大黄酸通过非活性氧依赖性内质网应激通路诱导L-02细胞凋亡

Rhein induces apoptosis in L-02 cells via reactive oxygen species-independent endoplasmic reticulum stress pathway

研究大黄酸对人正常肝细胞L-02的凋亡作用及探讨其机制。应用MTT法评价大黄酸对L-02细胞的活性抑制作用;流式细胞术检测大黄酸诱导L-02细胞的凋亡;DCFH-DA荧光探针检测细胞内活性氧(ROS)含量;实时荧光定量PCR(RT-q PCR)和Western blot技术检测细胞内质网应激相关基因和蛋白的表达变化;并探讨抗氧化剂N-乙酰半胱氨酸(NAC)对内质网应激蛋白及caspase-4、caspase-3的影响。实验结果显示:大黄酸能够呈剂量和时间依赖性抑制L-02细胞活性,增加L-02细胞凋亡率;诱导细胞ROS水平增高,NAC预给药可显著降低其水平;大黄酸能显著上调L-02细胞内的葡萄糖调节蛋白78(GRP 78)、活化转录因子4(ATF-4)和C/EBP同源蛋白(CHOP)基因的mRNA水平;显著增加GRP 78,磷酸化c-jun氨基末端激酶(p-JNK),CHOP蛋白表达;NAC不能抑制大黄酸诱导的GRP 78,p-JNK,CHOP蛋白表达上调,亦不能抑制大黄酸诱导的caspase-4及caspase-3活性增加。表明大黄酸能够诱导L-02细胞凋亡,其作用机制与非活性氧依赖性的内质网应激通路有关。

This research aimed to investigate the apoptotic effect of rhein on human primary liver cells（ L-02） and the underlying mechanisms. MTT assay was used to detect the inhibitory activity of rhein on L-02 cells. Rheininduced apoptosis in L-02 cells was evaluated by flow cytometry. Intracellular reactive oxygen species（ ROS） was detected by DCFH-DA fluorescent probe. Endoplasmic reticulum stress-related gene and protein expression were detected by real-time quantitative PCR（ RT-q PCR） and Western blot. The effect of antioxidant N-acetylcysteine（ NAC） on these proteins, cysteinyl aspartate specific proteinase 4（ caspase-4） and cysteinyl aspartate specific proteinase 3（ caspase-3） activity was explored. The results showed that rhein inhibited the viability of L-02 cells in a dose-dependent and time-dependent manners. The apoptosis rate of L-02 cells in rhein-treated groups was increased significantly. Rhein induced generation of ROS which was blocked by NAC. The expressions of GRP78,ATF-4,CHOP mRNA and the expressions of GRP 78,p-JNK,CHOP proteins were significantly increased in rhein-treated groups. However, NAC could not attenuate the expressions of GRP 78, p-JNK, CHOP proteins,caspase-4,caspase-3 activity induced by rhein significantly. In conclusion,rhein induces apoptosis in L-02 cells via a reactive oxygen species-independent endoplasmic reticulum stress pathway.