阿霉素联合氢化可的松诱导慢性肾脏病肾阳虚证大鼠模型的建立与评价及真武汤干预机制研究＊

目的 探索应用阿霉素联合氢化可的松诱导建立慢性肾脏病肾阳虚证大鼠模型的方法与评价及真武汤干预的机制。方法 采用阿霉素联合氢化可的松注射诱导建立慢性肾脏病肾阳虚证大鼠模型。52只SPF级雄性6-8周龄SD大鼠,随机分为正常组10只,模型组42只。大鼠适应性饲养2周,模型组大鼠阿霉素3 mg·kg^-1尾静脉注射,第4周4 mg·kg^-1剂量第2次注射,模型成功后随机分为阿霉素组,氢化可的松组与真武汤组。第12周,氢化可的松组与真武汤组进行氢化可的松10 mg·kg^-1肌肉注射,真武汤组按照每日5.39 g/kg进行真武汤干预。在实验中记录一般情况,称取体质量,采取BCA法检测24 h尿蛋白定量,采取红外体温计测量体温,采取游泳耐力法检测体力耐力。实验第24周取材,摘取肾脏,脾脏,睾丸称量并计算脏器指数;大鼠肾组织HE、Masson染色分析病理形态学变化;大鼠血清采用比色法检测Scr、BUN、UA、TC、TG、TP、ALB、SOD;采用酶联免疫吸附法检测CORT、T3、T4、TSH、FSH、LH、T、E2水平。结果 与正常组比较,氢化可的松组大鼠一般情况出现弓背、戗毛、蜷缩、喜卧懒动,精神不振等肾阳虚表现;24 h尿蛋白大量增加（P < 0.01）;与氢化可的松模型相比,真武汤组肾阳虚表现出现好转,24 h尿蛋白降低（P < 0.05）,体温升高（P < 0.05）;HE结果显示氢化可的松组肾小球发生球囊壁粘连,系膜区增宽,肾小管扩张或萎缩,大量蛋白管型,肾间质炎性细胞浸润;Masson结果显示氢化可的松组肾小球节段性硬化,肾小管与肾间质发生纤维化;真武汤组HE结果显示蛋白管型减少,球囊壁粘连减轻,肾间质炎性细胞浸润减少,Masson结果显示肾小球纤维化减轻,肾小管与肾间质纤维化减轻;与正常组相比,氢化可的松肾功能异常,血脂升高,蛋白显著降低;CORT降低,T3、T4降低（P < 0.05,P < 0.01）,TSH明显升高（P < 0.01）;SOD明显降低（P < 0.01）;T明显降低,FSH、LH、E2显著降低（P < 0.05,P < 0.01）;与氢化可的松组相比,真武汤组血肌酐与尿酸下降,血清蛋白上升（P < 0.05）;CORT上升,T3明显升高（P < 0.05）、T4升高、TSH下降,SOD显著升高（P < 0.01）、T、E2、FSH、LH出现升高。结论 阿霉素联合氢化可的松可以成功建立慢性肾脏病肾阳虚证动物模型。真武汤可能通过改善肾功能、作用于下丘脑肾上腺轴、甲状腺轴和性腺轴对慢性肾脏病肾阳虚证起到很好的干预作用。

Establishment and Evaluation of Adriamycin Combined with Hydrocortisone Induced Rat Model of Kidney-yang Deficiency Syndrome in Chronic Kidney Disease and Intervention Mechanism of Zhenwu Decoction

Objective To explore the method and evaluation of the application of adriamycin combined with hydrocortisone to induce the establishment of the rat model of kidney-yang deficiency syndrome in chronic kidney disease and the mechanism of intervention by Zhenwu Decoction.Methods Adriamycin combined with hydrocortisone injection was used to induce the rat model of kidney-yang deficiency syndrome in chronic kidney disease. A total of 52 SPF male SD rats aged 6 to 8 weeks were randomly divided into normal group (n = 10) and model group (n = 42). The rats were fed for 2 weeks, and the model group was injected with adriamycin 3 mg·kg^-1 through tail vein, and the model group was injected with adriamycin 4 mg·kg^-1 again at the 4^th week. After success, the model group was randomly divided into adriamycin group, hydrocortisone group and Zhenwu Decoction group. At the 12^th week, hydrocortisone group and Zhenwu Decoction group were given hydrocortisone 10 mg·kg^-1 intramusculature injection, and Zhenwu Decoction group was given Zhenwu Decoction intervention at 5.39 g·kg·d^-1. General conditions were recorded in the experiment, body weight was measured. 24-hour urinary protein quantity was measured by BCA method. Body temperature was measured by infrared thermometer, and physical endurance was measured by swimming endurance method. At the 24^th week of the experiment, the kidney, spleen and testicles were collected and weighed and the organ index was calculated. HE and Masson staining were used to analyze the pathomorphological changes of renal tissue of rats. Scr, BUN, UA, TC, TG, TP, ALB and SOD were detected by colorimetric method. The levels of CORT, T3, T4, TSH, FSH, LH, T and E2 were detected by enzyme linked immunosorbent assay.Results Compared with normal group, the rats in hydrocortisone group generally had the symptoms of kidney-yang deficiency, such as arching back, hair dishevelment, crouching, less movement and lassitude; 24-hour urinary protein was significantly increased (P < 0.01); Compared with hydrocortisone model, kidney-yang deficiency syndrome of Zhenwu Decoction group improved, 24-hour urinary protein decreased (P < 0.05), and body temperature increased (P < 0.05); HE results showed that in the hydrocortisone group, glomerular balloon wall adhesion, mesangial area widened, renal tubules dilated or atrophied, a large number of protein tubules, tubular epithelial vacuoloid degeneration, and renal interstitial inflammatory cell infiltration occurred. Masson''s results showed that the hydrocortisone group had segonal glomerular sclerosis. The renal tubules and interstitium were fibrotic. In Zhenwu Decoction group, HE results showed decreased protein tubules, decreased adhesion of balloon wall, decreased inflammatory cell infiltration in renal interstitium, and Masson results showed decreased glomerular fibrosis, renal tubules and renal interstitium fibrosis. The renal function of hydrocortisone group was abnormal, blood lipid was increased and protein was significantly decreased compared with the normal group. CORT was decreased, and T3 and T4 were decreased (P < 0.05, P < 0.01), and TSH was significantly increased (P < 0.01); SOD was significantly decreased (P < 0.01); T was significantly decreased, and FSH, LH and E2 were significantly decreased (P < 0.05, P < 0.01). Compared with hydrocortisone group, the rats in Zhenwu Decoction group had serum creatinine and uric acid were decreased, serum protein was increased (P < 0.05); CORT was increased, T3 was significantly increased (P < 0.05), T4 was increased, and TSH was decreased. SOD was significantly increased (P < 0.01), and T, E2, FSH and LH were increased.Conclusion Adriamycin combined with hydrocortisone can successfully establish the animal model of kidney-yang deficiency syndrome in chronic kidney disease. Zhenwu Decoction may play a good role in the intervention of kidney-yang deficiency syndrome in chronic kidney disease by improving renal function and acting on hypothalamic-adrenal axis, hypothalamic-thyroid axis and hypothalamic-gonadal axis.