目的制备伏立诺他(SAHA)羟丙基-β-环糊精包合物(SAHA-CD)滴眼液，观察其对小鼠碱烧伤角膜新生血管(CNV)的抑制作用。
方法使用羟丙基-β-环糊精包合的方法制备SAHA质量分数分别为0.1%、0.2%和0.4%的SAHA-CD滴眼液，采用高效液相色谱法测定滴眼液中SAHA含量。取SPF级昆明小鼠75只，建立右眼角膜碱烧伤模型，采用随机数字表法随机将小鼠分为5个组，每组15只，其中0.1% SAHA-CD组、0.2%SAHA-CD组、0.4%SAHA-CD组和地塞米松组，造模后即刻分别给予相应药物点眼，模型对照组即刻给予0.9%氯化钠注射液点眼，每日4次，每次5 μl，连续6 d。荧光素钠染色观察角膜上皮愈合情况并采用EyeStudio软件计算角膜上皮缺损面积。制作角膜铺片并采用ImageJ软件计算CNV的长度和面积。采用苏木精-伊红染色法观察角膜组织病理特征。采用酶联免疫吸附试验(ELISA)法检测角膜组织中血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)和基质金属蛋白酶9(MMP-9)质量浓度。
结果制备的质量分数为0.1%、0.2%和0.4%的SAHA-CD滴眼液中SAHA含量分别为标示量的97.62%、98.33%和98.14%。造模后，各组模型眼角膜水肿、混浊。给药第6天，各组CNV长度和面积总体比较差异均有统计学意义( F＝7.655、8.802，均 P<0.01)，其中0.2%SAHA-CD组、0.4%SAHA-CD组和地塞米松组CNV面积显著小于模型对照组，0.1%SAHA-CD组、0.2%SAHA-CD组和地塞米松组CNV长度显著小于模型对照组，差异均有统计学意义(均 P<0.05)。给药第3天和第6天，各组角膜中VEGF、bFGF和MMP-9蛋白相对表达量总体比较差异均有统计学意义(第3天： F＝6.345、7.149、18.650，均 P<0.01；第6天： F＝6.749、5.105、5.023，均 P<0.01)，其中0.2%SAHA-CD组角膜中VEGF、bFGF和MMP-9蛋白表达量均低于0.1%SAHA-CD组、0.4%SAHA-CD组和模型对照组，差异均有统计学意义(均 P<0.05)。
结论SAHA-CD滴眼液可抑制小鼠碱烧伤后CNV的形成和发展。
ObjectiveTo prepare vorinostat encapsulated hydroxypropyl-β-cyclodextrin (SAHA-CD) eye drops and investigate its inhibitory effect on corneal neovascularization (CNV) induced by alkali burns in mouse.
MethodsThe SAHA-CD eye drops at concentrations of 0.1%, 0.2%and 0.4%were prepared by inclusion technology with hydroxypropyl-β-cyclodextrin, and the content was assayed by high performance liquid chromatography.Seventy-five SPF mice with alkali burn-induced CNV were randomized into 0.1%SAHA-CD group, 0.2%SAHA-CD group, 0.4%SAHA-CD group, dexamethasone group and normal control group according to a random number table, 15 for each group, among which the SAHA-CD groups and dexamethasone group were treated with corresponding drugs, and model control group was treated with normal saline immediately after modeling, four times a day and five microliters each time, lasting for six days.The healing of corneal epithelium was examined with a slit lamp microscope after fluorescein sodium staining, and the areas of cornea epithelial defects were calculated using Eyestudio software.The corneal flat mount was prepared, and the length and areas of CNV were calculated with ImageJ software.The histology of mouse corneas was observed through hematoxylin and eosin staining.The expression level of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metalloproteinase-9 (MMP-9) in cornea were measured with enzyme linked immunosorbent assay (ELISA) kits.The use and care of animals complied with the ARVO statement and this study protocol was approved by the Experimental Animal Ethics Committee of Henan Eye Institute (No.HNEECA-2020-01).
ResultsThe actual drug contents of the 0.1%, 0.2% and 0.4%SAHA-CD eye drops were 97.62%, 98.33%and 98.14%of the labeled amount.The cornea showed edema and opacification after modeling.On the sixth day after treatment, significant differences were found in the length and areas of CNV among various groups ( F=7.655, 8.802; both at P<0.01).The areas of CNV in 0.2%SAHA-CD, 0.4%SAHA-CD and dexamethasone groups were significantly smaller than model control group, and the length of CNV in 0.1%SAHA-CD, 0.2%SAHA-CD and dexamethasone groups were significantly smaller than model control group (all at P<0.05).On the third and sixth day following modeling, significant differences in the expression levels of VEGF, bFGF and MMP-9 were found among the five groups (third day: F=6.345, 7.149, 18.650; all at P<0.01; sixth day: F=6.749, 5.105, 5.023; all at P<0.01), and the expression levels of VEGF, bFGF and MMP-9 in 0.2%SAHA-CD group were significantly lower than those in 0.1%SAHA-CD group, 0.4%SAHA-CD group and model control group (all at P<0.05).
ConclusionsSAHA-CD eye drops can inhibit alkali burn-induced CNV in mouse.
杨晶晶,梁珍,卢萍,等. 伏立诺他羟丙基-β-环糊精滴眼液对小鼠碱烧伤角膜新生血管的抑制作用[J]. 中华实验眼科杂志,2022,40(07)：609-616.
DOI:10.3760/cma.j.cn115989-20200807-00566版权归中华医学会所有。
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杨晶晶：酝酿和设计实验、实验研究、采集数据、分析和解释数据、撰写文章；梁珍、卢萍：实施研究、采集数据、数据统计分析；张振：实施研究、采集数据；张俊杰：酝酿和设计实验、对文章的知识性内容作批判性审阅及定稿
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