当归芍药散对血管性痴呆模型大鼠认知功能的影响及机制

Effect of Danggui Shaoyao San on cognitive function of vascular dementia model rats and its mechanisms

目的探讨当归芍药散(Danggui Shaoyao San,DSS)对血管性痴呆(vascular dementia,VD)大鼠认知功能的影响及其机制。方法50只SPF级雄性SD大鼠(6~7周龄)按照随机数字表法分为假手术组、模型组、尼莫地平组(9.45 mg·kg^(-1))、DSS低剂量组(1.6 g·kg^(-1))和DSS高剂量组(6.4 g·kg^(-1)),每组10只。采用双侧颈总动脉永久性结扎法建立VD大鼠模型;造模7 d后大鼠按照分组分别灌胃给药,1次/d,连续28 d。采用Morris水迷宫实验检测VD大鼠的学习记忆能力;ELISA法检测大鼠海马组织氧化应激产物丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)和活性氧(reactive oxygen species,ROS)水平;Western blot法检测海马组织凋亡相关蛋白Bcl-2、Bax、Caspase-3及瘦素受体/糖元合成激酶3β/微管相关蛋白tau(leptin receptor/glycogen synthase kinase 3βmicrotubule-associated protein tau,LEP-R/GSK-3β/tau)信号通路相关蛋白表达情况。采用GraphPad Prism 9.0软件对数据进行统计分析,多组间比较采用单因素方差分析和重复测量方差分析,进一步两两比较采用SNK-q检验。结果水迷宫实验结果显示,五组大鼠逃避潜伏期的时间和分组交互作用不显著(F=1.223,P>0.05),分组主效应和时间主效应均显著(F=74.65,18.32;均P<0.05)。第5天时,尼莫地平组、DSS低剂量组和DSS高剂量组逃避潜伏期均低于模型组(q=14.425,7.477,21.392,均P<0.05),且DSS高剂量组低于尼莫地平组[(15.28±2.46)s,(22.78±3.31)s,q=6.966,P<0.05]。五组大鼠穿越平台次数差异有统计学意义(F=17.331,P<0.05),尼莫地平组及DSS高低剂量组的穿越平台次数均高于模型组(q=6.789,10.635,5.270,均P<0.05),且DSS高剂量组的穿越平台次数高于尼莫地平组[(6.84±1.63)次,(5.22±1.75)次,q=3.846,P<0.05]。ELISA结果显示,五组大鼠海马组织MDA、ROS和SOD表达水平均差异有统计学意义(F=49.338,38.518,15.440,均P<0.05)。DSS高剂量组海马组织MDA水平、ROS水平低于模型组(q=16.061,13.541,均P<0.05)和尼莫地平组(q=4.317,5.162,均P<0.05),SOD水平高于模型组(q=8.179,P<0.05)和尼莫地平组(q=4.135,P<0.05)。Western blot结果显示,五组大鼠凋亡相关蛋白Bcl-2/Bax、Caspase-3表达均差异有统计学意义(F=30.692,43.384,均P<0.01)。DSS高剂量组Bcl-2/Bax表达水平高于模型组(q=10.562,P<0.05)和尼莫地平组(q=3.820,P<0.05),Caspase-3表达水平均低于模型组(q=12.139,P<0.05)和尼莫地平组(q=7.734,P<0.05)。五组大鼠海马组织LEP-R、p-GSK-3β、p-S404 tau和p-S202 tau表达水平均差异有统计学意义(F=80.927,59.230,159.784,105.923,均P<0.01)。尼莫地平组、DSS高剂量组LEP-R、p-GSK-3β蛋白水平均高于模型组(q=16.275,20.104,均P<0.05;q=12.942,17.257,均P<0.05),两组p-S404 tau、p-S202 tau表达水平均低于模型组(q=19.121,27.456,均P<0.05;q=17.559,22.780,均P<0.05)。DSS高剂量组LEP-R((0.98±0.15),(0.86±0.14))、p-GSK-3β((0.95±0.16),(0.82±0.13))蛋白水平均高于尼莫地平组(q=3.829,4.314,均P<0.05),p-S404 tau[(0.41±0.03),(0.58±0.07)]、p-S202 tau[(0.48±0.05),(0.59±0.06)]表达水平均低于尼莫地平组(q=8.335,5.220,均P<0.05)。结论DSS可改善VD模型大鼠的认知功能,其机制可能与降低氧化应激水平、抑制神经元凋亡、上调LEP-R/GSK-3β/tau信号通路有关。

Objective To investigate the effects of Danggui Shaoyao San(DSS)on cognitive function and neuronal apoptosis in vascular dementia(VD)rats.Methods Fifty SPF grade male SD rats aged 6-7 weeks were randomly divided into sham operation group,model group,positive drug group(nimodipine group,9.45 mg·kg^(-1)),DSS low-dose group(1.6 g·kg^(-1)),DSS high-dose group(6.4 g·kg^(-1))according to random number table,with 10 rats in each group.The VD rat model was established by permanent ligation of bilateral common carotid arteries.Seven days after modeling,the rats in different groups were administrated by gavage according to corresponding interventions,once a day,for 28 days.Morris water maze test was used to evaluate the learning and memory ability of rats.The levels of malondialdehyde(MDA),superoxide dismutase(SOD)and reactive oxygen species(ROS)in hippocampal area of rat brain were detected by ELISA.The protein expressions of apoptosis-related proteins Bcl-2,Bax,cleaved Caspase-3 and leptin receptor/glycogen synthase kinase 3βmicrotubule-associated protein tau(LEP-R/GSK-3β/tau)signaling pathway were detected by Western blot.GraphPad Prism 9 software was used for statistical analysis of data,repeated measure ANOVA and one-way ANOVA were used for comparison between multiple groups,and SNK-q test was used for further pairwise comparison.Results The results of water maze experiment showed that the time and group interaction of escape latency of the five groups were not significant(F=1.223,P>0.05),the main effect of group and time were significant(F=74.65,18.32,both P<0.05).On the 5th day,the escape latency of nimodipine group,DSS low-dose group and DSS high-dose group were lower than that of model group(q=14.425,7.477,21.392,all P<0.05),and that of DSS high-dose group was lower than that of nimodipine group((15.28±2.46)s,(22.78±3.31)s,q=6.966,P<0.05).There was statistically significant difference in the number of crossing platforms of rats in 5 groups(F=17.331,P<0.05).The numbers of platform crossing in nimodipine group and DSS high-dose group were higher than that in model group(q=6.789,10.635,5.270,all P<0.05),and the number of platform crossing in DSS high-dose group was higher than that in nimodipine group((6.84±1.63),(5.22±1.75),q=3.846,P<0.05).ELISA results showed that the levels of MDA,ROS and SOD in hippocampal tissues of rats in 5 groups were significantly different(F=49.338,38.518,15.440,all P<0.05).The levels of MDA and ROS in hippocampus of DSS high-dose group were lower than those of model group(q=16.061,13.541,both P<0.05)and nimodipine group(q=4.317,5.162,both P<0.05),SOD level of DSS high-dose group was higher than those of model group(q=8.179,P<0.05)and nimodipine group(q=4.135,P<0.05).Western blot results showed that the levels of apoptosis-related proteins Bcl-2/Bax and Caspase-3 were significantly different in the 5 groups(F=30.692,43.384,both P<0.01).The level of Bcl-2/Bax in DSS high-dose group was higher than that in model group(q=10.562,P<0.05)and nimodipine group(q=3.820,P<0.05),the level of Caspase-3 was lower than those of model group(q=12.139,P<0.05)and nimodipine group(q=7.734,P<0.05).The levels of LEP-R,p-GSK-3β,p-S404 tau and p-S202 tau expression level in hippocampal tissues of the 5 group were significantly different(F=80.927,59.230,159.784,105.923,all P<0.01).The levels of LEP-R and p-GSK-3βprotein in nimodpine group and DSS high-dose group were higher than those in model group(q=16.275,20.104,both P<0.05;q=12.942,17.257,both P<0.05),the levels of p-S404 Tau and p-S202 Tau in the two groups were lower than those in model group(q=19.121,27.456,both P<0.05;q=17.559,22.780,both P<0.05).The levels of LEP-R(0.98±0.15),(0.86±0.14))and p-GSK-3β((0.95±0.16)s,(0.82±0.13))in DSS high-dose group were higher than those in nimodipine group(q=3.829,4.314,both P<0.05),the levels of p-S404 Tau((0.41±0.03)s,(0.58±0.07))and p-S202 Tau((0.48±0.05)s,(0.59±0.06))in DSS high-dose group were lower than those of nimodipine group(q=8.335,5.220,both P<0.05).Conclusion DSS can improve the cognitive function of VD rats,and the mechanism may be related with reducing oxidative stress level,inhibiting neuronal apoptosis,and upregulating LEP-R/GSK-3β/Tau signaling pathway.