Department of Cardiovascular Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, 250062, Shandong, China.
Department of Cardiovascular Surgery, Weifang People's Hospital, Weifang, 261000, Shandong, China.
Stem Cell Res Ther. 2020 Dec 9;11(1):526. doi: 10.1186/s13287-020-02041-7.
Bone marrow mesenchymal stem cell (BM-MSC) has been shown to treat pulmonary arterial hypertension (PAH). However, excessive reactive oxygen species (ROS) increases the apoptosis of BM-MSCs, leading to poor survival and engraft efficiency. Thus, improving the ability of BM-MSCs to scavenge ROS may considerably enhance the effectiveness of transplantation therapy. Mammalian Ste20-like kinase 1 (Mst1) is a pro-apoptotic molecule which increases ROS production. The aim of this study is to uncover the underlying mechanisms the effect of Mst1 inhibition on the tolerance of BM-MSCs under HO condition.
Mst1 expression in BM-MSCs was inhibited via transfection with adenoviruses expressing a short hairpin (sh) RNA directed against Mst1 (Ad-sh-Mst1) and exposure to HO. Cell viability was detected by Cell Counting Kit 8 (CCK-8) assay, and cell apoptosis was analyzed by Annexin V-FITC/PI, Caspase 3 Activity Assay kits, and pro caspase 3 expression. ROS level was evaluated by the ROS probe DCFH-DA, mitochondrial membrane potential (ΔΨm) assay, SOD1/2, CAT, and GPx expression. Autophagy was assessed using transmission electron microscopy, stubRFP-sensGFP-LC3 lentivirus, and autophagy-related protein expression. The autophagy/Keap1/Nrf2 signal in HO-treated BM-MSC/sh-Mst1 was also measured.
Mst1 inhibition reduced ROS production; increased antioxidant enzyme SOD1/2, CAT, and GPx expression; maintained ΔΨm; and alleviated cell apoptosis in HO-treated BM-MSCs. In addition, this phenomenon was closely correlated with the autophagy/Keap1/Nrf2 signal pathway. Moreover, the antioxidant pathway Keap1/Nrf2 was also blocked when autophagy was inhibited by the autophagy inhibitor 3-MA. However, Keap1 or Nrf2 knockout via siRNA had no effect on autophagy activation or suppression.
Mst1 inhibition mediated the cytoprotective action of mBM-MSCs against HO-induced oxidative stress injury. The underlying mechanisms involve autophagy activation and the Keap1/Nrf2 signal pathway.
骨髓间充质干细胞（BM-MSC）已被证明可治疗肺动脉高压（PAH）。然而，过量的活性氧（ROS）会增加 BM-MSC 的凋亡，导致其存活率和植入效率差。因此，提高 BM-MSC 清除 ROS 的能力可能会极大地增强移植治疗的效果。哺乳动物 Ste20 样激酶 1（Mst1）是一种促凋亡分子，可增加 ROS 的产生。本研究旨在揭示 Mst1 抑制在低氧（HO）条件下对 BM-MSC 耐受性的影响的潜在机制。
通过转染表达针对 Mst1 的短发夹（sh）RNA 的腺病毒（Ad-sh-Mst1）和暴露于 HO 来抑制 BM-MSC 中的 Mst1 表达。通过细胞计数试剂盒 8（CCK-8）测定法检测细胞活力，并通过 Annexin V-FITC/PI、Caspase 3 活性测定试剂盒和前 Caspase 3 表达分析细胞凋亡。通过 ROS 探针 DCFH-DA、线粒体膜电位（ΔΨm）测定法、SOD1/2、CAT 和 GPx 表达评估 ROS 水平。使用透射电子显微镜、stubRFP-sensGFP-LC3 慢病毒和自噬相关蛋白表达评估自噬。还测量了 HO 处理的 BM-MSC/sh-Mst1 中的自噬/Keap1/Nrf2 信号。
Mst1 抑制减少了 ROS 的产生；增加抗氧化酶 SOD1/2、CAT 和 GPx 的表达；维持了 ΔΨm；并减轻了 HO 处理的 BM-MSCs 中的细胞凋亡。此外，这种现象与自噬/Keap1/Nrf2 信号通路密切相关。此外，当自噬被自噬抑制剂 3-MA 抑制时，抗氧化途径 Keap1/Nrf2 也被阻断。然而，通过 siRNA 敲除 Keap1 或 Nrf2 对自噬的激活或抑制没有影响。
Mst1 抑制介导了 mBM-MSC 对 HO 诱导的氧化应激损伤的细胞保护作用。潜在的机制涉及自噬的激活和 Keap1/Nrf2 信号通路。
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