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
법서지이대대서급성고안압시망막신경절세포적보호작용급기궤제
Neuroprotective effect of fasudil for retinal ganglion cells and its mechanism research in rat acute elevated intraocular pressure
  • 万方数据
    中华眼视光学与视觉科学杂志 중화안시광학여시각과학잡지
    CHINESE JOURNAL OF OPTOMETRY OPHTHALMOLOGY AND VISUAL SCIENCE
    2011年 3期 191-197 ,共7页

    张菁%胡义珍

    장정%호의진

    神经保护药%rho相关激酶%视网膜神经节细胞%眼内压%模型,动物,大鼠 神經保護藥%rho相關激酶%視網膜神經節細胞%眼內壓%模型,動物,大鼠
    신경보호약%rho상관격매%시망막신경절세포%안내압%모형,동물,대서
    Neuroprotective agents%rho-Asociated kinase%Retinal ganglion cells%Intraocular pressure%Model,animal,rat
    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.
    목적 관찰법서지이대대서급성고안압시망막신경절세포(RGCs)적보호작용병탐토기궤제.방법 실험연구.24지SD대서피수궤분입정상조(N조)、모형조(M조)、모형+린산완충액조(MP조)、모형+법서지이조(F조),정상조불주임하처리,후삼조제작급성고안압모형(생리염수관주법).기중MP조화F조우조모전1주、당천급기후매천분별복강주사린산완충액(PBS) 25 mg/kg화법서지이25 mg/kg,각조우조모후7 d취안구화심장혈,채용TUNEL법측정RGCs조망지수(AI)반영RGCs조망정황,면역조화법관찰각조시망막중Rho격매-2(ROCK-2)화내피소(ET-1)적분포,병용흡광도치(OD)반영각자표체량;채용Western blotting법측정각조시망막중린산화적기구단백린산매파단위(p-MYPT-1)적표체,방사면역법측정혈장중ET-1함량,혈액류변의측량불동전변솔하적전혈점도、홍세포취집지수(BCAI)화홍세포압적(HCT).각지표조간비교채용단인소방차분석,량량비교채용LSD-t검험.결과 각조RGCs AI차이구유통계학의의(F=402.041,P=0.000),기중F조RGCs AI위33.3%±2.0%,소우M조(64.3%±2.2%)혹MP조(62.5%±2.2%)(P<0.05).N조시망막적절세포층(GCL)중부산재궤개ROCK-2화ET-1양성세포,M、MP화F조ROCK-2양성세포분포우GCL、내총상층(IPL)、내핵층(INL)、외총상층(OPL)화외핵층(ONL)중,이ET-1양성세포분포우전4층,ONL중무표체,차량자재M화MP조중적OD치고우N조(N、M급MP조ROCK-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)(P균<0.01),F조OD치(ROCK-2:0.37±0.04;ET-1:0.35±0.06)저우M혹MP조(P균<0.05),단잉고우N조(P균<0.05).ROCK-2화ET-1재각조표체차이구유통계학의의(F=82.862、56.491,P=0.000).Western blotting검측발현각조시망막중P-MYPT-1표체차이구유통계학의의(F=606.236,P=0.000),M화MP조표체량분별위0.522±0.013화0.520±0.013,고우N조(0.263±0.014)(P<0.01),F조표체량(0.302±0.015)저우M혹MP조(P<0.05),단잉고우N조(P<0.05).방사면역법검측발현각조혈장ET-1함량차이유통계학의의(F=8.750,P=0.000),M화MP조함량균위(96±10)pg/ml,고우N조[(72±10)pg/ml](P<0.05),F조함량위(78+10)pg/ml,저우M혹MP조(P<0.05),단잉고우N조(P<0.05).혈액류변의검측발현각조저절、중절、고절상태하전혈점도,BCAI화HCT차이유통계학의의(F=7.086、4.279、14.780、37.351、143.264,P균<0.05),차각지표중M혹MP조교N조고(P<0.05),F조교M혹MP조저(P<0.05),단잉고우N조(P<0.05).결론 법서지이가이억제대서급성고안압모형중RGCs적조망,대RGCs유보호작용,추측기궤제가능여억제ROCK-2、감소p-MYPT-1、감소기동단백-기구단백교련、억제평활기수축、감소축혈관인자ET-1표체、강저혈점도유관.
    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.
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