黄芪甲苷对脂多糖刺激单核细胞的保护作用及其机制研究

A study on protection and mechanism of astragaiosideⅣon monocytes stimulated by lipopolysaccharide

目的 观察黄芪甲苷对脂多糖（LPS）刺激的单核细胞能否起保护作用及其机制.方法 以LPS刺激体外培养的大鼠单核细胞,首先观察LPS诱导单核细胞发生凋亡的最佳浓度,再将黄芪甲苷按浓度梯度（分别为1 000、300、100、30、10、3μg/ml）加入细胞培养上清液中,测定其最大无毒浓度（TD0）及半数中毒浓度（TD50）;并以不同浓度黄芪甲苷干预,测定黄芪甲苷的半数有效浓度（ED50）和95%有效浓度（ED95）.采用异硫氰酸荧光索/藻红蛋白标记的膜联蛋白V（Annexin V-FITC/PI）染色和赫斯特荧光染料（Hoechst33258）荧光单染,流式细胞仪观察黄芪甲苷对LPS所致细胞凋亡的保护作用.结果 LPS在单核细胞培养上清液中的浓度为100 ng/ml时可以诱导细胞凋亡,凋亡率为（43.67±7.14）%.黄芪甲苷的TD0〉1 000μg/ml时,TD50无法测出,其ED50和ED95分别为27.31μg/ml和101.29μg/ml;用100μg/ml黄芪甲苷进行干预后,培养的细胞贴壁良好,核浓缩、核着边等现象显著减少,凋亡率明显下降（P〈O.01）.结论 黄芪甲苷对LPS刺激后的单核细胞有保护作用,其机制与抑制细胞凋亡有关.

Objective To investigate the protective effect and mechanism of astragaloside IV on monocytes stimulated by lipopolysaccharide （LPS）. Methods LPS was introduced into culture medium of monocytes to come into being inflammation model in vitro. And the optimal concentration of inducing monoctye apoptosis was investigated. The concentrations of astragaloside Ⅳ used in this experiment were 1 000, 300, 100, 30, 10, 3 μg/ml, respectively. Then the data of maximum nontoxic dose （TD0）, 50% toxic dose （TD50）, effective dose in 50% （ED50） and effective dose in 95% （ED95） were calculated by DAS software. Annexin V-fluorescein isothiocyanate/phycoerythrin （FITC/PI） staining and Hoechst 33258 fluorescence staining were used, and flow cytometry was applied to observe the protective effects of Astragaloside IV on LPS-induced apoptosis. Results The phenomenon of cell apoptosis was significant when the concentration of LPS in supernatant fluid reached 100 ng/ml, the rate of apoptosis being （43.67 ± 7.14）%. The TD0 of astragalosideiV on monocytes was higher than 1 000 μg/ml and the datum of TD50 could not be obtained. The data of ED30 and ED95 of astragaloside Ⅳ was 27.31 μg/ml and 101.29 μg/ml, respectively. Astragaloside Ⅳ （100 μg/ml） could significantly inhibit the occurrence of apoptosis induced by LPS; the cultured monocytes well adherent to the waii and the phenomena of apoptosis reduced remarkably, such as, chromatin margination and nuclear condensation; the rate of apoptosis was decreased obviously （P〈0.01）. Conclusion Astragaloside IV exhibits the protective effect on monocytes infected by LPS. The mechanism is related to its inhibitory effect on LPS-induced monocyte apoptosis.