法舒地尔对大鼠急性高眼压视网膜神经节细胞的保护作用及其机制

目的 观察法舒地尔对大鼠急性高眼压视网膜神经节细胞(RGCs)的保护作用并探讨其机制.方法 实验研究.24只SD大鼠被随机分入正常组(N组)、模型组(M组)、模型+磷酸缓冲液组(MP组)、模型+法舒地尔组(F组),正常组不做任何处理,后三组制作急性高眼压模型(生理盐水灌注法).其中MP组和F组于造模前1周、当天及其后每天分别腹腔注射磷酸缓冲液(PBS) 25 mg/kg和法舒地尔25 mg/kg,各组于造模后7 d取眼球和心脏血,采用TUNEL法测定RGCs凋亡指数(AI)反映RGCs凋亡情况,免疫组化法观察各组视网膜中Rho激酶-2(ROCK-2)和内皮素(ET-1)的分布,并用吸光度值(OD)反映各自表达量;采用Western blotting法测定各组视网膜中磷酸化的肌球蛋白磷酸酶靶单位(p-MYPT-1)的表达,放射免疫法测定血浆中ET-1含量,血液流变仪测量不同剪变率下的全血黏度、红细胞聚集指数(BCAI)和红细胞压积(HCT).各指标组间比较采用单因素方差分析,两两比较采用LSD-t检验.结果 各组RGCs AI差异具有统计学意义(F=402.041,P=0.000),其中F组RGCs AI为33.3%±2.0%,少于M组(64.3%±2.2%)或MP组(62.5%±2.2%)(P<0.05).N组视网膜的节细胞层(GCL)中仅散在几个ROCK-2和ET-1阳性细胞,M、MP和F组ROCK-2阳性细胞分布于GCL、内丛状层(IPL)、内核层(INL)、外丛状层(OPL)和外核层(ONL)中,而ET-1阳性细胞分布于前4层,ONL中无表达,且两者在M和MP组中的OD值高于N组(N、M及MP组ROCK-2:0.21±0.03、0.52±0.06、0.54±0.03;ET-1:0.22±0.05、0.51±0.03、0.51±0.04)(P均<0.01),F组OD值(ROCK-2:0.37±0.04;ET-1:0.35±0.06)低于M或MP组(P均<0.05),但仍高于N组(P均<0.05).ROCK-2和ET-1在各组表达差异具有统计学意义(F=82.862、56.491,P=0.000).Western blotting检测发现各组视网膜中P-MYPT-1表达差异具有统计学意义(F=606.236,P=0.000),M和MP组表达量分别为0.522±0.013和0.520±0.013,高于N组(0.263±0.014)(P<0.01),F组表达量(0.302±0.015)低于M或MP组(P<0.05),但仍高于N组(P<0.05).放射免疫法检测发现各组血浆ET-1含量差异有统计学意义(F=8.750,P=0.000),M和MP组含量均为(96±10)pg/ml,高于N组[(72±10)pg/ml](P<0.05),F组含量为(78+10)pg/ml,低于M或MP组(P<0.05),但仍高于N组(P<0.05).血液流变仪检测发现各组低切、中切、高切状态下全血黏度,BCAI和HCT差异有统计学意义(F=7.086、4.279、14.780、37.351、143.264,P均<0.05),且各指标中M或MP组较N组高(P<0.05),F组较M或MP组低(P<0.05),但仍高于N组(P<0.05).结论 法舒地尔可以抑制大鼠急性高眼压模型中RGCs的凋亡,对RGCs有保护作用,推测其机制可能与抑制ROCK-2、减少p-MYPT-1、减少肌动蛋白-肌球蛋白交联、抑制平滑肌收缩、减少缩血管因子ET-1表达、降低血黏度有关.

Neuroprotective effect of fasudil for retinal ganglion cells and its mechanism research in rat acute elevated intraocular pressure

Objective To investigate the neuroprotective effect of fasudil for retinal ganglion cells and its mechanism research in rat acute elevated intraocular pressure (IOP). Methods Experimental study. Twenty-four SD rats were divided into 4 groups at random: N group (normal), M group(model), MP group (model+PBS: began PBS i.p. 25mg/kg qd a week before the operation) and F group (model+Fasudil: began Fasudil i.p. 25 mg/kg qd a week before the operation). Excavating their eyeballs and collectting blood from their hearts 7th day after operation, which was established by increasing IOP to 110 mmHg (lasting 60 minutes) through intra-anterior chamber infusion of saline solution, TUNEL was employed to observe apoptosis of retinal ganglion cells (RGCs) through apoptosis index (AI), immuno-histological assay to carry out on paraffin sections of retina and to research the distribution and expression with average optical density (OD) of Rho-associated kinase-2 (ROCK-2) and endothelin-1 (ET-1), Western blotting to view the expression of phosphorylated-myosin phosphatase target subunit-1 (p-MYPT-1), radio-immunity assay to survey the content of ET-1 in blood plasma, and blood rheometer to measure the blood viscosities, blood cell aggregation index (BCAI) and hematocrit (HCT). The results were analyzed using one-way ANOVA and LSD-t test. Results In TUNEL: there was remarkable difference in RGCs AI among the 4 groups (F=402.041, P=0.000). AI in F group [(33.3±2.0)%] was obviously decreased compared with M [(64.3±2.2)%] or MP [(62.5±2.2)%] group (P＜ 0.05). In immuno-histological assay: in N group ROCK-2 or ET-1 was only distributed in ganglion cells layer (GCL) and not found in other layers. The distribution of ROCK-2 in M, MP or F group was in GCL, inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL) and outer nuclear layer (ONL), and ET-1 was in anterior 4 layers, but not ONL. Meanwhile, the OD of ROCK-2 and ET-1 in retina of M or MP group were obviously increased compared with in N group (the OD of ROCK-2 in N, M, MP groups were 0.21 ±0.03, 0.52±0.06, 0.54±0.03, respectively, and the OD of ET-1 were 0.22±0.05, 0.51±0.03, 0.51±0.04, respectively.) (P＜0.01). And OD of ROCK-2 and ET-1 in F group were prominently decreased compared with M or MP group (the OD of ROCK-2 and ET-1 in F group were 0.37 ± 0.04 and 0.35 ±0.06, respectively) (P＜0.05), but still noteworthily increased compared with N group (P＜0.05). There was remarkable difference in expression of ROCK-2 and ET-1 in retina of 4 groups (F=82.862, 56.491, P=0.000 for both). In Western blotting assay, there was remarkable difference in expression of p-MYPT-1 in retina of 4 groups (F=606.236, P=0.000). Meanwhile, they in M or MP group (0.522±0.013, 0.520±0.013) were obviously increased compared with that in N group (0.263±0.014) (P<0.01). And it in F group (0.302±0.015) was prominently decreased compared with M or MP group (P＜0.05), but still noteworthily increased compared with N group (P＜0.05). In radio-immunity assay: there was remarkable difference in content of ET-1 in blood plasma of 4 groups (F=8.750, P=0.000). Meanwhile, they in M or MP group [(96±10)pg/ml, (96±10)pg/ml] were obviously increased compared with in N group [(72±10)pg/ml] (P＜0.05). And it in F group [(78±10)pg/ml] was prominently decreased compared with M or MP group (P＜0.05), but still noteworthily increased compared with N group (P＜0.05). There was remarkable difference in three kinds of blood viscosity (1 l/s, 115 l/s, 300 l/s), BCAI and HCT of the 4 groups (F=7.086, 4.279, 14.780, 37.351, 143.264, P＜0.05). Meanwhile, they in M or MP groups were obviously increased compared with that in N group (P＜0.05). And they in F group were prominently decreased compared with M or MP group (P＜ 0.05), but still noteworthily increased compared with N group (P＜0.05). Conclusion Fasudil could protect RGCs in rat acute elevated IOP and its mechanism may be related to inhibiting ROCK-2, decreasing p-MYPT-1, reducing actin-myosin cross link, restraining smooth muscle contraction, diminishing ET-1, depressing blood viscosity.