基于阿霉素肾病大鼠疾病行为对肾阳虚证候的研究

Study on the Chinese medicine symptoms of kidney yang deficiency based on sickness behaviour in rats with adriamycin nephropathy

目的研究阿霉素肾病大鼠的疾病行为和肾阳虚证经典证候发生的共性基础和关联。方法SD大鼠经尾静脉2次注射阿霉素(4.0、3.5 mg·kg^(-1),间隔1周)构建肾阳虚证肾病综合征的病证结合模型。建模成功后,随机分为阿霉素组(ADR组)、皮质酮组(CORT组)和氢化可的松组(HYD组),每组12只;另取12只正常大鼠作为正常组。同时,HYD组灌胃给予25 mg·kg^(-1)·d^(-1)HYD持续14 d建立单纯下丘脑-垂体-肾上腺皮质(HPA)轴抑制的肾阳虚证模型。CORT组给予25μg·mL^(-1)皮质酮替代饮水,其他3组均给予正常饮水,持续6周。用酶联免疫吸附试验法检测尿17-羟基类固醇水平,用蛋白质印迹法检测下丘脑、肾组织中糖皮质激素受体(GR)、核因子-κB(NF-κB)蛋白的表达水平。结果正常组、ADR组、CORT组和HYD组的尿17-羟基类固醇水平分别为(19.14±1.94)、(10.07±1.62)、(20.30±1.55)和(14.23±2.37)μg·L^(-1);下丘脑中GR蛋白相对表达水平分别为0.63±0.05、0.11±0.05、0.85±0.08和0.35±0.06,NF-κB蛋白相对表达水平分别为0.06±0.03、0.96±0.03、0.59±0.01和0.23±0.04;肾组织中GR蛋白相对表达水平分别为0.94±0.06、0.06±0.02、0.40±0.02和0.09±0.08,NF-κB蛋白相对表达水平分别为0.07±0.05、0.81±0.12、0.72±0.07和0.49±0.08。正常组的上述指标与ADR组和HYD组比较,在统计学上差异均有统计学意义(P<0.05,P<0.01);除肾组织NF-κB蛋白相对表达水平外,CORT组的上述指标与ADR组比较,在统计学上差异均有统计学意义(均P<0.01)。结论HPA轴功能障碍、GR损伤伴随炎症反应激活是肾阳虚经典证候和“虚寒”特征疾病行为的中西医结合共同基础。

Objective To study the common basis and association between sickness behaviour and occurrence of classical symptoms of kidney yang deficiency of rats with adriamycin nephropathy.Methods The SD rats were given adriamycin by tail-vein injection for 2 times(4.0 and 3.5 mg·kg^(-1),one week apart)to construct the model of nephrotic syndrome with Chinese medicine symptom of kidney yang deficiency.After successful modeling,the model rats were randomly divided into adriamycin group(ADR group),corticosterone group(CORT group)and hydrocortisone group(HYD group),with 12 rats per group;another 12 normal rats were taken as normal group.In the HYD group,25 mg·kg^(-1)·d^(-1)HYD was administered for 14 d to establish kidney yang deficiency model with simple hypothalamus-pituitary-adrenal cortex(HPA)axis inhibition.CORT group was adding 25μg·mL^(-1)corticosterone to the water for 6 weeks,and the others drinking water supplied.The levels of urinary 17-hydroxy steroid were measured by enzyme linked immunosorbent assay.Glucocorticoid receptor(GR)and nuclear factor-kappa B(NF-κB)protein expression levels in kidney and hypothalamus were detected by Western blotting.Results In the normal,ADR,CORT and HYD groups,the urinary 17-hydroxy steroid levels were(19.14±1.94),(10.07±1.62),(20.30±1.55)and(14.23±2.37)μg·L^(-1);the relative expression levels of GR protein in hypothalamic were0.63±0.05,0.11±0.05,0.85±0.08 and 0.35±0.06;the relative expression levels of NF-κB protein in hypothalamic were 0.06±0.03,0.96±0.03,0.59±0.01 and 0.23±0.04;the relative expression levels of GR protein in kidney tissue were 0.94±0.06,0.06±0.02,0.40±0.02 and 0.09±0.08;the relative expression levels of NF-κB protein in kidney tissue were 0.07±0.05,0.81±0.12,0.72±0.07 and 0.49±0.08,respectively.Compared with the ADR and HYD groups,the above indexes in the normal group were statistically significant(P<0.05,P<0.01).And compared with the ADR group,the relative expression levels of NF-κB protein in renal tissue with CORT group were not statistically significant(P>0.05),but the other indexes in CORT group were statistically significant(all P<0.01).Conclusion HPA axis dysfunction with GR damaged and activated inflammatory levels are the common basis for the combination of typical symptoms of kidney yang deficiency and sickness behaviour which characterised by deficiency and cold syndrome".