NADPH氧化酶4在1型糖尿病模型小鼠角膜病变中的致病作用及其机制  

Pathogenic role and mechanism of NADPH oxidase 4 in type 1 diabetic keratopathy mouse

作　　者：赵文心 张弦 覃亚周 张明[1] 高宁[1] 秦莉[1] 李晶明[1] Zhao Wenxin;Zhang Xian;Qin Yazhou;Zhang Ming;Gao Ning;Qin Li;Li Jingming(Department of Ophthalmology,First Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710061,China;Nanchang Bright Eye Hospital,Nanchang 330029,China)

机构地区：[1]西安交通大学第一附属医院眼科,西安710061 [2]南昌普瑞眼科医院,南昌330029

出　　处：《中华实验眼科杂志》2024年第7期602-612,共11页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金(81740158、81460163、81300786、82000862);陕西省重点研发计划一般项目(2024SF-YBXM-322、2021SF-156);陕西省青年科技新星项目(2016KJXX-12);陕西省自然科学基金(2016JM8029);西安交通大学第一附属医院科研发展基金(HX201970、2021ZXY-10、2022QN-25)。

摘　　要：目的探讨还原型烟酰胺腺嘌呤二核苷酸氧化酶4(Nox4)在1型DM模型小鼠角膜病变中的致病作用及其可能机制。方法选择Nox4基因敲除(Nox4^(-/-))纯合子雄性小鼠40只,以鼠龄、性别匹配的野生型C57BL/6(Nox4^(+/+))小鼠120只作为对照。采用随机数字表法分别将2种小鼠随机分为DM组和非DM组,DM组小鼠采用链脲佐菌素腹腔内注射法构建1型DM模型。采用随机数字表法分别将Nox4^(+/+)小鼠DM组和非DM组分为普通饲料喂养小鼠和添加Nox4抑制剂GKT137831(GKT)饲料喂养小鼠。于DM造模后第16周采用酚红棉线法检测各组小鼠泪液分泌量;采用荧光素钠染色评分法评估角膜上皮完整性;采用激光扫描共聚焦显微镜观察角膜基质层神经纤维密度变化;采用CellROX荧光探针检测角膜上皮中活性氧簇(ROS)含量;采用免疫荧光染色法检测小鼠角膜上皮中E-Cadherin蛋白和核因子-κB(NF-κB)蛋白表达变化;采用角膜铺片TUBB3染色法检测角膜中央区神经纤维密度。结果Nox4^(+/+)小鼠DM组和非DM组泪液分泌量分别为(2.40±1.18)和(5.30±1.02)mm/min,差异有统计学意义(P<0.01);Nox4^(-/-)小鼠DM组泪液分泌量为(4.19±0.63)mm/min,明显多于Nox4^(+/+)小鼠DM组,差异有统计学意义(P<0.05);普通饲料喂养小鼠与GKT添加饲料喂养小鼠DM组泪液分泌量分别为(2.23±0.83)和(4.02±0.71)mm/min,差异有统计学意义(P<0.01)。与Nox4^(+/+)小鼠非DM组比较,Nox4^(+/+)小鼠DM组角膜荧光素染色评分显著升高,角膜神经纤维密度显著降低,角膜上皮中ROS荧光强度明显增强,E-Cadherin蛋白表达荧光强度减弱,NF-κB蛋白表达荧光强度增强。Nox4^(-/-)或GKT添加饲料喂养小鼠DM组与非DM组比较角膜上皮中ROS荧光增强,E-Cadherin蛋白表达荧光减弱。Nox4^(-/-)和GKT添加饲料喂养小鼠DM组角膜上皮细胞中NF-κB蛋白荧光强度均较弱,与非DM组强度一致。角膜铺片免疫荧光染色显示,Nox4^(+/+)小鼠DM组中TUBB3染�Objective To investigate the pathogenic role and possible mechanism of NADPH oxidase 4(Nox4)in type 1 diabetic keratopathy mouse models.Methods Forty Nox4 knockout(Nox4^(-/-))heterozygous male mice were selected and 120 age-and sex-matched wild-type C57BL/6(Nox4^(+/+))mice were selected as controls.Nox4^(-/-)and Nox4^(+/+)mice were randomized into diabetic group(DM group)and non-DM group by random number method.Type 1 DM model was established in DM groups by intraperitoneal injection of streptozotocin.The DM and non-DM groups of Nox4^(+/+)mice were randomized into regular feed group and Nox4 inhibitor GKT137831(GKT)supplementary feed group by random number method.At 16 weeks after modeling,tear secretion of mice in different groups was measured by the phenol red thread test.Corneal epithelial integrity was evaluated by fluorescent staining.Changes in corneal never fiber density were observed by the in vivo laser scanning confocal microscopy.Reactive oxygen species(ROS)products in corneal epithelium were assayed by CellROX staining.The expressions of E-Cadherin and nuclear factor-κB(NF-κB)proteins were detected by immunofluorescence staining.Central corneal nerve fiber density was examined by flatmount staining with TUBB3 antibody.The use and care of laboratory animals complied with ARVO statement.The study protocol was approved by Laboratory Animal Care Committee of Xi'an Jiaotong University(No.XJTULAC201301).Results In Nox4^(+/+)mice,the tear secretion was(2.40±1.18)mm/minute in DM group,which was significantly less than(5.30±1.02)mm/minute in non-DM group(P<0.01).The tear secretion was(4.19±0.63)mm/minute in DM group of Nox4^(-/-)mice,which was significantly more than that in DM group of Nox4^(+/+)mice(P<0.05).Significant difference was found between(2.23±0.83)mm/minute of regular feed group and(4.02±0.71)mm/minute of GKT supplementary feed group(P<0.01).In Nox4^(+/+)mice,the DM group showed significantly increased corneal staining score,reduced corneal nerve fiber density,increased fluorescence intensit

关 键 词：糖尿病/并发症 角膜病变 还原型烟酰胺腺嘌呤二核苷酸氧化酶 氧化应激 酶抑制剂/治疗作用 疾病模型 近交系C57BL小鼠 

分 类 号：R-332[医药卫生] R587.2R772.2