安徽医科大学学报

目的 本研究通过人工模拟的干燥环境(空气湿度<30%)联合皮下注射氢溴酸东莨菪碱诱导混合型干眼症(DED)小鼠模型，并探究维生素B₁₂滴眼液如何调控DED小鼠角膜基质金属蛋白酶9(MMP-9)和肿瘤坏死因子α(TNF-α)的表达水平。方法C57BL/6雌鼠50只，10只小鼠为A组(空白组)，其余40只小鼠采用干燥环境(空气湿度<30%)联合皮下注射氢溴酸东莨菪碱诱导DED小鼠模型，分别对所有小鼠进行基础泪液分泌实验(SIT)、泪膜破裂时间(TBUT)和角膜荧光素染色评分(CFS)检测，最终选择符合造模标准的小鼠入组，并随机分为B组(模型组)、C组(PBS组)、D组(维生素B₁₂组)，药物处理第0、7、14、21天对各组小鼠进行TBUT、SIT、CFS及角膜炎症程度的评估，从宏观角度分析维生素B₁₂滴眼液治疗DED的疗效。药物处理21 d后，采用颈椎脱臼法处死所有小鼠并取角膜组织，进行HE染色观察各组小鼠角膜组织的形态学变化，ELISA法和免疫组织化学染色检测各组小鼠角膜组织中MMP-9及TNF-α的表达水平。结果 干燥环境(空气湿度<30%)联合皮下注射氢溴酸东莨菪碱诱导DED小鼠的DED相关指标的变化：SIT显著减少、TBUT显著缩短、角膜荧光素点染增加及CFS均显著升高(P<0.05)，表明DED模型成功建立，共30只DED小鼠入组。DED模型建立成功且未进行药物干预时，B组、C组、D组之间TBUT、SIT、角膜荧光素点染情况及CFS差异均无统计学意义；B组、C组、D组分别与A组比较，SIT减少、TBUT缩短、角膜荧光素点染明显和CFS均增加，且各项指标的差异均有统计学意义(P<0.05)。药物干预后，D组(维生素B₁₂组)各项指标分别与B组、C组比较：SIT明显增加、TBUT显著延长、角膜荧光素点染减少，角膜较前清亮，荧光素点染减少，CFS降低，且差异均有统计学意义(P<0.05)。ELISA和免疫组织化学染色结果均显示，与A组相比，B组和C组角膜组织中MMP-9和TNF-α的表达量均显著升高(P<0.05);D组角膜组织中MMP-9和TNF-α的表达量较B组和C组均有所下降，但仍较A组高(P<0.05)。结论 MMP-9及TNF-α两种炎症因子均参与DED的发生发展。维生素B₁₂滴眼液可能通过促进角膜上皮修复改善眼表不适症状。维生素B₁₂滴眼液可能通过降低小鼠角膜组织中MMP-9及TNF-α的表达，增加泪液分泌量，延长泪膜破裂时间，减少角膜荧光素点染，减轻角膜炎症反应，从而达到治疗DED的目的。

Objective To investigate how vitamin B₁₂ eye drops regulate MMP-9 and TNF-α expression levels in the corneas of dry eye disease(DED) mouse model induced by a combination of an artificially simulated dry envi‑ronment(air humidity<30%) and subcutaneous injection of hyoscine hydrobromide.Methods Fifty C57 BL/6 fe‑male mice were used in the study.10 mice formed A group(blank control), while the remaining 40 mice were sub‑jected to dry eye disease induction through subcutaneous injection of scopolamine hydrobromide in a controlled en‑vironment with<30% air humidity.All mice underwent schirmer I test(SIT), tear break-up time(TBUT), and corneal fluorescence staining score(CFS)evaluations.Mice meeting modeling criteria were randomly assigned to three groups: B group(model group), C group(PBS control), and D group(vitamin B₁₂ group).0, 7, 14, and 21 days after treatment, each group received TBUT, SIT, CFS, and corneal inflammation assessments.Macro‑scopic analysis evaluated the efficacy of vitamin B₁₂ eye drops in treating dry eye.After 21 days of treatment, all mice were euthanized via cervical dislocation and their corneas analyzed using HE staining to examine morphologi‑cal changes.ELISA and immunohistochemical methods were employed to detect MMP-9 and TNF-α expression lev‑els in corneal tissues across groups.Results In dry environments(air humidity<30%), subcutaneous injection of scopolamine hydrobromide induced changes in dry eye-related indicators in mice: SIT significantly decreased, TBUT significantly shortened, corneal fluorescein staining increased, and CFS all significantly elevated(P<0.05), indicating successful establishment of the dry eye model.A total of 30 dry eye mice were enrolled.When the dry eye model was successfully established without drug intervention, no statistically significant differences were observed between B group, C group and D group in terms of TBUT, SIT, corneal fluorescein staining, or CFS.Compared with A group, B group, C group and D group showed statistically significant differences in re‑duced SIT, shortened TBUT, increased corneal fluorescein staining, and elevated CFS(P<0.05).After drug in‑tervention, D group(vitamin B₁₂ group) exhibited significantly increased SIT, prolonged TBUT, reduced corneal fluorescein staining, clearer corneas, decreased fluorescein staining, and lower CFS compared with B and C groups(P<0.05).ELISA and immunohistochemical staining results showed that compared with A group, B and C groups exhibited significantly increased expression of MMP-9 and TNF-α in corneal tissues(P<0.05).D group showed decreased expression of MMP-9 and TNF-α compared with B and C groups but remained higher than A group(P<0.05).Conclusion The expression of MMP-9 and TNF-α in the corneal tissues of mice with dry eye is elevated, suggesting that both inflammatory factors, MMP-9 and TNF-α, are involved in the development of dry eye.Vitamin B₁₂ eye drops may improve the symptoms of ocular surface discomfort by promoting the repair of the corneal epithe‑lium.Vitamin B₁₂ eye drops may increase tear secretion, prolong tear film break up time, reduce corneal fluores‑cein staining, and alleviate the inflammatory reaction of the cornea by decreasing the expression of MMP-9 and TNF-α in the corneal tissues of the mice, thus playing a therapeutic role in the treatment of dry eye disease.