黄芪多糖对急性高眼压大鼠视网膜神经节细胞保护作用的实验研究
发布时间：2018-09-08 13:41
【摘要】：目的:青光眼已经成为世界上主要的致盲眼病之一，其典型表现是视乳头的凹陷性萎缩与视野的特征性缺损和缩小。目前关于青光眼视神经萎缩的发病机制还不十分清楚，传统上有两种学说：机械压力学说和血管缺血学说。机械压迫学说认为眼压作用于筛板直接压迫视神经纤维，阻碍了轴浆流的运输，轴突蛋白的生成和移动减少，导致细胞代谢受损；血管缺血学说强调视神经纤维由于缺血缺氧及失去周围组织的保护而发生损害。目前认为是机械压迫和血供障碍共同参与了青光眼视神经的损害，而眼压的升高进一步加重视网膜神经血管的调节障碍，促成了视神经的损害。阻止视神经损害、保护视功能的方法目前主要是通过药物和手术的方法降低眼压以减缓压力对视神经的损害，，已有明显的效果，这一途径得到共识。然而，单纯降眼压并不能完全制止青光眼视神经的损害，仍有一部分患者视野和视神经在不断受损。因此，人们越来越关注加强视神经保护的治疗，找到一种能保护视神经的药物，阻断和/或延缓神经节细胞原发性或继发性损伤，已成为当今青光眼研究的焦点和热点。 视网膜神经节细胞（retinal ganglion cells RGCS）的凋亡是一种高度调控的细胞死亡，是青光眼视神经损伤的最终通路。本实验通过制作高眼压大鼠模型，探讨黄芪多糖对大鼠急性高眼压状态下视网膜神经节细胞的保护作用，从而揭示具有活血化瘀功效的中药制剂在改善微循环，提高机体抗氧化能力，改善青光眼视神经轴浆流等方面具有明显的效果，为临床青光眼视神经保护的治疗提供实验基础和理论依据。 方法：选用大连医科大学实验动物中心供给的SD雌性大鼠60只（其中已剔除实验过程中死亡3只，丢失1只），随机分为4组，正常对照组（C组）、高眼压模型对照组（M组）、低浓度黄芪多糖治疗组（N组）、高浓度黄芪多糖治疗组（H组），每组各15只。各组大鼠每日实验处理如下：C组和M组给予2.5ml生理盐水灌胃，N组给予黄芪多糖500mg/kg（体重）2.5ml灌胃，H组给予黄芪多糖2000mg/kg（体重）2.5ml灌胃。用药2周后，除正常对照组，其余三组均双眼前房注射甲基纤维素方法制作急性高眼压模型，模型确定成功后的第5天过量麻醉处死，取眼球做石蜡切片，行HE染色观察视网膜形态结构变化，免疫组化检测caspase-3表达，TUNEL(TdT-mediated dUTP nick-end labeling)染色观察节细胞凋亡。利用Image Pro Plus5.1软件测量视网膜内外颗粒层厚度、神经纤维层厚度，计数阳性染色的节细胞和凋亡节细胞所占节细胞总数百分比，使用SPSS16.0统计软件包采用单因素方差分析(ANOVA)，组间两两比较采用t检验进行统计学分析，以P＜0.05为差异具有统计学意义。 结果：C组大鼠视网膜各层组织结构清晰，RGCs排列密集，细胞核边界清楚，caspase-3极少表达。M组大鼠视网膜全层、内外颗粒层及神经纤维层均高度水肿，RGCs排列疏松，细胞核肿胀，caspase-3在视网膜节细胞层和内颗粒层均有大量表达。N组大鼠视网膜各层组织结构清晰，节细胞整齐密集，caspase-3在视网膜节细胞层表达显著减少，N组较M组差异有统计学意义（P＜0.001）。H组大鼠视网膜内外颗粒层细胞基质仍然水肿，神经纤维层厚度较M组并无明显好转，H组较M组差异无统计学意义（P＞0.05），caspase-3阳性细胞数明显减少，RGCs凋亡率降低，H组较M组差异有统计学意义（P＜0.05、P＜0.001）。 结论：1、高眼压可以导致视网膜全层水肿、RGCs排列疏松、数目减少、细胞核肿胀、神经节细胞发生调亡。2、一定浓度的黄芪多糖对视网膜神经节细胞有显著的保护作用。3、高浓度的黄芪多糖虽然能够部分改善视网膜神经节细胞的凋亡，但应当注意其毒性作用的损害和影响。
[Abstract]:Objective: Glaucoma has become one of the leading blindness diseases in the world. Its typical manifestation is the concave atrophy of the optic papilla and the characteristic defect and narrowing of the visual field. The theory of vascular ischemia emphasizes that the optic nerve fibers are damaged by ischemia and hypoxia and loss of protection of the surrounding tissues. It is also involved in the damage of optic nerve in glaucoma, and the increase of intraocular pressure further aggravates the disturbance of retinal nerve and blood vessel regulation and contributes to the damage of optic nerve. However, simple hypotension does not completely stop the damage of the optic nerve in glaucoma. There are still some patients whose visual field and optic nerve are constantly damaged. Secondary injury has become the focus and hotspot of glaucoma research.
The apoptosis of retinal ganglion cells (RGCS) is a highly regulated cell death and the ultimate pathway of glaucoma optic nerve injury. In this study, the protective effect of Astragalus Polysaccharide on retinal ganglion cells in rats with acute ocular hypertension was investigated by making a rat model of ocular hypertension. The traditional Chinese medicine preparation of blood stasis removing effect has obvious effect on improving microcirculation, antioxidant capacity and axoplasmic flow of optic nerve in glaucoma, which provides experimental and theoretical basis for the treatment of clinical glaucoma optic nerve protection.
Methods: Sixty female SD rats supplied by Experimental Animal Center of Dalian Medical University were randomly divided into 4 groups: normal control group (group C), high intraocular pressure model control group (group M), low concentration astragalus polysaccharide treatment group (group N) and high concentration astragalus polysaccharide treatment group (group H). Rats in group C and group M were given 2.5 ml normal saline, group N 500 mg/kg (body weight) 2.5 ml Astragalus polysaccharide, group H 2000 mg/kg (body weight) 2.5 ml Astragalus polysaccharide. After 2 weeks of administration, except for the normal control group, the other three groups were injected methylcellulose into the anterior chamber of both eyes to make acute ocular hypertension model. On the 5th day after the operation, the rats were sacrificed under excessive anesthesia, and the eyeballs were taken for paraffin section. The morphological and structural changes of retina were observed by HE staining, the expression of Caspase-3 was detected by immunohistochemistry, and the apoptosis of ganglion cells was observed by TUNEL staining. The positive staining ganglion cells and apoptotic ganglion cells accounted for hundreds of fractions of ganglion cells. SPSS16.0 statistical software package was used for single factor analysis of variance (ANOVA).
Results: In group C, the structure of retinal layers was clear, RGCs were densely arranged, the nuclear boundaries were clear, and caspase-3 was rarely expressed. In group M, the retinal layers were highly edematous, the RGCs were loosely arranged, the nuclei were swollen, and caspase-3 was highly expressed in ganglion cells and inner granular layers. The expression of Caspase-3 in ganglion cell layer of retina was significantly decreased in group N compared with group M (P The number of Caspase-3 positive cells and the apoptosis rate of RGCs in group H were significantly lower than those in group M (P < 0.05, P < 0.001).
Conclusion: 1. High intraocular pressure can lead to retinal edema, loose RGCs arrangement, reduced number, nuclear swelling, ganglion cell apoptosis. 2. A certain concentration of Astragalus polysaccharide has a significant protective effect on retinal ganglion cells. 3. High concentration of Astragalus polysaccharide can partly improve the apoptosis of retinal ganglion cells, but should be. Attention should be paid to the damage and effects of its toxic effects.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.1;R775
[Abstract]:Objective: Glaucoma has become one of the leading blindness diseases in the world. Its typical manifestation is the concave atrophy of the optic papilla and the characteristic defect and narrowing of the visual field. The theory of vascular ischemia emphasizes that the optic nerve fibers are damaged by ischemia and hypoxia and loss of protection of the surrounding tissues. It is also involved in the damage of optic nerve in glaucoma, and the increase of intraocular pressure further aggravates the disturbance of retinal nerve and blood vessel regulation and contributes to the damage of optic nerve. However, simple hypotension does not completely stop the damage of the optic nerve in glaucoma. There are still some patients whose visual field and optic nerve are constantly damaged. Secondary injury has become the focus and hotspot of glaucoma research.
The apoptosis of retinal ganglion cells (RGCS) is a highly regulated cell death and the ultimate pathway of glaucoma optic nerve injury. In this study, the protective effect of Astragalus Polysaccharide on retinal ganglion cells in rats with acute ocular hypertension was investigated by making a rat model of ocular hypertension. The traditional Chinese medicine preparation of blood stasis removing effect has obvious effect on improving microcirculation, antioxidant capacity and axoplasmic flow of optic nerve in glaucoma, which provides experimental and theoretical basis for the treatment of clinical glaucoma optic nerve protection.
Methods: Sixty female SD rats supplied by Experimental Animal Center of Dalian Medical University were randomly divided into 4 groups: normal control group (group C), high intraocular pressure model control group (group M), low concentration astragalus polysaccharide treatment group (group N) and high concentration astragalus polysaccharide treatment group (group H). Rats in group C and group M were given 2.5 ml normal saline, group N 500 mg/kg (body weight) 2.5 ml Astragalus polysaccharide, group H 2000 mg/kg (body weight) 2.5 ml Astragalus polysaccharide. After 2 weeks of administration, except for the normal control group, the other three groups were injected methylcellulose into the anterior chamber of both eyes to make acute ocular hypertension model. On the 5th day after the operation, the rats were sacrificed under excessive anesthesia, and the eyeballs were taken for paraffin section. The morphological and structural changes of retina were observed by HE staining, the expression of Caspase-3 was detected by immunohistochemistry, and the apoptosis of ganglion cells was observed by TUNEL staining. The positive staining ganglion cells and apoptotic ganglion cells accounted for hundreds of fractions of ganglion cells. SPSS16.0 statistical software package was used for single factor analysis of variance (ANOVA).
Results: In group C, the structure of retinal layers was clear, RGCs were densely arranged, the nuclear boundaries were clear, and caspase-3 was rarely expressed. In group M, the retinal layers were highly edematous, the RGCs were loosely arranged, the nuclei were swollen, and caspase-3 was highly expressed in ganglion cells and inner granular layers. The expression of Caspase-3 in ganglion cell layer of retina was significantly decreased in group N compared with group M (P The number of Caspase-3 positive cells and the apoptosis rate of RGCs in group H were significantly lower than those in group M (P < 0.05, P < 0.001).
Conclusion: 1. High intraocular pressure can lead to retinal edema, loose RGCs arrangement, reduced number, nuclear swelling, ganglion cell apoptosis. 2. A certain concentration of Astragalus polysaccharide has a significant protective effect on retinal ganglion cells. 3. High concentration of Astragalus polysaccharide can partly improve the apoptosis of retinal ganglion cells, but should be. Attention should be paid to the damage and effects of its toxic effects.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.1;R775
【参考文献】
相关期刊论文 前10条
1 盛艳梅;孟宪丽;张艺;龙怡;;灯盏花素对体外高压致视网膜神经节细胞凋亡的影响[J];成都医学院学报;2007年01期
2 王宁利,孙兴怀,李静贞,王z牖
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