新型生物肽GC31对脂多糖诱导的非特异性角膜炎的抑制作用  

Inhibitory effect of a novel peptide GC31 on lipopolysaccharide-induced corneal inflammation

作　　者：朱邵品 金慧昳 杨晓璐[1] 夏欣[1] 许迅[1] 

机构地区：[1]上海交通大学附属第一人民医院眼科,200080

出　　处：《中华实验眼科杂志》2014年第9期791-796,共6页Chinese Journal Of Experimental Ophthalmology

基　　金：“十二五”国家科技重大专项项目（2011ZX09302-007-02）;国家自然科学基金项目（81273424、81170862、81300753）

摘　　要：背景 人体血栓调节蛋白中筛选的生物肽GC31具有天然抗炎活性,相比传统的眼部抗炎药物具有优势及临床转化价值,但目前对其具体的抗炎价值评估研究较少. 目的 研究GC31对脂多糖（LPS）诱导的非特异性角膜炎的抑制作用及可能机制.方法 按随机数字表法将SPF级8～10周龄雄性Wistar大鼠60只随机分为6个组.用于角膜基质内注射10 μl LPS（2g/L,溶于PBS）的方法诱导大鼠非特异性角膜炎模型,角膜中央出现云雾状混浊为造模成功,然后于GC31低剂量组、GC31高剂量组、地塞米松组大鼠结膜下分别注射125 μg GC31、250 μg GC31和250 μg地塞米松（均溶于25 μlPBS）,PBS对照组大鼠以同样方式注射PBS,而空白对照组大鼠不给予任何干预.注射后24 h裂隙灯显微镜下观察大鼠角膜炎症表现,并按照Anand的标准进行炎症评分,然后摘除大鼠眼球,对角膜组织进行组织病理学观察;采用免疫组织化学法检测各组大鼠角膜中核因子-KB（NF-KB） p65的表达;用ELISA法检测各组大鼠角膜组织中白细胞介素-6（IL-6）和肿瘤坏死因子-α （TNF-α）蛋白的变化;采用实时荧光定量PCR（real-time PCR）法检测各组大鼠角膜组织中IL-6 mRNA、TNF-α mRNA的表达量. 结果 空白对照组、PBS对照组、GC31低剂量组、GC31高剂量组和地塞米松组大鼠眼部炎症评分差异有统计学意义（F=301.238,P=0.000）,其中GC31高剂量组大鼠角膜炎症评分为1.85±0.36,明显低于模型组的2.90±0.43,差异有统计学意义（t＇=-5.144,P=0.000）,地塞米松组大鼠角膜炎症评分为1.28±0.36,低于GC31高剂量组,差异有统计学意义（t＇=-3.931,P=0.000）.角膜组织病理学检查显示,GC31高剂量组和地塞米松组大鼠角膜组织炎性细胞浸润较模型组明显减轻;免疫组织化学法检测显示,GC31低剂量组、GC31高剂量组以及地塞米松组大鼠角膜组织中NF-κB p65阳性细胞较模型组大鼠减少;ELISA法测定Background Most anti-inflammation eyedrops are limited in clinical application owing to multiple adverse effects.A novel peptide GC31 derived from human thrombomodulin has a natural anti-inflammatory activity.Compared with conventional anti-inflammatory eyedrops,GC31 possesses more advantages and potential clinical transforming value.However,relevant study is still lack.Objective The purpose of this study was to evaluate the anti-inflammatory effect of GC31 and the possible mechanisms.Methods Sixty SPF male Wistar rats aged 8-10 weeks were randomized into 6 groups using randomized number table.Non-specific keratitis models were established in 40 rats by intrastromal injection of 10 μl of lipopolysaccharide （LPS） dissolved in PBS.Different doses of GC31 （125 μg or 250 μg） or dexamethason soluble in PBS were sunconjunctically injected in the experimental eyes respectively in the low dose GC31 group,high dose of GC31 group and the dexamethason group,and 10 μl of PBS was used in the same way in the PBS control group.No drug was injected in the model group,and the normal rats were employed as the blank control group.The corneas were examined by slit lamp microscope and were scored based on the criteria of Anand 24 hours after injection.Then the corneas were collected for histopathological examination.Expression of nuclear factor-κB （NF-κB） p65 in the corneas was detected using immunochemistry.Expressions of interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） proteins were assayed using ELISA.Real-time PCR was used to detect the expressions of IL-6 mRNA and TNF-α mRNA.The use and care of the experimental animals followed Regulation for the Administration of Affair Concerning Experiment animals by State Science and Techonology Commission.Results A significant difference was seen in the ocular inflammatory scores among the six groups （F =301.238,P =0.000）.The inflammatory scores were significantly lower in the high dose of GC31 group than those in the model group （1.85 ± 0.36 vers

关 键 词：角膜炎 治疗 人体血栓调节蛋白 活性生物肽GC31 炎性因子 动物模型 抗炎药物 脂多糖 

分 类 号：R772.21[医药卫生—眼科]