硫辛酸和白藜芦醇在对乙酰氨基酚致HepG2细胞损伤中的抗氧化作用

Comparative Protective Effect of Lipoic Acid and Resveratrol on Acetaminophen-Induced Cellular Oxidative Damage in HepG2 Cells

硫辛酸(lipoic acid,LA)和白藜芦醇(resveratrol,RES)是两种重要的天然抗氧化活性因子,具有很好的抗氧化能力;对乙酰氨基酚(acetaminophen,APAP)是一种常见的解热镇痛药物,其过量可能导致氧化应激和肝脏损伤。本实验以体外细胞模型研究LA和RES对APAP引起的肝细胞损伤的抗氧化作用,并初步探讨其抗氧化机制,为APAP所致肝损伤的防治提供技术依据。首先通过APAP诱导建立HepG2细胞肝损伤模型;并采用CCK-8法检测不同浓度LA和RES对APAP诱导的HepG2细胞损伤的影响;运用细胞抗氧化活性法(cellular antioxidant activity,CAA)比较LA和RES的抗氧化活性;运用流式细胞术对细胞凋亡情况进行检测;酶标法检测丙二醛(malondialdehyde,MDA)含量以及谷胱甘肽(glutathione,GSH)质量浓度和超氧化物歧化酶(superoxide dismutase,SOD)的活力;实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qPCR)和Western blot技术检测凋亡相关基因Bax、Bcl-2、caspase-3的表达。CCK-8结果显示LA和RES均可提高APAP所致的氧化损伤的细胞活力,且LA比RES效果更明显;CAA结果显示LA抑制二氯荧光(dichlorofluorescein,DCF)生成的能力比RES强;流式结果表明LA和RES均可抑制氧化应激引起的细胞凋亡,且LA组比RES组抑制效果更明显;酶标法检测结果和qPCR以及Western Blot结果均表明LA和RES是通过降低MDA含量,提高GSH和SOD活力,上调Bcl-2表达量,下调Bax和caspase-3表达量来抑制APAP引起的细胞凋亡,且LA比RES抑制效果更明显。综上,对于APAP引起的肝损伤,LA比RES起到的保护作用更明显;其对APAP引起的肝损伤保护作用机制与减轻氧化应激反应和抑制细胞凋亡有关。

Lipoic acid(LA) and resveratrol(RES) are two important natural antioxidant agents. Acetaminophen(APAP) is a common analgesic drug. However, excess administration of APAP may cause oxidative stress and liver damage. In this paper, an in vitro cell model was used to compare the antioxidant effects of RES and LA on APAP-induced oxidative damage in HepG2 cells, and to explore the underlying mechanisms. The aim of this study was to provide a technical basis for the prevention and treatment of liver damage caused by acetaminophen. The effects of different concentrations of LA and RES on APAP-induced apoptosis in HepG2 cells were detected by Cell Counting Kit-8(CCK-8) method. The antioxidant activities of LA and RES were comparatively evaluated using cellular antioxidant activity(CAA) method. Flow cytometry was used to detect apoptosis. The content of malondialdehyde(MDA) and the activity of glutathione(GSH) and superoxide dismutase(SOD) were detected by enzyme labeling method. Quantitative real-time polymerase chain reaction(qPCR) and Western blot were used to detect the expression of the apoptosis-related genes Bax, Bcl-2 and caspase-3. Cytotoxicity test results showed that both resveratrol and lipoic acid could increase the viability of cells, and LA was more effective than RES. CAA results showed that LA inhibited dichlorofluorescein(DCF) production more strongly than did RES. The results of flow cytometry showed that both LA and RES could inhibit apoptosis induced by oxidative stress, and the effect of LA was stronger than that of RES. The results of enzyme labeling, qPCR and Western blot showed that LA and RES inhibited APAP-induced apoptosis by decreasing MDA content, increasing GSH and SOD activity, up-regulating Bcl-2 expression, and down-regulating Bax and caspase-3 expression, with LA being more effective than RES. To sum up, compared with RES, LA had a more protective effect on APAP-induced liver injury, and the underlying mechanism may be related to reducing oxidative stress and inhibiting apoptosis.