活血荣络方对Aβ_(25-35)损伤神经胶质细胞Wnt5α、Fz5、CaMKⅡ表达的影响

The Effects of Huoxuerongluofangonthe Expressions of Wnt5,Fz5 and CaMKⅡ in Aβ_(25-35) Injured Astrocytes

目的：旨在通过细胞实验,从Wnt5a-Fz5-Ca MKⅡ通路揭示活血荣络方治疗阿尔茨海默病（AD）的分子机制,为中医药治疗阿尔茨海默病提供新思路、新方法。方法：分别将Aβ25-35损伤星形胶质细胞铺板,以（2.53）×104/孔的密度接种到96孔板中,并随机分为6组,分别为2倍活血荣络方组、1倍活血荣络方组、0.5倍活血荣络方组、多奈哌齐组、s FRP组、空白组;待细胞的融合率为85%90%时,含药组分别给予相应的含药血清,s FRP组给予s FRP,空白组予以空白血清。取生长对数期细胞加相应刺激分别处理12、24、48、72 h。分别取细胞上清液分离细胞,用MTT法检测细胞毒力,用流式细胞术检测细胞凋亡;用化学免疫法检测Wnt5、Fz5、Ca MKⅡ的含量。结果：各组OD值随着时间的延长而增高,2倍活血荣络方组72 h OD值明显高于12 h（P〈0.05）,而空白组、s PRF组、2倍、0.5倍活血荣络方组、多奈哌齐组4个时间点见无明显差异（P〉0.05）;各给药组细胞活性均高于空白组（P〈0.05）,其中2倍活血荣络方组细胞活性最高;s FRP组细胞活性低于空白组（P〈0.05）。各组细胞凋亡数目随着时间的延长而增高,其中空白组72 h细胞凋亡比例高于12 h（P〈0.05）,s PRF组72 h细胞凋亡比例明显高于12 h;2倍、1倍活血荣络方组、0.5倍活血荣络方组、多奈哌齐组4个时间点见无明显差异（P〉0.05）;各给药组细胞凋亡比例明显低于空白组及s PRF组,其中2倍活血荣络方组细胞活性最高;s FRP组细胞凋亡比例高于空白组（P〈0.05）。各给药组Wnt5α、Fz5、Ca MKⅡ的表达均高于空白组（P〈0.05）,其中2倍活血荣络方组Wnt5α表达明显高于空白组（P〈0.01）,空白组明显高于s PRF组（P〈0.05）;其它给药组间比较：2倍活血荣络方组明显高于1倍活血荣络方组、多奈哌齐组及0.5倍活血荣络方组（P〈0.01）,1倍活血荣络方组与多奈哌齐组Wnt5α表达程度无明显差异（P〉0.05）,此两组Wnt5α表达均高于0.5倍活血荣络方组（P〈0.05）。结论：活血荣络方能有效提高Wnt5α、Fz5、Ca MKⅡ的表达,减轻神经毒性的积累及细胞凋亡反应的发生,保护神经胶质细胞,延缓AD发病进展,其中以2倍活血荣络方效果最佳。

Objective：This cell experiment aims to reveal the molecular mechanism of Huoxuerongluofang treating Alzheimer＇s disease from WntSa- Fz5 -CaMKⅡ pathway （AD）, and to provide new ideas and methods for the treatment of Alzheimer＇s disease in Chinese traditional medicine. Methods： Aβ25 -35 injured astrocyteswere bedboarded, and vaccinated into96 pore plates according to （2.5 -3 ） ×104 hole seeding density, then randomly divided into 6 groups, they were 2 times the Huoxuerongluofang group, 1 times theHuoxuerongluofang group, 0.5 times theHuoxuerongluofang group, donepezil group, sFRP group and blank group; When the fusion rate of cells reached 85% - 90%, drug groups were given corresponding drug serum, sFRP group was given sFRP, blank group was given blank serum. The cells were treated at 12 h, 24 h, 48 h, and 72 h respectively. Cell supernatant was taken to separate the cells, and the cytotoxicity was detected by MTT method. Cell apoptosis was detected by flow cytometry. The contents of WntS, Fz5 and CaMKII were detected by the method of chemical immunoassay. Results ： The OD value increased with the extension of time, the OD value of 2 times Huoxuerongluofang group at 72 h OD was significantly higher than that at 12 h （P 〈0. 05）, and which of the control group, sPRF group,2 times,0. 5 times the Huoxuerongluofang group, donepezil group was not significantly different （ P 〉 O. 05 ） among the 4 time points. Cell activities ofgroupswhich were given the treating drugs were higher than the con- trol group （ P 〈 O. 05 ）, especially cell activity of 2 times theHuoxuerongluofang group was the highest; cell activity of sFRP group cells was lower than that of the control group （P 〈 0. 05）. The number of apoptotic cells increased with the extension of time, the apoptosis ratio of the blank control group at 72 h was higher than that at 12 h （P 〈 0.05 ）, the apoptosis ratio of sPRF 72 h group was significantly higher than that of 12 h;which of 2 times, 1 times the Huoxuerongluofang group, 0. 5 times of Huoxuerongluofang group, donepezil group was no difference among the 4 time points （ P 〉 0. 05 ） ; The apoptosis ratio of each group using treating drugs was significantly lower than control group and sPRF group, which of 2 times the Huoxuerongluofanggroup was lowest; sFRP group the apoptosis ratio was higher than control group （P 〈 0.05）. The expressions of Wnt5α and Fz5 and CaMKII in each group using drugs were higher than those in control group （ P 〈0. 05 ）, Wnt5 expressions of 2 times theHuoxuerongluofang groupwere significantly higher than the control group （ P 〈0.01 ）,which of the blank group was significantly higher than sPRF group （P 〈 0. 05 ）;which of 2 times the Huoxuerongluofang group was higher than 1 times Huoxuerongluofang group, donepezil group and 0. 5 times huoxuerongluoang group （P 〈 0. 01 ）, there was no significant difference between the 1 times theHuoxuerongluofang group and donepezil group（ P 〉 0. 05 ）, the Wnt5 expressions of the two group were higher than 0. 5 times Huoxuerongluofang group （ P 〈 0. 05 ）. Conclusion： Huoxuerongluofang could effectively improve the expressions of Wnt5, Fz5, CaMKII, reduce the accumulation of neurotoxicity and the apoptosis reaction, and could protect the glial cells, delay the onset of AD,2 times the Huoxuerongluofangwould be the best dose.