摘要
目的:探讨沿阶草酮甲(OPA)对干眼症(DED)小鼠的治疗作用及机制.方法:将小鼠分为5组:NC组(n=12)、DED组(n=14)、L-OPA组(n=14)、M-OPA组(n=14)和H-OPA组(n=14).NC组为正常对照小鼠,其他组小鼠为苯扎氯铵诱导的干眼症模型小鼠.NC组和DED组小鼠腹腔注射1％二甲基亚砜(DMSO),L-OPA组、M-OPA组和H-OPA组小鼠分别腹腔注射10、20和40mg/kg/d的OPA,共治疗4周.治疗结束后,检测各组小鼠的泪液分泌量,并进行角膜荧光素钠染色、结膜过碘酸-雪夫(PAS)染色和角膜TUNEL染色.通过ELISA法测定泪液中的白细胞介素-1β(IL-1β)、细胞间黏附分子-1(ICAM-1)和肿瘤坏死因子-α(TNF-α)水平.通过RT-qPCR检测角膜Bax和Bcl-2 mRNA水平.通过Western blot或免疫组化染色检测结膜K10和白细胞共同抗原(LCA)蛋白表达水平.结果:与NC组比较,DED组的泪液分泌量和泪膜破裂时间(BUT)降低(P＜0.05),角膜荧光素钠染色分级升高(P＜0.05),泪液IL-1β、ICAM-1和TNF-α水平均升高(P＜0.05),结膜杯状细胞数量降低(P＜0.05),角膜TUNEL阳性率和Bax mRNA相对表达量均升高(P＜0.05),角膜Bcl-2 mRNA相对表达量降低(P＜0.05),角膜K10蛋白相对表达量升高(P＜0.05),结膜LCA蛋白相对表达量升高(P＜0.05).与DED组比较,L-OPA组、M-OPA组和H-OPA组的泪液分泌量和BUT升高(P＜0.05),角膜荧光素钠染色分级降低(P＜0.05),泪液IL-1β、ICAM-1和TNF-α水平均降低(P＜0.05),结膜杯状细胞数量升高(P＜0.05),角膜TUNEL阳性率和Bax mRNA相对表达量均降低(P＜0.05),角膜Bcl-2 mRNA相对表达量升高(P＜0.05),角膜K10蛋白相对表达量降低(P＜0.05),结膜LCA蛋白相对表达量降低(P＜0.05).结论:沿阶草酮甲在治疗苯扎氯铵诱导的干眼症小鼠中效果显著,其机制可能与抑制白细胞介导的炎症反应以及促进杯状细胞增殖有关.
Abstract
Objective:To explore the therapeutic effect and mechanism of Ophiopogonanone A(OPA)on dry eye syndrome(DED)mice.Methods:Mice were divided into five groups:NC group(n=12),DED group(n=14),L-OPA group(n=14),M-OPA group(n=14)and H-OPA group(n=14).The NC group was normal control mice,and the other groups were mice with dry eye disease induced by benzalkonium chloride.Mice in NC group and DED group were intraperitoneally injected with 1％dimethyl sulfoxide(DMSO),while mice in L-OPA group,M-OPA group and H-OPA group were intraperitoneally injected with OPA of 10,20 and 40 mg/kg/d,respectively.The rats were treated for 4 weeks.After treatment,the tear secretion of each group was measured,and corneal fluorescein sodium staining,conjunctival Periodic Acid-Schiff stain(PAS)and corneal TUNEL staining were performed.The levels of interleukin-1β(IL-1β),intercellular adhesion molecule-1(ICAM-1)and tumor necrosis factor-α(TNF-α)in tears were measured by ELISA method.The levels of Bax and Bcl-2 mRNA in cornea were measured by RT-qPCR.The protein expression of conjunctival K10 and leukocyte common antigen(LCA)was detected by Western blot or immunohistochemical staining.Results:Compared with NC group,tear secretion and tear break-up time(BUT)decreased(P＜0.05),corneal fluorescein sodium staining grade increased(P＜0.05),tear IL-1β,ICAM-1 and TNF-α levels increased(P＜0.05),the number of conjunctival goblet cells decreased(P＜0.05),corneal TUNEL positive rate and Bax mRNA relative expression increased(P＜0.05),corneal Bcl-2 mRNA relative expression decreased(P＜0.05),corneal K10 protein relative expression increased and conjunctival LCA protein relative expression increased(P＜0.05).Compared with DED group,tear secretion and BUT in L-OPA,M-OPA and H-OPA groups increased(P＜0.05),corneal fluorescein sodium staining grade decreased(P＜0.05),tear IL-1β,ICAM-1 and TNF-α levels decreased(P＜0.05),conjunctival goblet cell number increased(P＜0.05),corneal TUNEL positive rate and Bax mRNA relative expression decreased(P＜0.05),corneal Bcl-2 mRNA relative expression increased(P＜0.05),corneal K10 protein relative expression decreased and conjunctival LCA protein relative expression decreased(P＜0.05).Conclusion:Ophiopogonanone A is effective in the treatment of benzalkonium chloride-induced dry eye disease in mice,and its mechanism may be related to the inhibition of leukocyte-mediated inflammation and the promotion of goblet cell proliferation.
基金项目
国家自然科学基金项目(82070964)
陕西省大学生创新创业训练计划项目(S202311840062)
西安医学院第二附属医院2022年度院级科研项目(22KY0111)
NETL
NSTL
万方数据