氢饱和生理盐水对大鼠硒性白内障抑制作用的初步观察及机制研究

发布时间：2018-08-04 17:09

【摘要】：白内障是一种发病率、致盲率均居当今国内外眼病之首的疾病，严重影响患者的视力健康和生活质量。研究其发病机制和药物防治，具有重要的意义和价值。流行病学及实验室研究表明，氧化损伤是其发生发展的主要机制之一。氢气是一种优质的抗氧化剂，可选择性中和强毒性活性氧自由基，具有干扰自由基的生成，清除已生成的自由基，调节细胞代谢活性，无毒副作用等优点。硒性白内障发病机制主要为氧化损伤，本实验通过建立硒性白内障动物模型来初步研究氢饱和生理盐水对亚硒酸钠性白内障的作用机制，为临床药物治疗白内障提供新思路。目的：初步研究氢饱和生理盐水对SD大鼠亚硒酸钠性白内障的影响及相关机制。方法： 1氢饱和生理盐水对大鼠硒性白内障作用的初步观察选用7日龄健康SD大鼠68只，根据体重按随机数字表法把动物随机分为6组。分别为正常对照组（A，n=12），白内障组（B，n=12），氢水预防组（C，n=12），氢水全程治疗组（D，n=12），氢水晚期治疗组（E，n=12），氢水组（F，n=8）。白内障组、氢水预防组、氢水全程治疗组和氢水晚期治疗组大鼠12日龄按25μmol/kg体重单次颈背部皮下注射2mmol/L亚硒酸钠生理盐水。氢水预防组大鼠8日龄～12日龄腹腔注射氢饱和生理盐水5ml/kg，每日1次，连续5d；氢水全程治疗组大鼠8日龄～17日龄腹腔注射氢饱和生理盐水5ml/kg，每日1次，连续10d；氢水晚期治疗组大鼠17日龄～21日龄腹腔注射氢饱和生理盐水5ml/kg，每日1次，连续5d；氢水组大鼠8日龄～17日龄腹腔注射氢饱和生理盐水5ml/kg，每日1次，连续10d。于26日龄裂隙灯显微镜下观察大鼠晶状体混浊程度，测定晶状体中水溶性蛋白含量，SOD活性及GSH、MDA含量。所得数据进行统计学分析，以确定可能抑制白内障的作用时间。 2氢饱和生理盐水对大鼠硒性白内障的影响机制的研究根据第一部分实验结果，进一步研究氢饱和生理盐水对大鼠硒性白内障的影响机制。选用7日龄健康SD大鼠60只，根据体重按随机数字表法把动物随机分为3组。分别为正常对照组（A，n=20），白内障组（B，n=20），氢水全程治疗组（C，n=20）。白内障组和氢水全程治疗组大鼠12日龄按25μmol/kg体重单次颈背部皮下注射2mmol/L亚硒酸钠生理盐水。氢水全程治疗组大鼠8日龄～17日龄腹腔注射氢饱和生理盐水5ml/kg，每日1次，连续10d。于26日龄裂隙灯显微镜下观察大鼠晶状体混浊程度，测定晶状体中水溶性蛋白含量，SOD、CAT、Gpx、GR、GST活性及MDA、GSH、总巯基含量，以及视网膜中SOD、CAT、Gpx、GR活性及GSH、MDA含量。所得数据进行统计学分析。结果： 1氢饱和生理盐水对大鼠硒性白内障作用的初步观察 (1)晶状体混浊程度的观察：正常对照组和氢水组大鼠在整个实验过程中晶状体始终透明均为0级；白内障组16.67%（4/24）晶状体Ⅴ级混浊，83.33%（20/24）晶状体Ⅵ级混浊；氢水预防组25%（6/24）晶状体Ⅴ级混浊，75%（18/24）晶状体Ⅵ级混浊；氢水全程治疗组33.33%（8/24）晶状体Ⅲ级混浊，41.67%（10/24）晶状体Ⅳ级混浊，16.67%（4/24）晶状体Ⅴ级混浊，8.33%（2/24）晶状体Ⅵ级混浊；氢水晚期治疗组29.17%（7/24）晶状体Ⅴ级混浊，70.83%（17/24）晶状体Ⅵ级混浊。氢水预防组、氢水晚期治疗组与白内障组比较，晶状体混浊程度无明显差异，统计学检验结果显示差异无统计学意义(χ~2=1.06，P0.05)。氢水全程治疗组与白内障组比较，晶状体混浊的发生发展相对延缓，混浊程度明显低于白内障组，统计学检验结果显示差异有统计学意义(χ~2=30.48，P0.05)。 (2)晶状体各项生化指标比较：各组晶状体中水溶性蛋白含量，SOD活性及GSH、MDA含量比较，白内障组、氢水预防组、氢水晚期治疗组较正常对照组、氢水组晶状体中水溶性蛋白含量，SOD活性及GSH含量均明显下降(P0.05)，氢水全程治疗组水溶性蛋白含量，SOD活性及GSH含量下降不明显，且明显高于白内障组(P0.05)，差异有统计学意义，但氢水预防组、氢水晚期治疗组水溶性蛋白含量，SOD活性及GSH含量较白内障组无明显改变(P0.05)；白内障组、氢水预防组、氢水晚期治疗组较正常对照组、氢水组晶状体中MDA含量明显升高(P0.05)，氢水全程治疗组MDA含量升高不明显，且明显低于白内障组(P0.05)，差异有统计学意义，但氢水预防组、氢水晚期治疗组MDA含量较白内障组无明显改变(P0.05)。氢水组与正常对照组比较，生化指标无改变(P0.05)。 2氢饱和生理盐水对大鼠硒性白内障的影响机制的研究 (1)晶状体混浊程度的观察：正常对照组大鼠在整个实验过程中晶状体始终透明均为0级；白内障组20%（8/40）晶状体Ⅳ级混浊，25%（10/40）晶状体Ⅴ级混浊，55%（22/40）晶状体Ⅵ级混浊；氢水全程治疗组5%（2/40）晶状体Ⅱ级混浊，25%（10/40）晶状体Ⅲ级混浊，45%（18/40）晶状体Ⅳ级混浊，15%（6/40）晶状体Ⅴ级混浊，10%（4/40）晶状体Ⅵ级混浊。氢水全程治疗组与白内障组比较，晶状体混浊的发生发展相对延缓，混浊程度明显低于白内障组，统计学检验结果显示差异有统计学意义(χ~2=44.06，P0.05)。 (2)晶状体各项生化指标比较：各组晶状体中水溶性蛋白含量，SOD、CAT、Gpx、GR、GST活性及MDA、GSH、总巯基含量比较，白内障组较正常对照组晶状体中水溶性蛋白含量，SOD、CAT、Gpx、GR、GST活性及GSH、总巯基含量均明显下降(P0.05)，氢水全程治疗组水溶性蛋白含量，SOD、CAT、Gpx、GR、GST活性及GSH、总巯基含量下降不明显，且明显高于白内障组(P0.05)，，差异有统计学意义；白内障组较正常对照组晶状体中MDA含量明显升高(P0.05)，氢水全程治疗组MDA含量升高不明显，且明显低于白内障组(P0.05)，差异有统计学意义。 (3)视网膜各项生化指标比较：各组大鼠视网膜中SOD、CAT、Gpx、GR活性及GSH、MDA含量比较，正常对照组、白内障组、氢水全程治疗组各项生化指标无明显区别，差异无统计学意义(P0.05)。结论： 1.氢饱和生理盐水可减轻亚硒酸钠对晶状体的氧化损伤，对亚硒酸钠性白内障有一定的抑制作用，但无明显的预防作用，对于已形成的白内障无逆转作用，对晶状体无明显的毒性作用。 2.氢饱和生理盐水可以提高晶状体组织中水溶性蛋白含量，SOD、CAT、Gpx、GR、GST活性及GSH、总巯基含量，减少MDA生成，从而减轻晶状体的氧化损伤，对亚硒酸钠性白内障的发生和发展有一定的抑制和延缓作用。 3.氢饱和生理盐水对大鼠视网膜正常氧化还原状态的平衡无明显影响。创新点：首次探讨了氢饱和生理盐水对大鼠硒性白内障抑制作用及相关机制,为临床药物治疗白内障提供新思路。
[Abstract]:The incidence of cataract is a disease rate, and the rate of blindness is the first disease at home and abroad, which seriously affects the health and quality of life of the patients. It is of great significance and value to study the pathogenesis and drug control of the patients. The epidemiological and laboratory studies show that the oxidative damage is one of the main mechanisms of its development. High quality antioxidants, which can selectively neutralize strong toxic reactive oxygen radicals, interfere with the generation of free radicals, remove the generated free radicals, regulate cell metabolic activity, and have no side effects. The pathogenesis of selenite cataract is mainly oxidative damage. In this experiment, a selenium induced cataract animal model was developed to study the initial hydrogen satiety. And the physiological mechanism of sodium selenite in the treatment of selenite induced cataract, so as to provide new ideas for clinical medicine in the treatment of cataract.
Objective:
Objective to investigate the effect of hydrogen saturated saline on selenite induced cataract in SD rats and its mechanism.
Method:
Effect of 1 hydrogen saturated saline on selenium induced cataract in rats
68 healthy SD rats of 7 days of age were randomly divided into 6 groups according to their weight and random numbers. They were normal control group (A, n=12), cataract group (B, n=12), C (n=12), whole hydrogen water treatment group (D, n=12), hydrogen water advanced treatment group (E, n=12), hydrogen water group, cataract group, hydrogen water prevention group and whole hydrogen water treatment group. Rats in the late treatment group of hydrogen and water were injected with 2mmol/L sodium selenite subcutaneous injection at the 12 day of 25 mol/kg body weight on the back of the neck. The hydrogen water prevention group was intraperitoneally injected with hydrogen saturated saline 5ml/kg at the age of 8 to 12 days of age, 1 times a day, and continuous 5D, and hydrogen saturated saline 5ml/ was injected into the group of rats at the age of 8 days to 17 days of hydrogen water treatment group, 5ml/ Kg, 1 times a day and continuous 10d; the rats in the late treatment group of hydrogen and water were intraperitoneally injected with hydrogen saturated saline 5ml/kg at the age of 17 to 21 days of age, 1 times a day and continuous 5D. The hydrogen saturated rats were intraperitoneally injected with hydrogen saturated saline 5ml/kg, 1 times a day, and observed the degree of lens opacities in the rat lens under the 26 day Gap light microscope, and the degree of lens opacity was observed under the 26 day Gap light microscope. The contents of water-soluble protein, SOD activity, GSH and MDA in the crystalline lens were determined by statistical analysis.
Effect of 2 hydrogen saturated saline on selenium induced cataract in rats
According to the results of the first part of the experiment, we further studied the mechanism of the effect of hydrogen saturated saline on selenium induced cataract in rats. 60 healthy SD rats of 7 day old were randomly divided into 3 groups according to the weight according to the random number table. They were normal control group (A, n=20), cataract group (B, n=20), C (n=20), cataract group and cataract group. The rats were injected with 2mmol/L sodium selenite under the neck and back of 25 mol/kg body weight at 12 days of age. The rats were injected with hydrogen saturated saline 5ml/kg from 8 days to 17 days of age of hydrogen water treatment group, 1 times a day, and the degree of crystalline lens opacities was observed under the 26 day old slit lamp microscope, and the crystal shape was measured. The content of water-soluble protein in body, SOD, CAT, Gpx, GR, GST activity and MDA, GSH, total sulfhydryl content, and SOD in the retina, CAT, Gpx, GR activity and GSH, with statistical analysis.
Result:
Effect of 1 hydrogen saturated saline on selenium induced cataract in rats
(1) the observation of the degree of lens opacity:
In the normal control group and the hydrogen water group, the MICROTEK body was all the 0 level in the whole experiment, the cataract group 16.67% (4/24) lens V turbidity, 83.33% (20/24) lens VI turbidity, the hydrogen water prevention group 25% (6/24) lens V grade turbidity, 75% (18/24) crystal VI turbidity, and the 33.33% (8/24) lens III of the hydrogen water treatment group. Turbidity grade, 41.67% (10/24) lens IV turbidity, 16.67% (4/24) lens V turbidity, 8.33% (2/24) lens VI turbidity, 29.17% (7/24) lens V turbidity in late hydrogen water treatment group and 70.83% (17/24) lens VI turbidity. Hydrogen water prevention group, hydrogen water advanced treatment group and cataract group, the degree of lens opacities had no significant difference. The statistical test showed that the difference was not statistically significant (x ~2=1.06, P0.05). Compared with the cataract group, the development and development of lens opacities were relatively delayed, and the degree of turbidity was significantly lower than that of the cataract group. The statistical results showed that the difference was statistically significant (x ~2=30.48, P0.05).
(2) the comparison of the biochemical indexes of the lens:
The content of water-soluble protein, SOD activity and GSH, MDA content, cataract group, hydrogen water prevention group, and hydrogen water advanced treatment group were compared with normal control group. The water soluble protein content, SOD activity and GSH content in the hydrogen water group were significantly decreased (P0.05), the content of water soluble protein in the whole treatment group of hydrogen water, the decrease of SOD activity and GSH content in the hydrogen water treatment group. It was not obvious, and obviously higher than the cataract group (P0.05), the difference was statistically significant, but the water soluble protein content, the SOD activity and the GSH content in the hydrogen water treatment group were not significantly changed (P0.05), the cataract group, the hydrogen water prevention group, the late hydrogen water treatment group were compared with the normal control group, and the MDA content in the hydrogen water group was obvious. Increase (P0.05), MDA content in the hydrogen water treatment group increased not significantly, and obviously lower than the cataract group (P0.05), the difference was statistically significant, but the hydrogen water prevention group, the late hydrogen water treatment group MDA content than the cataract group was not significantly changed (P0.05). Hydrogen water group and normal control group, biochemical indexes did not change (P0.05).
Effect of 2 hydrogen saturated saline on selenium induced cataract in rats
(1) the observation of the degree of lens opacity:
In the normal control group, the MICROTEK body was all the 0 level in the whole experiment, the cataract group 20% (8/40) lens IV turbidity, 25% (10/40) lens V grade turbidity, 55% (22/40) lens VI turbidity, 5% (2/40) lens II turbidity, 25% (10/40) lens III turbidity, 45% (18/40) lens IV mixing in the whole course of hydrogen water treatment group. Turbidity, 15% (6/40) lens V grade turbidity, 10% (4/40) lens VI turbidity. Compared with cataract group, the occurrence and development of lens opacities were relatively delayed, and the degree of turbidity was significantly lower than that of cataract group. Statistical results showed that the difference was statistically significant (x ~2=44.06, P0.05).
(2) the comparison of the biochemical indexes of the lens:
The content of water-soluble protein, SOD, CAT, Gpx, GR, GST activity and MDA, GSH, total sulfhydryl content, the content of water-soluble protein in cataract group compared with that of normal control group, SOD, CAT, Gpx, GR, GST activity and total sulfhydryl content decreased significantly. And GSH, the total sulfhydryl content decreased not obviously, and obviously higher than the cataract group (P0.05), the difference was statistically significant. The content of MDA in the cataract group was significantly higher than that in the normal control group (P0.05). The increase of MDA content in the whole hydrogen water treatment group was not obvious, and it was significantly lower than the cataract group (P0.05), the difference was statistically significant.
(3) the comparison of the biochemical indexes of the retina:
The activities of SOD, CAT, Gpx, GR and GSH and MDA in the retina of rats were compared. There was no significant difference in the biochemical indexes between the normal control group, the cataract group and the whole treatment group of hydrogen water (P0.05).
Conclusion:
1. hydrogen saturated saline can reduce the oxidative damage of sodium selenite to the lens and inhibit the sodium selenite cataract, but it has no obvious preventive effect. It has no reverse effect on the formed cataract and has no obvious toxic effect on the lens.
2. hydrogen saturated saline can improve the content of water-soluble protein in lens tissue, SOD, CAT, Gpx, GR, GST activity and GSH, total sulfhydryl content, reduce MDA generation, thus reduce the oxidative damage of the lens, and have certain inhibition and delay effect on the occurrence and development of sodium selenite cataract.
3. hydrogen saturated saline had no significant effect on the balance of retinal redox state in rats.
Innovation: The inhibitory effect of hydrogen-saturated saline on selenium-induced cataract in rats and its related mechanisms were discussed for the first time, which provided a new idea for clinical treatment of cataract.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R776.1

【参考文献】