清心开窍方有效成分对AD大鼠学习记忆能力及其海马区Bax、Bcl-2、Caspase-3和βAPP表达的影响

Effect of Active Ingredients of Qingxin Kaiqiao Recipe on Spatial Learning and Memory Capacities,and Expressions of Bax,Bcl-2,Caspase-3,and βAPP in Hippocampus of AD Model Rats

目的探讨清心开窍方中皂苷、挥发油及多糖有效成分对淀粉样蛋白(Aβ)1-40海马注射诱导痴呆(Alzheimer's disease,AD)大鼠海马区Bax、Bcl-2、Caspase-3及βAPP表达的影响。方法选取112只雄性SD大鼠,随机分为7组,每组16只,分别为正常组、假手术组、模型组、安理申组、皂苷组、挥发油组、多糖组。采用海马注射Aβ_(1-40)诱导AD大鼠模型。造模后第2天开始灌胃,正常组、假手术组、模型组给予等量双蒸水灌胃,安理申组[盐酸多奈哌齐片,1.67 mg/(kg·d)]、皂苷组[9 m L/(kg·d)]、挥发油组[3.33 m L/(kg·d)]、多糖组[8.33 m L/(kg·d)]灌胃,每天1次,连续2周(上午10:00)。灌胃结束后,采用Morris水迷宫测试大鼠的空间学习记忆能力;采用TUNEL染色检测海马CA1区细胞凋亡;采用免疫组化、实时定量荧光PCR及WB法检测AD大鼠海马区Bax、Bcl-2、Caspase-3及βAPP表达。结果造模前各组大鼠同一时间点的逃避潜伏期、穿台次数比较,差异均无统计学意义(P>0.05),且逃避潜伏期随时间推移逐渐缩短。造模后,与模型组比较,除挥发油组、多糖组外,安理申组、皂苷组逃避潜伏期均缩短,穿台次数显著增多(P<0.05,P<0.01);与模型组比较,皂苷组、挥发油组、多糖组大鼠海马CA1区凋亡细胞数量明显减少(P<0.05,P<0.01);Bcl-2表达上调,Bax、Caspase-3、βAPP表达下调,Bcl-2/Bax比值明显提高(P<0.05,P<0.01)。结论清心开窍方3种有效成分能不同程度地改善AD大鼠的学习记忆能力,其机制可能与降低海马区Bax、Caspase-3及βAPP表达,提高Bcl-2表达,抑制AD大鼠海马区内细胞凋亡有关。

Objective To observe the effects of active ingredients of Q ingxin K aiqiao Recipe（ QKR）,such as saponins,volatile oils,effective compositions of polysaccharides,on expressions of Bcl-2 associated X protein（ Bax）, B-cell lym phom a-2（ Bcl-2）, cysteinyl aspartate specific proteinase-3（ Caspase-3）,and β-amyloid precursor protein（ βAPP） in hippocampus of Aβ1-4 0-induced Alzheim er＇ s disease（ AD） m odel rats. M ethods Totally 1 1 2 m ale Sprague-Dawley（ SD） rats were random ly divided into 7 groups,i. e.,the norm al control group,the sham-operation group,the m odel group,the Aricept group,the saponin group,the volatile oil group,the polysaccharide group,1 6 in each group. TheAD rat m odel was established by injecting Aβ1-4 0from bilateral hippocam pus. Equal volum e of double distilled water was adm inistered to rats by gastrogavage in the norm al control group,the sham-operation group,the m odel group from the 2 nd day after m odeling,once per day for 2 successive weeks（ at 1 0 ：0 0 am）. Aricept（ Donepezil H ydrochloride Tablet,1. 67 mg / kg per day）, saponin（ 9 mL / kg per day）,benzene（ 3. 33 mL / kg per day）,and polysaccharides（ 8. 33 mL / kg per day） was adm inistered to rats by gastrogavage to the Aricept group,the saponin group,the volatile oil group,the polysaccharides group,respectively,once per day for 2 successive weeks（ at 1 0 ：0 0 am）. By the end of gastrogavage spatial learning and m em ory capacities were detected using M orris water m aze（ MWZ）. Apoptosis in hippocam pal CA1 region was detected using TUNEL staining. Expressions of Bax,Bcl-2,Caspase-3,and βAPP were m easured via Real-tim e fluorescent quantitative PCR,Western blot,and im m unohistochem istry,respectively. Results There was no statistical difference in pre-m odeling escape latency and tim es of crossing platform s am ong groups at the sam e tim e point（P〉0.05）. Besides,escape latency was gradually shortened as tim e went by. Com pared with the m odel group,escape latency was shortened,and tim es of crossing platform s was significantly increased in the Aricept group and the saponin group（P〈0.05,P 〈0. 01）. Compared with the m odel group,the am ount of apoptotic cells in hippocam pal CA1 region was obviously reduced（P〈0.05,P〈 0. 01）,expressions levels of Bax,Caspase-3,and βAPP were down-regulated,Bcl-2 / Bax ratio was obviously elevated in the saponin group,the volatile oil group,the polysaccharide group（P〈0.05,P〈 0. 01）. Conclusion Three active ingredients（ spaonins,benzene,and polysaccharides） of QKR could im prove spatial m em ory and learning capacities to different degrees, which m ight be possibly achieved by decreasing expressions of Bax,Caspase-3,βAPP in hippocampal CA1 region,elevating Bcl-2 expression,and inhibiting apoptosis in hippocampus.