Department of Biomedical Sciences, University of North Dakota School of Medicine & Health Sciences, Grand Forks, North Dakota, United States of America.
Department of Surgery, University of North Dakota School of Medicine & Health Sciences, Grand Forks, North Dakota, United States of America.
PLoS One. 2024 Aug 16;19(8):e0304010. doi: 10.1371/journal.pone.0304010. eCollection 2024.
M64HCl, which has drug-like properties, is a water-soluble Focal Adhesion Kinase (FAK) activator that promotes murine mucosal healing after ischemic or NSAID-induced injury. Since M64HCl has a short plasma half-life in vivo (less than two hours), it has been administered as a continuous infusion with osmotic minipumps in previous animal studies. However, the effects of more transient exposure to M64HCl on monolayer wound closure remained unclear. Herein, we compared the effects of shorter M64HCl treatment in vitro to continuous treatment for 24 hours on monolayer wound closure. We then investigated how long FAK activation and downstream ERK1/2 activation persist after two hours of M64HCl treatment in Caco-2 cells. M64HCl concentrations immediately after washing measured by mass spectrometry confirmed that M64HCl had been completely removed from the medium while intracellular concentrations had been reduced by 95%. Three-hour and four-hour M64HCl (100 nM) treatment promoted epithelial sheet migration over 24 hours similar to continuous 24-hour exposure. 100nM M64HCl did not increase cell number. Exposing cells twice with 2-hr exposures of M64HCl during a 24-hour period had a similar effect. Both FAK inhibitor PF-573228 (10 μM) and ERK kinase (MEK) inhibitor PD98059 (20 μM) reduced basal wound closure in the absence of M64HCl, and each completely prevented any stimulation of wound closure by M64HCl. Rho kinase inhibitor Y-27632 (20 μM) stimulated Caco-2 monolayer wound closure but no further increase was seen with M64HCl in the presence of Y-27632. M64HCl (100 nM) treatment for 3 hours stimulated Rho kinase activity. M64HCl decreased F-actin in Caco-2 cells. Furthermore, a two-hour treatment with M64HCl (100 nM) stimulated sustained FAK activation and ERK1/2 activation for up to 16 and hours 24 hours, respectively. These results suggest that transient M64HCl treatment promotes prolonged intestinal epithelial monolayer wound closure by stimulating sustained activation of the FAK/ERK1/2 pathway. Such molecules may be useful to promote gastrointestinal mucosal repair even with a relatively short half-life.
M64HCl 具有类药性，是一种水溶性黏着斑激酶 (FAK) 激活剂，可促进缺血或 NSAID 诱导损伤后的小鼠黏膜愈合。由于 M64HCl 在体内的血浆半衰期较短（不到两小时），因此在以前的动物研究中，它通过渗透压微型泵以持续输注的方式给药。然而，M64HCl 更短暂的暴露对单层伤口闭合的影响仍不清楚。在此，我们比较了体外更短时间的 M64HCl 治疗与持续 24 小时治疗对单层伤口闭合的影响。然后，我们研究了在 Caco-2 细胞中用 M64HCl 处理两小时后，FAK 激活和下游 ERK1/2 激活能持续多长时间。通过质谱法测量的洗涤后立即的 M64HCl 浓度证实，M64HCl 已从培养基中完全去除，而细胞内浓度已降低 95%。三小时和四小时的 100 nM M64HCl（100 nM）治疗促进了上皮片迁移，在 24 小时内与持续 24 小时暴露相似。100 nM M64HCl 不会增加细胞数量。在 24 小时期间用 2 小时的 M64HCl 暴露两次，也有类似的效果。FAK 抑制剂 PF-573228（10 μM）和 ERK 激酶（MEK）抑制剂 PD98059（20 μM）在没有 M64HCl 的情况下均降低了基础伤口闭合，并且每种抑制剂均完全阻止了 M64HCl 对伤口闭合的任何刺激。Rho 激酶抑制剂 Y-27632（20 μM）刺激 Caco-2 单层伤口闭合，但在存在 Y-27632 的情况下，M64HCl 没有进一步增加。M64HCl（100 nM）治疗 3 小时可刺激 Rho 激酶活性。M64HCl 减少 Caco-2 细胞中的 F-肌动蛋白。此外，用 M64HCl（100 nM）处理两小时可刺激 FAK/ERK1/2 通路的持续激活，分别持续长达 16 小时和 24 小时。这些结果表明，短暂的 M64HCl 治疗通过刺激 FAK/ERK1/2 通路的持续激活，促进了肠道上皮单层伤口的长时间闭合。即使半衰期相对较短，此类分子也可能有助于促进胃肠道黏膜修复。
