褪黑素对视网膜缺血再灌注损伤大鼠视网膜细胞凋亡的影响及机制探讨

The effect of melatonin on retinal apoptosis in rats with ischemia-reperfusion injury

目的 观察褪黑素对视网膜缺血再灌注损伤（RIRI）大鼠视网膜细胞凋亡的影响，并探讨其可能机制。 方法 健康无眼疾雄性Sprague-Dawley成年大鼠54只，采用随机数字表法随机分为正常对照组、RIRI组及褪黑素组，分别为6、24、24只。RIRI组及褪黑素组大鼠采用线栓法建立RIRI模型。褪黑素组大鼠按1.25 ml/kg剂量注射4 mg/ml褪黑素，RIRI组大鼠注射等量生理盐水。正常对照组大鼠不作干预。建模后6、24 h及3、7 d，RIRI组及褪黑素组分别取6只大鼠用于实验。制作视网膜切片，苏木精伊红（HE）染色观察各组大鼠视网膜组织形态变化；免疫组织化学染色计数活性半胱氨酸天冬氨酸蛋白酶（Caspase）-3、核转录因子NF-E2 相关因子2（Nrf2）、血红素氧合酶（HO）-1 阳性细胞数量。采用一般线性回归分析法分析建模后不同时间点褪黑素组与RIRI组活性Caspase-3阳性细胞数差值与Nrf2、HO-1阳性细胞数差值的相关性。 结果 HE染色观察发现，正常对照组大鼠视网膜各层结构清晰完整，细胞排列整齐规则。RIRI组大鼠视网膜水肿明显，视网膜内层变厚，视网膜神经节细胞（RGC）数量减少。褪黑素组大鼠视网膜各层细胞排列较整齐，形态较规则。建模后6、24 h及3、7 d，与RIRI组比较，褪黑素组大鼠视网膜内层厚度增厚（F=16.710、62.303、68.389、57.132，P＜0.01），RGC数量增加（F=24.250、11.624、14.155、32.442，P＜0.05），差异均有统计学意义。免疫组织化学染色观察发现，建模后6、24 h及3、7 d，与RIRI组比较，褪黑素组大鼠视网膜活性Caspase-3阳性细胞数量明显减少（F=49.118、134.173、76.225、18.385，P＜0.01），Nrf2（F=11.041、31.480、59.246、6.740，P＜0.05）、HO-1（F=128.993、21.606、51.349、8.244，P＜0.05）阳性细胞数量明显增加，差异均有统计学意义。相关性分析结果显示，建模后不同时间点褪黑素组与RIRI组活性Caspase-3阳性细胞数差值与Nrf2、HO-1阳性细胞数差值均呈直线相关（r2=0.810、0.730，P＜0.01）。 结论 褪黑素可减轻RIRI大鼠视网膜细胞凋亡；其机制可能与促进Nrf2、HO-1蛋白表达，抑制活性Caspase-3蛋白表达有关。

Objective To observe the effect of melatonin （MT） on retinal apoptosis in rats with ischemia-reperfusion injury （RIRI）. Methods A total of 54 male healthy Sprague-Dawley adult rats were randomly divided into the normal control （CON） group （6 rats）, RIRI group （24 rats） and MT group （24 rats）. The rats of RIRI and MT group were induced using suture-occluded methods to establish RIRI model. The rats of MT group were injected with MT in the left carotid artery 30 minutes after RIRI, and RIRI group was injected with the same amount of saline. On 6, 24 hours and 3, 7 days after RIRI, the morphological changes of retina were evaluated by hematoxylin and eosin （HE） staining; the effects of MT on retinal cell apoptosis and Nrf2, HO-1 proteins were examined by immunohistochemistry staining. The correlation between active Caspase-3 and Nrf2 protein, active Caspase-3 and HO-1 protein in MT group were analyzed by linear regression analysis. Results HE staining results showed that the morphology of retinal cells was regular and retinal cells were well arranged in the CON and MT group. In the RIRI group, both the thickness of inner retinal layer and the number of retinal ganglion cells （RGC） were decreased. On 6, 24 hours and 3, 7 days after RIRI, the thickness of inner retinal layer （F=16.710, 62.303, 68.389, 57.132; P〈0.01） and RGC number （F=24.250, 11.624, 14.155, 32.442; P〈0.05） in MT group were more than those in RIRI group. Immunohistochemistry staining results showed that less active Caspase-3＋ cells were observed in MT group as compared with those in RIRI group at each time points （F=49.118, 134.173, 76.225, 18.385; P〈0.01）. There were more Nrf2＋ （F=11.041, 31.480, 59.246, 6.740; P〈0.05） and HO-1＋ cells （F=128.993, 21.606, 51.349, 8.244; P〈0.05） in MT group as compared with those in RIRI group at each time points. Linear regression analysis results showed that the difference of active Caspase-3＋ cells were all linearly correlated with the Nrf2＋ cells and HO-1＋cells in the MT group （r2=0.810, 0.730; P〈0.01）. Conclusion MT could reduce retinal cell apoptosis in RIRI rats, and its mechanism may be associated with increased Nrf2 and HO-1 expression, reduced active Caspase-3 expression.