氧化应激在孕期糖尿病致子代大鼠肾脏多巴胺D1受体功能障碍中的作用

Impact of oxidative stress on renal dopamine D1 receptor dysfunction in offspring of diabetic rat dams

目的探讨氧化应激在孕期糖尿病致子代大鼠肾脏多巴胺D1受体功能障碍中的作用。方法Sprague Dawley(SD)孕鼠10只,采用随机数字表法分为糖尿病组(孕期第0天腹腔注射链脲佐菌素35 mg/kg)和对照组(注射等体积的0.9%的生理盐水),每组5只。子代大鼠分为4组,分别为孕期对照子代大鼠溶剂组(对照子代溶剂组)、孕期对照子代大鼠抗氧化组(对照子代抗氧化组)、孕期糖尿病子代大鼠溶剂组(糖尿病子代溶剂组)和孕期糖尿病子代大鼠抗氧化组(糖尿病子代抗氧化组),每组10只。子代大鼠从4周龄开始,溶剂组给予正常饮水,抗氧化组给予抗氧化剂tempol(1.0 mmol/L溶于饮用水)饮水,直至研究结束期(20周龄)。无创鼠尾测压仪连续检测子代大鼠血压。检测子代大鼠肾脏氧化应激指标超氧化物歧化酶(SOD)和丙二醛(MDA)活性及多巴胺D1受体激动剂fenoldopam介导的尿钠排泄功能。检测子代大鼠肾脏G蛋白偶联受体激酶(GRK)2、GRK4及多巴胺D1受体的蛋白表达及磷酸化水平。结果糖尿病子代溶剂组大鼠平均动脉压明显高于对照子代溶剂组(P=0.013),而糖尿病子代抗氧化组大鼠平均动脉压则明显低于糖尿病子代溶剂组(P=0.038)。糖尿病子代溶剂组大鼠药物期尿流速和尿钠排泄率均明显低于对照组子代溶剂组(P均<0.01),糖尿病子代抗氧化组大鼠尿流速和尿钠排泄率则均明显高于糖尿病子代溶剂组(P均<0.01),而对照组子代抗氧化组与对照子代溶剂组比较差异则均无统计学意义(P均>0.05)。糖尿病子代溶剂组大鼠肾脏组织MDA活性明显高于对照子代溶剂组(P<0.01),SOD活性则明显低于对照子代溶剂组(P=0.013)。而糖尿病子代抗氧化组大鼠肾脏组织组MDA活性则明显低于糖尿病子代溶剂组(P<0.01),SOD活性明显高于糖尿病子代溶剂组(P=0.035)。糖尿病子代溶剂组大鼠肾脏GRK2和GRK4蛋白表达水平均明显高于对照子代溶剂组(P均<0.01),而糖尿病子代抗氧化组大鼠肾脏GRK2和GRK4蛋白表达水平则均明显低于糖尿病子代溶剂组(P均<0.01)。4组子代大鼠多巴胺D1受体蛋白表达水平差异无统计学意义(P=0.735)。糖尿病子代溶剂组大鼠肾脏多巴胺D1受体磷酸化水平明显高于对照子代溶剂组(P<0.01),而糖尿病子代抗氧化组大鼠肾脏多巴胺D1受体磷酸化水平则明显低于糖尿病子代溶剂组(P<0.01)。结论氧化应激参与了孕期糖尿病大鼠子代肾脏多巴胺D1受体功能障碍。

Objective To explore the effects of oxidative stress on renal dopamine D1 receptor dysfunction in offspring of diabetic rat dams. Methods The pregnant Sprague Dawley (SD) rats (n=10) were randomly divided into the diabetic group (a single intraperitoneal injection of 35 mg/kg streptozotocin on day 0 of gestation) and control group (injected with the equal volume of 0.9% saline on day 0 of gestation) according to the random number table (n=5 each group). The offspring rats were divided into 4 groups including offspring of control dams treated with vehicle, offspring of control dams treated with antioxidant, offspring of diabetic dams treated with vehicle and offspring of diabetic dams treated with antioxidant (n=10 each group). After birth, the offspring rats were treated with normal drinking water or antioxidant (tempol, 1.0 mmol/L) from the age of 4 weeks until the end of the study (20 weeks). The blood pressure was monitored continuously by non-invasive tail-cuff method. The renal oxidative markers including superoxide dismutase (SOD) and malondialdehyde (MDA) activity and D1 receptor agonist (fenoldopam)-mediated urinary and sodium excretion were detected. Furthermore, the protein expression of renal G protein-coupled receptor kinase 2 (GRK2), GRK4, dopamine D1 receptor and the phosphorylation level of D1 receptor were detected. Results The mean arterial pressure of offspring from the diabetic dams treated with vehicle was significantly higher than that of offspring from control dams treated with vehicle (P=0.013), while the mean arterial pressure of offspring from diabetic dams treated with antioxidant was significantly lower than that of offspring from the diabetic dams treated with vehicle (P=0.038). The fenoldopam-mediated urinary flow and urinary sodium excretion rate were significantly lower in offspring of diabetic dams treated with vehicle than those in offspring of control dams treated with vehicle (P<0.01), which were significantly higher in offspring of diabetic dams treated with antioxidant as compared to offspring of diabetic dams treated with vehicle (both P<0.01). There was no significant difference in fenoldopam-mediated urinary flow and urinary sodium excretion rate in offspring of control dams treated with antioxidant or vehicle (urinary flow: P=0.772;urinary sodium excretion rate: P=0.716). Compared with offspring of control dams treated with vehicle, the renal MDA activity was significantly increased, while the SOD activity was significantly decreased in offspring of diabetic dams treated with vehicle (MDA: P<0.01;SOD: P=0.013). The renal MDA activity was significantly decreased, while the SOD activity was significantly increased in offspring of diabetic dams treated with antioxidant in comparison with offspring of diabetic dams treated with vehicle (MDA: P<0.01;SOD: P=0.035).The renal GRK2 and GRK4 protein expression in offspring of diabetic dams treated with vehicle were significantly higher than those in offspring of control dams treated with vehicle (P<0.01), while the expression levels of renal GRK2 and GRK4 in offspring of diabetic dams treated with antioxidant were significantly downregulated compared with offspring of diabetic dams treated with vehicle (P<0.01). There was no significant difference in the protein expression of dopamine D1 receptor among 4 groups (P=0.735). The level of dopamine D1 receptor phosphorylation in offspring of diabetic dams treated with vehicle was significantly higher than that in offspring of control dams treated with vehicle (P<0.01), while the dopamine D1 receptor phosphorylation level was significantly lower in offspring of diabetic dams treated with antioxidant compared to that in offspring of diabetic dams treated with vehicle (P<0.01). Conclusion Oxidative stress is involved in the dopamine D1 receptors dysfunction in the offspring of diabetic dams.