Department of Ophthalmology, The First Affiliated Hospital of University of South China, Hengyang, Hunan 421001, P.R. China.
Medical College, University of South China, Hengyang, Hunan 421001, P.R. China.
Int J Mol Med. 2018 Jul;42(1):71-80. doi: 10.3892/ijmm.2018.3580. Epub 2018 Mar 22.
Bradykinin (BK) has been demonstrated to induce proliferation in several types of cell in ex vivo corneas. However, the mechanisms underlying the action of BK on corneal endothelial cells (CECs) remain largely unknown. The present study aimed to investigate the effect of BK on rabbit corneal endothelial cell (RCEC) proliferation, and assess the involvement of the zonula occludens‑1(ZO‑1)/ZO‑1associated nucleic acid binding protein (ZONAB) pathway. Cell proliferation and cell cycle distribution was analyzed following treatment with BK (0.01, 0.1,1.0 or 10.0 µM) for the indicated time intervals (24, 48, 72 and 96 h), or following BK treatment combined with transfection of ZONAB‑small interfering (si)RNA for 72 h. In addition, the expression of tight junction ZO‑1, nuclear ZONAB, proliferating cell nuclear antigen(PCNA) and cyclin D1 were evaluated using western blotting or immunofluorescence. BK treatment was demonstrated to induce time‑ and concentration‑dependent cell proliferation and cell cycle progression, along with the upregulation of tight junction ZO‑1 and nuclear ZONAB, as well as PCNA and cyclin D1 protein expression. Furthermore, knockdown with ZONAB‑siRNA inhibited cell proliferation, induced cell cycle arrest and downregulated PCNA and cyclin D1 protein expression. ZONAB knockdown therefore successfully reversed the increase in proliferation induced by BK treatment. Taken together, these results suggested that BK stimulated RCEC proliferation, potentially via the ZO‑1/ZONAB pathway. The signaling paradigm disclosed in the present study potentially serves as an important therapeutic target for cornea regeneration and transplantation.
缓激肽（BK）已被证明可在离体角膜中的几种细胞类型中诱导增殖。然而，BK 对角膜内皮细胞（CEC）作用的机制在很大程度上仍不清楚。本研究旨在探讨 BK 对兔角膜内皮细胞（RCEC）增殖的影响，并评估紧密连接蛋白 1（ZO-1）/ZO-1 相关核酸结合蛋白（ZONAB）通路的参与情况。用 BK（0.01、0.1、1.0 或 10.0 μM）处理细胞，分别在 24、48、72 和 96 h 后分析细胞增殖和细胞周期分布，或用 BK 处理并转染 ZONAB 小干扰（si）RNA 72 h 后分析细胞增殖和细胞周期分布。此外，用 Western blot 或免疫荧光法检测紧密连接 ZO-1、核 ZONAB、增殖细胞核抗原（PCNA）和细胞周期蛋白 D1 的表达。结果表明，BK 处理可诱导时间和浓度依赖性的细胞增殖和细胞周期进程，同时上调紧密连接 ZO-1 和核 ZONAB，以及 PCNA 和细胞周期蛋白 D1 蛋白表达。此外，用 ZONAB-siRNA 敲低可抑制细胞增殖，诱导细胞周期停滞，并下调 PCNA 和细胞周期蛋白 D1 蛋白表达。因此，ZONAB 敲低成功逆转了 BK 处理诱导的增殖增加。综上所述，这些结果表明，BK 通过 ZO-1/ZONAB 通路刺激 RCEC 增殖。本研究揭示的信号范式可能为角膜再生和移植提供重要的治疗靶点。
