泽泻汤对高热量饮食诱导认知功能损害模型小鼠的认知功能及脑组织神经炎症的影响

Effects of Zexie Decoction(泽泻汤)on Cognitive Function and Neuroinflammation of Brain Tissue in Mouse Models with High-Calorie Diet-Induced Cognitive Impairment

目的探究泽泻汤对高热量饮食介导的认知功能损害的影响及可能机制。方法将27只C57BL/J小鼠随机分成对照组、模型组和泽泻汤组,每组9只。模型组和泽泻汤组小鼠均采用高脂饲料喂养建立高热量饮食诱导认知功能损害小鼠模型,泽泻汤组在高脂饲料喂养的同时给予0.36 g/(kg·d)泽泻汤灌胃,对照组和模型组小鼠给予等体积生理盐水灌胃,持续8周。每周同一时间记录小鼠体质量;干预结束后,各组小鼠进行口服葡萄糖耐量测试、胰岛素耐量测试;利用莫利斯水迷宫实验、旷场实验、新物体识别实验、Y迷宫测试对小鼠进行认知行为学检测;核磁共振波谱检测小鼠左侧海马、下丘脑、皮层神经代谢物质N-乙酰天门冬氨酸(NAA)、胆碱(CHO)、乳酸(Lac)、肌酸(Cr)、脂质(Lip)、肌醇(mI)的表达;蛋白质免疫印迹法检测小鼠脑组织突触素(SYN)、突触体相关蛋白25(SNAP-25)、突触后致密蛋白95(PSD-95)、肿瘤坏死因子α(TNF-α)、核转录因子κB p65(NF-κB p65)及磷酸化核转录因子κB p65(p-NF-κB p65)的表达;尼氏染色检测海马神经元的形态变化。结果与对照组比较,模型组小鼠体质量、口服葡萄糖耐量测试血糖和胰岛素耐量测试血糖水平均升高;模型组小鼠在莫利斯水迷宫实验中第3天、第4天的移动总路程和逃避潜伏期增加、在平台区域停留时间和穿越平台次数减少,在旷场实验中模型组小鼠进入中心区域的次数、中心区域停留时间与总时间的比值、中心区域活动路程与总路程的比值显著减少,在新物体识别实验中模型组小鼠新物体识别频次和识别指数显著降低,Y迷宫测试中模型组小鼠自发交替率明显降低(P<0.05或P<0.01);模型组小鼠左侧海马、下丘脑和皮层的CHO/Cr、NAA/Cr明显降低,mI/Cr、Lac/Cr和Lip/Cr明显升高;脑组织中SYN/β-actin、SNAP-25/β-actin、PSD-95/β-actin值显著降低,p-NF-κB p65/NF-κB p65、TNF-α/β-actin值显著升高(P<0.05或P<0.01)。与模型组比较,泽泻汤组小鼠上述指标均显著改善(P<0.05或P<0.01)。尼氏染色结果显示,与对照组比较,模型组小鼠海马齿状回神经元排列散乱稀疏,密度明显降低,细胞核有固缩,尼氏小体数量减少,染色变浅;与模型组比较,泽泻汤组海马齿状回区域神经元密度增加,细胞核皱缩改善,细胞间隙缩小,排列稍紧密。结论泽泻汤对高热量饮食诱导的认知功能损害小鼠的认知功能有改善效果,可能与重构糖代谢平衡,抑制神经炎症,减少神经元损伤,增强突触可塑性有关。

Objective To investigate the mechanism of Zexie Decoction(泽泻汤)in inhibiting neuroinflamma⁃tion and improving cognitive impairment mediated by high-calorie diet.Methods Twenty seven C57BL/J mice were randomly divided into control group(n=9),model group(n=9)and Zexie Decoction group(n=9).The mice in the model group and the Zexie Decoction group were fed with high-calorie diet to establish the model of cognitive im⁃pairment.Meanwhile,the mice in Zexie Decoction group were also fed with 0.36 g(/kg·d)Zexie Decoction,and the mice in the control group and model group were fed with the same volume of normal saline for 8 weeks.The body weight of mice was recorded at the same time every week;after intervention,oral glucose tolerance test(OGTT)and insulin tolerance test(ITT)commenced;the cognitive level of mice was detected by Morris water maze,open field test,new object recognition test and Y maze;magnetic resonance spectroscopy(MRS)was used to detect the expression of N-acetylaspartate(NAA),choline(CHO),lactic acid(Lac),creatine(Cr),lipid(Lip),and myoInositol(mI)in left hippocampus,hypothalamus and cortex.Western blotting was used to detect the expression of synaptophysin(SYN),synaptosome associated protein-25(SNAP-25),postsynaptic dense protein-95(PSD-95),tumor necrosis factor-α(TNF-α),nuclear factor kappa B(NF-κB p65)and its phosphorylated form(P-NF-B p65)in mouse brain;Nissl's staining was used to detect the morphological changes of hippocampal neurons.Results Com⁃pared with the control group,body mass,blood glucose in oral glucose tolerance test,and blood glucose in insulin tolerance test increased in the model group;in the Morris water maze experiment,the total distance travelled and es⁃cape latency of the model group mice increased,the time spent in the platform area and the number of times traversing the platform decreased on days 3 and 4;in the open-field experiment,the number of times the model group mice entered the central area,the ratio of the time in the central area to the total time,and the ratio of the distance trav⁃elled in the central area to the total distance significantly decreased;in the new object recognition test,the frequency of new object recognition and recognition index were significantly lower in the model group mice;in the Y-maze test,the spontaneous alternation rate of mice in the model group was significantly lower(P<0.05 or P<0.01);in the left hip⁃pocampus,hypothalamus,and cortex of mice in the model group,the CHO/Cr,NAA/Cr significantly decreased,and the mI/Cr,Lac/Cr and Lip/Cr significantly increased;SYN/β-actin,SNAP-25/β-actin and PSD-95/β-actin values sig⁃nificantly decreased,and p-NF-κB p65/NF-κB p65 and TNF-α/β-actin values significantly increased in brain tissue(P<0.05 or P<0.01).Compared with the model group,the above indexes of mice in the Zexie Decoction group sig⁃nificantly improved(P<0.05 or P<0.01).The results of Nissl staining showed that compared with the control group,the neurons in the dentate gyrus of the hippocampus in the model group were scattered and sparsely arranged,the density was significantly reduced,the nuclei of the cells had consolidation and shrinkage,the number of Nissl vesicles was reduced,and the staining became lighter;compared with the model group,the density of neurons in the hippo⁃campal dentate gyrus region of the Zexie Decoction group increased,the wrinkling of nuclei improved,the cell gap narrowed,and the arrangement was slightly tight.Concusion The ameliorative effect of Zexie Decoction on cognitive function in mice with high-calorie diet-induced cognitive impairment may relate to the restructuring of glucose metabo⁃lism homeostasis,inhibition of neuroinflammation,reduction of neuronal damage,and enhancement of synaptic plasticity.