益智方拆方不同配比对Aβ_(25～35)诱导大鼠神经元过氧化及凋亡的影响

Influences of disassemble formulas of Yizhi Fang with different combinations on peroxiation and apoptosis of neurons induced by Aβ_(25-35) in rats

目的比较益智方补虚药组和化浊药组不同比例配伍对β淀粉样蛋白25～35(Aβ25～35)毒性导致细胞过氧化及凋亡的影响,探讨益智方中补虚药和化浊药减轻Aβ毒性的作用机制及其合理配伍比例。方法培养原代大鼠皮层神经元,加入Aβ25～35复制阿尔茨海默病(AD)细胞模型。根据药性把益智方拆分为补虚药组和化浊药组,设立正常对照组、益智方组、补虚药组、化浊药组、补虚化浊2∶1配伍组、补虚化浊1∶2配伍组,加入各对应含药组大鼠血清,无Aβ25～35为空白对照组。24 h后比色法测定计算各组细胞存活率、乳酸脱氢酶(LDH)漏出率、超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量;免疫组化方法显示天冬氨酸特异性半胱氨酸蛋白酶-3(Caspase3)的表达情况;末端脱氧核苷酸转移酶介导的缺口末端标记测定(Tunel)法显示凋亡细胞。结果 20μmol/L Aβ25～35组细胞存活率降低、LDH漏出率及MDA含量增加、SOD活性降低,Caspase3表达增强,细胞凋亡增多(P<0.01)。各含药大鼠血清能提高细胞存活率、降低LDH漏出率及MDA含量,增加细胞SOD活性,减轻Caspase3表达,减少凋亡细胞(P<0.01或P<0.05)。其中益智方组和补虚药组效果最好(P<0.01或P<0.05),补虚化浊2∶1配伍组、补虚化浊1∶2配伍组不如益智方组和补虚药组(P<0.01或P<0.05),化浊药组效果较弱(P<0.01或P<0.05)。结论 Aβ25～35具有神经毒性,导致神经细胞发生过氧化损伤和过度凋亡,益智方补虚药组在抑制细胞过氧化和凋亡方面优于化浊药组,化浊药需配伍补虚药提高疗效,故补虚药在抑制Aβ25～35神经毒性方面发挥了主要作用。

Objective To compare the influences of disassemble formulas of Yizhi Fang with different combinations on peroxidative and apoptosis of cells induced by A[325_35, and to investigate the mechanism of Chinese medicinal with actions of tonifying deficiency （Buxu medicinal ） and those with actions of resolving turbidity （Huazhuo medicinal） in reducing the toxicity of A[3zs_35, as well as their reasonable combination proportion. Methods adding A^25_35 into cortical neurons and Huazhuo formula according to Huazhuo group, Bu-hua 2 ： 1 group The cell model of Alzheimer＇ s disease （AD） was established by of primary cultured rat. Yizhi Fang was modified into Buxu formula medicinal nature. Normal group, Quanfang and Bu-hua 1：2 group were established and group, Buxu group, the medicated serum samples were added into corresponding groups respectively. Blank control group was not given A^25_35 （non-AI3 group） and model group was given AI325-35 （A~ group）. The neurocyte survival rate, leakage rate of lactate dehydrogenase （LDH）, activity of superoxide dismutase （SOD） and malondialdehyde （MDA） content were detected and calculated by using chromatometry in all groups after 24 hours. The expression of 3 was observed by using immunohistochemical method. The apoptosis of ceils was observed by using TUNEL assay. Results In A[3 group （20 μmol/L）, the survival rate of neurocyte and activity of SOD decreased, and LDH leakage rate, MDA content increased, expression of Caspase3 and apoptotic cell counting increased （P 〈 0. 01 ）. The medicated rat serum samples could increase the survival rate of neurocyte, decrease LDH leakage rate and MDA content, improve the expression of Caspase3, and reduce apoptotic cells （P 〈0.01 or P 〈0.05）. The effectiveness was the best in Quanfang group and Buxu group （P 〈0.01 or P 〈0.05）, secondly in Bu-hua 2：1 group and Bu-hua 1：2 group （P 〈0.01 or P 〈0.05）, and weaker in Huazhuo group （P 〈 0. O1 or P 〈 0.05 ）. Conclusion A[325_35 is neurotoxic to cause the peroxidation injury and excessive apoptosis of neurocyte. The Buxu medicinal is better than Huazhuo medicinal in inhibiting peroxidation and apoptosis, while Huazhuo medicinal should be combined with Buxu medicinal for improving curative effect. Buxu medicinal plays a major role in inhibiting neurotoxieity of AI325 -35-