CD36在高糖诱导的大鼠肾小球系膜细胞凋亡中的作用及机制

The effects and underlying mechanism of CD36 in high glucose- induced rat glomerularmesangial cells apoptosis

目的观察高糖对大鼠肾小球系膜细胞CD36抗原表达的影响，探讨其与系膜细胞氧化应激和凋亡的关系。方法大鼠。肾小球系膜细胞分为对照组（5．6mmol/L葡萄糖培养基），甘露醇组（24．2mmol/L甘露醇＋5．6mmol/L葡萄糖培养基），高糖组（30mmol/L葡萄糖MEM培养基）、anti-CD36受体封闭组（anti-CD36＋30mmol/L葡萄糖MEM培养基）。采用激光共聚焦显微镜检测细胞内活性氧（Reactive Oxygen Species，ROS）水平。收集细胞培养上清液，测定丙二醛（MDA）、8-羟基脱氧尿苷（8-OHDG）及谷胱甘肽过氧化酶（GSH．PX）含量。流式AnnexinV-FITC/PI双染法检测细胞凋亡。用RT-PCR和Western印迹法检测肾小球系膜细胞CD36、促凋亡基因Bax、抑凋亡基因Bel-2的mRNA及蛋白的表达。结果大鼠。肾小球系膜细胞表达CD36抗原，高糖培养24h时CD36蛋白表达水平最高。对照组与甘露醇组的ROS水平、细胞上清液中MDA、8-OHDG、GSH-PX水平、早晚期凋亡率、CD36抗原、Bax及Bcl-2的mRNA及蛋白表达差异无统计学意义（均P〉0．05）。与对照组相比，高糖组ROS水平、细胞上清液中MDA、8-OHDG水平、早晚期凋亡率、CD36和Bax的mRNA及蛋白表达水平增高，而细胞上清液中GSH-PX水平、Bcl-2的mRNA及蛋白表达降低，差异均有统计学意义（均P〈0．05）。与高糖组相比，anti-CD36受体封闭组CD36的mRNA及蛋白表达差异无统计学意义（P〉0．05），ROS水平、细胞上清液中MDA、8-OHDG水平、早晚期凋亡率、BaxmRNA及蛋白表达减少，而细胞上清液GSH．PX水平、Bcl-2mRNA及蛋白表达增高，差异有统计学意义（均P〈0．05）。相关分析证实细胞内ROS水平与细胞凋亡率及CD36、Bax蛋白表达均呈正相关，与Bel-2蛋白表达呈负相关。结论肾小球系膜细胞表达CD36抗原，且高糖可以诱导CD36抗原的表达，其表达水平与细胞内氧化应激水平成正相关，CD36抗原参与高糖诱导的肾小球系膜细胞凋亡，机制可能是通过高糖环境中增加细胞内氧化应激水平来介导的。

Objective To investigate the effects and underlying mechanism of the scavenger receptor CD36 in high glucose- induced rat glomerular mesangial cells apoptosis. Methods The mesangial cells of rats were divided into 4 groups： control group （5.6 mmol/L glucose）, mannitol group （24.2 mmol/L mannitol＋ 5.6 mmol/L glucose）, high glucose group （30 mmol/L glucose）, CD36 mono- antibody group （30 mmol/L glueose＋CD36 mono-antibody）. The intracellular ROS level was detected by confocal microscopy with fluorescent probe CM H2DCFDA. MDA, GSH- PX, 8- OHDGA in cell supernatant were detected. Apoptosis was determined by flow cytometry followed by Annexin V-FITC/PIdouble stains. The expression of CD36, Bax and Bcl-2 were detected by RT-PCR and Western blotting. Results The expression of CD36 was detected in glomerular mesangial cells. The highest level was found in. high glucose group in 24 hours. There was no significant difference found between control group and mannitol group with respect to intraceilular ROS generation, MDA, 8-OHDG, GSH-PX level, apoptosis rate, expression of CD36, Bax and Bcl-2 （all P 〉 0.05）. There was no significant difference in the expression of CD36 between CD36 mono- antibody group and high glucose group （P 〉 0.05）. Compared to control group, the intracellular ROS generation, MDA and 8-OHDG levels, apoptosis rate, the expression of CD36 and Bax were significantly increased, the GSH-PX level and the expression of Bcl-2 were significantly lower in high glucose group （all P 〈 0.05）. Compared to the high glucose group, the intracellular ROS generation, MDA and 8-OHDG levels, apoptosis rate, the expression of Bax were suppressed but the GSH-PX level and the expression of Bcl-2 increased in CD36 mono-antibody group （all P 〈 0.05）. The intracellular ROS level was positively correlated with apoptosis rate, protein expression of CD36 and Bax gene, was negatively correlated with Bcl- 2 protein expression. Conclusions CD36 was involved in the high glucose induced apoptosis of mesangial cells which was potentially mediated by an increased level of oxidative stress.