丹参素经PI3K/Akt信号通路改善血管性痴呆模型小鼠认知功能的机制研究

Danshensu improvement of cognitive functions in a mouse model of vascular dementia via regulation of PI3K/Akt signaling

目的基于磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(Akt)信号通路探讨丹参素对血管性痴呆小鼠认知功能的改善情况。方法将60只SPF级昆明种小鼠按随机数字表法分为正常对照组、模型组、阳性对照组、丹参素低、中、高剂量组,每组10只。模型组、阳性对照组、丹参素低、中、高剂量组小鼠参照文献的方法构建血管性痴呆模型。建模成功后,从第1天开始丹参素各剂量组按10、20、30mg/kg鼠重腹腔注射丹参素混悬液,阳性对照组按2mg/kg鼠体腹腔注射尼莫地平注射液,正常对照组和模型组腹腔注射等体积生理盐水,每天1次,持续28天。给药结束后对小鼠进行水迷宫实验,检测小鼠血清脑损伤相关因子星形胶质源性蛋白(S-100β)、神经元特异性烯醇化酶(NSE)含量,苏木精-伊红(HE)染色观察小鼠脑组织结构,ELISA法检测小鼠脑组织PI3K、Akt蛋白水平。结果正常对照组小鼠脑组织结构正常,细胞排列整齐,无明显空泡结构;模型组可见明显的脑白质受损,细胞排列紊乱,视野中大量空泡结构;给予丹参素干预后,随着丹参素给药剂量的增加,小鼠脑组织细胞排列逐渐变整齐,空泡结构逐渐减少。与正常对照组比较,模型组小鼠逃避潜伏期延长[(46.36±3.12)s比(15.25±1.23)s,P<0.05];血清S-100β、NSE水平升高[S-100β:(1.54±0.11)ng/L比(0.65±0.03)ng/L、NSE:(4.36±0.12)ng/L比(2.13±0.15)ng/L,P均<0.05],脑组织PI3K、Akt蛋白表达水平显著降低[PI3K:(0.17±0.03)比(1.13±0.12)、Akt:(0.47±0.03)比(1.81±0.13),P均<0.05];与模型组比较,丹参素低、中、高剂量组和阳性对照组小鼠逃避潜伏期显著缩短[(40.78±3.54)s、(34.65±3.13)s、(26.31±2.01)s、(25.23±1.75)s比(46.36±3.12)s,P均<0.05],血清S-100β、NSE水平显著降低[S-100β:(1.31±0.12)ng/L、(1.01±0.08)ng/L、(0.70±0.05)ng/L、(0.71±0.03)ng/L比(1.54±0.11)ng/L、NSE:(3.78±0.18)ng/L、(2.95±0.13)ng/L、(2.31±0.17)ng/L、(2.23±0.15)ng/L比(4.36±0.12)ng/L,P均<0.05],脑组织PI3K、Akt蛋白表达水平显著升高[PI3K:(0.33±0.06)、(0.52±0.07)、(0.74±0.09)、(0.77±0.09)比(0.17±0.03),Akt:(0.72±0.07)、(1.04±0.09)、(1.40±0.11)、(1.42±0.10)比(0.47±0.03),P<0.05];随着丹参素给药剂量的增加,小鼠逃避潜伏期缩短,血清S-100β、NSE水平逐渐降低,脑组织PI3K、Akt蛋白表达水平逐渐升高,具有量-效关系(P均<0.05);丹参素高剂量组上述指标与阳性对照组比较,差异无统计学意义(P>0.05)。结论丹参素可能通过调节PI3K/Akt信号通路及相关细胞因子,改善血管性痴呆模型小鼠认知功能障碍。

Objective To assess the effects of Danshensu on the improvement of the cognitive function in vascular dementia mice and the underlying molecular events.Methods Sixty specific pathogen-free(SPF)Kunming mice were randomly divided into negative control,model,positive control,and model plus low,medium,and high doses of Danshensu groups(n=10 per group).The mouse model of vascular dementia was established according to a previous report and then treated with Danshensu suspension via intraperitoneal injection at a dose of 10,20,and 30mg/kg,respectively,while the positive control mice were injected with Nimodipine(2mg/kg)and the negative control mice were injected with the same amount of saline,once a day for consecutive 28 days.At the end of experiments,the water maze test was conducted and serum levels of brain injury-related factors astrocyte derived protein(S-100β)and neuron specific enolase(NSE)were detected using ELISA,while brain tissues were assessed using hematoxylin eosin(H&E)staining and PI3K and Akt proteins were measured using Western blot.Results The negative control group of mice showed normal structures of the brain,i.e.,the neurons were arranged in order and there was no obvious vacuole structure,whereas the model group of mice showed obvious damages in the white matter,e.g.,the neuron arrangement was altered and a large number of vacuoles occurred in the visual field;however,administration of various doses of Danshensu improved the brain tissue structures,e.g.,neurons obviously had normal arrangement and the vacuole structures were reduced.Furthermore,compared with the negative control group,the escape latency of model mice was increased[(46.36±3.12)vs.(15.25±1.23)s,P<0.05]and the serum levels of S-100βand NSE were also increased[S-100β:(1.54±0.11)vs.(0.65±0.03)ng/L,NSE:(4.36±0.12)vs.(2.13±0.15)ng/L,all P<0.05]whereas brain level of PI3K and Akt proteins was significantly decreased[(PI3K 0.17±0.03)vs.(1.13±0.12),Akt:(0.47±0.03)vs.(1.81±0.13),all P<0.05].In Addition,compared with the model group,the mouse escape latency in low,medium,and high doses Danshensu groups and positive control group was significantly shortened[(40.78±3.54),(34.65±3.13),(26.31±2.01),(25.23±1.75)vs.(46.36±3.12)s,respectively;all P<0.05]and serum levels of S-100βand NSE were significantly decreased[S-100β:(1.31±0.12),(1.01±0.08),(0.70±0.05),and(0.71±0.03)vs(1.54±0.11)ng/L,NSE:(3.78±0.18),(2.95±0.13),(2.31±0.17),and(2.23±0.15)vs.(4.36±0.12)ng/L,respectively;all P<0.05],whereas brain level of PI3K and Akt proteins was significantly increased[PI3K:(0.33±0.06),(0.52±0.07),(0.74±0.09),and(0.77±0.09)vs.(0.17±0.03),Akt:(0.72±0.07),(1.04±0.09),(1.40±0.11),(1.42±0.10)vs.(0.47±0.03),respectively;all P<0.05].The treatment responses were dose-dependent,i.e.,with the increase in Danshensu dosage,the mouse escape latency was shortened and serum levels of S-100βand NSE were gradually decreased,whereas brain level of PI3K and Akt proteins was gradually increased(all P<0.05).However,there was no difference in the above named indexes between the positive control group and the highest dose Danshensu group(P>0.05).Conclusion Danshensu treatment showed to improve the cognitive impairment in a mouse model of vascular dementia via regulation of the PI3K/Akt signaling pathway and related cytokine expression.