Wang Lu, Luo Ran, Zhao Yin-Xu, Zou Yu, Bu Ming, Lin Yu
College of Pharmacy, Qiqihar Medical University Qiqihar 161006, China.
Zhongguo Zhong Yao Za Zhi. 2024 Jun;49(12):3365-3372. doi: 10.19540/j.cnki.cjcmm.20240221.501.
This study aims to investigate the effect of ergosterol peroxide(EP) on the apoptosis of human hepatocellular carcinoma and its mechanism of action. The cell viability of HepG2 and SK-Hep-1 cells with 0(blank control), 2.5, 5, 10, 20, 40, and 80 μmol·L(-1) of EP after 24, 48, and 72 h of action was detected by using CCK-8 assay, and the half inhibitory concentrations(IC_(50)) at 24, 48, and 72 h were calculated. Formal experiments were performed to detect the effect of EP on intracellular reactive oxygen species(ROS) using DCFH-DA staining, the effect of EP on intracellular mitochondrial membrane potential using JC-1 staining, the number of apoptotic cells using Annexin V-FITC/PI double-staining after HepG2 cells were co-cultured with 0(blank control), 10, 20, 40 μmol·L(-1) EP for 48 h. The effects of EP at different concentrations on apoptotic morphology were detected using AO/EB staining. The effects of different concentrations of EP on the protein expression of mitochondrial apoptosis pathway-related proteins B cell lymphoma 2(Bcl-2), cytochrome C(Cyt-C), Bcl-2-related X protein(Bax), caspase-3, cleaved caspase-3, caspase-9, and cleaved caspase-9 were examined by using Western blot. The results showed that different concentrations of EP could inhibit the proliferation of hepatocellular carcinoma with concentration-and time-dependent trends. Compared with the blank control group, the ROS level in the EP-treated group increased significantly(P<0.05). The mitochondrial membrane potential decreased significantly(P<0.05). The total apoptosis rate increased significantly(P<0.05). The expression of Bcl-2 protein was significantly down-regulated, and the expression of Cyt-C, Bax, cleaved caspase-9, and cleaved caspase-3 were significantly up-regulated(P<0.05). In summary, EP may inhibit the proliferation of hepatocellular carcinoma by modulating the mitochondria-mediated apoptosis pathway and induce apoptosis.
本研究旨在探讨过氧化麦角甾醇（EP）对人肝癌细胞凋亡的影响及其作用机制。采用CCK-8法检测0（空白对照）、2.5、5、10、20、40和80 μmol·L⁻¹的EP作用24、48和72 h后HepG2和SK-Hep-1细胞的活力，并计算24、48和72 h的半数抑制浓度（IC₅₀）。进行正式实验，用DCFH-DA染色检测EP对细胞内活性氧（ROS）的影响，用JC-1染色检测EP对细胞内线粒体膜电位的影响，将HepG2细胞与0（空白对照）、10、20、40 μmol·L⁻¹的EP共培养48 h后，用Annexin V-FITC/PI双染法检测凋亡细胞数量。用AO/EB染色检测不同浓度EP对凋亡形态的影响。用蛋白质免疫印迹法检测不同浓度EP对线粒体凋亡途径相关蛋白B细胞淋巴瘤2（Bcl-2）、细胞色素C（Cyt-C）、Bcl-2相关X蛋白（Bax）、半胱天冬酶-3（caspase-3）、裂解的半胱天冬酶-3、半胱天冬酶-9和裂解的半胱天冬酶-9蛋白表达的影响。结果表明，不同浓度的EP可抑制肝癌细胞增殖，呈浓度和时间依赖性。与空白对照组相比，EP处理组的ROS水平显著升高（P<0.05）。线粒体膜电位显著降低（P<0.05）。总凋亡率显著升高（P<0.05）。Bcl-2蛋白表达显著下调，Cyt-C、Bax、裂解的半胱天冬酶-9和裂解的半胱天冬酶-
