白藜芦醇对碱烧伤小鼠角膜纤维化反应的抑制作用

目的　探索白藜芦醇（RSV）对碱烧伤小鼠角膜纤维化反应的抑制作用。方法　选取SPF级、健康6~8周龄雄性C57BL/6J小鼠48只，按照随机数字表法分为正常对照组、模型组和RSV组，每组16只。模型组和RSV组小鼠右眼建立角膜碱烧伤模型，左眼不做任何处理。RSV组小鼠用2 g·L^-1RSV溶液滴眼，模型组小鼠用相同剂量的羟丙基-β-环糊精溶液滴眼，连续滴眼 21 d。正常对照组小鼠不做碱烧伤和滴眼干预。碱烧伤后21 d，裂隙灯显微镜观察各组小鼠角膜混浊情况，对角膜混浊程度进行评分；通过HE染色观察各组小鼠角膜组织结构变化；采用免疫荧光染色观察各组小鼠角膜组织中α平滑肌肌动蛋白（α-SMA）和Ⅲ型胶原纤维蛋白α1链（COL3A1）的表达；利用Western blot检测各组小鼠角膜组织中转化生长因子-β1（TGF-β1）、α-SMA、COL3A1蛋白相对表达水平。结果　碱烧伤后21 d，RSV组小鼠角膜混浊评分为（2.25±0.89）分，明显低于模型组［（3.25±0.71）分］，差异有统计学意义（P=0.026）。HE染色结果显示，模型组小鼠角膜基质中细胞增多，给予RSV干预后细胞相对减少。免疫荧光染色结果显示，RSV组小鼠角膜组织中α-SMA和COL3A1的表达均弱于模型组。Western blot检测结果显示,RSV组小鼠角膜组织中TGF-β1（0.60±0.17）、α-SMA（0.27±0.05）、COL3A1（0.49±0.21）蛋白相对表达水平均显著低于模型组［TGF-β1（1.48±0.50)、α-SMA(0.55±0.18)、COL3A1(1.32±0.87）］，差异均有统计学意义（均为P＜0.05）。结论　在小鼠角膜碱烧伤中，RSV可以有效抑制角膜组织中TGF-β1的表达及肌成纤维细胞的活化，减少COL3A1沉积，从而抑制角膜的纤维化反应，进而减轻角膜混浊程度。

Inhibition of resveratrol on corneal fibrosis in alkali-burned mice

Objective　To investigate the inhibitory effect of resveratrol (RSV) on corneal fibrosis in alkali-burned mice.Methods　Totally forty-eight SPF healthy 6-8 week-aged male C57BL/6J mice were randomly divided into normal control group, model group and RSV group, with 16 mice in each group. The alkali burn model was established in the right eye of mice in the model group and RSV group, and the left eye was not treated. The mice in RSV group were treated with 2 g·L^-1 RSV eye drops and those in model group were treated with the same dose hydroxypropyl-β-cyclodextrin eye drops for 21 d. The normal control group was not intervened by alkali burn model or eye drops. At 21 d after corneal alkali burn, corneal opacity condition was observed by slit-lamp microscope and the degree of corneal opacity was scored. The changes in corneal tissue structure were observed by hematoxylin-eosin (HE) staining. Immunofluorescence staining was used to evaluate the expression of α smooth muscle actin (α-SMA) and collagen type Ⅲ Alpha 1 Chain (COL3A1) in corneal tissue，and Western blot was used to evaluate the relative expression levels of transforming growth factor-β1 (TGF-β1), α-SMA and COL3A1. Results　At 21 d after corneal alkali burn, the corneal opacity score of RSV group was (2.25±0.89) scores, which was significantly lower than that in model group［(3.25±0.71) scores］, and the difference was statistically significant (P=0.026). HE staining results showed that the cells in corneal stroma increased in model group and decreased after RSV intervention. Immunofluorescence staining results showed that the expression levels of α-SMA and COL3A1 in corneal tissue of RSV group were lower than those in the model group. In addition, the relative protein expression level detected by Western blot showed that the TGF-β1 in the RSV group was 0.60±0.17, which was significantly lower than that in model group (1.48±0.50); the α-SMA in RSV group was 0.27±0.05, which was significantly lower than that in model group (0.55±0.18); and the COL3A1 in RSV group was 0.49±0.21, which was significantly lower than that in model group (1.32±0.87), and the differences were statistically significant (all P<0.05).Conclusion　RSV can effectively inhibit the expression of TGF-β1, the activation of myofibroblasts and reduce the deposition of COL3A1 in alkali-burned corneal tissue, thereby inhibiting corneal fibrosis and reducing corneal opacity.