STAT3抑制剂改善小鼠干眼症的机制研究

Investigation on the Mechanism of STAT3 Inhibitor on Mice with Dry Eye

目的探讨信号转导与转录激活因子(signal transducer and activator of transcription 3,STAT3)抑制剂对小鼠干眼症的作用及其机制。方法采用随机分组对照研究的方法,将36只C57BL/6J小鼠随机分为3组,即对照组、模型组和实验组,每组12只。模型组和实验组小鼠双眼结膜囊内滴入5μl 0.2%苯扎溴铵(benzalkonium chloride,BAC)溶液,连续14天,每天2次,构建干眼症模型。造模结束后第2天,对照组和模型组小鼠结膜囊内滴入5μl磷酸盐缓冲液,实验组滴入5μl STAT3抑制剂,持续7天,每天3次。治疗结束后,小鼠行角膜荧光素染色评分、酚红棉试验和泪膜破裂时间及糖原染色分别对角膜上皮屏障功能、泪液分泌和结膜杯状细胞密度进行定量。实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测小鼠眼表组织中炎性细胞因子水平;Western blot法检测各组小鼠眼表组织总蛋白和细胞核内磷酸化的STAT3(p-STAT3)表达及微管相关蛋白1轻链(light chain 3,LC3)表达。结果与对照组比较,模型组小鼠角膜屏障功能破坏、泪液体积减少、泪膜破裂时间缩短、结膜中杯状细胞减少;眼表组织中IL-1β、IL-6、γ-干扰素(γ-Interferon,IFN)和肿瘤坏死因子-α(tumor necrosis factor,TNF-α)的mRNA表达均增加;胞质及细胞核内p-STAT3表达明显增高,LC3B-Ⅱ/LC3B-Ⅰ也明显增高。比较模型组,实验组小鼠角膜屏障功能有一定恢复,泪液体积和泪膜破裂时间均明显增加;上述炎性细胞因子水平均显著下降;p-STAT3表达和LC3B-Ⅱ/LC3B-Ⅰ均显著下降。结论STAT3激活参与干眼症的发病,STAT3抑制剂可以通过抑制眼表组织炎症和过度自噬治疗干眼症。

Objective To investigate the effect and mechanism of STAT3 inhibitor on dry eye in mice.Methods Thirty-six C57BL/6J mice were randomly divided into 3groups:control group,model group and experimental group,with 12mice in each group.Mice in model group and experimental group were given 5μl 0.2%BAC solution in conjunctival sac of both eyes for 14days,twice a day,to make dry eye model.After treatment,corneal epithelial barrier function,tear secretion and conjunctival goblet cell density were quantified by fluorescein staining score,phenolic cotton test,beak-up time(BUT)of tear film and Periodic Acid-Schiff stain,respectively.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect inflammatory factors in mouse ocular surface tissues.The expression of p-STAT3 in total protein and in the nucleus and LC3 protein were detected by Western blot.Results Compared with the control group,the mice in model group showed damaged corneal barrier function,reduced tear volume,shortened BUT and decreased Goblet cells were reduced in the conjunctiva.The mRNA levels of IL-1β,IL-6,γ-IFN and TNF-αin conjunctival tissue increased.The expression of p-STAT3 in cytoplasm and in the nucleus increased significantly and the ratio of LC3B-Ⅱ/LC3B-Ⅰalso increased.Compared with model group,corneal barrier function of mice in experimental group was restored to some extent,tear volume and tear film BUT increased significantly;the levels of the above inflammatory factors decreased significantly.The expression of p-STAT3 and LC3B-Ⅱ/LC3B-Ⅰratio decreased significantly.Conclusion STAT3 activation is involved in the pathogenesis of dry eye,and STAT3 inhibitors can treat dry eye by inhibiting inflammation and excessive autophagy in ocular surface tissues.