mTOR通路抑制剂雷帕霉素对真菌性角膜炎小鼠角膜瘢痕化的影响

Effect of mTOR pathway inhibitor Rapamycin on corneal scarring in mice with fungal keratitis

目的探讨mTOR通路抑制剂雷帕霉素对真菌性角膜炎小鼠角膜瘢痕化的影响。方法取96只SPF级C57BL/6J雄性小鼠,随机分为雷帕霉素组和对照组,每组各48只。两组小鼠同时建立真菌性角膜炎模型。雷帕霉素组模型制作前1 d按6.0 mg·kg-1雷帕霉素对小鼠进行腹腔注射预处理,之后按0.2 g·L^-1浓度在结膜下注射5μL,持续3 d;对照组注射PBS溶液。造模后对各组小鼠进行角膜临床评分,Western blot和实时荧光定量PCR分别检测造模后各组小鼠不同时间角膜LC-3Ⅱ、α-SMA和TGF-β1表达情况。结果造模后24 h、48 h、72 h、96 h、120 h、144 h、336 h,对照组小鼠角膜临床评分均明显高于雷帕霉素组,差异均有统计学意义(均为P<0.05)。造模后144 h、216 h雷帕霉素组小鼠角膜LC-3Ⅱ表达上调,LC-3Ⅱ蛋白和mRNA相对表达量与对照组相比,差异均有统计学意义(均为P<0.05);而在造模后72 h及336 h两组LC-3Ⅱ蛋白和mRNA相对表达量差异均无统计学意义(均为P>0.05)。与对照组相比,造模后144 h、216 h雷帕霉素组小鼠角膜α-SMA蛋白及mRNA相对表达量下降,差异均有统计学意义(均为P<0.05);造模后144 h、336 h雷帕霉素组TGF-β1 mRNA相对表达量亦下降,与对照组相比,差异均有统计学意义(均为P<0.05)。结论雷帕霉素通过促进自噬作用下调角膜瘢痕化相关因子的表达,减轻了真菌性角膜炎模型小鼠角膜瘢痕化程度。

Objective To investigate the effect of mTOR pathway inhibitor Rapamycin on corneal scarring in mice with fungal keratitis.Methods Ninety-six male SPF C57BL/6J mice were randomly divided into Rapamycin group and control group,48 mice in each group.Fungal keratitis models were established in mice of both groups.The mice in the Rapamycin group were pretreated with 6.0 mg·kg-1 Rapamycin intraperitoneally the day before modeling,and then injected with 5μL subconjunctivally at a concentration of 0.2 g·L^-1 for 3 days;mice in control group received injection of PBS solution.The corneal clinical scores were recorded for mice in each group after modeling,and the expressions of LC-3Ⅱ,α-SMA and TGF-β1 at different times in each group were detected by Western blot and real-time quantitative RT-PCR.Results The corneal clinical scores in the control mice were significantly higher than those in the Rapamycin group at 24 h,48 h,72 h,96 h,120 h,144 h,and 336 h after modeling,and the differences were statistically significant(all P<0.05).Compared with the control group,the corneal LC-3Ⅱexpression was up-regulated,and differences were found in the relative expressions of LC-3Ⅱprotein and mRNA in Rapamycin group at 144 h and 216 h after modeling(all P<0.05).How-ever,there was no significant difference in the relative expression of LC-3Ⅱprotein and mRNA between the two groups at 72 h and 336 h after modeling(both P>0.05).Compared with the control group,the relative expression levels ofα-SMA protein and mRNA in the cornea of mice in Rapamycin group decreased at 144 h and 216 h after modeling,and the differences were statistically significant(both P<0.05).The relative expression levels of TGF-β1 mRNA in the Rapamycin group also decreased at 144 h and 336 h after modeling,and the differences were statistically significant compared with the control group(both P<0.05).Conclusion Rapamycin can reduce the scarring degree of fungal keratitis mice by promoting autophagy and down-regulating the expression of scarring-related factors.