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李玲,李点,张婷,姚蕾,李炎.养阴润目丸对苯扎氯铵诱导的干眼兔模型结膜上皮细胞焦亡的影响[J].湖南中医药大学学报,2023,43(9):1570-1575[点击复制] |
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| 养阴润目丸对苯扎氯铵诱导的干眼兔模型结膜上皮细胞焦亡的影响 |
| 李玲,李点,张婷,姚蕾,李炎 |
| (湖南中医药大学中医学院, 湖南 长沙 410208;湖南中医药大学第一附属医院, 湖南 长沙 410007) |
| 摘要: |
| 目的 研究养阴润目丸对干眼兔结膜上皮细胞焦亡的调控作用。方法 将40只雌雄各半的健康普通级新西兰兔采用随机数字表法，取8只作为空白组，剩余32只均采用双眼局部滴注0.1%苯扎氯铵溶液制备干眼模型，模型成功后随机分为空白组，模型组，养阴润目丸低、中、高剂量组，每组8只。养阴润目丸低、中、高剂量组分别按剂量0.435、0.869、1.738 g·(kg·d)-1给予养阴润目丸水溶液灌胃。连续灌胃2周后，分别进行泪液分泌试验（Schirmer's test, SIT）、泪膜破裂时间（break up time, BUT）和角膜荧光素染色评分；取兔结膜上皮细胞，透射电镜下观察结膜上皮的细胞形态；Western blot检测结膜中NOD样受体家族核苷酸结合寡聚化结构域样受体蛋白3（NOD-like receptor family pyrin domain-containing protein 3, NLRP3）、半胱氨酸天冬氨酸特异性蛋白酶-1（cysteine aspartate-specific proteinase-1, Caspase-1）、消化道皮肤素D（Gasdermin D, GSDMD）蛋白表达；ELISA法检测兔血清中炎症因子白细胞介素-1β（interleukin-1β, IL-1β）、白细胞介素-18（interleukin-18, IL-18）含量。结果 与空白组相比，模型组实验兔SIT明显减少、泪膜BUT明显缩短、角膜荧光素染色评分明显升高（P<0.01，P<0.05）；兔结膜上皮细胞超微结构可见明显肿胀、变形、破裂、皱缩等细胞损伤；兔结膜上皮细胞中NLRP3、Caspase-1、GSDMD的蛋白表达明显升高（P<0.05）；兔血清中IL-1β、IL-18含量明显增多（P<0.05）。与模型组相比，养阴润目丸各剂量组SIT明显增多、泪膜BUT明显延长、角膜荧光素染色评分明显降低（P<0.05）；兔结膜上皮细胞中NLRP3、Caspase-1、GSDMD的蛋白表达明显降低（P<0.05）；兔血清中IL-1β、IL-18含量明显减少（P<0.05）。结论 养阴润目丸可以通过降低干眼兔结膜上皮细胞中NLRP3、Caspase-1、GSDMD蛋白表达，抑制结膜上皮细胞发生焦亡，减少炎症细胞因子IL-1β、IL-18分泌，从而阻断相关级联反应，起到增加泪液分泌量、延长泪膜BUT等作用，最终治疗苯扎氯铵诱导的干眼。 |
| 关键词: 干眼 养阴润目丸 苯扎氯铵 结膜上皮细胞 细胞焦亡 |
| DOI：10.3969/j.issn.1674-070X.2023.09.004 |
| 投稿时间：2023-03-01 |
| 基金项目:国家自然科学基金面上项目（81674030）；教育部产学合作协同育人项目（22100443904328）。 |
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| Effects of Yangyin Runmu Pill on pyroptosis of conjunctival epithelial cell induced by benzalkonium chloride in a rabbit dry-eye model |
| LI Ling,LI Dian,ZHANG Ting,YAO Lei,LI Yan |
| (College of Chinese Medicine, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;The First Hospital of Hunan University of Chinese medicine, Changsha, Hunan 410007, China) |
| Abstract: |
| Objective To study the effects of Yangyin Runmu Pill (YYRMP) on pyroptosis of conjunctival epithelial cells in dry-eye rabbits. Methods Eight rabbits were randomly selected among 20 male and 20 female New Zealand rabbits as the blank group. The remaining 32 rabbits were used to prepare the dry-eye models by local infusion of 0.1% benzalkonium chloride solution into both eyes. After successful modeling, they were randomly subdivided into blank group, model group, and low-, medium-, and high- dose YYRMP groups, with 8 rabbits in each group. The low-, medium-, and high-dose YYRMP groups were treated with YYRMP solution by gavage at dosages of 0.435, 0.869, and 1.738 g·(kg·d)-1, respectively. After continuous gavage for 2 weeks, Schirmer's test (SIT), break up time (BUT), and corneal fluorescence staining score were performed respectively; rabbit conjunctival epithelial cells were observed by transmission electron microscopy. The expressions of NOD-like receptor family pyrin domain-containing protein 3 (NLRP3), cysteine aspartate-specific proteinase-1 (Caspase-1), and Gasdermin D (GSDMD) protein in the conjunctiva were checked by Western blot; the content of serum inflammatory factors interleukin-1β (IL-1β) and interleukin-18 (IL-18) in rabbit serum was determined by ELISA. Results Compared with the blank group, the experimental rabbits in the model group showed significantly lower SIT, shorter tear film BUT, and higher fluorescence staining scores (P<0.01, P<0.05); the ultrastructure of rabbit conjunctival epithelial cells showed obvious swelling, deformation, rupture, shrinkage, and other cellular damage; the protein expressions of NLRP3, Caspase-1, and GSDMD and the content of IL-1β, IL-18 increased (P<0.05). Compared with the model group, the experimental rabbits in YYRMP groups showed higher SIT, longer tear film BUT, and lower fluorescence staining scores (P<0.05); the protein expressions of NLRP3, Caspase-1, and GSDMD in rabbit conjunctival epithelial cells were reduced (P<0.05); the content of IL-1β and IL-18 decreased (P<0.05). Conclusion YYRMP can reduce the expressions of NLRP3, Caspase-1, and GSDMD proteins in conjunctival epithelial cells of dry-eye rabbits, inhibit conjunctival epithelial cell apoptosis, and reduce the secretion of IL-1β and IL-18, thereby blocking related cascade reactions. YYRMP increases tear secretion, prolongs tear film BUT, and ultimately treats dry eyes induced by benzalkonium chloride. |
| Key words: dry eye Yangyin Runmu Pill benzalkonium chloride conjunctival epithelial cells pyroptosis |
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