乙酰唑胺对圆锥角膜成纤维细胞胶原代谢的影响

Effects of Acetazolamide on Collagen Metabolism in Keratoconus Fibroblasts

圆锥角膜是以角膜中央变薄、向前突出成圆锥形为特征的一种角膜疾病,胶原代谢异常是其发病原因之一。通过探究乙酰唑胺药物对圆锥角膜成纤维细胞胶原合成的影响及机制,为圆锥角膜疾病的临床治疗提供新思路。采用100μmol/L乙酰唑胺溶液处理人圆锥角膜成纤维细胞,采用qPCR方法检测处理后胶原代谢基因的表达,分别采用ELISA、Western Blot检测总胶原及I型胶原的含量,采用荧光探针DCFH-DA检测细胞内ROS水平,采用分光光度法检测MDA含量及细胞总抗氧化能力T-AOC的变化。结果发现,乙酰唑胺处理后,圆锥角膜细胞中胶原合成及交联酶编码基因COLs、LOXs的表达显著升高,MMP2、MMP3、MMP9等胶原降解基因的表达均显著下降,I型胶原和总胶原含量降低。此外,细胞内抗氧化酶基因NQO-1表达升高,总抗氧化能力增强,ROS和MDA含量显著下降。结果表明,乙酰唑胺通过调节圆锥角膜成纤维细胞氧化应激水平,改善胶原的合成及交联,抑制胶原降解。

Keratoconus is characterized by the progressive thinning and protrusion of central cornea.Dysregulated collagen metabolism is one of the causes of this disease. In this study, the effect of acetazolamide on collagen synthesis in keratoconus fibroblasts and its mechanism were explored, which could provide a reference for the clinical treatment of keratoconus. The cultured keratoconus fibroblasts were treated with 100 μmol/L acetazolamide solution. The expression of collagen metabolism-related genes was then analyzed by qPCR. The collagen content was detected by ELISA and Western Blot. The intracellular ROS content was analyzed by fluorescent probe DCFH-DA. The changes of MDA content and the total antioxidant capacity were detected by spectrophotometry. It was suggested that after acetamide treatment, the expression of COLs and LOXs, of which coded enzymes paticipated in collagen synthesis and crosslinking, were significantly up-regulated, while the expression of collagen degradation genes including MMP2, MMP3, and MMP9 were significantly down-regulated. The contents of type I collagen and total collagens were both reduced by acetamide treatment. In addition, the total antioxidant capacity of cells and the expression of the antioxidant enzyme gene NQO-1 were significantly up-regulated, while the contents of ROS and MDA were significantly decreased. The results indicated that acetazolamide could induce the synthesis and inhibit the degradation of collagens in keratoconus fibroblasts by reducing the intracellular oxidative stress levels.