褪黑素受体激动剂Neu-P11促进兔眼组织AQP1的表达

Study on AQP1 expression in rabbit eye pro moted by melatonin receptor agonist Neu P11

目的观察褪黑素受体激动剂Neu-P11对眼压的影响以及水通道蛋白1(aquaporin protein 1,AQP1)在正常眼压兔眼组织中的表达,探讨药物Neu-P11降低眼压的作用以及其与AQP1的相关性。方法雄性新西兰大白兔30只(60眼),分为正常对照组和Neu-P11组,每组各15只(30眼)。采用局部麻醉下双眼局部点眼法,50μmol·L-1Neu-P11点眼,每3 d做一次眼压测量实验。1个月后处死动物,留取包含角膜、虹膜、睫状体、房角及前部巩膜的眼前段组织,用RT-PCR、免疫组织化学和Western blot技术检测AQP1转录和蛋白表达。结果与正常对照组比较,Neu-P11组在局部用50μmol·L-1Neu-P11点眼后0.5 h(t=3.990,P=0.004)、1 h(t=5.704,P=0.000)、2 h(t=7.393,P=0.000)、3 h(t=4.445,P=0.002)、4 h(t=2.869,P=0.021)眼压明显下降,差异均有统计学意义。50μmol·L-1Neu-P11点眼,可使兔眼压降低,1 h达到最低(11.99±2.45)mmHg(1 kPa=7.5 mmHg),与用药前比较差异有显著统计学意义(P=0.000),6 h后恢复正常水平(18.66±2.42)mmHg(P=0.365)。免疫组织化学染色结果显示:正常对照组AQP1仅分布于睫状体非色素上皮细胞、虹膜色素上皮细胞等少数细胞胞质和胞浆中,而Neu-P11组角膜内皮细胞、虹膜基质血管内皮细胞和色素上皮细胞、睫状体非色素上皮细胞、小梁网组织细胞和Schlemm管的胞膜和胞浆均可见阳性表达颗粒,且阳性反应细胞数明显增多。Neu-P11组较正常对照组眼前节组织AQP1 mRNA表达上调,AQP1蛋白表达增高(为正常对照组的3.77倍),两组差异均有显著统计学意义(均为P<0.01)。结论褪黑素受体激动剂Neu-P11能够降低兔眼压,并能够使兔眼组织中AQP1的表达增高。

Objective To observe effects of melatonin receptor agonist Neu-P11 on intraocular pressure and aquaporin-1 （AQP1） expression in rabbit eye with normal intraocular pressure,and to investigate the effects of Neu-Pll on lowering intraocular pressure and its correlation with AQP1. Methods 50 male New Zealand rabbits were randomly and evenly divided into 2 groups：normal control group and Neu-P11 group. Rabbits were dropped 50 μmol · L-1 Neu-Pll into the eye by routine eye drops under local anaesthesia and intraocular pressure was tested every 3 days. Rabbits were killed one month later, and eye anterior segment including cornea, iris, ciliary body, chamber angle and anterior sclera was taken out, and AQP1 transcription and expression were tested by RT-PCR,immunohistochemistry and Western blot technology. Results After rabbits in group Neu-Pll were eye dropped locally with 50 μmol · L-1 Neu-Pll 0.5 h （t=3.990,P=O.O04） ,1 h（t =5.704,P =0. 000） ,2 h（t =7. 393,P =0. 000） ,3 h（t = 4. 445 ,P = 0.002 ） ,4 h（ t = 2. 869 ,P = 0. 021 ）, intraocular pressure significantly dropped, compared with normal control group, and the difference had statistical significance. In- traocnlar pressure dropped by 50 μmol ·L-1 Neu-P11 eye drop, and 1 h later reached the lowest level （11.99 ±2.45）mg （1 kPa =7.5 mmHg） ,and the difference between before and after medication was of statistical significance （ P = 0. 000 ）. 6 h later, intraocular pressure returned to the normal level （ 18.66 ± 2.42 ） mmHg （ P = 0. 365 ）. The result of immunohistochemical staining indicated that AQP1 only distributed in cytoplasm of a few non-pigment epithelial cells of ciliary body and iris pigment epithelium in normal control group ,while in Neu-Pl 1 group ,positive expressive articles could be seen in cell membrane and cytoplasm of corneal endothelial cells, iris stromal vascular endothelial cells and iris pigment epithelium, non-pigment epithelial cells of ciliary body, trabecular meshwork cells and Schlemm cavity, and the number of positive articles significantly in-creased. AQP1 mRNA expression in anterior segment raised in Neu-P11 group, compared with normal control group, and AQP1 expression increased 3.77 times of that in normal control group. The difference was found statistically significant （P 〈 0.01 ）. Conclusion Melatonin receptor agonist Neu-P11 may reduce intraocular pressure in rabbits and raise the AQP1 expression in eye tissues of rabbits.