介孔二氧化硅纳米颗粒经氧化应激所致肾细胞毒性及GSK-3β在其中的作用

Mesoporous Silica Nanoparticles-Induced Oxidative Stress Involved with GSK-3β Caused Renal Cytotoxicity

研究氧化应激及GSK-3β在介孔二氧化硅纳米颗粒(MSNs)诱导肾细胞毒性中的作用及机制,以及抗氧化剂N-乙酰半胱氨酸(NAC)在此毒性中的保护作用。选用NRK-52E大鼠的肾小管上皮细胞,将其直接或用1μmol/L NAC预处理,然后暴露于400μg/mL MSNs中。采用MTT法测定细胞活力,采用JC-1荧光染色法检测线粒体膜电势,采用western blot法检测GSK-3β等蛋白水平,并测定抗氧化物SOD、GSH和CAT活性。NRK-52E细胞暴露于MSNs内24 h即出现严重的细胞毒性,其IC_(50)为(438.6±7.1)μg/mL。400μg/mL MSNs作用24 h后,细胞的存活率下降到47.57%±2.03%,胞内SOD、GSH、CAT活性水平分别下降到39.74%±2.23%、51.42%±3.08%、46.05%±3.71%(P<0.001)。400μg/mL MSNs还可显著活化GSK-3β,崩解线粒体ΔΨ_m,促进线粒体Cyt C漏出,并剪切激活Caspase-3,引起细胞死亡(P<0.001);1μmol/L NAC干预后可显著缓解400μg/mL MSNs引起的这些变化(P<0.01)。MSNs通过激活GSK-3β信号通路来诱导肾细胞氧化应激损伤,NAC可以改善线粒体功能,提高细胞抗氧化能力,减轻此损伤。

The purpose of the research is to investigate the roles of oxidative stress and GSK-3β in mesoporous silica nanoparticles（MSNs）-induced nephrotoxicity, and the potential protective effects of N-acetylcysteine（NAC）. The NRK-52 E cells were exposed to 400 μg/mL MSNs, or were pre-treated with 1 μmol/L NAC followed by MSNs. After the treatments, the viability of NKR-52 E cells was determined using a 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide（MTT） assay. The fluorescent probe JC-1 was used to determine the mitochondrial membrane potential（ΔΨm）. The levels of GSK-3β related proteins were measured by using western blot. And the activities of superoxide dismutase（SOD）, glutathione（GSH） and catalase（CAT） were detected to evaluate the antioxidant effects. After 24 h of exposure, MSNs produced severe cytotoxicity in the NRK-52 E cells with an IC（50） value of（438.6±7.1） μg/mL. After treatment with 400 μg/mL MSNs for 24 h, the rate of NRK-52 E cell viability was significantly decreased to 47.57%±2.03%, and the activities of SOD, GSH, CAT were respectively decreased to 39.74%±2.23%、51.42%±3.08%、46.05%±3.71%（P〈0.001）. 400 μg/mL MSNs also significantly activated the GSK-3β pathway and subsequently triggered cell death by depolarizing the ΔΨm, which opened the mitochondrial permeability transition pores, released cytochrome c（Cyt C） and, ultimately, activated caspase-3（P〈0.001）. And the 400 μg/mL MSNs-induced significantly toxic injuries of NRK-52 E cells could be attenuated by the pretreatment of NAC（P〈0.01）. MSNs induced renal cytotoxicity via oxidative stress, which was associated with up-regulation GSK-3β activation. NAC can attenuate mitochondrial dysfunction, enhance the antioxidative ability of renal cells and prevent oxidative stress injury induced by MSNs.