Double Knockdown of PHD1 and Keap1 Attenuated Hypoxia-Induced Injuries in Hepatocytes.
Double Knockdown of PHD1 and Keap1 Attenuated Hypoxia-Induced Injuries in Hepatocytes.
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PHD1 和 Keap1 的双重敲低可减轻缺氧引起的肝细胞损伤
DOI：
10.3389/fphys.2017.00291
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发表时间：
2017
影响因子：
4
通讯作者：
Chen PS
中科院分区：
文献类型：
--
作者：
Liu J;Li Y;Liu L;Wang Z;Shi C;Cheng Z;Zhang X;Ding F;Chen PS
背景和目的：缺氧和氧化应激导致肝纤维化。在本实验中，我们利用小发夹RNA(ShRNA)干扰低氧肝细胞内氧感受器--脯氨酸羟基酶1(PhD1)和细胞内氧化应激感受器--海带样ECH相关蛋白1(Keap1)，以探讨PHD1和Keap1的功能。方法：建立CCl4诱导的大鼠肝纤维化模型，检测肝组织中Phd1、低氧诱导因子-1α(HIF-1α)、低氧诱导因子-2α(HIF-2α)、KEAP1、核因子-红系P45相关因子2(NRF2)的表达。同时，体外构建共转染PhD1和Keap1shRNAs的AML12细胞，检测细胞内氧化应激、细胞凋亡率和细胞存活率。采用实时定量聚合酶链式反应(qRT-PCR)和免疫印迹分析促纤维化分子的表达。采用双抗体夹心酶联免疫吸附试验(EL ISA)检测血清α-1型I型胶原(COL1A1)水平。最后，用低氧AML12肝细胞培养上清液培养大鼠肝星状细胞(HSC-T6)，检测肝纤维化相关分子、细胞凋亡和细胞增殖水平。结果：肝纤维化大鼠肝组织中缺氧缺氧诱导因子-1α和缺氧诱导因子-2α表达上调，而Phd1的表达与对照组比较差异无统计学意义。氧化应激信号Keap1在肝细胞中的表达增加，而Keap1下游主要分子之一Nrf2的表达减少。在体外，AML12肝细胞中Phd1和Keap1的双下调表现为低氧诱导的氧化应激和细胞凋亡的减少，而且这些低氧的AML12细胞表现出细胞活力的增加和促纤维化分子的下调表达。此外，在低氧双基因敲除CM中培养的HSC-T6细胞显示出纤维化相关分子的下调，细胞增殖减少，并促进了细胞凋亡。结论：双重敲除Phd1和Keap1基因可减轻缺氧和氧化应激对肝细胞的损伤，进而抑制HSC的激活，为防治肝纤维化提供了新的治疗策略。
Background and Aims: Hypoxia and oxidative stress contribute toward liver fibrosis. In this experiment, we used small hairpin RNA (shRNA) to interfere with the intracellular oxygen sensor—prolyl hydroxylase 1 (PHD1) and the intracellular oxidative stress sensor—kelch-like ECH associated protein 1 (Keap1) in the hypoxic hepatocytes in order to investigate the function of PHD1and Keap1. Methods: We first established the CCl4-induced liver fibrosis model, subsequently, the levels of the PHD1, hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), Keap1, and nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) were detected in liver tissues. Simultaneously, AML12 cells co-transfected with PHD1 and Keap1shRNAs were constructed in vitro, then the intracellular oxidative stress, the proportion of cells undergoing apoptosis, and cell viability were measured. The expression of pro-fibrogenic molecules were analyzed via quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The level of alpha-1 type I collagen (COL1A1) was determined using an enzyme-linked immunosorbent assay (ELISA). Finally, serum-free “conditioned medium” (CM) from the supernatant of hypoxic AML12 hepatocytes was used to culture rat hepatic stellate cells (HSC-T6), and the levels of fibrosis-related molecules, apoptosis, and cell proliferation were determined. Results: The marker of hypoxia—HIF-1α and HIF-2α in the livers with fibrosis were upregulated, however, the increase in PHD1 expression was not statistically significant in comparison to the control group. Sign of oxidative stress—Keap1 was increased, while the expression of Nrf2, one of the Keap1 main downstream molecules, was reduced in the hepatocytes. And in vitro, the double-knockdown of PHD1 and Keap1 in AML12 hepatocytes presented with decreased hypoxia-induced oxidative stress and apoptosis, furthermore, these hypoxic AML12 cells showed the increased cell viability and the doweregulated expression of pro-fibrogenic molecules. In addition, HSC-T6 cells cultured in the hypoxic double-knockdown CM demonstrated the downregulation of fibrosis-related molecules, diminished cell proliferation, and enhanced apoptosis. Conclusions: Our study demonstrated that double-knockdown of PHD1 and Keap1 attenuated hypoxia and oxidative stress induced injury in the hepatocytes, and subsequently inhibited HSC activation, which offers a novel therapeutic strategy in the prophylaxis and treatment of liver fibrosis.