广谱机械力敏感离子通道抑制剂钌红调控眼压的实验研究

Effects of a broad-spectrum mechanosensitive ion channels inhibitor ruthenium red on intraocular pressure

目的研究广谱机械力敏感离子通道抑制剂钌红对眼压的调控作用。设计实验研究。研究对象人原代小梁网细胞、C57BL/6J小鼠(2月龄)10只。方法分离培养人原代小梁网细胞,通过细胞形态和免疫荧光技术鉴定细胞类型;利用膜片钳技术记录机械力刺激下小梁网细胞机械力敏感电流的变化,并观察广谱机械力敏感离子通道抑制剂钌红对电流的抑制作用;将10只小鼠随机分为2组(每组5只):第1组(对照组)小鼠双眼前房内分别注射2 μl生理盐水,第2组(钌红组)小鼠双眼前房内分别注射2 μl 20 μM钌红。注射前日测量基础眼压,注射后每日测量眼压,并于注射12天后在小鼠每眼的前房设定15、25、35 mmHg压力下各灌注15 min生理盐水的方式测量房水流畅系数及注入速率。主要指标机敏电流(pA)、眼压(mmHg)和房水流畅系数(μl/min/mmHg)。结果钌红组给药后机械力敏感电流(-67.6±30.2pA)显著低于给药前(-309.7±65.9pA)(P=0.003);注射10天后,钌红组眼压为(14.67±2.11)mmHg,低于生理盐水组(17.85±3.66mmHg)(P=0.0604);注射12天后,钌红组房水流畅系数(0.01058±0.00236 μl/min/mmHg)明显低于生理盐水组(0.00324±0.00135μl/min/mmHg)(P=0.0273);灌注压为15mmHg时,钌红组注入速率(0.37±0.15μl/min)高于生理盐水组(0.28±0.05μl/min)(P=0.6278)。结论钌红有效抑制人小梁网细胞机械力敏感电流,降低小梁网房水引流功能;同时,钌红也可有效抑制房水生成速率,为青光眼治疗提供新思路。

Objective To study the effects of broad-spectrum mechanosensitive ion channels inhibitor ruthenium red on the regulation of intraocular pressure. Design Experimental research. Participants Human primary trabecular meshwork (TM) cells and 10 C57BL/6J mice. Methods Human primary TM cells were isolated and characterized based on cell morphology and “biomarkers” expression. Mechanosensitive currents in TM cells were detected by patch-clamp recording. A broad-spectrum mechanosensitive ion channel inhibitor, ruthenium red (RR) was applied to inhibit mechanosensitive currents in TM cells. 10 C57BL/6J mice were randomly divided into 2 groups. Saline (control group, n=5) or RR (RR group, n=5) was injected into the anterior chambers of mice, respectively. Intraocular pressure (IOP) was measured daily and outflow facility was measured at 12 days after injection. Main Outcome Measures mechanosensitive currents (pA), IOP (mmHg) and aqueous humor outflow facility (μl/min/mmHg). Results Mechanosensitive currents of RR group (-67.6±30.2 pA) was significantly lower than the mechanosensitive currents before administration(-309.7±65.9 pA)(P=0.003). At the 10th day after the injection, IOP was decreased in RR group (14.67±2.11 mmHg) compared to vehicle control (17.85±3.66 mmHg)(P=0.0604). At 12 days after the injection, outflow facility in RR group mice (0.01058 ± 0.00236 μl/min/mmHg) was decreased significantly compared to that in controls (0.00324 ± 0.001352 μl/min/mmHg)(P=0.0273). The pumping rate in RR group mice(0.37±0.15 μl/min)was higher than that in controls (0.28±0.05 μl/min)(P=0.6278)to maintain certain level of pressure (15 mmHg). Conclusion Mechanosensitive currents in human primary TM cells can be inhibited effectively by RR. Outflow facility can be significantly reduced by RR. Meanwhile, aqueous humor production rate can be regulated by RR, which result in a reduction of IOP in mice. These results provide a novel therapeutic approach to lower IOP in glaucoma.