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润目灵护眼液治疗苯扎氯铵诱导的干眼症及其作用机制研究

宋健涛 刘鑫 祁兴华 张巧 陈忆菁 朱慧 裴科 段煜 蔡皓

宋健涛, 刘鑫, 祁兴华, 张巧, 陈忆菁, 朱慧, 裴科, 段煜, 蔡皓. 润目灵护眼液治疗苯扎氯铵诱导的干眼症及其作用机制研究[J]. 南京中医药大学学报, 2023, 39(1): 70-78. doi: 10.14148/j.issn.1672-0482.2023.0070
引用本文: 宋健涛, 刘鑫, 祁兴华, 张巧, 陈忆菁, 朱慧, 裴科, 段煜, 蔡皓. 润目灵护眼液治疗苯扎氯铵诱导的干眼症及其作用机制研究[J]. 南京中医药大学学报, 2023, 39(1): 70-78. doi: 10.14148/j.issn.1672-0482.2023.0070
SONG Jian-tao, LIU Xin, QI Xing-hua, ZHANG Qiao, CHEN Yi-jing, ZHU Hui, PEI Ke, DUAN Yu, CAI Hao. Investigation on Treatment of Xerophthalmia Induced by Benzalkonium Chloride with Runmuling Eye-Drops and Its Underlying Mechanism[J]. Journal of Nanjing University of traditional Chinese Medicine, 2023, 39(1): 70-78. doi: 10.14148/j.issn.1672-0482.2023.0070
Citation: SONG Jian-tao, LIU Xin, QI Xing-hua, ZHANG Qiao, CHEN Yi-jing, ZHU Hui, PEI Ke, DUAN Yu, CAI Hao. Investigation on Treatment of Xerophthalmia Induced by Benzalkonium Chloride with Runmuling Eye-Drops and Its Underlying Mechanism[J]. Journal of Nanjing University of traditional Chinese Medicine, 2023, 39(1): 70-78. doi: 10.14148/j.issn.1672-0482.2023.0070

润目灵护眼液治疗苯扎氯铵诱导的干眼症及其作用机制研究

doi: 10.14148/j.issn.1672-0482.2023.0070
基金项目: 

南京中医药大学南京健康广场健康产业开放课题 NJJKGC202009

详细信息
  • 中图分类号: R285.5

Investigation on Treatment of Xerophthalmia Induced by Benzalkonium Chloride with Runmuling Eye-Drops and Its Underlying Mechanism

  • 摘要:   目的  探究润目灵护眼液经超声雾化机雾化后对过度蒸发性干眼症的治疗效果及作用机制。  方法  16只新西兰兔随机分为空白对照组、模型组、原方组和考察组,每组4只, 采用苯扎氯铵滴眼法诱导过度蒸发性兔干眼症模型, 经超声雾化机给予润目灵护眼液, 通过检测兔眼泪液的分泌量、泪膜的破裂时间和角膜的荧光染色, 以评价润目灵组方对兔干眼症模型的干预作用。免疫荧光染色法检测兔眼结膜黏蛋白-5亚型AC(MUC5AC)的表达情况; 在光学显微镜下观察兔眼角膜和结膜的病理情况; ELISA法检测兔眼泪腺和房水中TNF-α和IL-6的含量。Western blot法检测兔眼角膜中JNK/P38通路以及MMP-9和MUC5AC蛋白的表达水平, 以评价润目灵护眼液对兔干眼症模型的治疗作用。  结果  经HPLC测定,原方组中绿原酸、金丝桃苷、木犀草苷的浓度分别为0.123 63、0.017 25、0.095 83 mg·mL-1。通过优化提取工艺,考察组中绿原酸、金丝桃苷、木犀草苷的含量显著提升,浓度分别为0.225 18、0.042 11、0.183 10 mg·mL-1。与模型组相比,原方组与考察组兔眼泪液的分泌量增加(P < 0.05),兔眼泪膜的稳定性增强(P < 0.05),且考察组泪液分泌量高于原方组(P < 0.05);与模型组相比,原方组与考察组兔眼结膜中的MUC5AC表达水平升高(P < 0.05),且考察组中被MUC5AC抗体染色的结膜细胞多于原方组(P < 0.05);与模型组相比,原方组与考察组中兔眼角膜及结膜中的上皮细胞得到修复,兔眼泪腺和房水中的炎症因子含量降低(P < 0.01),且考察组房水中IL-6的表达水平低于原方组(P < 0.05);与模型组相比,原方组与考察组兔眼角膜中的JNK/P38通路相关蛋白表达水平降低(P < 0.05,P<0.01)、MUC5AC表达水平升高(P < 0.05, P < 0.01)以及MMP-9表达水平降低(P < 0.05,P < 0.01)。同时,与原方组相比,考察组兔眼角膜中的P-P38/P38、P-P65/P65、MMP-9以及MUC5AC的改善效果更加明显(P < 0.05)。  结论  干眼症的发病机制可能与炎症通路具有相关性, 其中润目灵方中含有的金丝桃苷、木犀草苷以及绿原酸等成分可以增加兔眼泪液的分泌量, 维持泪膜的稳定性, 并有效改善眼表的损伤和抑制眼表炎症的表达, 从而达到治疗干眼症的效果。

     

  • 图  1  润目灵组方对兔眼泪液分泌量的影响

    注: 与空白对照组相比, ΔP < 0.05;与模型组相比, *P < 0.05, * *P < 0.01;与原方组相比, #P < 0.05, ##P < 0.01。x±s, n=4。

    Figure  1.  Effects of Runmuling formula on tear secretion in rabbit eyes

    图  2  润目灵组方对干眼症模型兔眼泪膜稳定性的影响

    注: 与空白对照组相比, ΔP < 0.05;与模型组相比, *P < 0.05。x±s, n=4。

    Figure  2.  Effects of Runmuling formula on tear film stability in rabbit xerophthalmia model

    图  3  造模第14天荧光素钠试纸染色不同程度的眼表损伤

    Figure  3.  Varying degrees of ocular surface damage after staining with sodium fluorescein test strip at d14 of modeling

    图  4  润目灵组方对干眼症模型兔眼角膜荧光染色评分的影响

    注: 与空白对照组相比, ΔP < 0.05, ΔΔP < 0.01;与模型组相比, *P < 0.05, * *P < 0.01。x±s, n=4。

    Figure  4.  Effects of Runmuling formula on scores of corneal fluorescence staining in rabbit xerophthalmia model

    图  5  润目灵组方对干眼症模型兔眼结膜中MUC5AC表达水平的影响

    注: 与空白对照组相比, ΔΔP < 0.01;与模型组相比, *P < 0.05, * *P < 0.01;与原方组相比, #P < 0.05。x±s, n=4。

    Figure  5.  Effects of Runmuling Formula on expression levels of conjunctival MUC5AC in rabbit xerophthalmia model

    图  6  润目灵组方对干眼症模型兔眼角膜、球结膜和睑结膜的影响

    Figure  6.  Effects of Runmuling formula on cornea, bulbar conjunctiva and palpebral conjunctiva in rabbit xerophthalmia model

    图  7  润目灵组方对干眼症模型兔眼泪腺、房水及哈氏腺中TNF-α及IL-6表达水平的影响

    注: 与空白对照组相比,△△P < 0.01;与模型组相比, *P < 0.05, * *P < 0.01;与原方组相比, #P < 0.05。x±s, n=4。

    Figure  7.  Effects of Runmuling formula on expression levels of TNF-α and IL-6 in lacrimal gland, aqueous humor, and Harderian gland in rabbit xerophthalmia model

    图  8  润目灵组方对干眼症模型兔眼角膜中JNK/P38通路相关蛋白表达水平的影响

    注: C.空白对照组;M.模型组;Y.原方组;K.考察组;与空白对照组相比, ΔΔΔP < 0.001; 与模型组相比, *P < 0.05, * *P < 0.01;与原方组相比, #P < 0.05。x±s, n=4。

    Figure  8.  Effects of Runmuling formula on expression levels of JNK/P38 pathway related proteins in cornea in rabbit xerophthalmia model

    图  9  润目灵组方对干眼症模型兔眼角膜中MMP-9和MUC5AC蛋白表达水平的影响

    注: C.空白对照组;M.模型组;Y.原方组;K.考察组;与空白对照组相比, ΔΔP < 0.01, ΔΔΔP < 0.001; 与模型组相比, *P < 0.05, * *P < 0.01;与原方组相比, #P < 0.05。x±s, n=4。

    Figure  9.  Effects of Runmuling formula on expression levels of MMP-9 and MUC5AC proteins in cornea in rabbit xerophthalmia model

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出版历程
  • 收稿日期:  2022-08-08
  • 网络出版日期:  2023-01-18
  • 发布日期:  2023-01-10

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