摘要
目的探讨人参健脑益智方(RJYF)通过调节抗核转录因子-κB2(p52)信号通路抑制抗NOD样受体蛋白3(NLRP3)炎症小体,从而改善老年痴呆神经元损伤的作用机制。方法将30只SD大鼠按随机数字表法分为假手术(sham)组、β淀粉样蛋白25-35(Aβ25-35)组、Aβ25-35+RJYF-L组、Aβ25-35+RJYF-H组和Aβ25-35+阳性药物(Donepezil)组,每组6只。sham组注射5μL生理盐水;Aβ25-35组注射5μL Aβ25-35溶液;Aβ25-35+RJYF-L组、Aβ25-35+RJYF-H组和Aβ25-35+Donepezil组分别对Aβ25-35组大鼠灌胃3.6 g/kg、7.2 g/kg剂量的RJYF和0.92 mg/kg剂量的Donepezil。将18只大鼠按随机数字表法分为对照组、RJYF-L组和RJYF-H组,每组6只。对照组灌胃生理盐水;RJYF-L组和RJYF-H组分别灌胃3.6 g/kg和7.2 g/kg剂量的RJYF。将神经元细胞分为八组,分别为control组、model组、model+NS组、model+DS-L组、model+DS-H组、model+DS-H+oe-NC组、model+DS-H+oe-p52组、model+DS-H+NLRP3激活剂(Nigericin)组。control组是神经元细胞正常培养;model组是神经元细胞与Aβ25-35共培养;model+NS组、model+DS-L组、model+DS-H组是model组细胞分别与对照组、RJYF-L组和RJYF-H组含药血清共培养;model+DS-H+oe-NC组、model+DS-H+oe-p52组是在model+DS-H组细胞中分别转染oe-NC、oe-p52;model+DS-H+Nigericin组是使用Nigericin处理model+DS-H组。Morris水迷宫评估大鼠认知功能。苏木精伊红染色评估脑组织病理。免疫组织化学检测p52的表达。原位末端转移酶标记检测组织中细胞凋亡。细胞计数试剂盒检测细胞活性。流式细胞术检测细胞凋亡。Western blot检测p52、NLRP3、含CARD结构的凋亡相关斑点样蛋白(ASC)和活化的半胱氨酸蛋白酶1(c-caspase 1)的表达。结果与sham组相比,Aβ25-35组认知功能发生障碍,神经元出现不规则的细胞形态,神经元死亡增加,p52、NLRP3、ASC和c-caspase 1升高。与Aβ25-35组相比,Aβ25-35+RJYF-L组、Aβ25-35+RJYF-H组和Aβ25-35+Donepezil组认知功能和细胞形态得到改善,神经元死亡减少,p52、NLRP3、ASC和c-caspase 1降低。与control组相比,model组细胞活性降低[(54.96±4.33)%比(100.00±6.31)%,P<0.01],凋亡增加[(18.05±1.35)%比(6.49±1.24)%,P<0.01],p52、NLRP3、ASC和c-caspase 1增加。与model+NS组相比,model+DS-L组和model+DS-H组细胞活性增加[(71.03±6.61)%、(81.71±6.22)%比(54.16±3.72)%,P<0.05或P<0.01],凋亡减少[(14.64±0.73)%、(11.15±1.60)%比(18.67±0.92)%,P<0.05或P<0.01],p52、NLRP3、ASC和c-caspase 1降低。与model+DSH组相比,model+DS-H+Nigericin组细胞活性降低[(119.74±8.57)%比(141.79±9.86)%,P<0.05],细胞凋亡增加[(13.64±0.69)%比(10.02±0.73)%,P<0.01],NLRP3、ASC和c-caspase 1增加。与model+DS-H+oe-NC组相比,model+DS-H+oe-p52组细胞活性降低[(123.89±7.47)%比(150.68±9.48)%,P<0.05],凋亡增加[(15.15±1.06)%比(11.2±1.65)%,P<0.05],p52、NLRP3、ASC和c-caspase 1升高。结论RJYF调节p52抑制NLRP3炎症小体,减轻神经元损伤,改善阿尔茨海默病(AD)的认知功能。
Objective To investigate the mechanism of Renshen Jiannao Yizhi Formula(RJYF)alleviating neuronal damage in Alzheimer's disease(AD)by regulating the nuclear transcription factor-kB2(p52)signaling pathway to inhibit the activation of the NOD-like receptor protein 3(NLRP3)inflammasome.Methods Thirty SpragueDawley rats were randomly divided into five groups(6 rats per group)using a random number table:the sham group,theβ-amyloid25-35(Aβ25-35)group,the Aβ25-35+RJYF low-dose(RJYF-L)group,the Aβ25-35+RJYF high-dose(RJYF-H)group,and the Aβ25-35+positive control(Donepezil)group.The sham group was injected with 5μL of normal saline,while the Aβ25-35 group was injected with 5μL of Aβ25-35 solution.Rats in the Aβ2535+RJYF-L group,Aβ25-35+RJYF-H group,and Aβ25-35+Donepezil group previously treated with Aβ25-35 were administered RJYF at doses of 3.6 g/kg and 7.2 g/kg,or Donepezil at 0.92 mg/kg,via intragastric gavage.Additionally,18 rats were randomly divided into three groups:the control,RJYF-L,and RJYF-H groups(n=6 per group)using a random number table.The control group received an equivalent volume of normal saline via gavage,while the RJYF-L and RJYF-H groups were administered RJYF at doses of 3.6 g/kg and 7.2 g/kg,respectively.Neuron cells were divided into 8 groups:control group,model group,model+NS group,model+DS-L group,model+DS-H group,model+DS-H+oe-NC group,model+DS-H+oe-p52 group,and model+DS-H+Nigericin group.The control group was under normal conditions,and the model group was co-cultured with Aβ25-35.The model+NS,model+DS-L,and model+DS-H groups were Aβ25-35-treated cells co-cultured with control rat serum,low-dose RJYFmedicated serum,and high-dose RJYF-medicated serum,respectively.Cells in the model+DS-H+oe-NC and model+DS-H+oe-p52 groups were established by transfecting cells in the model+DS-H group with an empty overexpression vector(oe-NC)and or a p52 overexpression plasmid(oe-p52),respectively.For the model+DS-H+Nigericin group,cells in the model+DS-H group were further treated with Nigericin,a NLRP3 activator.The Morris water maze was used to evaluate the cognitive function of rats.Hematoxylin and eosin staining was used to detect the histopathological changes in brain tissue,and immunohistochemical staining was used to detect the expression of p52.Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay was used to detect apoptosis in tissues.Cell Counting Kit-8 was used to detect cell viability.Flow cytometry was performed to measure apoptosis.Western blotting was performed to measure the protein expression levels of p52,NLRP3,apoptosis-associated speck-like containing a CARD(ASC),and cleaved-cysteinyl aspartate-specific proteinase 1(c-caspase 1).Results Compared with the sham group,the Aβ25-35 group showed significant cognitive impairment,irregular neuronal morphology,and increased neuronal death.Furthermore,the protein expression levels of p52,NLRP3,ASC,and c-caspase 1were markedly elevated.In contrast,compared with the Aβ25-35 group,the Aβ25-35+RJYF-L,Aβ25-35+RJYFH,and Aβ25-35+Donepezil group showed improved cognitive function and neuronal morphology,while reducing neuronal death.These improvements were accompanied by a significant downregulation of p52,NLRP3,ASC,and c-caspase 1 expression.Compared with the control group,the model group showed significantly decreased cell activity[(100.00±6.31)s vs.(54.96±4.33)s,P<0.05]and increased apoptosis[(6.49±1.24)s vs.(18.05±1.35)s,P<0.05].Furthermore,the protein expression levels of p52,NLRP3,ASC,and c-caspase 1 were markedly elevated.Compared with the model+NS group,the model+DS-L and model+DS-H group showed increased cell activity[(71.03±6.61)s,(81.71±6.22)s vs.(54.16±3.72)s,P<0.05]and decreased apoptosis[(14.64±0.73)s,(11.15±1.60)s vs.(18.67±0.92)s,P<0.05].However,the protein expression levels of p52,NLRP3,ASC,and c-caspase 1 were significantly downregulated.Compared with the model+DS-H group,the model+DS-H+Nigericin group showed decreased cell activity[(119.74±8.57)s vs.(141.79±9.86)s,P<0.05]and increased apoptosis[(13.64±0.69)s vs.(10.02±0.73)s,P<0.05].Furthermore,the protein expression levels of NLRP3,ASC,and c-caspase 1 were markedly elevated.Compared with the model+DS-H+oe-NC group,the model+DS-H+oe-p52 group showed decreased cell activity[(123.89±7.47)s vs.(150.68±9.48)s,P<0.05]and increased apoptosis[(15.15±1.06)s vs.(11.2±1.65)s,P<0.05].Furthermore,the protein expression levels of p52,NLRP3,ASC,and c-caspase 1 were markedly elevated.Conclusion RJYF exerts neuroprotective effects by inhibiting NLRP3 inflammasome via regulation of p52,thereby alleviating neuronal injury and improving cognitive function in AD.
作者
谢立全
陈远园
杨聘
XIE Liquan;CHEN Yuanyuan;YANG Pin(Department of Geratology,Hangzhou Hospital of Traditional Chinese Medicine,Hangzhou 310007,Zhejiang)
出处
《浙江中西医结合杂志》
2026年第2期133-140,共8页
Zhejiang Journal of Integrated Traditional Chinese and Western Medicine
基金
浙江省中医药科技计划项目(No.2023ZL537)。
