Twining S S, Schulte D P, Zhou X, Wilson P M, Fish B L, Moulder J E
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226, USA.
Curr Eye Res. 1997 Feb;16(2):158-65. doi: 10.1076/ceyr.16.2.158.5085.
Vitamin A deficiency alters the transparency of the cornea due to epithelial cell keratinization and increases the susceptibility of the cornea to ulceration. The purpose of this study was to determine the effect of vitamin A deficiency on rat corneal matrix metalloproteinases and serine proteinases.
Four dietary groups of male WAG/RijMCW rats were prepared: (1) Vitamin A deficient rats were raised on a casein-based retinoid deficient diet; (2) Retinol repleted rats were raised on the retinoid deficient diet. On the eighty-sixth day on this diet, the rats were fed retinyl palmitate and then given free access to the retinyl palmitate-supplemented control diet; (3) The weight-matched, pair-fed rats were restricted in their intake of the retinyl palmitate-supplemented diet so that their weight gain matched that of the A-rats; (4) The non-restricted rats were given free access to the retinyl palmitate-supplemented diet. The animals were killed at the late plateau stage for weight of the deficiency (102-106 days). Zymography was used to study proteinases in the corneal extracts.
Vitamin A deficient and control rat corneas contain multiple matrix metalloproteinases and serine proteinases. The matrix metalloproteinases at 90/92 kDa (gelatinase B) and 66/63/57 kDa (gelatinase A) were significantly decreased in the corneas of the vitamin A deficient rats relative to the control corneas. Corneas from the four groups of rats contained 76, 45, 38, 28 and 22 kDa proteinases that cleaved casein. Only the vitamin A deficient corneas contained a 50 kDa casein cleaving enzyme. The 76, 45, 38 and 28 kDa serine proteinases were significantly lower in the vitamin A deficient corneas. The major 22 kDa enzyme was not altered by the deficiency. All casein cleaving proteinases were inhibited by phenylmethylsulfonyl fluoride and chymostatin except for a minor 76 kDa band. The activity of this band was not altered by inhibitors for the other classes of proteinases, ethylenediaminetetraacetic acid, E-64 or pepstatin. The concentrations of the 61, 52 and 40 kDa plasminogen activators were not altered by the deficiency.
Alterations in corneal proteinases under vitamin A deficiency conditions may be involved in the characteristic changes observed in the cornea under vitamin A deficiency conditions: decreased exfoliation of epithelial cells, increased levels of keratofibrils in the corneal keratocytes, increased stromal keratocyte degradation and increased susceptibility towards ulceration.
维生素A缺乏会因上皮细胞角化而改变角膜透明度，并增加角膜溃疡的易感性。本研究的目的是确定维生素A缺乏对大鼠角膜基质金属蛋白酶和丝氨酸蛋白酶的影响。
制备四组以雄性WAG/RijMCW大鼠为实验对象的饮食组：（1）维生素A缺乏的大鼠以基于酪蛋白的类视黄醇缺乏饮食饲养；（2）视黄醇补充组大鼠以类视黄醇缺乏饮食饲养。在这种饮食的第86天，给这些大鼠喂食棕榈酸视黄酯，然后让它们自由摄取补充了棕榈酸视黄酯的对照饮食；（3）体重匹配的配对喂养大鼠限制其对补充了棕榈酸视黄酯饮食的摄入量，以使它们的体重增加与维生素A缺乏组大鼠相匹配；（4）非限制组大鼠自由摄取补充了棕榈酸视黄酯的饮食。在缺乏状态的晚期平台期（102 - 106天）处死动物。用酶谱法研究角膜提取物中的蛋白酶。
维生素A缺乏组和对照组大鼠角膜含有多种基质金属蛋白酶和丝氨酸蛋白酶。与对照角膜相比，维生素A缺乏组大鼠角膜中90/92 kDa（明胶酶B）和66/63/57 kDa（明胶酶A）的基质金属蛋白酶显著降低。四组大鼠的角膜含有能切割酪蛋白的76、45、38、28和22 kDa蛋白酶。只有维生素A缺乏组的角膜含有一种50 kDa的酪蛋白切割酶。维生素A缺乏组角膜中76、45、38和28 kDa的丝氨酸蛋白酶显著降低。主要的22 kDa酶不受缺乏状态的影响。除了一条较小的76 kDa条带外，所有酪蛋白切割蛋白酶都被苯甲基磺酰氟和抑肽酶抑制。这条带的活性不受其他类蛋白酶抑制剂、乙二胺四乙酸、E - 64或胃蛋白酶抑制剂的影响。61、52和40 kDa纤溶酶原激活剂的浓度不受缺乏状态的影响。
维生素A缺乏条件下角膜蛋白酶的改变可能与维生素A缺乏条件下在角膜观察到的特征性变化有关：上皮细胞脱落减少、角膜角膜细胞中角原纤维水平增加、基质角膜细胞降解增加以及溃疡易感性增加。
