摘要
目的:探讨β-谷甾醇对阿尔茨海默病(AD)模型小鼠认知功能的改善作用,并阐明其可能的分子机制。方法:72只小鼠随机分为对照组、假手术组、AD组、盐酸多奈哌齐(0.6 mg·kg^(-1))及低(1.0 mg·kg^(-1))和高(4.0 mg·kg^(-1))剂量β-谷甾醇组,每组12只。小鼠侧脑室注射β淀粉样蛋白_(1-42)(Aβ_(1-42))建立AD模型,采用β-谷甾醇进行干预治疗,自主活动实验观察各组小鼠自由活动次数,筑巢行为实验观察各组小鼠筑巢行为评分,新物体辨别实验观察各组小鼠对新物体辨别时间和新旧物体辨别时间的比值(DR),Morris水迷宫实验观察各组小鼠逃避潜伏期和跨越平台次数,实时荧光定量PCR(RT-qPCR)法检测各组小鼠海马组织中白细胞介素17A(IL-17A)和p53 mRNA表达水平,Western blottig法检测各组小鼠海马组织中IL-17A、p53、Caspase-3、cleaved Caspase-3、B细胞淋巴瘤2(Bcl-2)和Bcl-2相关X蛋白(Bax)蛋白表达水平,酶联免疫吸附测定(ELISA)法检测各组小鼠海马组织中超氧化物歧化酶(SOD)活性和谷胱甘肽(GSH)、肿瘤坏死因子α(TNF-α)及IL-17A水平。结果:筑巢行为实验,与对照组比较,AD组小鼠筑巢行为评分明显降低(P<0.01);与AD组比较,盐酸多奈哌齐组和低剂量β-谷甾醇组小鼠筑巢行为评分升高(P<0.05)。新物体辨别实验,与对照组比较,AD组小鼠对新物体辨别时间明显减少(P<0.01),DR明显降低(P<0.01);与AD组比较,盐酸多奈哌齐组和高剂量β-谷甾醇组小鼠对新物体辨别时间明显增加(P<0.01),低剂量β-谷甾醇组小鼠DR升高(P<0.05)。Morris水迷宫实验,训练第3和5天,与对照组比较,AD组小鼠逃避潜伏期明显延长(P<0.01);与AD组比较,盐酸多奈哌齐组、低和高剂量β-谷甾醇组小鼠逃避潜伏期明显延长(P<0.05或P<0.01)。与对照组比较,AD组小鼠跨越平台次数明显增加(P<0.01);与AD组比较,盐酸多奈哌齐组、低和高剂量β-谷甾醇组跨越平台次数明显减少(P<0.01)。RT-qPCR法检测,与对照组比较,AD组小鼠海马组织中IL-17A和p53mRNA表达水平均明显升高(P<0.01);与AD组比较,低和高剂量β-谷甾醇组小鼠海马组织中IL-17A mRNA表达水平降低(P<0.05),盐酸多奈哌齐组和高剂量β-谷甾醇组小鼠海马组织中p53 mRNA表达水平降低(P<0.05)。Western blottig法检测,与对照组比较,AD组小鼠海马组织中IL-17A、p53和cleaved Caspase-3蛋白表达水平及Bax/Bcl-2比值明显升高(P<0.01);与AD组比较,低剂量β-谷甾醇组小鼠海马组织中IL-17A和p53蛋白表达水平及Bax/Bcl-2比值明显降低(P<0.05或P<0.01),盐酸多奈哌齐组小鼠海马组织中cleaved Caspase-3蛋白表达水平明显降低(P<0.01),高剂量β-谷甾醇组小鼠海马组织中IL-17A、cleaved Caspase-3蛋白表达水平和Bax/Bcl-2比值明显降低(P<0.05或P<0.01)。ELISA法检测,与对照组比较,AD组小鼠海马组织中SOD活性和GSH水平明显降低(P<0.01),TNF-α和IL-17A水平明显升高(P<0.01);与AD组比较,高剂量β-谷甾醇组小鼠海马组织中SOD活性和GSH水平明显升高(P<0.01),IL-17A水平降低(P<0.05),低剂量β-谷甾醇组小鼠海马组织中TNF-α水平降低(P<0.05)。结论:β-谷甾醇可减轻神经炎症并抑制细胞凋亡,进而改善AD模型小鼠的认知功能,其作用机制可能与抑制IL-17-p53信号通路有关。
Objective:To discuss the improvement effect ofβ-sitosterol on the cognitive function of the Alzheimer’s disease(AD)model mice,and to elucidate its possible molecular mechanism.Methods:Sixty mice were randomly divided into control group,sham operation group,AD group,donepezil hydrochloride(0.6 mg·kg^(-1))group,low dose(1.0 mg·kg^(-1))ofβ-sitosterol group,and high dose(4.0 mg·kg^(-1))ofβ-sitosterol group;and there were 12 mice in each group.The mice were injected withβamyloid protein _(1-42)(Aβ_(1-42))to establish the AD models,andβ-sitosterol interventional treatment was conducted.The free activity numbers of the mice in various groups were observed by autonomous activity experiment;the scores of nesting behavior of the mice in various groups were observed by nesting behavior experiment;the identification time of new objects and the discrimination ratios(DR)of new and old objects of the mice in various groups were observed by new object discrimination experiment;Morris water maze experiment was used to observe the escape latencyies and numbers of crossing platform of the mice in various groups;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of interleukin-17A(IL-17A)and p53 mRNA in hippocampus tissue of the mice in various groups;Western blotting method was used to detect the expression levels of IL-17A,p53,Caspase-3,cleaved Caspase-3,B-cell lymphoma 2(Bcl-2),and Bcl-2 associated X protein(Bax)in hippocampus tissue of the mice in various groups;enzyme-linked immunosorbent assay(ELISA)was used to detect the activities of superoxide dismutase(SOD)and levels of glutathione(GSH),tumor necrosis factorα(TNF-α),and IL-17A in hippocampus tissue of the mice in various groups.Results:The nesting behavior experiment results showed that compared with control group,the score of nesting behavior of the mice in AD group was significantly decreased(P<0.01);compared with AD group,the scores of nesting behavior of the mice in donepezil hydrochloride group and low dose ofβ-sitosterol group were increased(P<0.05).The new object discrimination experiment results showed that compared with control group,the identification time of new objects of the mice in AD group was decreased(P<0.01),and DR was decreased(P<0.01);compared with AD group,the identification time of new objects of the mice in donepezil hydrochloride group and high dose ofβ-sitosterol group were significantly increased(P<0.01),and DR was increased(P<0.05).The Morris water maze experiment results showed that on the 3rd and 5th days after training,compared with control group,the escape latency of the mice in AD group was significantly increased(P<0.01);compared with AD group,the escape latencies of the mice in latendonepezil hydrochloride group and low and high doses ofβ-sitosterol groups were increased(P<0.05 or P<0.01).Compared with control group,the number of crossing platforms of the mice in AD group was increased(P<0.01);compared with AD group,the numbers of crossing platform of the mice in donepezil hydrochloride group and low and high doses ofβ-sitosterol groups were decreased(P<0.01).The RT-qPCR results showed that compared with control group,the expression levels of IL-17A and p53 mRNA in hippocampus tissue of the mice in AD group were significantly increased(P<0.01);compared with AD group,the expression levels of IL-17A mRNA in hippocampus tissue of the mice in low and high doses ofβ-sitosterol groups were decreased(P<0.05),while the expression levels of p53 mRNA in hippocampus tissue of the mice in donepezil hydrochloride group and high dose ofβ-sitosterol group were significantly decreased(P<0.05).The Western blotting results showed that compared with control group,the expression levels of IL-17A,p53,and cleaved Caspase-3 proteins and the Bax/Bcl-2 ratio in hippocampus tissue of the mice in AD group were significantly increased(P<0.01);compared with AD group,the expression levels of IL-17A and p53 proteins and Bax/Bcl-2 ratio in hippocampus tissue of the mice in low dose ofβ-sitosterol group were significantly decreased(P<0.05 or P<0.01),while the expression levels of IL-17A and p53 proteins and Bax/Bcl-2 ratio in hippocampus tissue of the mice in donepezil hydrochloride group and high dose ofβ-sitosterol group were decreased(P<0.01).The ELISA results showed that compared with control group,the SOD activity and GSH level in hippocampus tissue of the mice in AD group were decreased(P<0.01),while the levels of TNF-α,IL-17A and IL-17A in hippocampus tissue of the mice were increased(P<0.01);compared with AD group,the SOD activity and GSH level in hippocampal tissue of the mice in high dose ofβ-sitosterol group were significantly increased(P<0.01),while the IL-17A level was decreased(P<0.05),and the level of TNF-αin hippocampus tissue of the mice in low dose ofβ-sitosterol group was decreased(P<0.05).Conclusion:β-sitosterol can reduce the neuroinflammation and inhibit the apoptosis,thereby improving the cognitive function of the AD model mice;its mechanism may be related to inhibiting the IL-17-p53 signaling pathway.
作者
王星烨
孔祥日
金梦丽
王冰梅
黎明全
WANG Xingye;KONG Xiangri;JIN Mengli;WANG Bingmei;LI Mingquan(Laboratory of Integrated Chinese and Western Medicine,School of Integrated Chinese and Western Medicine,Changchun University of Chinese Medicine,Changchun 130117,China;Department of Neurology,Third Affiliated Hospital,Changchun University of Chinese Medicine,Changchun 130118,China;Department of Endocrinology,School of Chinese Medicine,Changchun University of Chinese Medicine,Changchun 130117,China;Department of Physiology,School of Clinical Medicine,Changchun University of Chinese Medicine,Changchun 130117,China)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2023年第3期599-607,共9页
Journal of Jilin University：Medicine Edition
基金
吉林省科技厅科技发展计划项目(20210101194JC,20200404085YY,20200404065YY)。
