Department of Pharmacy, Kyushu University Hospital, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan.
Atherosclerosis. 2010 Jan;208(1):112-8. doi: 10.1016/j.atherosclerosis.2009.07.033. Epub 2009 Jul 23.
Hepatotoxicity is one of the major complaints that occur during lipid-lowering therapy with 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors, known as statins. We reported earlier that lipophilic but not hydrophilic statins induce apoptosis through inhibition of mevalonate biosynthesis cascade in Chang liver cells. The present study was designed to determine the role for small G protein RhoA in the hepatocytotoxicity of statins.
Statin-induced hepatocytotoxicity in HepG2 cells were assessed by WST-8 cell viability assay, JC-1 mitochondrial membrane potential assay and caspase-3/7 activity assay. Cytosolic RhoA was detected by Western blotting and RhoA activation was measured by ELISA.
The lipophilic atorvastatin but not the hydrophilic pravastatin induced the mitochondrial membrane depolarization and the activation of caspase-3/7, which led to cell injury. Supplementation of mevalonate or geranylgeranyl pyrophosphate (GGPP) but not farnesyl pyrophosphate (FPP) reversed these cellular events and cell death induced by atorvastatin. Atorvastatin induced a translocation of RhoA protein into the cytosol and inhibited the activity of the protein. In addition, atorvastatin reduced mitochondrial membrane potential, which was mimicked by GGTase inhibitor GGTI-2147 or the specific RhoA inhibitor such as toxin B and C3 exoenzyme. However, only a few cells revealed mitochondrial membrane depolarization and a loss of viability after exposure to the Rho-kinase inhibitors such as Y-27632 and hydroxy fasudil.
RhoA inactivation and to a lesser extent Rho-kinase inhibition after depletion of GGPP is implicated in the etiology of mitochondrial membrane depolarization and subsequent caspase-dependent cell death induced by the lipophilic statin in HepG2 cells.
3-羟基-3-甲基戊二酰辅酶 A（HMG-CoA）还原酶抑制剂，即他汀类药物，是降脂治疗中常见的肝毒性反应的主要原因之一。我们之前报道过，亲脂性他汀类药物而非亲水性他汀类药物通过抑制甲羟戊酸生物合成级联反应诱导Chang 肝细胞凋亡。本研究旨在确定小 G 蛋白 RhoA 在他汀类药物肝毒性中的作用。
通过 WST-8 细胞活力测定、JC-1 线粒体膜电位测定和 caspase-3/7 活性测定评估他汀类药物诱导的 HepG2 细胞肝毒性。通过 Western blot 检测胞质 RhoA，通过 ELISA 检测 RhoA 激活。
亲脂性阿托伐他汀而非亲水性普伐他汀诱导线粒体膜去极化和 caspase-3/7 激活，导致细胞损伤。补充甲羟戊酸或香叶基香叶基焦磷酸（GGPP）而非法尼基焦磷酸（FPP）可逆转阿托伐他汀引起的这些细胞事件和细胞死亡。阿托伐他汀诱导 RhoA 蛋白向胞质易位并抑制其活性。此外，阿托伐他汀降低线粒体膜电位，这可被 GGTase 抑制剂 GGTI-2147 或特定的 RhoA 抑制剂如毒素 B 和 C3 外切酶模拟。然而，只有少数细胞在暴露于 Rho 激酶抑制剂如 Y-27632 和羟基法舒地尔后出现线粒体膜去极化和活力丧失。
GGPP 耗竭后 RhoA 失活，在一定程度上 Rho 激酶抑制，与亲脂性他汀类药物在 HepG2 细胞中诱导的线粒体膜去极化和随后的 caspase 依赖性细胞死亡有关。
