Liu Xiao-Xia, Xia Jing, Tang Jia-Feng, Zhou Ming-Hua, Chen Di-Long, Liu Ze-Hong
Traditional Chinese Medicine Department, Jiangjin Central Hospital of Chongqing, Chongqing 404120, China.
Chongqing Three Gorges Medical College, Chongqing 402260, China.
Zhongguo Zhong Yao Za Zhi. 2017 Jan;42(1):146-151. doi: 10.19540/j.cnki.cjcmm.20161222.053.
To study the effect of ginseng saponin Rh₂ in inducing apoptosis of human leukemia K562 cells, and explore its mechanism from the aspect of autophagy pathway. CCK-8 assay was used to examine the growth inhibition of human leukemia cell lines K562 treated with ginsenoside Rh₂; flow cytometry (FCM) was used to detect cell apoptosis; Hoechst staining was used to observe the changes of cell morphological apoptosis; Acridine and MDC staining were used to detect the effects of the Rh₂ on autophagy; Western blot and RT-PCR were used to detect the expression levels of the proteins closely associated with autophagy and apoptosis. In order to study the effect of autophagy in proliferation and apoptosis, we used the autophagy inhibitor (3-MA).CCK-8 indicated that Rh₂ at low concentration could effectively inhibit the proliferation of leukemia cellsin dose- and time-dependent manners in K562 cells; FCM indicated that Rh₂ induced apoptosis; Hoechest staining showed that K562 cells had typical apoptotic morphological changes by treated Rh₂; Acridine and MDC staining showed that Rh₂ enhanced the green fluorescence and a large number of acidic autophagy vesicles were present; Western blot and RT-PCR results showed that Rh₂ increased the expression levels of Beclin-1, LC3A, LC3B, activated Caspase-3 and p-p38 in K562 cells; application of autophagy inhibitors(3-MA) could weaken the inhibition effect of Rh₂ on proliferation and induction effect on apoptosis in K562 cells. Ginsenoside Rh₂ inhibited the proliferation and induced apoptosis probably through activating p-p38, and inducing cell autophagy signaling pathway in K562 cells.
研究人参皂苷Rh₂诱导人白血病K562细胞凋亡的作用，并从自噬途径方面探讨其机制。采用CCK-8法检测人参皂苷Rh₂处理后人白血病细胞株K562的生长抑制情况；采用流式细胞术（FCM）检测细胞凋亡；采用Hoechst染色观察细胞形态学凋亡变化；采用吖啶橙和MDC染色检测Rh₂对自噬的影响；采用蛋白质免疫印迹法（Western blot）和逆转录-聚合酶链反应（RT-PCR）检测与自噬和凋亡密切相关蛋白的表达水平。为研究自噬在增殖和凋亡中的作用，我们使用了自噬抑制剂（3-MA）。CCK-8结果表明，低浓度Rh₂能以剂量和时间依赖性方式有效抑制K562细胞中白血病细胞的增殖；FCM结果表明，Rh₂诱导细胞凋亡；Hoechest染色显示，经Rh₂处理的K562细胞出现典型的凋亡形态学变化；吖啶橙和MDC染色显示，Rh₂增强了绿色荧光，且存在大量酸性自噬小泡；Western blot和RT-PCR结果显示，Rh₂增加了K562细胞中Beclin-1、LC3A、LC3B、活化的Caspase-3和p-p38的表达水平；应用自噬抑制剂（3-MA）可减弱Rh₂对K562细胞增殖的抑制作用及其对凋亡的诱导作用。人参皂苷Rh₂可能通过激活p-p38并诱导K562细胞的自噬信号通路来抑制细胞增殖并诱导细胞凋亡。
