Key Laboratory of Laboratory Medicine of Jiangsu Province, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, People's Republic of China.
Key Laboratory of Laboratory Medicine of Jiangsu Province, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, People's Republic of China.
Cytotherapy. 2018 Jan;20(1):29-44. doi: 10.1016/j.jcyt.2017.08.002. Epub 2017 Sep 21.
On the basis of previous studies, exosomes secreted by human umbilical cord mesenchymal stromal cell (hucMSC-ex) could prevent and repair acute kidney injury induced by cisplatin in rats. However, its potential mechanism is still unclear. In the present study, the model with hucMSC-ex pretreated human renal tubular epithelial cell lines HK-2 that could prevent the injury of cisplatin was successfully established.
First, we pretreated the HK-2 cells with hucMSC-ex for 24 h. Cisplatin was then used to injure HK-2 cells. Gain and loss of function study were used to explore the role of 14-3-3ζ. The expression level of proliferating cell nuclear antigen (PCNA) was analyzed by immunofluorescence assay and Western blot. The number of apoptotic cells was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and flow cytometry analysis. The formation of autophagosomes was observed under super-resolution optical microscope. Western blot was used to analyze the expression levels of LC3B, P62, 14-3-3ζ and Bax.
Pretreating cells with hucMSC-ex could prevent the injury of cisplatin by reducing the number of apoptotic cells and increasing the expression level of PCNA. Simultaneously, the autophagic level was up-regulated. The application of autophagic inhibitor 3-methyladenine (3-MA) could reverse the protective effect of hucMSC-ex. The overexpression of 14-3-3ζ enhanced the autophagic level and protected the injury of cisplatin. The knock-down of 14-3-3ζ could reduce the autophagic level and enhance the disadvantage of cisplatin. The enhanced injury of cisplatin was reversed when the knock-down of 14-3-3ζ was replenished with hucMSC-ex.
14-3-3ζ transported by hucMSC-ex may up-regulate autophagic level in HK-2 cells, which can prevent the injury of cisplatin. This discovery provides the new theoretical basis for the prevention of cisplatin-induced nephrotoxicity by hucMSC-ex.
基于先前的研究，人脐带间充质基质细胞（hucMSC-ex）分泌的外泌体可预防顺铂诱导的大鼠急性肾损伤，并具有修复作用。然而，其潜在机制尚不清楚。在本研究中，成功建立了 hucMSC-ex 预处理人肾小管上皮细胞系 HK-2 的模型，该模型可预防顺铂造成的损伤。
首先，我们用 hucMSC-ex 预处理 HK-2 细胞 24 h，然后用顺铂损伤 HK-2 细胞。通过获得和丧失功能研究来探索 14-3-3ζ 的作用。通过免疫荧光法和 Western blot 分析增殖细胞核抗原（PCNA）的表达水平。通过末端脱氧核苷酸转移酶 dUTP 缺口末端标记法和流式细胞术分析检测凋亡细胞数量。在超分辨率光学显微镜下观察自噬体的形成。Western blot 用于分析 LC3B、P62、14-3-3ζ 和 Bax 的表达水平。
用 hucMSC-ex 预处理细胞可通过减少凋亡细胞数量和增加 PCNA 的表达水平来预防顺铂造成的损伤。同时，自噬水平上调。自噬抑制剂 3-甲基腺嘌呤（3-MA）的应用可逆转 hucMSC-ex 的保护作用。14-3-3ζ 的过表达增强了自噬水平并保护了顺铂造成的损伤。敲低 14-3-3ζ 可降低自噬水平并加重顺铂的不利影响。当用 hucMSC-ex 补充敲低 14-3-3ζ 时，可逆转顺铂造成的损伤加重。
hucMSC-ex 转运的 14-3-3ζ 可能会上调 HK-2 细胞中的自噬水平，从而预防顺铂造成的损伤。这一发现为 hucMSC-ex 预防顺铂诱导的肾毒性提供了新的理论依据。
