[1]向征,石赟懿,谭钢.一氧化氮(NO)对角膜神经再生的影响作用[J].眼科新进展,2022,42(10):769-774.[doi:10.13389/j.cnki.rao.2022.0158]
　XIANG Zheng,SHI Yunyi,TAN Gang.Effect of gas signal molecule nitric oxide on corneal nerve regeneration[J].Recent Advances in Ophthalmology,2022,42(10):769-774.[doi:10.13389/j.cnki.rao.2022.0158]
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一氧化氮(NO)对角膜神经再生的影响作用/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
42卷
期数:
2022年10期
页码:
769-774
栏目:
实验研究
出版日期:
2022-10-05

文章信息/Info

Title:
Effect of gas signal molecule nitric oxide on corneal nerve regeneration
作者:
向征; 石赟懿; 谭钢
421005　湖南省衡阳市，南华大学附属第一医院眼科
Author(s):
XIANG Zheng; SHI Yunyi; TAN Gang
Department of Ophthalmology,the First Affiliated Hospital,University of South China,Hengyang 421005,Hunan Province,China
关键词:
角膜碱烧伤; 一氧化氮; 角膜神经再生;  神经生长因子
Keywords:
corneal alkali burn;  nitric oxide;  corneal nerve regeneration;  nerve growth factor
分类号:
R772.2
DOI:
10.13389/j.cnki.rao.2022.0158
文献标志码:
A
摘要:
目的　探讨一氧化氮(NO)对角膜神经再生的影响作用。方法　本研究以亚硝酸钠（NaNO2）作为外源性NO供体，在细胞实验中以小鼠神经母细胞瘤细胞(Neuro-2a)为研究对象。采用不同浓度的NaNO2处理Neuro-2a细胞并筛选出NO的最佳神经营养浓度。在动物实验中，将30只SD 大鼠随机分组，10只作为NC组，其余20只大鼠建立角膜碱烧伤模型，再随机分为PBS组和NO组，每组10只。从碱烧伤当天开始，PBS组给予PBS治疗,NO组给予10.00 μmol·L-1 NaNO2与PBS混合治疗。用荧光素钠染色后观察并记录大鼠角膜上皮愈合情况，计算角膜上皮愈合率。用CCK-8检测细胞活性，流式细胞术检测细胞凋亡率，免疫荧光法检测细胞神经元标记物的表达。于大鼠角膜碱烧伤处理后7 d取大鼠角膜上皮组织，分别采用实时荧光定量PCR和Western blot法检测每组角膜上皮中神经元标志物βⅢ-微管蛋白和神经生长因子(NGF)、胶质细胞源性神经营养因子(GDNF)、睫状神经营养因子(CNTF)的表达水平。结果　10.00 μmol·L-1 NaNO2处理Neuro-2a细胞24 h后可显著提高细胞活性；使用浓度为0.00 μmol·L-1、10.00 μmol·L-1、1000.00 μmol·L-1的NaNO2处理Neuro-2a细胞24 h后，测得细胞凋亡率分别为18.60%、13.00%、19.48%；与0.00 μmol·L-1组相比，10.00 μmol·L-1组细胞凋亡率降低(P<0.01)。与0.00 μmol·L-1组相比，10.00 μmol·L-1组Neuro-2a细胞βⅢ-微管蛋白、MAP2和SMI312三种神经元标志物相对表达量均增加(均为P<0.05) 。碱烧伤后1 d、3 d、7 d，与PBS组相比，NO组大鼠角膜上皮愈合率均升高(均为P<0.05) 。与NC组相比，PBS组和NO组大鼠角膜组织各神经营养因子mRNA的相对表达量均增高(均为P<0.05)。与PBS组相比，NO组大鼠角膜组织NGF、GDNF、CNTF mRNA的相对表达量均增高(均为P<0.05)。Western blot检测结果显示：碱烧伤后7 d,与NC组相比，PBS组和NO组大鼠角膜组织βⅢ-微管蛋白、NGF、GDNF、CNTF的蛋白表达水平均增高(均为P<0.05)；与PBS组相比，NO组大鼠角膜组织βⅢ-微管蛋白、NGF、GDNF、CNTF蛋白的表达水平均明显增高，差异均有统计学意义(均为P<0.05)。结论　气体信号分子NO能促进神经细胞的生长以及相关神经元标志物的表达；在大鼠角膜碱烧伤模型中，局部应用外源性NO进行治疗，可对角膜上皮和角膜神经产生明显的营养作用。
Abstract:
Objective　To investigate the effect of the gas signal molecule nitric oxide (NO) on corneal nerve regeneration. Methods　In this study, sodium nitrite (NaNO2) was used as an exogenous NO donor. In cell experiments, mouse neuroblastoma cells (Neuro-2a) were selected and treated with different concentrations of NaNO2 to screen out the optimal neurotrophic concentration of NO. In animal experiments, 30 SD rats were selected, and 10 of them were randomly divided into the normal control (NC) group. The remaining 20 rats were used to establish corneal alkali burn models and then were randomly divided into the PBS group and the NO group, with 10 rats in each group. From the day of alkali burn, rats in the PBS group were treated with PBS, and rats in the NO group were treated with 10.00 μmol·L-1 NaNO2 mixed with PBS. After fluorescein sodium staining, the healing of corneal epitheliums in rats was observed and recorded, and its rate was calculated. The cell viability was detected by CCK-8, the cell apoptosis by flow cytometry, and the expression of neuronal markers by immunofluorescence. Rat corneal epithelial tissues were collected seven days after alkali burn, and the expression levels of βⅢ-tubulin, nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF) in them were measured by real-time quantitative polymerase chain reaction and Western blot. Results　The viability of Neuro-2a cells treated with 10.00 μmol·L-1 NaNO2 for 24 h was significantly increased. The apoptosis of Neuro-2a cells treated with 0.00 μmol·L-1, 10.00 μmol·L-1, and 1000.00 μmol·L-1 NaNO2 for 24 h was 18.60%, 13.00%, and 19.48%, respectively. Compared with the 0.00 μmol·L-1 group, the apoptosis rate in the 10.00 μmol·L-1 group decreased (P<0.01), while the relative expression levels of βⅢ-tubulin, microtubule-associated protein 2 and SMI 312 increased (all P<0.05).Compared with the PBS group, the corneal epithelial healing rate in the NO group significantly improved one, three and seven days after alkali burn (all P<0.05). Compared with the NC group, the relative mRNA expression of each neurotrophic factor in the PBS and NO groups increased (all P<0.05). Compared with the PBS group, the relative mRNA expression of NGF, GDNF and CNTF in the NO group increased (all P<0.05). Western blot results showed that seven days after alkali burn, the protein expression of βIII-tubulin, NGF, GDNF and CNTF in the PBS and NO groups increased compared with the NC group (all P<0.05). Compared with the PBS group, the protein expression of βIII-tubulin, NGF, GDNF and CNTF in the NO group increased significantly (all P<0.05). Conclusion　The gas signal molecule NO can promote the growth of nerve cells and the expression of related neuronal markers. In the rat corneal alkali burn model, local application of exogenous NO can produce obvious nutritional effects on the corneal epitheliums and nerves.

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备注/Memo

备注/Memo:
国家自然科学基金项目（编号：81100648，82171031）；湖南省自然科学基金资助项目（编号：2021JJ30620）；湖南省卫生健康委员会科技计划资助项目（编号：20201966）
更新日期/Last Update: 2022-10-05