Biological Sciences, Inflammation Research Program, Allergan, Inc., Irvine, California 92612, USA.
Invest Ophthalmol Vis Sci. 2013 Jul 16;54(7):4717-33. doi: 10.1167/iovs.13-11681.
To investigate the role of mitochondrial permeability transition pore (MPTP) and effect of cyclosporin A (CsA) on inflammatory apoptosis of human conjunctival epithelial cells (IOBA-NHC) and T cells.
IOBA-NHC and Jurkat cells were stimulated with IFNγ, TNFα, αFas, or PMA/αCD3, in the presence or absence of CsA. MPTP was determined using the calcein-cobalt technique. Mitochondrial membrane potential (ΔΨm) was measured with JC-1. Apoptosis was quantified by Annexin V/PI staining. Apoptosis mediators were evaluated by flow cytometry or Western blot.
In IOBA-NHC, TNFα, and IFNγ induced MPTP opening, ΔΨm loss, and increased cell apoptosis. This was accompanied by upregulation of Fas/FasL; Bax; and caspase-3, -8, and -9 activation. Addition of CsA prevented IOBA-NHC from cell death by blocking MPTP opening, ΔΨm loss, Fas/FasL, and caspase activation. In PMA/αCD3-activated Jurkat T cells, MPTP opening and ΔΨm loss were increased along with cell apoptosis and upregulated Fas/FasL/caspase expressions. CsA further promoted T-cell apoptosis, ΔΨm loss, and upregulation of Fas/FasL/caspase.
Inflammation induces aberrant MPTP opening, resulting in an increased apoptosis in conjunctival epithelial cells. CsA protected IOBA-NHC from cell death by blocking both intrinsic and extrinsic apoptosis pathways. CsA promoted T-cell apoptosis via upregulating Fas/FasL and caspase activities with a minimal effect on MPTP. The findings suggest that the differential effect of CsA on T cells versus ocular surface resident epithelial cells may contribute to its therapeutic efficacy in treating ocular inflammation such as dry eye disease.
研究线粒体通透性转换孔（MPTP）的作用及环孢素 A（CsA）对人结膜上皮细胞（IOBA-NHC）和 T 细胞炎症凋亡的影响。
用 IFNγ、TNFα、αFas 或 PMA/αCD3 刺激 IOBA-NHC 和 Jurkat 细胞，同时存在或不存在 CsA。用钙黄绿素-钴技术测定 MPTP。用 JC-1 测定线粒体膜电位（ΔΨm）。用 Annexin V/PI 染色定量细胞凋亡。用流式细胞术或 Western blot 评估凋亡介质。
在 IOBA-NHC 中，TNFα 和 IFNγ 诱导 MPTP 开放、ΔΨm 丧失和细胞凋亡增加。这伴随着 Fas/FasL；Bax；和 caspase-3、-8 和 -9 的激活。CsA 通过阻断 MPTP 开放、ΔΨm 丧失、Fas/FasL 和 caspase 激活来防止 IOBA-NHC 细胞死亡。在 PMA/αCD3 激活的 Jurkat T 细胞中，MPTP 开放和 ΔΨm 丧失增加，同时伴有细胞凋亡和 Fas/FasL/caspase 表达上调。CsA 进一步促进了 T 细胞凋亡、ΔΨm 丧失和 Fas/FasL/caspase 的上调。
炎症诱导异常的 MPTP 开放，导致结膜上皮细胞凋亡增加。CsA 通过阻断内在和外在凋亡途径来保护 IOBA-NHC 细胞免于死亡。CsA 通过上调 Fas/FasL 和 caspase 活性来促进 T 细胞凋亡，对 MPTP 的影响最小。这些发现表明，CsA 对 T 细胞与眼表固有上皮细胞的不同作用可能有助于其在治疗干眼症等眼部炎症中的治疗效果。
