Umetsu Araya, Ida Yosuke, Sato Tatsuya, Watanabe Megumi, Tsugeno Yuri, Furuhashi Masato, Hikage Fumihito, Ohguro Hiroshi
Department of Ophthalmology, School of Medicine, Sapporo Medical University, Sapporo 060-8556, Japan.
Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University, Sapporo 060-8556, Japan.
Bioengineering (Basel). 2022 Jul 19;9(7):327. doi: 10.3390/bioengineering9070327.
The additive effects of an α2-adrenergic agonist, brimonidine (BRI), on the pan-ROCK inhibitor (ROCK-i), ripasudil (Rip), and the ROCK2-I, KD025, on adipogenic differentiation (DIF+) were examined using two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells. The following analyses were carried out: (1) lipid staining (2D and 3D), (2) real-time measurements of cellular metabolism (2D), (3) mRNA expression of DIF+ related genes and extracellular matrix molecules (ECMs) including collagen (Col)-1, -4, and -6, and fibronectin (Fn), and (4) the sizes and physical properties of the 3D spheroids. The findings indicate that DIF+ induced (1) a substantial enhancement in lipid staining and enhanced expression of the Pparγ and Fabp4 genes, (2) significantly larger and softer 3D spheroids, and (3) down-regulation of Col1 and Fn and up-regulation of Col4 and Col6 genes. Treatment with Rip alone caused a significant enhancement in adipogenesis of both the 2D and 3D cultured 3T3-L1 cells and in the physical properties of the 3D spheroids; these effects were substantially inhibited by BRI, and the effects induced by BRI or KD025 were not insignificant. These collective findings indicate that the addition of BRI inhibited the Rip-induced enhancement of DIF+ in 3T3-L1 cells, presumably by modulating ROCK1 signaling.
使用3T3-L1细胞的二维或三维（2D或3D）培养，研究了α2肾上腺素能激动剂溴莫尼定（BRI）对泛ROCK抑制剂（ROCK-i）瑞巴派特（Rip）以及ROCK2抑制剂KD025在脂肪生成分化（DIF+）方面的相加作用。进行了以下分析：（1）脂质染色（2D和3D），（2）细胞代谢的实时测量（2D），（3）DIF+相关基因和细胞外基质分子（ECM）的mRNA表达，包括胶原蛋白（Col）-1、-4和-6以及纤连蛋白（Fn），以及（4）3D球体的大小和物理性质。研究结果表明，DIF+诱导（1）脂质染色显著增强以及Pparγ和Fabp4基因表达增加，（2）3D球体明显更大且更软，以及（3）Col1和Fn基因下调，Col4和Col6基因上调。单独使用Rip处理导致2D和3D培养的3T3-L1细胞的脂肪生成以及3D球体的物理性质显著增强；这些作用被BRI显著抑制，并且BRI或KD025诱导的作用并非不显著。这些共同发现表明，添加BRI抑制了Rip诱导的3T3-L1细胞中DIF+的增强，可能是通过调节ROCK1信号传导实现的。
