<i>In vitro</i> evaluation of cytotoxic, oxidative, genotoxic, and apoptotic activities of physodic acid from <i>Pseudevernia furfuracea</i> in HepG2 and THLE2 cells

In the present study, cytotoxic (apoptotic and necrotic), antioxidant, prooxidant, genotoxic, and apoptosis-related genes expression modulatory effects of physodic acid (PA) isolated from Pseudevernia furfuracea on cancerous (HepG2) and healthy (THLE2) human liver cells were evaluated. Cytotoxic effects PA obtained from P. furfuracea on HepG2 and THLE2 cells were tested via MTT and LDH analyses. In order to determine genotoxic effect of PA, 8-OH-dG levels in the cells were measured. In addition, apoptotic or necrotic effects of PA on the hepatic cells were determined by qRT-PCR analysis. Cytotoxicity analyses revealed the cytotoxic effect of PA on HepG2 cells, which is much higher than THLE2 cells. Gene expression studies demonstrated the apoptotic effects of PA on HepG2 cells through both extrinsic and intrinsic pathways. PA increased the antioxidant capacity of THLE2 cells with increasing concentrations, which describes its antioxidant power. Additionally, none of the tested levels of PA caused oxidative DNA damage on THLE2 cells, but its high concentration increased genetic damages on HepG2. Taken together, P. furfuracea lichen and its metabolite, PA, would be a promising alternative for complementary treatments against liver cancer.本研究评估了从粉芽扁枝衣（Pseudevernia furfuracea）中分离得到的扁枝衣酸（physodic acid, PA）对人肝癌细胞系（HepG2）与正常肝细胞系（THLE2）的细胞毒性（含凋亡与坏死效应）、抗氧化、促氧化、遗传毒性及凋亡相关基因表达调控活性。采用MTT法与乳酸脱氢酶（lactate dehydrogenase, LDH）分析法，检测了PA对HepG2及THLE2细胞的细胞毒性作用。为明确PA的遗传毒性，本研究检测了细胞内8-羟基脱氧鸟苷（8-hydroxy-2'-deoxyguanosine, 8-OH-dG）的水平。此外，通过实时荧光定量PCR（quantitative real-time polymerase chain reaction, qRT-PCR）分析，确定了PA对肝细胞的凋亡与坏死效应。细胞毒性分析结果显示，PA对HepG2细胞的杀伤活性显著高于其对THLE2细胞的作用。基因表达研究证实，PA可通过外源性及内源性两条通路诱导HepG2细胞凋亡。PA可呈浓度依赖性提升THLE2细胞的抗氧化能力，验证了其抗氧化活性。此外，各受试浓度的PA均未对THLE2细胞造成氧化性DNA损伤，但高浓度PA可诱导HepG2细胞产生遗传损伤。综上，粉芽扁枝衣（P. furfuracea）地衣及其活性代谢产物PA有望成为肝癌辅助治疗的潜在候选方案。