ABSTRACT

Mesenchymal stromal cells (MSCs) have received attention as promising therapeutic agents for the treatment of various diseases. However, poor post-transplantation viability is a major hurdle in MSC-based therapy, despite encouraging results in many inflammatory disorders. Recently, three dimensional (3D)-cultured MSCs (MSC_3D) were shown to have higher cell survival and enhanced anti-inflammatory effects, although the underlying mechanisms have not yet been elucidated. In this study, we investigated the molecular mechanisms by which MSC_3D gain the potential for enhanced cell viability. Herein, we found that macroautophagy/autophagy was highly induced and ROS production was suppressed in MSC_3D as compared to 2D-cultured MSCs (MSC_2D). Interestingly, inhibition of autophagy induction caused decreased cell viability and increased apoptotic activity in MSC_3D. Furthermore, modulation of ROS production was closely related to the survival and apoptosis of MSC_3D. We also observed that HMOX1 (heme oxygenase 1) was significantly up-regulated in MSC_3D. In addition, gene silencing of HMOX1 caused upregulation of ROS production and suppression of the genes related to autophagy. Moreover, inhibition of HIF1A (hypoxia inducible factor 1 subunit alpha) caused suppression of HMOX1 expression in MSC_3D, indicating that the HIF1A-HMOX1 axis plays a crucial role in the modulation of ROS production and autophagy induction in MSC_3D. Finally, the critical role of autophagy induction on improved therapeutic effects of MSC_3D was further verified in dextran sulfate sodium (DSS)-induced murine colitis. Taken together, these results indicated that autophagy activation and modulation of ROS production mediated via the HIF1A-HMOX1 axis play pivotal roles in enhancing the viability of MSC_3D.

Abbreviations: 3D: three dimensional; 3MA: 3 methlyadenine; AMPK: AMP-activated protein kinase; Baf A₁: bafilomycin A₁; CFSE: carboxyfluorescein succinimidyl ester; CoCl₂: cobalt chloride; CoPP: cobalt protoporphyrin; DSS: dextran sulfate sodium; ECM: extracellular matrix; FOXO3/FOXO3A: forkhead box O3; HIF1A: hypoxia inducible factor 1 subunit alpha; HMOX1/HO-1: heme oxygenase 1; HSCs: hematopoietic stem cells; IL1A/IL-1α: interleukin 1 alpha; IL1B/IL-1β: interleukin 1 beta; IL8: interleukin 8; KEAP1: kelch like ECH associated protein 1; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; MSC_2D: 2D-cultured MSCs; MSC_3D: 3D-cultured MSCs; MSCs: mesenchymal stromal cells; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; PGE2: prostaglandin E2; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PINK1: PTEN induced kinase 1; ROS: reactive oxygen species; siRNA: small interfering RNA; SIRT1: sirtuin 1; SOD2: superoxide dismutase 2; SQSTM1/p62: sequestosome 1; TGFB/TGF-β: transforming growth factor beta.

摘要

间充质基质细胞（MSCs）作为治疗各种疾病的有前途的治疗剂而受到关注。然而，尽管在许多炎症性疾病中取得了令人鼓舞的结果，但移植后活力差是基于 MSC 治疗的主要障碍。最近，三维 (3D) 培养的 MSCs (MSC _3D ) 被证明具有更高的细胞存活率和增强的抗炎作用，尽管其潜在机制尚未阐明。_{在这项研究中，我们研究了 MSC 3D}获得增强细胞活力潜力的分子机制。在此，我们发现与_2D培养的 MSC（MSC _2D）。有趣的是，自噬诱导的抑制导致 MSC _3D中细胞活力降低和凋亡活性增加。_{此外，ROS 产生的调节与 MSC 3D}的存活和凋亡密切相关。_{我们还观察到 HMOX1（血红素加氧酶 1）在 MSC 3D}中显着上调。此外，HMOX1 的基因沉默导致 ROS 产生的上调和与自噬相关的基因的抑制。此外，HIF1A（缺氧诱导因子 1 亚基 α）的抑制导致 MSC _3D中 HMOX1 表达的抑制，表明 HIF1A-HMOX1 轴在调节 MSC _{3D中 ROS 产生和自噬诱导中起关键作用}. 最后，在硫酸葡聚糖钠 (DSS) 诱导的小鼠结肠炎中进一步验证了自噬诱导对改善 MSC _{3D治疗效果的关键作用。}总之，这些结果表明，通过HIF1A-HMOX1 轴介导的自噬激活和 ROS 产生的调节在增强 MSC _3D的活力中起关键作用。

缩写： 3D：三维；3MA：3甲基腺嘌呤；AMPK：AMP激活的蛋白激酶；Baf A ₁：巴弗洛霉素 A ₁；CFSE：羧基荧光素琥珀酰亚胺酯；CoCl ₂：氯化钴；CoPP：钴原卟啉；DSS：硫酸葡聚糖钠；ECM：细胞外基质；FOXO3/FOXO3A：叉头箱O3；HIF1A：缺氧诱导因子1亚基α；HMOX1/HO-1：血红素加氧酶 1；HSCs：造血干细胞；IL1A/IL-1α：白细胞介素 1 α；IL1B/IL-1β：白细胞介素 1 β；IL8：白细胞介素 8；KEAP1：kelch 样 ECH 相关蛋白 1；LAMP1：溶酶体相关膜蛋白 1；LAMP2：溶酶体相关膜蛋白 2；MSC _2D：二维培养的 MSC；MSC _3D: 3D 培养的 MSC；MSCs：间充质基质细胞；NFE2L2/NRF2：核因子，红系2样2；PGE2：前列腺素 E2；PIK3C3/VPS34：磷脂酰肌醇 3-激酶催化亚基 3 型；PINK1：PTEN 诱导激酶 1；ROS：活性氧；siRNA：小干扰RNA；SIRT1：sirtuin 1；SOD2：超氧化物歧化酶2；SQSTM1/p62：隔离体 1；TGFB/TGF-β：转化生长因子β。