摘要
目的观察普罗布考对氨基核苷嘌呤霉素(PAN)致小鼠永生化足细胞损伤模型哺乳类动物雷帕霉素靶蛋白(mTOR)及氧化应激水平的影响,探讨普罗布考的肾脏保护作用机制。方法 PAN刺激体外培养的足细胞24h,建立足细胞损伤模型。采用CCK-8方法检测不同浓度普罗布考作用下PAN损伤足细胞的细胞存活率。将足细胞随机分为正常组、PAN组(PAN 50mg/L)、普罗布考组(PAN 50mg/L+普罗布考40μmol/L),予以相应药物刺激24h。采用荧光酶标仪检测DCFH-DA荧光探针标记的足细胞中活性氧(ROS)水平的变化。采用荧光显微镜检测DCFH-DA荧光探针标记的足细胞荧光强度的变化。采用Western-blot方法检测各组足细胞mTOR蛋白表达水平。结果 CCK-8方法检测结果显示,PAN造成足细胞损伤后加入浓度为20、40、100μmol/L普罗布考均使足细胞活性上升,与0μmol/L普罗布考组比较,差异有显著性(F=1 959.00,P<0.05),其浓度为40μmol/L时作用最明显。荧光显微镜检测结果显示,PAN组足细胞内荧光强度增强。DCF荧光强度检测结果显示,PAN组细胞内ROS生成明显升高,与正常组比较,差异有显著性(F=33 525.19,P<0.05);普罗布考组ROS生成明显减少,与PAN组比较,差异有显著性(P<0.05)。Western-blot方法检测结果显示,PAN可使mTOR表达水平明显升高,与正常组比较,差异有显著性(F=179.55,P<0.05);普罗布考40μmol/L共处理细胞后,mTOR表达水平下降,与PAN组比较,差异有显著性(P<0.05)。结论普罗布考可通过下调PAN导致的mTOR激活,降低ROS生成,减轻氧化应激水平,从而抑制足细胞损伤。
Objective To evaluate the effect of probucol on level of roTOR and oxidative stress in mammals of puromycin aminonucleoside （PAN）-induced damage models and explore the mechanism of probucol in the protection of renal function. Methods Podocytes cultured in vitro were stimulated with PAN for 24 h to create a podocyte-damage model. The survival rate of PAN-damaged podocytes, under stimulation of different concentrations of probucol, was detected using CCK8 assay. The podocytes were then divided into normal group, PAN group （PAN 50 mg/L） and probucol group （PAN 50 mg/L＋ probucol 40μmol/L） and given corresponding stimulation for 24 h. ROS levels in cells labeled by DCFH-DA were measured using a spectrofluorometer. The changes of fluorescence intensity in the podocytes were determined using DCFH-DA. The expression of mTOR was measured applying Western-blot. Results The CCK-8 test results showed that the activity of PAN-injured podocytes increased after 20, 40, and 100 μmol/L probucol were added, compared with 0 μmol/L-probucol group, the difference was significant （F = 1 959.00,P〈 0.05）, the most obvious effect was noted when the concentration was 40 μmol/L. Fluorescence microscope detection results indica- ted the fluorescence intensity in podocytes of PAN group enhanced. DCF fluorescence intensity test indicated that, in PAN group, the formation of ROS was significantly increased, the difference was significant versus normal control group （F = 33 525.19, P 0.05）. In probucol group, the formation of ROS decreased, the difference was significant as compared with PAN group （P〈0.05）. Western-blot suggested that： PAN could notably increase the expression of mTOR, the difference was significant versus normal control （F = 179.55, P 〈 0.05）, after treated with 40 μmol/L of probucol, the expression of mTOR significantly decreased, compared with PAN group the difference was significant （P〈0.05）. Conclusion Probucol can protect podocytes through down regulation of mTOR activated
作者
刘丽秋
傅文
韩润鸿
王淑娟
Liqiu FU Wen HAN Runhong WANG Shujuan(The Affiliated Hospital of Qingdao University, Qingdao 266003, Chin)
出处
《青岛大学医学院学报》
CAS
2016年第6期651-654,共4页
Acta Academiae Medicinae Qingdao Universitatis
基金
青岛市科技局课题(11-2-3-1-(2)-nsh)
