复方黄甘对糖尿病肾病小鼠氧化应激和Klotho mRNA及蛋白表达的影响

Effect of Huang Gan formula on the oxidative stress and Klotho gene expression in mice with diabetic nephropathy

目的研究复方黄甘对糖尿病肾病小鼠氧化应激及肾组织Klotho表达的影响。方法按照体重将50只C57BL/6J雄鼠随机分为正常组、模型组、对照组、低高2个剂量实验组。除正常组外,其余各组均用高脂饲料和腹腔注射链脲佐菌素的方法建立糖尿病肾病模型。低高2个剂量实验组分别给予6,12g·kg^(-1)复方黄甘灌胃,对照组给予64 mg·kg^(-1)氯沙坦钾片灌胃,正常组和模型组给予等量0.9%Na Cl灌胃。5组小鼠连续给药4周。4周后,用全自动生化分析仪检测各组小鼠24 h尿蛋白、尿素氮(BUN)、血清肌酸酐(Scr)、三酰甘油(TG)、总胆固醇(TC);用酶联免疫吸附实验法检测各组小鼠肾组织氧化应激指标水平;用实时荧光定量聚合酶链式反应和Western blot法检测各组小鼠肾组织Klotho mRNA及蛋白表达水平。结果治疗后,正常组、模型组、对照组、低高2个剂量实验组的24 h尿蛋白分别为(2.02±0.33),(8.36±2.10),(3.07±1.93),(4.29±1.79),(3.13±2.19)mg·d^(-1);BUN分别为(12.03±0.94),(27.93±5.73),(15.73±2.36),(15.49±1.57),(14.13±2.33)mmol·L^(-1);Scr分别为(13.72±0.97),(48.36±2.31),(24.93±1.83),(25.71±1.79),(21.09±2.39)μmol·L^(-1);TG分别为(1.43±0.33),(16.92±4.01),(6.24±1.83),(7.09±2.61),(6.37±2.33)mmol·L^(-1);TC分别为(2.03±0.42),(5.61±0.87),(3.97±0.53),(4.88±0.62),(4.31±0.28)mmol·L^(-1);丙二醛分别为(7.39±0.99),(16.20±2.03),(10.83±1.91),(10.07±1.87),(9.06±1.13)nmol·mg^(-1);超氧化物歧化酶水平分别为(120.31±9.58),(71.26±8.73),(100.23±10.55),(98.27±6.77),(109.73±11.18)U·mg^(-1);谷胱甘肽过氧化物酶水平分别为(103.27±7.48),(69.36±5.08),(92.73±10.72),(90.77±9.38),(96.58±8.23)μmol·g^(-1);Klotho mRNA表达分别为1.03±0.19,0.27±0.03,0.51±0.11,0.43±0.13,0.67±0.09;Klotho蛋白表达分别为1.58±0.24,0.45±0.03,0.83±0.11,1.25±0.08,1.39±0.12,正常组的上述指标与模型组、对照组、低、高剂量实验组比较差异均有统计学意义(均P<0.05),模型组的上述指标与低、高剂量实验组比较差异均有统计学意义(均P<0.05)。结论复方黄甘对糖尿病肾病小鼠具有肾保护作用,其机制可能与抑制氧化应激、升高Klotho基因表达有关。

Objective To investigate the effect of Huang Gan formula （HGF） on the oxidative stress and Klotho expression in mice with diabetic nephropathy. Methods Fifty C57BL/6J male mice were randomly divided into normal group, model group, control group, low - dose and high - dose experimental groups. Mice were induced into diabetic nephropathy by high fat diet and intraperitoneal injection of streptozotocin in other groups except the normal group. The low - dose and high - dose experimental groups were given a garage of 6, 12 g·kg^-1 HGF formula respectively. Control group were given a garage of 64 mg·kg^-1 losartan. Normal group and model group were given an equal volume of saline solution. All groups were continuously admi- nistrated for 4 weeks. After administration, 24 hour urinary protein, blood urea nitrogen （ BUN）, serum creatinine （Scr）, triglyceride （TG）, total cholesterol （TC） were detected by automatic biochemistry analyzer. The indicators of oxidative stress in renal tissues were determined using enzyme linked immunosorbent assay kit. The Klotho mRNA and protein expression level in renal tissue were detected by real -time polymerase chain reaction and Western blot. Results After administration, normal, model, control, low - and high -dose experimental groups＇ 24 hours urinary protein were （2.02 ±0. 33）, （8.36±2. 10）, （3.07±1.93） , （4.29±1.79）, （3.13±2. 19） mg·d^-1 ; BUN were （12.03±0.94）, （27.93±5.73）, （15.73±2.36）, （15.49±1.57） , （14.13±2.33） mmol·L^-1; Scr were （13.72±0.97）, （48.36±2.31）, （24.93±1.83）, （25.71±1.79）, （21.09±2.39） μmol·L^-1; TG were （1.43±0.33）, （16.92±4.01）, （6.24±1.83）, （7.09±2.61）, （6.37±2.33） mmol·L^-1; TC were （2.03±0.42）, （5.61±0.87）, （3.97±0.53）, （4.88±0.62） （4.31±0.28） mmol·L^-1; malondialdehyde （MDA） were （7.39±0.99）, （16.20±2.03）, （10.83±1.91）, （10.07±1.87）, （9.06±1.13） nmol·mg^-1; superoxide dismutase （SOD） were （ 120.31 ± 9.58 ）, （ 71.26± 8.73 ） , （ 100.23 ± 10.55 ）, （98.27±6.77）, （109.73 ± 11.18） U·mg^-1; glutathione peroxidase （GSH - Px） were （103.27 ± 7.48）, （69.36±5.08）, （92.73± 10. 72）, （90.77±9.38）, （96.58 ±8.23） μmol·g^-1. Klotho mRNA expression level were 1.03 ±0. 19, 0.27± 0.03, 0.51±0.11, 0.43± 0.13, 0.67± 0.09. Klotho protein expression level were 1.58±0.24, 0. 45 ± 0. 03, 0. 83 ± 0. 11, 1.25 ± 0. 08, 1.39± 0. 12. These indexes of normal group were significantly different from model group, control group, low - and high - dose experimental groups （ all P 〈 0.05 ）. Besides, there were significant differences between model group and low - and high - dose experimental groups （ all P 〈 0.05 ）. Conclusion HGF has a renoprotective effect on diabetic nephropathy and its mechanism may be related to the inhibition of oxidative stress and the increase of Klotho gene expression.