诱导性基因敲除导致的角膜水肿是由线粒体超氧阴离子诱导的Src 激酶激活引起的。

Corneal Edema in Inducible Knockout Is Initiated by Mitochondrial Superoxide Induced Src Kinase Activation.

机构信息

Vision Science Program, School of Optometry, Indiana University, Bloomington, IN 47405, USA.

出版信息

Cells. 2023 Jun 1;12(11):1528. doi: 10.3390/cells12111528.

DOI:10.3390/cells12111528

PMID:37296649

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10253072/

Abstract

PURPOSE

Inducible KO leads to corneal edema by disruption of the pump and barrier functions of the corneal endothelium (CE). The loss of Slc4a11 NH-activated mitochondrial uncoupling leads to mitochondrial membrane potential hyperpolarization-induced oxidative stress. The goal of this study was to investigate the link between oxidative stress and the failure of pump and barrier functions and to test different approaches to revert the process.

METHODS

Mice which were homozygous for Slc4a11 Flox and Estrogen receptor -Cre Recombinase fusion protein alleles at 8 weeks of age were fed Tamoxifen (Tm)-enriched chow (0.4 g/Kg) for 2 weeks, and controls were fed normal chow. During the initial 14 days, Slc4a11 expression, corneal thickness (CT), stromal [lactate], Na-K ATPase activity, mitochondrial superoxide levels, expression of lactate transporters, and activity of key kinases were assessed. In addition, barrier function was assessed by fluorescein permeability, ZO-1 tight junction integrity, and cortical cytoskeleton F-actin morphology.

RESULTS

Tm induced a rapid decay in Slc4a11 expression that was 84% complete at 7 days and 96% complete at 14 days of treatment. Superoxide levels increased significantly by day 7; CT and fluorescein permeability by day 14. Tight junction ZO-1 distribution and the cortical cytoskeleton were disrupted at day 14, concomitant with decreased expression of Cldn1, yet with increased tyrosine phosphorylation. Stromal lactate increased by 60%, Na-K ATPase activity decreased by 40%, and expression of lactate transporters MCT2 and MCT4 significantly decreased, but MCT1 was unchanged at 14 days. Src kinase was activated, but not Rock, PKCα, JNK, or P38Mapk. Mitochondrial antioxidant Visomitin (SkQ1, mitochondrial targeted antioxidant) and Src kinase inhibitor eCF506 significantly slowed the increase in CT, with concomitant decreased stromal lactate retention, improved barrier function, reduced Src activation and Cldn1 phosphorylation, and rescued MCT2 and MCT4 expression.

CONCLUSIONS

Slc4a11 KO-induced CE oxidative stress triggered increased Src kinase activity that resulted in perturbation of the pump components and barrier function of the CE.

摘要

目的

诱导型 KO 导致角膜内皮（CE）的泵和屏障功能障碍，从而引起角膜水肿。Slc4a11 NH 激活的线粒体解偶联导致的线粒体膜电位超极化诱导氧化应激。本研究的目的是研究氧化应激与泵和屏障功能障碍之间的联系，并测试不同的方法来逆转这一过程。

方法

8 周龄时，Slc4a11 基因纯合子 Flox 和雌激素受体-Cre 重组酶融合蛋白等位基因的小鼠用含有 Tamoxifen（Tm）的饲料（0.4 g/Kg）喂养 2 周，对照组则用普通饲料喂养。在最初的 14 天内，评估 Slc4a11 表达、角膜厚度（CT）、基质[乳酸]、Na-K ATP 酶活性、线粒体超氧化物水平、乳酸转运蛋白的表达和关键激酶的活性。此外，通过荧光素通透性、ZO-1 紧密连接完整性和皮质细胞骨架 F-肌动蛋白形态评估屏障功能。

结果

Tm 诱导 Slc4a11 表达迅速下降，7 天内下降 84%，14 天内下降 96%。超氧化物水平在第 7 天显著增加；CT 和荧光素通透性在第 14 天增加。紧密连接 ZO-1 分布和皮质细胞骨架在第 14 天被破坏，同时 Claudin1 的表达减少，但酪氨酸磷酸化增加。基质乳酸增加 60%，Na-K ATP 酶活性下降 40%，乳酸转运蛋白 MCT2 和 MCT4 的表达显著下降，但 MCT1 在第 14 天没有变化。Src 激酶被激活，但 Rock、PKCα、JNK 或 P38Mapk 没有被激活。线粒体抗氧化剂 Visomitin（SkQ1，靶向线粒体的抗氧化剂）和 Src 激酶抑制剂 eCF506 显著减缓 CT 的增加，同时减少基质乳酸的保留，改善屏障功能，减少 Src 激活和 Claudin1 磷酸化，并挽救 MCT2 和 MCT4 的表达。