Protective effect of probucol against oxidative cell damage induced by oxidized low-density lipoprotein and high glucose in human mesangial cells

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type:TOHO University Scholarly Publication

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Background: We previously reported that probucol decreases systemic oxidative stress and delays progressive loss of renal function in type 2 diabetes with nephropathy. Apoptosis is an important pathogenic factor for microvascular injury in diabetes. In the present study, we investigated the effect of probucol on oxidative damage induced by oxidized low-density lipoprotein (OxLDL) and high glucose in normal human mesangial cells (NHMCs). Methods: Messenger ribonucleic acid (mRNA) “p22phox" and protein “p47phox" expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits was measured by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. Production of intracellular reactive oxygen species (ROS) was measured by fluorescence-activated cell sorting. Apoptotic signaling was evaluated by measuring caspase-3 activity. Results: Both high glucose (500 mg/dl) and OxLDL enhanced intracellular ROS production. There were increases in the expression of p22phox mRNA and p47phox protein from NADPH oxidase subunits, and caspase-3 activity also increased. Probucol treatment inhibited increased expression of NADPH oxidase subunits and ROS production, as well as activation of caspase-3, induced by OxLDL and high glucose. Conclusions: Probucol appears to suppress oxidative damage and activation of apoptosis signaling induced by OxLDL and high glucose in NHMCs.目的:われわれは 2 型糖尿病患者においてプロブコールが酸化ストレスを減少させ腎機能障害の進展を抑制する報告をした.アポトーシスは糖尿病の微小血管障害の主要病因であり本研究では nicotinamide ade-nine dinucleotide phosphate(NADPH)oxidase 発現と reactive oxygen species(ROS)の産生およびアポトーシスに対するプロブコールの効果を正常人メサンギウム細胞(normal human mesangial cells:NHMCs)で検討した.方法:NHMCs は正常糖(100 mg/dl),高糖状態(500 mg/dl)にて培養し酸化 low-density lipoprotein(LDL),プロブコール(1,10μM)を添加した.NADPH oxidase サブユニットの messenger ribonucleic acid(mRNA) “p22phox",蛋白量“p47phox"はそれぞれ reverse transcriptase-polymerase chain reaction(RT-PCR),ウェスタンブロット法で,ROS 産生は fluorescence activated cell sorting(FACS)を用いアポトーシスはカスパーゼ 3 活性で評価した.結果:高糖状態および酸化 LDL いずれも p22phox,p47phox の発現が増加し ROS の産生およびカスパーゼ 3 の活性も高めた.プロブコールはこれらの変化を抑制した.考察:本研究は高糖,酸化 LDL 添加状態の NHMCs で,プロブコールが酸化反応とそれによるアポトーシスを抑制することを示唆した.

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