晚期氧化蛋白产物通过 NADPH 氧化酶途径诱导MC3 T3-E1细胞产生ROS

摘要

目的：研究晚期氧化蛋白产物( AOPP )通过 NADPH 氧化酶途经诱导小鼠成骨样细胞(MC3T3-E1)产生ROS。方法实验分为空白对照组、RSA(鼠血清白蛋白)组、AOPP组。培养MC3T3-E1细胞,加入RSA和不同浓度的AOPP,用2,7-二氯二氢荧光素二乙酯为荧光探针流式细胞仪检细胞内活性氧簇(ROS)的生成量。在阻断实验中,MC3T3-E1细胞分别与各种可能产生活性氧的酶的抑制剂孵育后加入AOPP,观察阻断剂对ROS生成的阻断作用。采用Western印迹和免疫荧光法观察NADPH氧化酶亚基的变化。结果分别采用50、100、200μg/ml AOPP刺激MC3T3-E1细胞,细胞ROS的生成随着AOPP浓度的升高而逐渐增加,其中200μg/ml AOPP产生的ROS最多(P<0.05)。 AOPP诱导MC3T3-E1生成ROS还表现为时间依赖性,AOPP刺激30 min即可引起ROS生成增加(P<0.05),3 h达峰。加入不同的酶阻断剂后,仅NADPH氧化酶抑制剂二苯碘鎓( DPI)、夹竹桃麻素( apocynin)、胞浆型超氧化物歧化酶C-SOD抑制了ROS的生成(P<0.05)。同时,AOPP刺激后p47phox有明显的膜迁移,并可诱导MC3T3-E1细胞Nox4蛋白表达上调。结论 AOPP通过NADPH氧化酶途径诱导MC3T3-E1细胞产生ROS,推测这可能是AOPP参与骨质疏松发病的机制。%Objective In the present study, we investigated the effects of advanced oxidation protein products(AOPP) on reactive oxygen species(ROS) production in murine osteoblastic MC3T3-E1 cells by NADPH oxidase enzymes pathway. Methods Experiments were divided into three groups, including control group, rats albumin(RSA) group, and AOPP group. Different concentrations of AOPP were added to the osteoblastic MC3T3-E1 cells culture medium. The production of ROS in MC3T3-E1 cells was measured by the fluorescence intensity of intracellular fluoroprobe ( DCFD ) . In order to verify the effect of enzyme of the production of ROS, the specific inhibitors of corresponding enzymes were added in the MC3T3-E1 cells which were cultured in the medium with AOPP. Finally, western blot and immunofluorescence were used to observe the changes of NADPH oxidase enzymes subunits. Results Different concentrations of AOPP (50,100,200μg/ml) induced MC3T3-E1 cells to produce different amount of ROS. The higher concentrations of AOPP were added, the more ROS were produced. Furthermore,200μg/ml AOPP induced the maximum amount of ROS production(P<0. 05). Meanwhile, AOPP induced MC3T3-E1 cells to produce different amount of ROS with a time-dependent manner. The peak amount of ROS production in MC3T3-E1 cells was observed in 3h when AOPP were added (P<0. 05). In addition, when specific inhibitors of corresponding enzymes were added in the MC3T3-E1 cells, the production of ROS were significantly suppressed by C-SOD, DPI, and apocynin(P<0. 05). On the other hand, AOPP can up-regulate the expression of Nox4 protein of the MC3T3-E1 cells, which is one of the subunits of NADPH oxidase enzymes. Meanwhile, AOPP can also induce the membrane migration of p47phox subunit. Conclusion AOPP induces osteoblastic MC3T3-E1 cells to produce ROS by NADPH oxidase enzymes pathway, and which may be one of the pathogenesis of AOPP involved in osteoporosis.