淫羊藿苷对肾阳虚小鼠肾保护作用的研究

Study on the protective effect of icariin on kidney of kidney-yang deficiency mice

目的 探讨基于哺乳动物雷帕霉素靶蛋白信号通路研究淫羊藿苷对肾阳虚小鼠肾的保护作用。方法 90只雄性小鼠,将小鼠随机分为6组,每组15只。正常组注射生理盐水,其余组注射氢化可的松建立小鼠肾阳虚模型,在此基础上低、中、高剂量实验组分别给予低、中、高剂量的淫羊藿苷(29.3,58.5和117.0 mg·kg^(-1)),阳性组给予1.3 g·kg^(-1)甲基睾酮,正常组和模型组蒸馏水灌胃。记录小鼠体质量,计算增重率。放射免疫法检测血清中三碘甲状腺原氨酸(T3)、四碘甲状腺原氨酸(T4)、促肾上腺皮质激素(ACTH)、促甲状腺激素(TSH)、皮质醇(COR)水平;苏木精-伊红(HE)染色观察肾组织病理情况;比色法检测肾组织中一氧化氮(NO)含量;实时荧光定量聚合酶链反应(RT-qPCR)检测睾丸组织中雄激素受体(AR)mRNA表达水平;蛋白质印迹法检测肾组织雷帕霉素靶蛋白(mTOR)蛋白表达。结果 正常组、模型组、阳性对照组、低剂量实验组、中剂量实验组、高剂量实验组的增重率分别为(29.37±1.20)%,(17.45±0.12)%,(23.44±2.01)%,(19.34±2.30)%,(25.87±2.21)%和(27.74±1.62)%;T3含量分别为(0.87±0.08),(0.58±0.06),(0.75±0.05),(0.50±0.05),(0.74±0.07)和(0.80±0.08) ng·mL^(-1);T4含量分别为(33.75±3.20),(18.16±1.37),(28.24±1.98),(16.20±1.12),(29.17±3.25)和(31.84±3.20)ng·mL^(-1);ACTH含量分别为(31.38±2.80),(17.25±2.05),(27.55±1.20),(16.33±1.47),(27.38±2.16)和(30.03±2.79)pg·mL^(-1);TSH含量分别为(0.85±0.08),(1.25±0.14),(1.00±0.11),(1.16±0.14),(1.02±0.10)和(0.93±0.09)IU·mL^(-1);COR含量分别为(7.20±0.33),(8.28±0.65),(7.38±0.57),(7.90±0.52),(7.34±0.48)和(7.27±041)ng·mL^(-1);NO含量分别为(0.55±0.05),(0.69±0.07),(0.60±0.06),(0.66±0.06),(0.58±0.05)和(0.57±0.04)μmol·g^(-1);AR mRNA表达水平分别为1.03±0.04,0.33±0.03,0.76±0.06,0.36±0.04,0.75±0.07和0.82±0.08;p-mTOR蛋白表达水平分别为0.30±0.03,0.87±0.08,0.44±0.04,0.82±0.08,0.43±0.04和0.36±0.03。以上指标,模型组与对照组比较,差异均有统计学意义(均P<0.05);阳性对照组以及中、高剂量实验组与模型组比较,差异均有统计学意义(P<0.05,P<0.01)。结论 淫羊藿苷通过抑制mTOR信号通路活性对肾阳虚小鼠起到肾保护的作用。

Objective To investigate the protective effect of icariin on kidneys in mice with kidney-yang deficiency based on mammlian targe of rapamycin signaling pathway.Methods Ninety male mice were randomly divided into 6 groups with 15 mice in each group.The norma group was injected with normal saline,and the other groups were injected with hydrocortisone to establish a model of kidney-yang deficiency in mice.On this basis,low,medium,high-dose experimental groups were given low,medium and high doses of icariin(29.3,58.5 and 117.0 mg·kg^(-1)),and the positive group was given 1.3 g·kg^(-1)methyltestosterone.The normal group and the model were given distilled water by gavage.The body weight of the mice was recorded and the weight gain rate was calculated.The levels of triiodothyronine (T3),tetraiodothyronine (T4),adrenocorticotropic hormone (ACTH),thyroid stimulating hormone (TSH) and cortisol (COR) in serum were detected by radioimmunoassay;the pathological tissue of kidney was observed by hematoxylin-eosin (HE) staining;the content of nitric oxide (NO) in kidney tissue was detected by colorimetric method,the expression level of androgen receptor (AR) mRNA in testis tissue was detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR);the expression of p-m TOR protein in renal tissue was detected by Western blotting method.Results The weight gain rates of normal group,model group,positive control group,low-dose experimental group,mediumdose experimental group and high-dose experimental group were as follows:(29.37±1.20)%,(17.45±0.12)%,(23.44±2.01)%,(19.34±2.30)%,(25.87±2.21)%,(27.74±1.62)%;T3 contents were (0.87±0.08),(0.58±0.06),(0.75±0.05),(0.50±0.05),(0.74±0.07),(0.80±0.08) ng·m L-1,respectively;T4contents were (33.75±3.20),(18.16±1.37),(28.24±1.98),(16.20±1.12),(29.17±3.25),(31.84±3.20) ng·m L-1,respectively;ACTH content was (31.38±2.80),(17.25±2.05),(27.55±1.20),(16.33±1.47),(27.38±2.16),(30.03±2.79) pg·m L-1,respectively;TSH contents were (0.85±0.08),(1.25±0.14),(1.00±0.11),(1.16±0.14),(1.02±0.10),(0.93±0.09) IU·m L-1,respectively;COR contents were (7.20±0.33),(8.28±0.65),(7.38±0.57),(7.90±0.52),(7.34±0.48),(7.27±041)ng·m L-1,respectively;NO contents were (0.55±0.05),(0.69±0.07),(0.60±0.06),(0.66±0.06),(0.58±0.05),(0.57±0.04)μmol·g^(-1),respectively;AR mRNA expression levels were 1.03±0.04,0.33±0.03,0.76±0.06,0.36±0.04,0.75±0.07,0.82±0.08,respectively;p-m TOR protein expression was0.30±0.03,0.87±0.08,0.44±0.04,0.82±0.08,0.43±0.04,0.36±0.03,respectively.Above indicators,the differences between the model group and the control group were statistically significant (all P<0.05);there were statistically significant differences between positive control group,medium and high dose experimental groups and model group (P<0.05,P<0.01).Conclusion Icariin inhibits the activity of m TOR signaling pathway and protects the kidneys of mice with kidney-yang deficiency.