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

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法测定表明,模型组大鼠角膜中IL-6及TNF-α蛋白质量浓度均明显升高,其中IL-6升高近3 000倍,而GC31高剂量组角膜中IL-6及TNF-α蛋白质量浓度明显低于模型组,差异均有统计学意义（t=-3.361,P=0.001;t＇=-3.151,P=0.002）,GC31低剂量组角膜中IL-6与TNF-α蛋白质量浓度低于模型组,差异均有统计学意义（t=-2.626,P=0.009;t＇=-2.310,P=0.017）,地塞米松组大鼠角膜中IL-6及TNF-α蛋白质量浓度明显低于GC31高剂量组,差异均有统计学意义（t=-3.361,P=0.001;t＇=-3.360,P=0.000）.此外,实时定量PCR法检测表明各组大鼠IL-6 mRNA及TNF-α mRNA的表达和比较趋势与其蛋白表达相同,差异均有统计学意义（P＜0.01）. 结论 活性生物肽GC31对LPS诱导的非特异性角膜炎具有抑制作用,250μg GC31抑制炎症反应的作用明显强于125 μg GC31,其作用机制主要是下调组织中炎性因子的表达.

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 versus 2.90± 0.43） （t＇ =-5.144,P =0.000） ; and the scores in the dexamethason group was lower than those in the high dose of GC31 group（t＇ =-3.931,P=0.000）.Infiltration of inflammatory cells in corneal tissue was milder in the high dose of GC31 and the dexamethason group compared with the model group.The positive response for NF-κB p65 was obviously weaker in the rat corneas in the low and high dose of GC31 groups and the dexamethason group in comparison with the model group.The contents of IL-6 and TNF-α proteins in the corneas were significantly reduced in the low and high dose of GC31 group and the dexamethason group compared with the model group （low dose group：t=-2.626,P=0.009;t＇=-2.310,P=0.017.high dose group：t =-3.361,P=0.001 ;t＇=-3.151,P=0.002）,and the contents of IL-6 and TNF-α proteins in the dexamethason group were lower than those in the high dose of GC31 group （t=-3.361,P=0.001;t＇=-3.360,P=0.000）.In addition,the expression trend and compared results of IL-6 mRNA and TNF-α mRNA among the groups were similar to those of the IL-6 and TNF-α proteins （all at P〈0.01）.Conclusions GC31 suppresses LPS-induced corneal inflammation response by downregulating the expression of inflammatory eytokines.The effect is more dominant in the doses of 250 μg than that in the doses of 125 μg.