左旋精氨酸与褪黑激素对大鼠肾脏缺血再灌注损伤的保护作用

Protective effect of L-Arginine plus melatonin on renal ischemia/reperfusion injury in rats

目的:诱导型一氧化氮合酶来源的一氧化氮与内皮型一氧化氮合酶来源的一氧化氮的平衡对维持血流动力学稳定及内环境动态平衡有重要作用。观察左旋精氨酸与褪黑激素对肾脏缺血再灌注损伤中诱导型一氧化氮合酶与内皮型一氧化氮合酶的影响。方法:实验于2006-07/2007-03在中山大学医学院动物实验中心完成。①实验分组:健康雄性清洁级SD大鼠60只,体质量170～210g,随机数字表法分为缺血再灌注组、左旋精氨酸治疗组、褪黑激素治疗组、联合治疗组,每组15只。②实验方法:建立大鼠肾脏缺血再灌注损伤模型,术前1h各组大鼠腹腔内注射生理盐水及相应药物,每隔24h重复注射1次,连续5d。③实验评估:观察各组术前及术后1,3,5d血肌酐、诱导型一氧化氮合酶和内皮型一氧化氮合酶的变化规律及肾脏病理组织学的改变,并采用Paller法对术后第3天肾小管进行评分。结果:纳入大鼠60只,均进入结果分析。①血肌酐的表达:术后第1天联合治疗组水平显著低于缺血再灌注组(P<0.05);术后第3,5天左旋精氨酸治疗组、褪黑激素治疗组与联合治疗组显著低于缺血再灌注组(P<0.05)。②内皮型一氧化氮合酶的表达:术后第3天缺血再灌注组比术前降低(P<0.05);术后第3天和第5天左旋精氨酸治疗组、联合治疗组高于缺血再灌注组(P<0.05)。③诱导型一氧化氮合酶的表达:术后第1天缺血再灌注组与术前相比升高,第3天达到高峰,第5天仍高于术前(P<0.05);褪黑激素治疗组、联合治疗组术后第3天与第5天明显低于缺血再灌注组(P<0.05)。④血肌酐水平与诱导型一氧化氮合酶的表达呈正相关,r=0.57,P<0.01,与内皮型一氧化氮合酶/诱导型一氧化氮合酶呈负相关,r=-0.61,P<0.01。⑤肾小管损伤Palller法评分:依次为联合治疗组<褪黑激素治疗组<左旋精氨酸治疗组<缺血再灌注组。结论:通过调高内皮型一氧化氮合酶的表达、抑制诱导型一氧化氮合酶的表达可以减轻肾脏缺血再灌注损伤,联合应用左型精氨酸与褪黑激素即通过以上途径更有效的起到对肾脏缺血再灌注损伤的保护作用。

AIM: The balance of inducible nitric oxide synthase derived nitric oxide and endothelial nitric oxide synthase derived nitric oxide plays an important role in maintaining the stability of haemodynamics and the dynamic balance of internal environment. This article is intended to investigate the effects of L-Arginine and melatonin on inducible nitric oxide synthase and endothelial nitric oxide synthase in rats undergoing renal ischemia/reperfusion injury. METHODS: The experiments were conducted at the Center of the Animal Experiment of Medical College of Sun Yat-sen University from July 2006 to March 2007. ①Grouping: 60 healthy male SD rats with the body mass of 170-210 g were randomized into four groups, ischemia/reperfusion group, L-Arginine group, melatonin group and combination group with 15 in each group. ②Project: The ischemia/reperfusion rat models were built. Rats of each group were injected with saline and corresponding drugs intraperitoneally one hour before operation, once every 24 hours for 5 days. ③Evaluation: Changes in serum creatinine, inducible nitric oxide synthase and endothelial nitric oxide synthase as well as pathohistology of kidney before operation, 1, 3 and 5 days after operation were observed. Renal tubule was determined at day 3 after operation by Paller score. RESULTS: Totally 60 rats were involved in the result analysis. ①Serum creatinine level was significantly lower in the combination group than the ischemia/reperfusion group at day 1 after operation (P < 0.05). Serum creatinine level was significantly lower in the L-Arginine group, melatonin group and combination group than the ischemia/reperfusion group at days 3 and 5 after operation(P < 0.05). ②Expression of endothelial nitric oxide synthase decreased in the ischemia/reperfusion group at day 3 after operation than before operation(P < 0.05). The expression of endothelial nitric oxide synthase was higher in the L-Arginine group and combination group than the ischemia/reperfusion group at days 3 and 5 after operation(P < 0.05). ③The expression of inducible nitric oxide synthase was significantly higher in the ischemia/reperfusion group on the first day than before operation. The expression on the third day reached the peak, and was also significantly higher on the fifth day than that before operation (P < 0.05). The expression was significantly lower in the melatonin group and combination group than the ischemia/reperfusion group at days 3 and 5(P < 0.05). ④Serum creatinine level was positively correlated with the expression of inducible nitric oxide synthase (r =0.57,P < 0.01), but negatively correlated with endothelial nitric oxide synthase/inducible nitric oxide synthase (r =-0.61,P < 0.01). ⑤The Paller's score was combination group, melatonin group, L-Arginine group and ischemia/reperfusion group from lower to higher in order. CONCLUSION: Renal ischemia/reperfusion injury can be relieved by elevating the expression of endothelial nitric oxide synthase and inhibiting the expression of inducible nitric oxide synthase. L-Arginine and melatonin combination can attenuate renal ischemia/reperfusion injury by upper approaches.