Meng Bo, Li Hongyi, Sun Xian, Qu Wei, Yang Binbin, Cheng Fang, Shi Liping, Yuan Huiping
Department of Ophthalmology, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150086, P.R. China.
Department of Ophthalmology and Otorhinolaryngology, Hospital of Heilongjiang University, Harbin, Heilongjiang 150080, P.R. China.
Mol Med Rep. 2017 Jul;16(1):617-624. doi: 10.3892/mmr.2017.6647. Epub 2017 May 30.
The purpose of the present study was to investigate the protective effect of the σ-1 receptor (Sig-1R) agonist (+)‑pentazocin (PTZ) on pressure-induced apoptosis and death of human trabecular meshwork cells (hTMCs). The expression levels of Sig‑1R and insulin receptor (InsR) were examined in hTMCs. Cells were cultured under a pressure of 0, 20, 40, 60 and 80 mmHg for 48 h, and under 80 mmHg for 44 h, after which the cells were treated with (+)‑PTZ (20 µM), N-(2-(3,4-dichlorophenyl)ethyl)-N‑methyl-2‑(dimethylamino) ethylamine (BD‑1063; 20 µM) administered 30 min prior to (+)‑PTZ, or BD‑1063 (20 µM) and then exposed to 80 mmHg again until the 48 h time‑point. The changes of the cells were observed by optical and electron microscopy, the apoptosis and death of hTMCs were detected by ethidium bromide/acridine orange dual staining assay and the expression of Sig‑1R and InsR by reverse transcription‑quantitative polymerase chain reaction and western blot analysis. The phosphorylation of extracellular signal‑regulated kinase (ERK), an important downstream protein of the InsR‑mitogen‑activated protein kinases (MAPK) signaling pathway, was also detected by western blot analysis when (+)‑PTZ and BD‑1063 were added to the 80 mmHg‑treated cells. Sig‑1Rs and InsRs were expressed in hTMCs. The apoptosis and death of hTMCs increased from 40 mmHg with 50% cell death when the pressure was at 80 mmHg and the structure of the cells noticeably changed. The expression of Sig‑1R and InsR increased along with the elevation of pressure. (+)‑PTZ decreased the apoptosis and death of hTMCs and increased the expression of Sig‑1R and InsR, and the phosphorylation of ERK. Such effects were blocked by BD‑1063. The present study suggested that Sig‑1R agonist (+)‑PTZ can protect hTMCs from pressure‑induced apoptosis and death by activating InsR and the MAPK signal pathway.
本研究的目的是探讨σ-1受体（Sig-1R）激动剂（+）-喷他佐辛（PTZ）对压力诱导的人小梁网细胞（hTMCs）凋亡和死亡的保护作用。检测hTMCs中Sig-1R和胰岛素受体（InsR）的表达水平。将细胞分别在0、20、40、60和80 mmHg压力下培养48 h，以及在80 mmHg压力下培养44 h，之后用（+）-PTZ（20 μM）、在（+）-PTZ给药前30分钟给予的N-（2-（3,4-二氯苯基）乙基）-N-甲基-2-（二甲基氨基）乙胺（BD-1063；20 μM）或BD-1063（20 μM）处理细胞，然后再次暴露于80 mmHg压力下直至48小时时间点。通过光学显微镜和电子显微镜观察细胞变化，通过溴化乙锭/吖啶橙双重染色法检测hTMCs的凋亡和死亡情况，通过逆转录定量聚合酶链反应和蛋白质免疫印迹分析检测Sig-1R和InsR的表达。当向80 mmHg处理的细胞中加入（+）-PTZ和BD-1063时，还通过蛋白质免疫印迹分析检测细胞外信号调节激酶（ERK）（InsR-丝裂原活化蛋白激酶（MAPK）信号通路的重要下游蛋白）的磷酸化情况。Sig-1Rs和InsRs在hTMCs中表达。当压力达到80 mmHg时，hTMCs的凋亡和死亡从40 mmHg时开始增加，细胞死亡达50%，细胞结构明显改变。Sig-1R和InsR的表达随压力升高而增加。（+）-PTZ可减少hTMCs的凋亡和死亡，并增加Sig-1R和InsR的表达以及ERK的磷酸化。这些作用被BD-1063阻断。本研究表明，Sig-1R激动剂（+）-PTZ可通过激活InsR和MAPK信号通路保护hTMCs免受压力诱导的凋亡和死亡。
