[四棱叶崖爬藤提取物对人肺癌H1299细胞凋亡的影响及其机制研究]

[Effect of extract of radix tetrastigma hemsleyani on apoptosis of human lung carcinoma H1299 cells and its mechanism study].

作者信息

Zhong Liang-Rui, Lin Shuang, Chen Wei-Fang, Wei Ke-Min

机构信息

Department of Oncology, Tongde Hospital of Zhejiang Province, Affiliated to Zhejiang Chinese Medical University, Hangzhou, China.

出版信息

Zhongguo Zhong Xi Yi Jie He Za Zhi. 2014 Nov;34(11):1354-8.

PMID:25566628

Abstract

OBJECTIVE

To study the effect of extract of Radix Tetrastigma hemsleyani on the proliferation and apoptosis of human lung carcinoma H1299 cells, and to explore its mechanisms. METHODS H1299 cells were treated with the extract of Radix Tetrastigma hemsleyani in different concentrations at different time points. Its inhibition on H1299 cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. The morphology of the H1299 cell was observed by inverted microscope. Changes of apoptosis were observed by Hoechst33258 methods. The apoptosis rate was detected by flow cytometry. Expression changes of apoptosis-related proteins pro-caspase-3, pro-caspase-9, cle-caspase-3, cle-caspase-9, and poly-ADP-ribose polymerase (PARP) were detected by Western blot.

RESULTS

Compared with the control group, the inhibition rate of H1299 cells increased after acted by 0.5, 1, 5, and 10 mg/mL extract of Radix Tetrastigma hemsleyani (P < 0.05, P < 0.01). The longer the acting time, the higher the inhibition rate (P < 0.01). Under inverted microscope, typical morphological changes could be seen and the number of H1299 cells was reduced. Under fluorescence microscope, dark stained nucleus and formed apoptotic body could be observed. Results of flow cytometry showed that the apoptosis rate was obviously dose-effect correlated with the concentration of extract of Radix Tetrastigma hemsleyani. Results of Western blot indicated that compared with the control. group, the protein expression of pro-caspase-3, pro-caspase-9, and PARP were down-regulated and that of cle-caspase-3, cle-caspase-9, and cle-PARP were up-regulated by 5 and 10 mg/mL extract of Radix Tetrastigma hemsleyani (P < 0.05).

CONCLUSIONS

Extract of Radix Tetrastigma hemsleyani had obvious effect in inhibiting the proliferation and inducing apoptosis of human lung carcinoma H1299 cells, which might be achieved by activating the expression of caspase protein.

摘要

目的

研究三叶崖爬藤提取物对人肺癌H1299细胞增殖和凋亡的影响，并探讨其作用机制。方法：用不同浓度的三叶崖爬藤提取物在不同时间点处理H1299细胞。采用细胞计数试剂盒-8（CCK-8）法检测其对H1299细胞增殖的抑制作用。用倒置显微镜观察H1299细胞的形态。采用Hoechst33258法观察凋亡变化。用流式细胞术检测凋亡率通过蛋白质免疫印迹法检测凋亡相关蛋白前半胱天冬酶-3、前半胱天冬酶-9、裂解的半胱天冬酶-3、裂解的半胱天冬酶-9和聚ADP核糖聚合酶（PARP）的表达变化。

结果

与对照组相比，0.5、1、5和10mg/mL三叶崖爬藤提取物作用后H1299细胞的抑制率升高（P<0.05，P<0.01）。作用时间越长，抑制率越高（P<0.01）。在倒置显微镜下可见典型的形态学变化，H1299细胞数量减少。在荧光显微镜下，可观察到细胞核染色加深并形成凋亡小体。流式细胞术结果显示，凋亡率与三叶崖爬藤提取物浓度呈明显的剂量效应关系。蛋白质免疫印迹法结果表明，与对照组相比，5和10mg/mL三叶崖爬藤提取物可使前半胱天冬酶-3、前半胱天冬酶-9和PARP的蛋白表达下调，使裂解的半胱天冬酶-3、裂解的半胱天冬酶-9和裂解的PARP的蛋白表达上调（P<0.05）。