Wang Bingying, Jia Haoyuan, Zhang Bin, Wang Juanjuan, Ji Cheng, Zhu Xueming, Yan Yongmin, Yin Lei, Yu Jing, Qian Hui, Xu Wenrong
Key Laboratory of Laboratory Medicine of Jiangsu Province, School of Medicine, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu, 212013, People's Republic of China.
The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu, People's Republic of China.
Stem Cell Res Ther. 2017 Apr 8;8(1):75. doi: 10.1186/s13287-016-0463-4.
The administration of cisplatin is limited due to its nephrotoxic side effects, and prevention of this nephrotoxicity of cisplatin is difficult. Mesenchymal stem cell (MSC)-derived exosomes have been implicated as a novel therapeutic approach for tissue injury. In this study, we demonstrated that the pretreatment of human umbilical cord MSC-derived exosomes (hucMSC-Ex) can prevent the development of cisplatin-induced renal toxicity by activation of autophagy in vitro and in vivo.
In vitro, rat renal tubular epithelial (NRK-52E) cells were pre-incubated with exosomes from hucMSC or HFL1 (human lung fibroblast cells; as control) for 30 min, and 3-methyladenine (an autophagic inhibitor) and rapamycin (an autophagic inducer) for 1 h before cisplatin treatment for 8 h, respectively. Cells were harvested for apoptosis assay, enzyme-linked immunosorbent assay (ELISA), Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). In vivo, we constructed cisplatin-induced acute kidney injury rat models. Prior to treatment with cisplatin for 0.5 h, hucMSC-Ex or HFL1-Ex were injected into the kidneys via the renal capsule. 3-methyladenine and rapamycin were injected under the kidney capsule before hucMSC-Ex. All animals were sacrificed at 3 days after cisplatin injection. Renal function, Luminex assay, tubular apoptosis and proliferation, and autophagy response were evaluated.
hucMSC-Ex inhibited cisplatin-induced mitochondrial apoptosis and secretion of inflammatory cytokines in renal tubular epithelial cells in vitro. hucMSC-Ex increased the expression of the autophagic marker protein LC3B and the autophagy-related genes ATG5 and ATG7 in NRK-52E cells. Rapamycin mimicked the effects of hucMSC-Ex in protecting against cisplatin-induced renal injury, while the effects were abrogated by the autophagy inhibitor 3-methyladenine in the animals.
Our findings indicate that the activation of autophagy induced by hucMSC-Ex can effectively relieve the nephrotoxicity of cisplatin. Therefore, pre-treatment of hucMSC-Ex may be a new method to improve the therapeutic effect of cisplatin.
顺铂因其肾毒性副作用而限制了其应用，且预防顺铂的这种肾毒性较为困难。间充质干细胞（MSC）来源的外泌体已被认为是一种治疗组织损伤的新方法。在本研究中，我们证明人脐带间充质干细胞来源的外泌体（hucMSC-Ex）预处理可通过在体外和体内激活自噬来预防顺铂诱导的肾毒性。
在体外，大鼠肾小管上皮（NRK-52E）细胞分别与hucMSC或HFL1（人肺成纤维细胞；作为对照）的外泌体预孵育30分钟，以及在顺铂处理8小时前分别与3-甲基腺嘌呤（一种自噬抑制剂）和雷帕霉素（一种自噬诱导剂）预孵育1小时。收集细胞进行凋亡检测、酶联免疫吸附测定（ELISA）、蛋白质免疫印迹法和定量实时聚合酶链反应（qRT-PCR）。在体内，我们构建了顺铂诱导的急性肾损伤大鼠模型。在顺铂处理0.5小时前，通过肾包膜将hucMSC-Ex或HFL1-Ex注入肾脏。在hucMSC-Ex之前，将3-甲基腺嘌呤和雷帕霉素注入肾包膜下。在顺铂注射后3天处死所有动物。评估肾功能、Luminex检测、肾小管凋亡和增殖以及自噬反应。
hucMSC-Ex在体外抑制了顺铂诱导的肾小管上皮细胞线粒体凋亡和炎性细胞因子分泌。hucMSC-Ex增加了NRK-52E细胞中自噬标记蛋白LC3B以及自噬相关基因ATG5和ATG7的表达。雷帕霉素模拟了hucMSC-Ex对顺铂诱导的肾损伤的保护作用，而在动物中自噬抑制剂3-甲基腺嘌呤消除了这些作用。
我们的研究结果表明，hucMSC-Ex诱导的自噬激活可有效减轻顺铂的肾毒性。因此，hucMSC-Ex预处理可能是提高顺铂治疗效果的一种新方法。
