板蓝根活性部位对细胞凋亡和RAW264.7表达IL-10、TNF-α的影响

Effect of active site of Radix Isatidis on cell apoptosis and expression of IL-10 and TNF-α in RAW264.7 cells

目的探讨板蓝根活性部位对Hep-2、Hela、U937、Jukart细胞的细胞凋亡和小鼠单核巨噬细胞白血病细胞RAW264.7表达细胞因子IL-10、TNF-α的影响。方法 0.5 g/L板蓝根活性部位作用于Hep-2、Hela、U937、Jukart 24 h,经PI染色,流式细胞术研究其对细胞凋亡的影响;100TCID50的单纯疱疹病毒(HSV-1)作用于U937,0.5 g/L药物处理24 h,流式细胞术研究其对细胞凋亡的影响。ELISA检测1 g/L、0.5 g/L、0.25 g/L药物对LPS处理小鼠单核巨噬细胞白血病细胞RAW264.7后IL-10、TNF-α表达的影响。结果①0.5 g/L板蓝根活性部位作用24 h对Hep-2、Hela、Jukart、U937细胞凋亡无明显影响,但HSV-1感染U937后药物抗病毒组较病毒组的细胞凋亡率由9.72%下降至6.71%,差异有统计学意义(P<0.01);药物作用于Hela细胞时引起其G1期阻滞(从68.76%延长至74.77%),S期缩短(从31.22%降至21.62%),与正常Hela细胞组比较,差异有统计学意义(P<0.05)。②LPS刺激RAW264.7后,与细胞对照组比较,LPS模型组细胞凋亡率升高,差异有统计学意义(P<0.01)。LPS刺激1 g/L、0.5 g/L、0.25 g/L药物组细胞凋亡率分别为19.35%、28.14%、7.52%,明显低于LPS模型组(70.42%),差异有统计学意义(P<0.01)。与细胞对照组比较,1 g/L药物对照组细胞凋亡率上升(由5.81%升至15.03%),差异有统计学意义(P<0.01),而0.5 g/L、0.25 g/L药物对照组无明显促进细胞凋亡的效应。与LPS刺激0.5 g/L、0.25 g/L药物组比较,0.5 g/L、0.25 g/L药物对照组的细胞凋亡率明显下降,差异有统计学意义(P<0.01);与细胞对照组比较,1 g/L药物对照组RAW264.7G1、S期缩短,G2期延长,差异有统计学意义(P<0.01);0.5 g/L药物对照组细胞G1期延长,S期缩短,差异有统计学意义(P<0.01)。③LPS刺激RAW264.7后,与细胞对照组比较,LPS模型组IL-10、TNF-α明显升高,差异有统计学意义(P<0.05);0.5 g/L、0.25 g/L药物对照组IL-10降低,1 g/L药物对照组TNF-α升高,差异有统计学意义(P<0.05)。与LPS模型组比较,LPS 1 g/L、0.5 g/L、0.25 g/L IL-10刺激药物组降低,差异有统计学意义(P<0.05)。结论板蓝根活性部位抑制HSV-1和LPS引起U937、RAW264.7的细胞凋亡,并24 h时下调LPS作用后RAW264.7的IL-10表达。

Objective To discuss the effect of active sites of Radix lsatidis on apoptosis of Hep-2, Hela, U937, Jukart and on expression of IL-10 and TNF-α in RAW264.7 cells. Methods Hep-2, Hela, U937 and Jukart were pretreated by 0.5 g/L active sites in Radix lsatidis for 24 hours, and the apoptosis were assayed by flow cytometry. U937, infected by HSV-1, was incubated with 0.5 g/L medicine for 24 hours, and the apoptosis were assayed by flow cytometry. The expression of IL-10 and TNF-α in RAW264.7 induced by LPS were detected by ELISA. Results （1）The result showed that 0.5 g/L active sites of Radix Isatidis did not affect the apoptosis of Hep-2, Hela, U937 and Jukart, but the apoptosis rate of U937 infected by HSV-1 was reduced from 9.72% to 6.71%, with significant difference between anti-viral group and viral group （P 〈 0.01 ） ; the G1 phase arrest caused by medicating the Hela cell, and the S phase was shortened, with significant difference between the normal Hela cell and infected Hela cell （P 〈 0.05 ）. （2）In medication group, the apoptosis rates were 19.35%, 28. 14% and 7.52% after LPS-induced apoptosis in RAW264.7 cells by 1 g/L, 0.5 g/L and 0.25 g/L medicine. The apoptosis rates in medication group were lower than that in LPS model group significantly （P 〈 0.01 ）. Compared with cell control group, the apoptosis rates in 1 g/L medication group was increased significantly （P 〈 0. 01 ）, and there was no significant apoptosis promoted effect in 0.5 g/L and 0.25 g/L medication groups. The apoptosis rates in 0.5 g/L and 0.25 g/L medication groups were decreased significantly （ P 〈 0.01 ） ; compared with cell control group, the G1 phase and S phase were shortened, while the G2 phase was extended in 1 g/L medication group, with significant difference between the two groups （P 〈0.01 ）. The G1 phase was extended, but the S phase was shortened in 0.5 g/L medication group significantly （P 〈 0.01 ）. （3）After stimulated RAW264.7 cells by LPS, compared with cell control group, the IL-10 and TNF-α in LPS group were increased significantly （ P 〈 0.05 ） ; the IL-10 in 0.5 g/L and 0.25 g/L medication groups were decreased significantly, and the TNF-α in 1 g/L medication group was increased significantly （P 〈 0.05）. Compared with LPS model group, the IL-10 in 1 g/L, 0.5 g/L and 0.25 g/L medication groups were decreased significantly （ P 〈 0.05 ）. Conclusion Active sites of Radix Isatidis can inhibited apoptosis in U937 and RAW264.7 cells infected by HSV-1 and LPS. It can decrease the expression of IL-10 in RAW264.7 cells after intervened by LPS for 24 hours.