黄芪多糖对急性高眼压大鼠眼压、视网膜、内外颗粒层、视神经纤维及caspase-3、视网膜神经节细胞凋亡影响随机平行对照研究
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摘要
[目的]观察黄芪多糖对急性高眼压大鼠眼压、视网膜、内外颗粒层、视神经纤维及caspase-3、视网膜神经节细胞凋亡影响。[方法]使用随机平行对照方法,将50只SD大鼠按随机数字表法分为5组,空白对照组、模型组、黄芪多糖组(高、中、低剂量组),10只/组。实验1d灌胃干预,黄芪多糖组黄芪多糖500、1000、2000mg/kg,空白对照组及模型组等体积生理盐水,连续14d,每周称重一次并随体重调整给药剂量。参照Kawasaki等建模方法,实验15d,对模型组及黄芪多糖组水合氯醛麻醉后,氯霉素滴眼液冲洗眼睛,滴盐酸奥布卡因麻醉眼部,手术显微镜下穿刺大鼠眼部前房,抽取0.1m L房水后注射1m L甲基纤维素,连续5d,同时滴注氯霉素滴眼液预防感染。检测眼压稳定>22mm Hg后确定模型成立。实验20d,过量麻醉处死后迅速摘除眼球,抽部分玻璃体与房水并甲醛溶液等量置换,4%多聚甲醛固定,24h后流水冲洗,乙醇脱水,石蜡包埋,切片。HE染色、免疫组织化学染色及TUNEL。观测视网膜病理图像、眼压、视网膜、内外颗粒层及视神经纤维层厚度及caspase-3表达、视网膜神经节细胞凋亡。[结果]实验第20d:黄芪多糖低剂量组视网膜组织结构清晰度提高,节细胞密集整齐,视网膜全层、视经纤维层及外颗粒层厚度均明显高于模型组。眼压、视网膜、内颗粒层、外颗粒层、视神经纤维模型组、黄芪多糖高、中、低剂量组均明显高于空白组(P<0.01),眼压黄芪多糖低中高各剂量组间呈量效递减(P<0.01),视网膜、内颗粒层、外颗粒层、视神经纤维呈量效递增(P<0.01)。caspase-3表达、视网膜节细胞凋亡率模型组、黄芪多糖高、中、低剂量组均明显高于空白组(P<0.01);黄芪多糖各剂量组均低于模型组(P<0.01),黄芪多糖各剂量组之间呈量效递减(P<0.01,P<0.05)。[结论]黄芪多糖对急性高眼压大鼠视网膜损伤具有保护作用,与用药剂量相关。
[Objective] To observe the protective effects of astragalus polysaccharide against damage onrat retinal. [Methods] The model rats were randomly divided into five groups, such as control group, model group and treatment group(500, 1000, 2000 mg/kg), with 10 rats in each. The astragalus polysaccharide at the daily dose of 500, 1000 and 2000 mg/kg were administered in the treatment group by gastrogavage with the control and model group administered saline in equal volume. The medication lasted for 14 days. The rats weight once every week to adjust the drug dosage. On the fifteen day, according to the modeling method of kawasaki,the model and treatment group were anaesthetized by chloralic hydras, and then take Chloramphenicol eye drop for washing and oxybuprocaine hydrochloride for anesthetization on eye. acute ocular hypertensionmodelwas established by anterior chamber injected with methyl cellulose production on the model and treatment group with the eye pressure>22 mmHg stability. The model rats were confirmed on the fifth day, sacrificedby excessive anesthesia and enucleated the eye ball. The tissues were fixed in 4%paraformaldehyde and embedded in paraffin, and then they were dyed with he, immunohistochemistry methods and TUNEL.Compared retinal morphology, detected caspase-3 expression, and measured retinal thickness and nerve fiber layer. Finally, calculate the percentage of apoptosis ganglion cells. [Results] As compared with the control group, Intraocular pressure, retina, inner granular layer, outer granular layer, optic nerve fiber, caspase-3 expression positive rate, and retinal cell apoptosis rateof model and treatment group show higher values,and there was a significant difference between them(P<0.01). RGCs were loosely arranged and caspase-3 expressed more abundantly(P<0.01). Compared with the model group, the retina structure of low dose group wererelative clear. Ganglion cells were dense and neat. But there was no significant difference improvement on the thickness of nerve fiber of the high dose group(P>0.05). The retina and optic nerve fibershave no significant difference improvement between the model and the high dosage treatment group(P>0.05).[Conclusion] Astragalus polysaccharide had protective effective on rat retinal with dose dependent.
[Objective] To observe the protective effects of astragalus polysaccharide against damage onrat retinal. [Methods] The model rats were randomly divided into five groups, such as control group, model group and treatment group(500, 1000, 2000 mg/kg), with 10 rats in each. The astragalus polysaccharide at the daily dose of 500, 1000 and 2000 mg/kg were administered in the treatment group by gastrogavage with the control and model group administered saline in equal volume. The medication lasted for 14 days. The rats weight once every week to adjust the drug dosage. On the fifteen day, according to the modeling method of kawasaki,the model and treatment group were anaesthetized by chloralic hydras, and then take Chloramphenicol eye drop for washing and oxybuprocaine hydrochloride for anesthetization on eye. acute ocular hypertensionmodelwas established by anterior chamber injected with methyl cellulose production on the model and treatment group with the eye pressure>22 mmHg stability. The model rats were confirmed on the fifth day, sacrificedby excessive anesthesia and enucleated the eye ball. The tissues were fixed in 4%paraformaldehyde and embedded in paraffin, and then they were dyed with he, immunohistochemistry methods and TUNEL.Compared retinal morphology, detected caspase-3 expression, and measured retinal thickness and nerve fiber layer. Finally, calculate the percentage of apoptosis ganglion cells. [Results] As compared with the control group, Intraocular pressure, retina, inner granular layer, outer granular layer, optic nerve fiber, caspase-3 expression positive rate, and retinal cell apoptosis rateof model and treatment group show higher values,and there was a significant difference between them(P<0.01). RGCs were loosely arranged and caspase-3 expressed more abundantly(P<0.01). Compared with the model group, the retina structure of low dose group wererelative clear. Ganglion cells were dense and neat. But there was no significant difference improvement on the thickness of nerve fiber of the high dose group(P>0.05). The retina and optic nerve fibershave no significant difference improvement between the model and the high dosage treatment group(P>0.05).[Conclusion] Astragalus polysaccharide had protective effective on rat retinal with dose dependent.
引文
[1]田发盈.黄芪的药理分析及应用研究[J].中国卫生标准管理,2016,7(20):148-149.
[2]张晗蕊.黄芪多糖抑制多聚谷氨酰胺蛋白毒性的分子机制研究[D].武汉:武汉大学,2011.
[3]全国动物实验动物标准化技术委员会.实验动物环境及设施:GB14925-2010[S].北京:中国标准出版社,2011.
[4]Kawasaki A,Han M H,Wei J Y,et al.Protective effect of arachidonic acid on glutamate neurotoxicity in rat retinal ganglioncells[J].Invest Ophthalmol Vis Sci,2002,43(6):1835-1842.
[5]汪晓磊,马建民,孟照洋,等.急性高眼压模型中p75NTR抑制剂对视网膜神经节细胞作用的研究[J].首都医科大学学报,2017,38(1):24-28.
[6]付金京.雌激素对大鼠慢性高眼压视网膜神经节细胞凋亡及caspase-3表达的影响[D].银川:宁夏医科大学,2012.
[7]胡丰平,张燕,董立红,等.不同手术方式治疗青光眼合并白内障患者的疗效比较[J].安徽医学,2016,37(2):175-177.
[8]申战省.Caspase-3抑制剂对兔外伤性视神经损伤后视网膜神经节细胞凋亡影响的实验研究[D].天津:天津医科大学,2009.
[9]韩光信.甲基纤维素[J].塑料工业,1978(3):79-80.
[10]祁晓君.丙戊酸钠在大鼠视神经损伤再生中的作用研究及对BDNF、GAP-43表达的影响[D].石家庄:河北医科大学,2014.
[11]魏春秀,罗旭昇,杨薇,等.大柴胡汤在眼科疾病治疗中的运用[J].广州中医药大学学报,2017,34(2):290-292.
[12]尹连荣,高健生.黄芪对高眼压大鼠视网膜神经节细胞的保护作用[J].北京中医药大学学报,2016,39(10):828-832.
[13]周舟.Caspase-3激活在缺氧诱导心肌细胞凋亡过程中的作用[D].重庆:第三军医大学,2002.
[2]张晗蕊.黄芪多糖抑制多聚谷氨酰胺蛋白毒性的分子机制研究[D].武汉:武汉大学,2011.
[3]全国动物实验动物标准化技术委员会.实验动物环境及设施:GB14925-2010[S].北京:中国标准出版社,2011.
[4]Kawasaki A,Han M H,Wei J Y,et al.Protective effect of arachidonic acid on glutamate neurotoxicity in rat retinal ganglioncells[J].Invest Ophthalmol Vis Sci,2002,43(6):1835-1842.
[5]汪晓磊,马建民,孟照洋,等.急性高眼压模型中p75NTR抑制剂对视网膜神经节细胞作用的研究[J].首都医科大学学报,2017,38(1):24-28.
[6]付金京.雌激素对大鼠慢性高眼压视网膜神经节细胞凋亡及caspase-3表达的影响[D].银川:宁夏医科大学,2012.
[7]胡丰平,张燕,董立红,等.不同手术方式治疗青光眼合并白内障患者的疗效比较[J].安徽医学,2016,37(2):175-177.
[8]申战省.Caspase-3抑制剂对兔外伤性视神经损伤后视网膜神经节细胞凋亡影响的实验研究[D].天津:天津医科大学,2009.
[9]韩光信.甲基纤维素[J].塑料工业,1978(3):79-80.
[10]祁晓君.丙戊酸钠在大鼠视神经损伤再生中的作用研究及对BDNF、GAP-43表达的影响[D].石家庄:河北医科大学,2014.
[11]魏春秀,罗旭昇,杨薇,等.大柴胡汤在眼科疾病治疗中的运用[J].广州中医药大学学报,2017,34(2):290-292.
[12]尹连荣,高健生.黄芪对高眼压大鼠视网膜神经节细胞的保护作用[J].北京中医药大学学报,2016,39(10):828-832.
[13]周舟.Caspase-3激活在缺氧诱导心肌细胞凋亡过程中的作用[D].重庆:第三军医大学,2002.