Antiapoptotic Effect of Acetylcholine in Fas-Induced Apoptosis in Human Keratocytes
Antiapoptotic Effect of Acetylcholine in Fas-Induced Apoptosis in Human Keratocytes
复制标题
DOI：
10.1167/iovs.16-19707
复制
发表时间：
2016-11-01
影响因子：
4.4
通讯作者：
Danielson, Patrik
中科院分区：
文献类型：
--
作者：
Sloniecka, Marta;Backman, Ludvig J.;Danielson, Patrik
目的.研究乙酰胆碱（acetylcholine，ACh）对Fas介导的体外培养人角膜基质细胞凋亡的抑制作用，并探讨其机制。从健康角膜分离原代人角膜细胞。用Fas配体（FasL）诱导角膜基质细胞凋亡。通过ELISA评估细胞死亡。用发光半胱天冬酶活性测定法测量半胱天冬酶-3、-7、-8和-9的活性。采用逆转录-定量（q）PCR检测核因子-κ B（NF-κ B）基因的表达。细胞色素c释放凋亡检测试剂盒提取线粒体和胞浆。细胞色素c的释放、Bid的裂解和B细胞淋巴瘤2（Bcl-2）的表达通过蛋白质印迹法测定。细胞死亡ELISA显示ACh能够减少Fas诱导的角膜基质细胞凋亡。效应半胱天冬酶-3和-7的活性分析表明，ACh，当加入Fas处理的细胞，降低这两种酶的激活。Fas处理的细胞中加入ACh后，起始caspase-8和-9的活性也降低。ACh的这种抗凋亡作用依赖于ACh浓度和毒蕈碱型ACh受体的激活。对ACh抗凋亡机制的分析表明，ACh下调FasL诱导的NF-κ B B RNA表达，上调抗凋亡蛋白Bcl-2的表达，下调促凋亡蛋白Bad的表达，减少细胞色素c的释放，阻止促凋亡蛋白Bid的裂解。乙酰胆碱在体外人原代角膜基质细胞Fas凋亡模型中具有抗凋亡作用因此，ACh可能通过调节其起始阶段在角膜伤口愈合中发挥作用。
PURPOSE. To investigate the possible antiapoptotic effect of acetylcholine (ACh) in Fas-mediated apoptosis of primary human keratocytes in vitro, and to explore the underlying mechanism.METHODS. Primary human keratocytes were isolated from healthy corneas. Fas ligand (FasL) was used to induce apoptosis in keratocytes. Cell death was assessed by ELISA. Activity of caspase-3, -7, -8, and -9 was measured with luminescent caspase activity assays. Expression of nuclear factor-kappa B (NF-kappa B) gene was assessed with RT-quantitative (q)PCR. Cytochrome c release apoptosis assay kit was used to extract mitochondria and cytosol. Cytochrome c release, cleavage of Bid, and expression of B-cell lymphoma 2 (Bcl-2) were determined by Western blot.RESULTS. Cell death ELISA revealed that ACh is able to reduce Fas-induced apoptosis in keratocytes. Analysis of the activity of effector caspases-3 and -7 showed that ACh, when added to Fas-treated cells, decreases the activation of both these enzymes. The activity of initiator caspases -8 and -9 also decreased when ACh was added to Fas-treated cells. This antiapoptotic effect of ACh was dependent on ACh concentration and activation of muscarinic ACh receptors. Analysis of the antiapoptotic mechanisms triggered by ACh showed that ACh downregulates expression of FasL-induced NF-kappa B RNA expression, upregulates expression of antiapoptotic protein Bcl-2, downregulates expression of proapoptotic protein Bad, reduces cytochrome c release, and prevents proapoptotic Bid protein cleavage.CONCLUSIONS. Acetylcholine has an antiapoptotic effect in a Fas-apoptosis model of human primary keratocytes in vitro. It is therefore possible that ACh may play a role in corneal wound healing, by modulating its initiation phase.