Luo Chaohuan, Lian Xueke, Hong Liangli, Zou Jingfang, Li Zhi, Zhu Yuzhang, Huang Tianliang, Zhang Yongneng, Hu Yaqiu, Yuan Huier, Wen Tengfei, Zhuang Wanling, Cai Bozhi, Zhang Xin, Hisatome Ichiro, Yamamoto Tetsuya, Huang Jiexiong, Cheng Jidong
Department of Internal Medicine, The First Affiliated Hospital of Shantou University Medical College, Shantou, China.
Cell Physiol Biochem. 2016;40(3-4):538-548. doi: 10.1159/000452567. Epub 2016 Nov 25.
BACKGROUND/AIMS: Hyperuricemia is part of the metabolic-syndrome cluster of abdominal obesity, impaired glucose tolerance, insulin resistance, dyslipidemia, and hypertension. Monocytes/macrophages are critical in the development of metabolic syndrome, including gout, obesity and atherosclerosis. However, how high uric acid (HUA) exposure affects monocyte/macrophage function remains unclear. In this study, we investigated the molecular mechanism of HUA exposure in monocytes/macrophages and its impact on oxidized low-density lipoprotein (oxLDL)-induced foam-cell formation in a human monocytic cell line, THP-1.
We primed THP-1 cells with phorbol-12-myristate-13-acetate (PMA) for differentiation, then exposed cells to HUA and detected the production of reactive oxygen species (ROS) and analyzed the level of phospho-AMPKα. THP-1 cells were pre-incubated with Compound C, an AMPK inhibitor, or N-acetyl-L-cysteine (NAC), a ROS scavenger, or HUA before PMA, to assess CD68 expression and phospho-AMPKα level. PMA-primed THP-1 cells were pre-treated with oxLDL before Compound C and HUA treatment. Western blot analysis was used to examine the levels of phospho-AMPKα, CD68, ABCG1, ABCA1, cyclooxygenase-2 (COX-2) and NF-κB (p65). Flow cytometry was used to assess ROS production and CD68 expression in live cells. Oil-red O staining was used to observe oxLDL uptake in cells.
HUA treatment increased ROS production in PMA-primed THP-1 cells; NAC blocked HUA-induced oxidative stress. HUA treatment time-dependently increased phospho-AMPKα level in PMA-primed THP-1 cells. The HUA-induced oxidative stress increased phospho-AMPKα levels, which was blocked by NAC. HUA treatment impaired CD68 expression during cell differentiation by activating the AMPK pathway, which was reversed by Compound C treatment. Finally, HUA treatment inhibited oxLDL uptake in the formation of foam cells in THP-1 cells, which was blocked by Compound C treatment. HUA treatment significantly increased the expression of ABCG1 and reversed the oxLDL-reduced ABCG1 expression but did not affect the expression of ABCA1, NF-κB (p65) or COX-2.
HUA exposure activated the ROS-AMPK pathway, impaired CD68 expression, and inhibited oxLDL-induced foam-cell formation in a human monocytic cell line, THP-1.
背景/目的：高尿酸血症是腹部肥胖、糖耐量受损、胰岛素抵抗、血脂异常和高血压等代谢综合征集群的一部分。单核细胞/巨噬细胞在包括痛风、肥胖和动脉粥样硬化在内的代谢综合征发展过程中起关键作用。然而，高尿酸（HUA）暴露如何影响单核细胞/巨噬细胞功能仍不清楚。在本研究中，我们在人单核细胞系THP-1中研究了HUA暴露在单核细胞/巨噬细胞中的分子机制及其对氧化型低密度脂蛋白（oxLDL）诱导的泡沫细胞形成的影响。
我们用佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）预处理THP-1细胞以使其分化，然后将细胞暴露于HUA并检测活性氧（ROS）的产生，并分析磷酸化AMPKα的水平。THP-1细胞在PMA处理前用AMPK抑制剂化合物C或ROS清除剂N-乙酰-L-半胱氨酸（NAC）或HUA进行预孵育，以评估CD68表达和磷酸化AMPKα水平。在化合物C和HUA处理前，用oxLDL对PMA预处理的THP-1细胞进行预处理。采用蛋白质免疫印迹分析检测磷酸化AMPKα、CD68,、ABCG1、ABCA1、环氧合酶-2（COX-2）和核因子κB（p65）的水平。采用流式细胞术评估活细胞中ROS的产生和CD68的表达。用油红O染色观察细胞对oxLDL的摄取情况。
HUA处理增加了PMA预处理的THP-1细胞中ROS的产生；NAC阻断了HUA诱导的氧化应激。HUA处理使PMA预处理的THP-1细胞中磷酸化AMPKα水平呈时间依赖性增加。HUA诱导的氧化应激增加了磷酸化AMPKα水平，而NAC可阻断这一作用。HUA处理通过激活AMPK途径损害细胞分化过程中的CD68表达，化合物C处理可逆转这一作用。最后，HUA处理抑制了THP-1细胞中泡沫细胞形成过程中对oxLDL的摄取，化合物C处理可阻断这一作用。HUA处理显著增加了ABCG1的表达，并逆转了oxLDL降低的ABCG1表达，但不影响ABCA1、NF-κB（p65）或COX-2的表达。
HUA暴露激活了ROS-AMPK途径，损害了CD68表达，并抑制了人单核细胞系THP-1中oxLDL诱导的泡沫细胞形成。
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