ASC),cysteine protease-1(Caspase-1)and interleukin-1 beta(IL-1β)were detected by Western blot,and the expression of IL-1β,tumor necrosis factor-α(TNF-α)and interleukin-18(IL-18)mRNA were detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR). Result: Compared with the blank control group,the memory and space exploration ability of the model group were significantly reduced(P<0.05),the number of hippocampal neurons decreased,the deposition of Aβ1-42 increased,the markers of actived MG Iba1,iNOS increased,the protein expression of Nlrp3,ASC,Caspase-1,IL-1β increased significantly(P<0.05),and the mRNA expression of IL-1β,IL-18,and TNF-α increased significantly(P<0.05). Compared with model group,the Chinese medicine group can improve the APP/PS1 mice’s space exploration ability and memory ability(P<0.05),increase the number of hippocampal neurons,reduce Aβ1-42 deposition,reduce the activation of MG,and reduce the protein expression of Nlrp3,ASC,Caspase-1 and IL-1β(P<0.05),and reduced the expression of IL-1β mRNA(P<0.05). Conclusion: Modified Shuyuwan can reduce the expression of IL-1β and other inflammatory factors in the hippocampus of APP/PS1 mice by inhibiting the Nlrp3/ASC/Caspase-1 pathway,and relieve nerve inflammation and pathological injury of AD.