Park Sang Mi, Jung Eun Hye, Kim Jae Kwang, Jegal Kyung Hwan, Park Chung A, Cho Il Je, Kim Sang Chan
MRC-GHF, Department of Herbal Formulation, College of Korean Medicine, Daegu Haany University, Gyeongsan, Republic of Korea.
College of Korean Medicine, Daegu Haany University, Daegu, Republic of Korea.
J Ginseng Res. 2017 Jul;41(3):392-402. doi: 10.1016/j.jgr.2017.01.012. Epub 2017 Jan 25.
Previously, we reported that Korean Red Ginseng inhibited liver fibrosis in mice and reduced the expressions of fibrogenic genes in hepatic stellate cells (HSCs). The present study was undertaken to identify the major ginsenoside responsible for reducing the numbers of HSCs and the underlying mechanism involved.
Using LX-2 cells (a human immortalized HSC line) and primary activated HSCs, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assays were conducted to examine the cytotoxic effects of ginsenosides. HO productions, glutathione contents, lactate dehydrogenase activities, mitochondrial membrane permeabilities, apoptotic cell subpopulations, caspase-3/-7 activities, transferase dUTP nick end labeling (TUNEL) staining, and immunoblot analysis were performed to elucidate the molecular mechanism responsible for ginsenoside-mediated cytotoxicity. Involvement of the AMP-activated protein kinase (AMPK)-related signaling pathway was examined using a chemical inhibitor and small interfering RNA (siRNA) transfection.
Of the 11 ginsenosides tested, 20-protopanaxadiol (PPD) showed the most potent cytotoxic activity in both LX-2 cells and primary activated HSCs. Oxidative stress-mediated apoptosis induced by 20-PPD was blocked by -acetyl-l-cysteine pretreatment. In addition, 20-PPD concentration-dependently increased the phosphorylation of AMPK, and compound C prevented 20-PPD-induced cytotoxicity and mitochondrial dysfunction. Moreover, 20-PPD increased the phosphorylation of liver kinase B1 (LKB1), an upstream kinase of AMPK. Likewise, transfection of LX-2 cells with LKB1 siRNA reduced the cytotoxic effect of 20-PPD. Thus, 20-PPD appears to induce HSC apoptosis by activating LKB1-AMPK and to be a therapeutic candidate for the prevention or treatment of liver fibrosis.
此前，我们报道过韩国红参可抑制小鼠肝纤维化，并降低肝星状细胞（HSCs）中纤维化相关基因的表达。本研究旨在确定负责减少肝星状细胞数量的主要人参皂苷及其潜在机制。
使用LX-2细胞（一种人永生化肝星状细胞系）和原代活化肝星状细胞，进行MTT（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四氮唑）试验，以检测人参皂苷的细胞毒性作用。进行HO生成、谷胱甘肽含量、乳酸脱氢酶活性、线粒体膜通透性、凋亡细胞亚群、半胱天冬酶-3/-7活性、转移酶dUTP缺口末端标记（TUNEL）染色和免疫印迹分析，以阐明人参皂苷介导的细胞毒性的分子机制。使用化学抑制剂和小干扰RNA（siRNA）转染来检测AMP激活的蛋白激酶（AMPK）相关信号通路的参与情况。
在测试的11种人参皂苷中，20-原人参二醇（PPD）在LX-2细胞和原代活化肝星状细胞中均表现出最强的细胞毒性活性。乙酰半胱氨酸预处理可阻断20-PPD诱导的氧化应激介导的凋亡。此外，20-PPD浓度依赖性地增加AMPK的磷酸化，化合物C可预防20-PPD诱导的细胞毒性和线粒体功能障碍。此外，20-PPD增加了AMPK的上游激酶肝激酶B1（LKB1）的磷酸化。同样，用LKB1 siRNA转染LX-2细胞可降低20-PPD的细胞毒性作用。因此，20-PPD似乎通过激活LKB1-AMPK诱导肝星状细胞凋亡，有望成为预防或治疗肝纤维化的候选药物。
