AMPK/MFF Activation: Role in Mitochondrial Fission and Mitophagy in Dry Eye.
AMPK/MFF Activation: Role in Mitochondrial Fission and Mitophagy in Dry Eye.
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AMPK/MFF激活：在干眼线粒体分裂和线粒体自噬中的作用。
DOI：
10.1167/iovs.63.12.18
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发表时间：
2022-11-01
影响因子：
4.4
通讯作者：
中科院分区：
文献类型：
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作者：
探讨线粒体形态和一磷酸腺苷活化蛋白激酶(AMPK)/线粒体分裂因子(MFF)在干眼症发病中的作用及其机制。永生化人角膜上皮细胞和原代角膜上皮细胞在高渗透压(HOP)下培养。C57BL/6雌性小鼠皮下注射东莨菪碱。用实时荧光定量聚合酶链式反应检测mRNA的表达。免疫印迹和免疫荧光染色检测蛋白表达。用激光共聚焦显微镜和透射电子显微镜观察线粒体形态。首先，HOP诱导HCECs线粒体氧化损伤，并伴有线粒体分裂和吞丝体增加。随后，由于HOP诱导的能量代谢功能障碍，AMPK/MFF通路蛋白表达增加。有趣的是，在HOP组中，AMPK通路通过增加动力蛋白相关蛋白1(Drp1)向线粒体外膜的募集来促进线粒体的分裂和有丝分裂。此外，AMPK基因敲除减弱了HOP引起的线粒体分裂和有丝分裂。AMPK激活引发线粒体分裂和有丝分裂。HOP应激时HCECs的线粒体分裂是通过MFF磷酸化介导的。MFf基因敲除可逆转HOP处理的人脐静脉内皮细胞线粒体碎裂和有丝分裂。在HOP存在的情况下，抑制MFF可保护内皮细胞免受氧化损伤、细胞死亡和炎症。最后，我们在体内检测了线粒体的分裂和AMPK通路的激活。AMPK/MFF通路通过正向调节线粒体分裂和有丝分裂，参与干眼症的发生。抑制线粒体分裂可以减轻干眼的氧化损伤和炎症反应，为干眼的治疗提供实验依据。
To assess the role of mitochondrial morphology and adenosine monophosphate–activated protein kinase (AMPK)/mitochondrial fission factor (MFF) in dry eye and the underlying mechanisms. Immortalized human corneal epithelial cells (HCECs) and primary HCECs were cultured under high osmotic pressure (HOP). C57BL/6 female mice were injected subcutaneously with scopolamine. Quantitative real-time PCR was used to measure mRNA expression. Protein expression was assessed by western blot and immunofluorescence staining. Mitochondrial morphology was observed by confocal microscopy and transmission electron microscopy. First, HOP induced mitochondrial oxidative damage to HCECs, accompanied by mitochondrial fission and increased mitophagy. Then, AMPK/MFF pathway proteins were increased consequent to HOP-induced energy metabolism dysfunction. Interestingly, the AMPK pathway promoted mitochondrial fission and mitophagy by increasing the recruitment of dynamin-related protein 1 (DRP1) to the mitochondrial outer membrane in the HOP group. Moreover, AMPK knockdown attenuated mitochondrial fission and mitophagy due to HOP in HCECs. AMPK activation triggered mitochondrial fission and mitophagy. Mitochondrial fission of HCECs stressed by HOP was mediated via MFF phosphorylation. MFF knockdown reversed mitochondrial fragmentation and mitophagy in HCECs treated with HOP. Inhibition of MFF protected HCECs against oxidative damage, cell death, and inflammation in the presence of HOP. Finally, we detected mitochondrial fission and AMPK pathway activation in vivo. The AMPK/MFF pathway mediates the development of dry eye by positively regulating mitochondrial fission and mitophagy. Inhibition of mitochondrial fission can alleviate oxidative damage and inflammation in dry eye and may provide experimental evidence for treating dry eye.