中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
9期
791-796
,共6页
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
角膜炎/治療%人體血栓調節蛋白%活性生物肽GC31%炎性因子%動物模型%抗炎藥物%脂多糖
각막염/치료%인체혈전조절단백%활성생물태GC31%염성인자%동물모형%항염약물%지다당
Corneal inflammation/treatment%Thrombomodulin%Peptide GC31%Inflammatory factor%Disease model/animal%Anti-inflammation drug%Lipopolysaccharide
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.
揹景 人體血栓調節蛋白中篩選的生物肽GC31具有天然抗炎活性,相比傳統的眼部抗炎藥物具有優勢及臨床轉化價值,但目前對其具體的抗炎價值評估研究較少. 目的 研究GC31對脂多糖(LPS)誘導的非特異性角膜炎的抑製作用及可能機製.方法 按隨機數字錶法將SPF級8～10週齡雄性Wistar大鼠60隻隨機分為6箇組.用于角膜基質內註射10 μl LPS(2g/L,溶于PBS)的方法誘導大鼠非特異性角膜炎模型,角膜中央齣現雲霧狀混濁為造模成功,然後于GC31低劑量組、GC31高劑量組、地塞米鬆組大鼠結膜下分彆註射125 μg GC31、250 μg GC31和250 μg地塞米鬆(均溶于25 μlPBS),PBS對照組大鼠以同樣方式註射PBS,而空白對照組大鼠不給予任何榦預.註射後24 h裂隙燈顯微鏡下觀察大鼠角膜炎癥錶現,併按照Anand的標準進行炎癥評分,然後摘除大鼠眼毬,對角膜組織進行組織病理學觀察;採用免疫組織化學法檢測各組大鼠角膜中覈因子-KB(NF-KB) p65的錶達;用ELISA法檢測各組大鼠角膜組織中白細胞介素-6(IL-6)和腫瘤壞死因子-α (TNF-α)蛋白的變化;採用實時熒光定量PCR(real-time PCR)法檢測各組大鼠角膜組織中IL-6 mRNA、TNF-α mRNA的錶達量. 結果 空白對照組、PBS對照組、GC31低劑量組、GC31高劑量組和地塞米鬆組大鼠眼部炎癥評分差異有統計學意義(F=301.238,P=0.000),其中GC31高劑量組大鼠角膜炎癥評分為1.85±0.36,明顯低于模型組的2.90±0.43,差異有統計學意義(t'=-5.144,P=0.000),地塞米鬆組大鼠角膜炎癥評分為1.28±0.36,低于GC31高劑量組,差異有統計學意義(t'=-3.931,P=0.000).角膜組織病理學檢查顯示,GC31高劑量組和地塞米鬆組大鼠角膜組織炎性細胞浸潤較模型組明顯減輕;免疫組織化學法檢測顯示,GC31低劑量組、GC31高劑量組以及地塞米鬆組大鼠角膜組織中NF-κB p65暘性細胞較模型組大鼠減少;ELISA法測定錶明,模型組大鼠角膜中IL-6及TNF-α蛋白質量濃度均明顯升高,其中IL-6升高近3 000倍,而GC31高劑量組角膜中IL-6及TNF-α蛋白質量濃度明顯低于模型組,差異均有統計學意義(t=-3.361,P=0.001;t'=-3.151,P=0.002),GC31低劑量組角膜中IL-6與TNF-α蛋白質量濃度低于模型組,差異均有統計學意義(t=-2.626,P=0.009;t'=-2.310,P=0.017),地塞米鬆組大鼠角膜中IL-6及TNF-α蛋白質量濃度明顯低于GC31高劑量組,差異均有統計學意義(t=-3.361,P=0.001;t'=-3.360,P=0.000).此外,實時定量PCR法檢測錶明各組大鼠IL-6 mRNA及TNF-α mRNA的錶達和比較趨勢與其蛋白錶達相同,差異均有統計學意義(P＜0.01). 結論 活性生物肽GC31對LPS誘導的非特異性角膜炎具有抑製作用,250μg GC31抑製炎癥反應的作用明顯彊于125 μg GC31,其作用機製主要是下調組織中炎性因子的錶達.
배경 인체혈전조절단백중사선적생물태GC31구유천연항염활성,상비전통적안부항염약물구유우세급림상전화개치,단목전대기구체적항염개치평고연구교소. 목적 연구GC31대지다당(LPS)유도적비특이성각막염적억제작용급가능궤제.방법 안수궤수자표법장SPF급8～10주령웅성Wistar대서60지수궤분위6개조.용우각막기질내주사10 μl LPS(2g/L,용우PBS)적방법유도대서비특이성각막염모형,각막중앙출현운무상혼탁위조모성공,연후우GC31저제량조、GC31고제량조、지새미송조대서결막하분별주사125 μg GC31、250 μg GC31화250 μg지새미송(균용우25 μlPBS),PBS대조조대서이동양방식주사PBS,이공백대조조대서불급여임하간예.주사후24 h렬극등현미경하관찰대서각막염증표현,병안조Anand적표준진행염증평분,연후적제대서안구,대각막조직진행조직병이학관찰;채용면역조직화학법검측각조대서각막중핵인자-KB(NF-KB) p65적표체;용ELISA법검측각조대서각막조직중백세포개소-6(IL-6)화종류배사인자-α (TNF-α)단백적변화;채용실시형광정량PCR(real-time PCR)법검측각조대서각막조직중IL-6 mRNA、TNF-α mRNA적표체량. 결과 공백대조조、PBS대조조、GC31저제량조、GC31고제량조화지새미송조대서안부염증평분차이유통계학의의(F=301.238,P=0.000),기중GC31고제량조대서각막염증평분위1.85±0.36,명현저우모형조적2.90±0.43,차이유통계학의의(t'=-5.144,P=0.000),지새미송조대서각막염증평분위1.28±0.36,저우GC31고제량조,차이유통계학의의(t'=-3.931,P=0.000).각막조직병이학검사현시,GC31고제량조화지새미송조대서각막조직염성세포침윤교모형조명현감경;면역조직화학법검측현시,GC31저제량조、GC31고제량조이급지새미송조대서각막조직중NF-κB p65양성세포교모형조대서감소;ELISA법측정표명,모형조대서각막중IL-6급TNF-α단백질량농도균명현승고,기중IL-6승고근3 000배,이GC31고제량조각막중IL-6급TNF-α단백질량농도명현저우모형조,차이균유통계학의의(t=-3.361,P=0.001;t'=-3.151,P=0.002),GC31저제량조각막중IL-6여TNF-α단백질량농도저우모형조,차이균유통계학의의(t=-2.626,P=0.009;t'=-2.310,P=0.017),지새미송조대서각막중IL-6급TNF-α단백질량농도명현저우GC31고제량조,차이균유통계학의의(t=-3.361,P=0.001;t'=-3.360,P=0.000).차외,실시정량PCR법검측표명각조대서IL-6 mRNA급TNF-α mRNA적표체화비교추세여기단백표체상동,차이균유통계학의의(P＜0.01). 결론 활성생물태GC31대LPS유도적비특이성각막염구유억제작용,250μg GC31억제염증반응적작용명현강우125 μg GC31,기작용궤제주요시하조조직중염성인자적표체.
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.
