二氮嗪对新生儿窒息后血清诱导人近曲肾小管上皮细胞损伤时线粒体凋亡途径的影响

摘要

目的 研究二氮嗪对新生儿窒息后血清诱导人近曲肾小管上皮细胞(HK-2)损伤时线粒体凋亡途径的影响.方法 取培养好的HK-2细胞,接种于6孔板中,分为对照组、窒息组、二氮嗪组.以体积分数为20%的新生儿窒息后24 h血清作为攻击血清;以终浓度100 mol/L的二氮嗪进行干预.采用免疫组化法检测细胞内天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)的表达;激光共聚焦显微镜下观察间接免疫荧光双染色Omi/HtrA2在细胞内的转位和线粒体膜电位的变化.结果 与对照组相比,窒息组HK-2细胞内caspase-3表达的吸光度(A)值明显增加(25.19±3.33比13.63±1.89,P<0.01),Omi/HtrA2转位率明显升高[(56.01±5.30)%比(37.59±5.60)%,P<0.01],线粒体膜红/绿荧光强度比值明显降低(0.79±1.42比1.82±0.23,P<0.01);与窒息组相比,二氮嗪组HK-2细胞caspase-3表达的A值(20.17±2.19)明显降低,Omi/HtrA2转位率[(46.91±2.70)%]明显降低,线粒体膜红/绿荧光强度比值(1.47±0.14)明显增加,但均未恢复至对照组水平(均P<0.01).结论 二氮嗪通过减少Omi/HtrA2在胞内转位、减少caspase-3表达、稳定线粒体膜电位,从而明显减轻新生儿窒息后血清诱导的HK-2细胞损伤.%Objective To study the influence of diazoxide on mitochondrial apoptosis pathway of human renal proximal tubular cells (HK-2 cells). Methods Cultured HK-2 cells were inoculated on 6-well plates, according to stochastic tables law, and they were divided into normal serum-treated group (NSTG), postasphyxial serum treatment group (PSTG), and postasphyxial serum and diazoxide treatment group (PSDTG). The serum from neonates 24 hours after asphyxia in a dilution of 20% (volume fraction) was used for challenge. Diazoxide in a final concentration of 100 mol/L, was used for intervention. The expression of caspase-3 was detected by immunohistochemical method. The translocation rate of Omi/HtrA2 and mitochondria membrane potential were determined by indirect immunofluorescence and confocal microscopy. Results Compared with that of NSTG, the expression of caspase-3 absorbance (A) value of HK-2 cells in PSTG was significantly increased (25.19±3.33 vs. 13.63±1.89, P<0.01), the translocation rate of Omi/HtrA2 of HK-2 cells in PSTG was significantly increased [(56.01±5.30)% vs. (37.59± 5.60)%, P<0.01], mitochondrial membrane red/green fluorescence intensity ratio was decreased significantly (0.79±1.42 vs. 1.82±0.23, P<0.01). Compared with the PSTG, the expression value of caspase-3 of HK-2 cells in PSDTG was significantly decreased (20.17±2.19), the translocation rate of Omi/HtrA2 of HK-2 cells in PSDTG was significantly decreased [(46.91±2.70)%], and mitochondrial membrane red/green fluorescence intensity ratio increased significantly (1.47±0.14), but did not recover to the same degree as that of the NSTG (all P<0.01). Conclusion The diazoxide may reduce the expression of caspase-3, intracellular translocation of Omi/HtrA2, and stability of mitochondrial membrane potential, thereby significantly alleviates HK-2 cells injury induced by postasphyxial-serum of neonate.