GSK-3β在介孔二氧化硅纳米粒致肾纤维化中的作用及机制研究

Mesoporous silica nanoparticles (MSNs) are a kind of new drug carrier system, formed through the interaction of inorganic precursor and organic surfactant, with the pore size and spacing in nanoscale (2-50 nm). Mesoporous silica nanomaterials shows great potential applications in biological engineering, but the potential toxicity has limited its applications. The mechanism of MSNs-induced nephrotoxicity has not been fully elucidated. Our previous studies demonstrated that MSNs exerted toxic action on mice tissues (2013,2014), and induced nephrotoxicity through the NF-κB inflammatory signalling pathway (2015, 2016). Recent results also indicated that MSNs could induce oxidative stress and GSK-3β activation in the NRK-52E cells and BALB/c mice, and pretreated with antioxidant or si-RNA-GSK-3β could significantly attenuate the MSNs-induced cell injury. Based on these research fruits, the function of mitochondrial, accumulation of ROS, the level of inflammation and fibrosis in NRK-52E cells and mice kidney will be detected as the biomarkers, which will be used to investigate the interaction between GSK-3β and mPTP protein or NF-κB, and the effects of GSK-3β-/- gene knockout on MSNs-induced nephrotoxicity. In addition, the genetic modification will be used to silence or amplify GSK-3β in NRK-52E cells, and an inhibitor or agonist of GSK-3β will also be used to change the level of GSK-3β. These experiments will be used to illustrate that MSNs induces nephrotoxicity by activating GSK-3β, while inhibiting the activity of GSK-3β can alleviate MSNs-induced oxidative stress, inflammation, fibrosis, and attenuate the nephrotoxicity. The research will clarify the importance and mechanisms of GSK-3β in MSNs-induced kidney injury, and provide a rational basis for seeking antidotes to MSNs-induced nephrotoxicity in clinical application.

介孔二氧化硅纳米粒(MSNs)具有经济方便、载药量大等优势，作为药用载体材料在生物医学工程中有突出的应用前景，然其潜在毒性已成为限制其应用的主要原因。目前MSNs肾毒性机制尚未彻底阐明。在我们前期工作观察到MSNs能引起动物肾组织出现炎症及纤维化等，及近期预实验发现MSNs能诱导肾细胞氧化应激、活化GSK-3β，预先使用抗氧化剂或干扰GSK-3β能提高肾细胞存活率的基础上，本项目拟在体内外，以线粒体、氧化应激、炎症及纤维化为指标，研究GSK-3β与线粒体膜孔蛋白、NF-κB间的相互作用，及GSK-3β敲除后对MSNs肾毒性的影响；并通过干扰或过表达GSK-3β，及将GSK-3β抑制剂或激动剂与MSNs联用，来论证MSNs肾毒性与GSK-3β活化相关，抑制此活化能对抗氧化应激，缓解炎症和纤维化，减轻肾毒性之假说。本项目将深度解析GSK-3β在MSNs肾毒性中的作用机制，为其临床减毒提供依据。

介孔二氧化硅纳米粒(MSNs)具有经济方便、载药量大等优势，作为药物递送系统在生物医学工程中有突出的应用前景，然其潜在毒性已成为限制其临床应用的主要原因。尽管有多项研究报道MSNs可引起肾脏毒性，但是此肾毒性机制尚未彻底阐明。本研究从细胞和动物水平，探讨GSK-3β、FOXO3a、NF-κB信号通路在MSNs所致肾毒性中的作用。结果显示，在肾脏组织与细胞内，MSNs可以抑制SOD、GSH、CAT活性，引起ROS堆积，崩解线粒体电势ΔΨm，促进炎症因子TNF-α、IL-1β、IL-6释放，造成氧化应激损伤、炎症反应，诱导细胞凋亡而降低NRK-52E细胞存活率，引起小鼠肾组织病变。这可能与MSNs激活GSK-3β、抑制FOXO3a相关。活化的GSK-3β可诱导肾脏细胞线粒体膜孔开放，促进线粒体Cyt C漏出并剪切激活Caspase-3，引起细胞凋亡；还可促进NF-κB转位入核，诱导其下游靶蛋白TNF-α、IL-1β、IL-6的表达，增加FN、TGF-β含量，引起炎症反应与纤维化。预先使用抗氧化剂NAC抑制GSK-3β活性或沉默GSK-3β后，均可显著缓解MSNs引起的上述毒性反应。此外，预先使用芹菜素，可提高被MSNs所抑制的FOXO3a活性。芹菜素通过激活FOXO3a，促进其转位入核，既可诱导其下游靶蛋白SOD与CAT表达，还可破坏NF-κB与其相应的DNA位点结合，抑制NF-κB的转录作用。而在沉默FOXO3a后，芹菜素对MSNs所致肾毒性的保护作用被显著减弱。本项目的研究结果从新的角度揭示了MSNs引起肾毒性的分子机制，为临床MSNs肾毒性的防治提供了新的思路与靶点。