黑逍遥散调控Fas/FasL/Caspase-8/Caspase-3信号通路对AD大鼠神经元细胞凋亡的影响  

Hei Xiaoyaosan Regulates Fas/FasL/Caspase-8/Caspase-3 Signaling Pathway to Inhibit Neuronal Apoptosis in AD Rats

作　　者：王虎平[1,2,3] 陈怡琴 杨娇 胡韵韵 吕育洁 孟志鹏 WANG Huping;CHEN Yiqin;YANG Jiao;HU Yunyun;LYU Yujie;MENG Zhipeng(Gansu University of Chinese Medicine,Lanzhou 730000,China;Gansu Key Laboratory of Traditional Chinese Medicine(TCM)Excavation and Innovative Transformation,Lanzhou 730000,China;Gansu Engineering Laboratory for New Products of TCM,Lanzhou 730000,China)

机构地区：[1]甘肃中医药大学,兰州730000 [2]甘肃省中医方药挖掘与创新转化重点实验室,兰州730000 [3]甘肃省中药新产品创制工程实验室,兰州730000

出　　处：《中国实验方剂学杂志》2024年第17期18-26,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81960828,82160862);甘肃省自然科学基金项目(22JR11RA113);甘肃中医药大学中医学学科导师基金项目[甘中医大科发(2022)13号];第五批全国中医临床优秀人才研修项目(国中医药人教函〔2022〕239号);首批陇原青年英才项目(中共甘肃省委人才工作领导小组〔2022〕5号)。

摘　　要：目的:探究黑逍遥散调控肿瘤坏死因子受体超家族成员6(Fas)/Fas配体(FasL)/胱天蛋白酶-8(Caspase-8)/胱天蛋白酶-3(Caspase-3)信号通路干预神经元细胞凋亡防治阿尔茨海默病(AD)的作用及机制。方法:选用4月龄SPF级SD雄性大鼠90只,随机选取10只作为空白组,10只作为假手术组(双侧海马各注射生理盐水1μL),其余70只双侧海马各注射β淀粉样蛋白1-42(Aβ1-42)1μL溶液复制AD模型。筛选出造模成功的大鼠50只,随机分为模型组、盐酸多奈哌齐组(0.45 mg·kg^(-1))及黑逍遥散高、中、低剂量组(15.30、7.65、3.82 g·kg^(-1))。连续灌胃42 d,1次/d。原位末端标记法(TUNEL)染色观察大鼠皮层和海马神经元细胞凋亡情况,免疫组化法(IHC)检测大鼠海马组织B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测Fas、FasL、Fas相关死亡结构域蛋白(Fadd)mRNA表达,蛋白免疫印迹法(Western blot)检测Fas、FasL、Fadd、Caspase-3、切割型(cleaved)Caspase-3、Caspase-8、cleaved Caspase-8相关蛋白的表达。结果:与空白组和假手术组比较,模型组大鼠皮层和海马区细胞凋亡率显著升高(P<0.01),Bax水平显著增高(P<0.01),Bcl-2水平显著下调(P<0.01),海马组织Fas、FasL、Fadd mRNA表达显著提高(P<0.01),Fas、FasL、Fadd、cleaved Caspase-3、cleaved Caspase-8蛋白表达显著增高(P<0.01);与模型组比较,盐酸多奈哌齐组及黑逍遥散高、中剂量组大鼠皮层和海马区细胞凋亡率明显下降(P<0.05,P<0.01),Bax水平显著降低(P<0.01),Bcl-2水平显著提高(P<0.01),海马组织Fas、FasL、Fadd mRNA表达明显下调(P<0.05,P<0.01),Fas、FasL、Fadd、cleaved Caspase-3、cleaved Caspase-8蛋白表达显著下调(P<0.01);黑逍遥散低剂量组大鼠皮层和海马区细胞凋亡率明显下降(P<0.05,P<0.01),Bcl-2表达水平显著降低(P<0.01),海马组织FasL、Fadd mRNA表达明显下调(P<0.05),Fas、FasL、Fadd、cleaved Caspase-3、cleaveObjective:To explore the effect and mechanism of Hei Xiaoyaosan in regulating the tumor necrosis factor receptor superfamily member 6(Fas)/Fas ligand(FasL)/cysteine protease-8(Caspase-8)/cysteine protease-3(Caspase-3)signaling pathway to intervene in neuronal apoptosis and prevent Alzheimer's disease(AD).Method:Ninety SPF-grade SD male rats of 4 months old were selected and randomly grouped as follows:10 rats in the blank group,10 rats in the sham group(bilateral hippocampus injected with 1μL normal saline),and 70 rats in the modeling group[bilater hippocampus injected with 1μL amyloid-beta protein 1-42(Aβ1-42)solution for the modeling of AD].Fifty successfully modeled rats were selected and randomly assigned into model,donepezil hydrochloride(0.45 mg·kg^(-1)),and high-,medium-,and low-dose(15.30,7.65,3.82 g·kg^(-1))Hei Xiaoyaosan groups.Rats were administrated with corresponding agents by gavage once a day for 42 days.Terminal-deoxynucleoitidyl transferase-mediated nick end labeling(TUNEL)was employed to observe the apoptosis of neurons in the cortex and hippocampus,and immunohistochemistry(IHC)was used to detect the expression of B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax)in the hippocampus.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was employed to determine the mRNA levels of Fas,FasL,and Fas-associated protein with death domain(Fadd).Western blot was used to determine the protein levels of Fas,FasL,Fadd,Caspase-3,cleved Caspase-3,Caspase-8,and cleved Caspase-8.Result:Compared with the blank group and sham group,the model group showed increased apoptosis rate in the cortex and hippocampus(P<0.01),elevated Bax level(P<0.01),lowered Bcl-2 level(P<0.01),up-regulated mRNA levels of Fas,FasL,and Fadd in the hippocampus(P<0.01),and up-regulated protein levels of Fas,FasL,Fadd,cleaved Caspase-3,and cleaved Caspase-8(P<0.01).Compared with the model group,donepezil hydrochloride and Hei Xiaoyaosan at high and medium doses decreased the apoptosis rate in the cortex a

关 键 词：黑逍遥散 阿尔茨海默病 肿瘤坏死因子受体超家族成员6(Fas)/Fas配体(FasL)/胱天蛋白酶-8(Caspase-8)/胱天蛋白酶-3(Caspase-3)信号通路 细胞凋亡 

分 类 号：R2-0[医药卫生—中医学] R33R289R742.82