Institute of Burn Research, State Key Laboratory of Trauma, Burn and Combined Injury, Key Laboratory of Proteomics of Chongqing, Southwest Hospital, The Third Military Medical University (Army Medical University), Chongqing 400038, China; School of Nursing, The Third Military Medical University (Army Medical University), Chongqing 400038, China.
Department of Plastic and Reconstructive Surgery, Southwestern Hospital, The Third Military Medical University (Army Medical University), Chongqing 400038, China.
Nitric Oxide. 2018 Aug 1;78:1-10. doi: 10.1016/j.niox.2018.04.001. Epub 2018 Apr 23.
Nitric oxide (NO) has emerged as a critical molecule in wound healing, but the mechanism underlying its activity is not well defined. Here, we explored the effect of NO on the de-adhesion of epidermal stem cells (ESCs) and the mechanism involved in this process.
The effects of NO on isolated human and mouse ESCs cultured in the presence of different concentrations of the NO donor S-nitroso-N-acetyl penicillamine (SNAP) were evaluated in cell de-adhesion assays mediated by integrin β and collagen IV. Subsequently, changes in the expression of integrin β1 and the phosphorylation of Talin in response to different doses of SNAP were detected by Western blot analysis and real-time PCR in vitro. Furthermore, the roles of various soluble guanylyl cyclase (sGC)- and protein kinase G (PKG)-specific inhibitors and agonists in the effects of NO on ESC de-adhesion, integrin β1 expression and Talin phosphorylation were analysed. Moreover, the effects of NO on integrin β1 expression and sGC/cGMP/PKG signalling-mediated wound healing were detected in vivo using 5-bromo-2-deoxyuridine (BrdU) label-retaining cells (LRCs) in a scald model and an excision wound healing model, respectively.
SNAP promoted primary human and mouse ESC de-adhesion in a concentration-dependent manner in the integrin β1-and collagen IV-mediated adhesion assay, and this effect was suppressed by the sGC and PKG inhibitors. Additionally, integrin β1 expression and Talin phosphorylation at serine 425 (S425) were negatively correlated with SNAP levels, and this effect was blocked by the sGC and PKG inhibitors. Moreover, the roles of NO in integrin β1 expression and cGMP signalling pathway-mediated wound healing were confirmed in vivo.
Our data indicate that the stimulatory effects of NO on ESC de-adhesion related to integrin β1 expression and Talin phosphorylation were mediated by the cGMP signalling pathway, which is likely involved in wound healing.
一氧化氮（NO）已成为伤口愈合过程中的关键分子，但它的作用机制尚未完全明确。本研究旨在探讨 NO 对表皮干细胞（ESCs）去黏附的影响及其作用机制。
采用整合素β和胶原 IV 介导的细胞去黏附实验，评估不同浓度的一氧化氮供体 S-亚硝基-N-乙酰青霉胺（SNAP）对分离培养的人源和鼠源 ESCs 的影响。随后，通过 Western blot 分析和实时 PCR 检测不同剂量 SNAP 作用下整合素β1表达和 Talin 磷酸化的变化。此外，分析了各种可溶性鸟苷酸环化酶（sGC）和蛋白激酶 G（PKG）特异性抑制剂和激动剂在 NO 对 ESC 去黏附、整合素β1表达和 Talin 磷酸化作用中的作用。此外，通过 5-溴-2-脱氧尿嘧啶（BrdU）标记保留细胞（LRC）在烫伤模型和切除伤口愈合模型中，检测 NO 对整合素β1表达和 sGC/cGMP/PKG 信号通路介导的伤口愈合的影响。
SNAP 以浓度依赖性方式促进整合素β1和胶原 IV 介导的黏附实验中，原代人源和鼠源 ESC 的去黏附，而 sGC 和 PKG 抑制剂可抑制这一作用。此外，整合素β1表达和 Talin 在丝氨酸 425（S425）的磷酸化与 SNAP 水平呈负相关，而 sGC 和 PKG 抑制剂可阻断这一作用。此外，体内实验证实了 NO 在整合素β1表达和 cGMP 信号通路介导的伤口愈合中的作用。
本研究数据表明，NO 对 ESC 去黏附的刺激作用与整合素β1表达和 Talin 磷酸化有关，是通过 cGMP 信号通路介导的，可能参与了伤口愈合过程。
