肾衰养真颗粒对CRF营养不良大鼠的药效学研究
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摘要
研究背景
营养不良是慢性肾衰竭(chronic renal failure, CRF)临床常见的重要并发症,尽管肾脏替代治疗在不断发展,但终末期肾脏疾病(ESRD)患者的死亡率仍然很高,而且在众多死亡因素中,营养不良占重要地位。据统计,在ESRD患者群中,营养不良发生率约为20%-50%。营养不良可以导致患者免疫功能低下、贫血、频发感染,甚至导致多脏器衰竭,其发生不仅影响CRF患者的预后,而且与其病死率增高有关。引起CRF营养不良的原因很多,主要包括以下几个方面：1.营养物质摄入减少；2.代谢及内分泌紊乱：因尿毒症毒素、代谢性酸中毒、内分泌紊乱、炎症、脂质代谢紊乱等引起的蛋白质分解增加与合成减少；3.CRF贫血；4.透析过程中丢失蛋白质和氨基酸。随着透析技术的日益改进和完善,维持性透析病人增加,CRF营养不良的发生率进一步提高。目前临床上常使用α-酮酸(商品名：开同)进行治疗,但其价格昂贵,难以普遍应用。其它治疗如静滴白蛋白、能量合剂、水溶性维生素,注射重组生长激素等,疗效短暂且有限,并不能从本质上改善CRF患者的蛋白质合成,使用受限。传统的中药复方汤剂有不同程度的改善CRF营养不良的作用,但服用不方便,疗效不统一,质量难以控制。而且至今为止,国内尚无治疗CRF营养不良的中药新药问世。因此,开发有效的防治CRF营养不良的药物,具有十分重要的社会价值和意义,其潜在的市场和经济效益也相当可观。
祖国医学认为,CRF营养不良其病机属于脾肾衰败,气血亏虚,浊毒内阻。在治疗上若单纯益气养血,则有助邪碍脾,关门留寇之弊,若单纯逐瘀降浊,则又耗伤正气,损伤脾胃,故须降浊排毒与益气养血并用,攻补兼施,方奏气血双补、通腑降浊之功。在此原则指导下,导师魏连波教授据多年临床经验在人参养荣汤基础上筛选药物组方,研制出治疗CRF营养不良的中药新药—肾衰养真颗粒,方由黄芪、白术、当归等六味中药组成,有补益气血、通腑降浊之用。我们前期临床观察已证明肾衰养真颗粒纠正CRF营养不良的总有效率达81.7%,显著优于开同对照组(有效率63.2%,P<0.01),并能很好的改善CRF贫血。因此,对其进一步开发利用具有重要的社会意义和经济价值。
本课题拟按照中药新药6(1)类申报要求,通过建立两种CRF营养不良大鼠模型,研究肾衰养真颗粒对模型大鼠的肾脏保护作用及营养状态的改善作用,从而完成肾衰养真颗粒治疗两种CRF营养不良模型大鼠的主要药效学研究。
目的
1.建立5/6肾切除CRF营养不良大鼠模型及腺嘌呤CRF营养不良大鼠模型,动态观察两种模型CRF营养不良的发生时间和营养状况；
2.观察CRF营养不良模型大鼠尿素氮(BUN)、血肌酐(Scr)、24h尿蛋白(24hUpro)等肾功能指标及血红蛋白(Hb)、白蛋白(ALB)、前白蛋白(PA)、胰岛素样生长因子1(IGF-1)、促红细胞生成素(EPO)、转铁蛋白(Tf)、甲状旁腺激素(PTH)等相关营养指标的变化,以及肾衰养真颗粒对上述指标的影响,探讨其改善CRF营养不良的作用机制,从而按照中药新药分类6.1的技术要求,完成其主要药效学研究。
方法
制备两种CRF营养不良大鼠模型。模型一：采用5/6肾切除联合4%酪蛋白饲料喂养制作5/6肾切除CRF营养不良大鼠模型；模型二：用含0.5%腺嘌呤加4%酪蛋白的饲料喂养制作腺嘌呤CRF营养不良大鼠模型。动态观察两种模型CRF营养不良的发生时间和相关指标的变化,符合CRF营养不良模型条件的大鼠随机分为正常对照组、模型对照组、开同(KT)阳性对照组、肾衰养真汤剂对照组和肾衰养真颗粒(SSYZ)高、中、低剂量组。给药治疗8周,观察大鼠的一般状况、体重、摄食,检测红细胞(RBC)计数、Hb、红细胞比积(Hct)、24hUPro、Scr、BUN、ALB、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、甘油三脂(TG)、PA、IGF-1、EPO、Tf、PTH及血钾(K)、钠(Na)、钙(Ca)、磷(P)的含量,留取大鼠肾脏,观察大鼠肾脏外观,光镜(HE染色)观察肾组织病理学改变。
结果
1.两种模型制备情况5/6肾切除CRF营养不良大鼠模型造模8周末,47只大鼠符合CRF营养不良模型,检测Scr: (123.43±18.00)μmol/L, BUN: (23.68±5.65)mmol/L,24Upro:(62.91±5.12)mg/24h, ALB:(30.02±1.79)g/L,体重：(328.04±35.62)g,与正常组体重(418.50±31.97)g比较平均减少20%以上。造模过程中共死亡29只,存活率为61.84%。腺嘌呤致CRF营养不良大鼠模型造模8周末,62只大鼠符合CRF营养不良模型,检测Scr：(113.02±17.27)μmol/L, BUN: (28.30±4.34)mmol/L,24Upro:(68.65±5.79)mg/24h, ALB:(29.38±2.74)g/L,体重：(276.45±32.26)g,与正常组体重(418.50±31.97)g比较平均减少20%以上。造模过程中共死亡14只,存活率为81.58%。两种模型制备过程中,正常组大鼠精神状况良好,活泼好动,反应灵敏,毛色润泽,食欲良好,尿量正常,体重逐渐增加。CRF营养不良模型大鼠精神萎靡,反应迟钝,毛竖枯槁无光泽,动作迟缓,弓背蜷体震颤,食欲减退,尿量减少,体重增加缓慢,与正常组之间差异显著(P<0.05)。
2.干预后各组一般状况的比较干预8周末,两种模型均见：与模型组比较,各治疗组大鼠一般状况显著改善,体重亦显著增加(P<0.05)。
3.各组肾功能指标的比较两种模型均见：干预8周末,与模型组相比,各治疗组大鼠24hUPro、Scr、BUN均显著降低(P<0.05)；在5/6肾切除致CRF营养不良大鼠模型中：与KT组相比,SSYZ各剂量组及汤剂组大鼠24hUPro、BUN水平无显著差异(P>0.05),而Scr水平显著低于KT组(P<0.05)；在腺嘌呤致CRF营养不良大鼠模型中：与KT组相比,SSYZ中剂量组大鼠24hUPro、Scr水平显著低于KT组(P<0.05), SSYZ高剂量组Scr水平亦显著低于KT组,其余各组之间无统计学差异。SSYZ低剂量组与汤剂组大鼠BUN水平显著高于KT组(P<0.05),余各组间无统计学差异。
4.各组营养指标的比较
在5/6肾切除CRF营养不良大鼠模型中：与模型组相比,SSYZ各剂量组与汤剂组RBC计数均显著增加(P<0.05),而KT组未见显著增加；各治疗组大鼠Hb、Hct、ALB、PA、IGF-1、EPO、Tf水平均显著高于模型组(P<0.05), PTH显著降低(P<0.05),各治疗组大鼠血脂水平及电解质水平亦较模型组显著改善。
在腺嘌呤CRF营养不良大鼠模型中：与模型组相比,各治疗组大鼠血RBC计数、Hb、Hct、ALB、PA、IGF-1、EPO、Tf;水平均显著优于模型组(P<0.05),PTH显著降低,各治疗组大鼠血脂水平及电解质水平亦较模型组改善显著。
5.各组肾脏病理变化的比较
在5/6肾切除CRF营养不良大鼠模型中：正常组肾小球、肾小管及间质结构清晰,排列分布及形态正常。模型组大鼠肾小球代偿性肥大,可见较多肾小球呈节段性硬化,系膜增宽；部分肾小管扩张,变性,坏死,脱落；间质呈慢性淋巴细胞浸润。各治疗组也见部分肾小球肥大,肾小球基底膜(glomerular basement membrance, GBM)增厚,肾小球系膜增宽,但病变范围小且较轻,部分肾小球局灶性硬化,肾小管上皮细胞部分脱落,间质见少量淋巴细胞浸润,肾小管损害明显减轻。各治疗组中SSYZ高中剂量效果较SSYZ低剂量及汤剂组为好,KT组效果不及SSYZ中剂量组。
在腺嘌呤CRF营养不良大鼠模型中：正常组大鼠肾脏组织结构如常,肾小球、肾小管间质无病理改变,管腔内无结晶物沉积。模型组大鼠肾小球数目明显减少,肾小球萎缩,囊腔扩张,肾小球系膜细胞中度增生,肾小管萎缩、坏死、管腔明显扩大,管腔内有大量棕黑色针状结晶,可见蛋白管型、细胞管型,肾间质重度纤维化,成纤维细胞明显增多,大量炎性细胞浸润。各治疗组病理改变较模型组为轻：SSYZ颗粒组肾小球系膜细胞轻度增生,肾小管上皮细胞比较完整,肾小管轻度扩张,管腔内有少量针状结晶,肾间质轻度炎性细胞浸润,轻度纤维化。KT组肾小球系膜细胞轻度增生,少量肾小管上皮细胞脱落,肾小管中度扩张,肾小管中有少量针状结晶,肾间质纤维化较轻。
结论
本课题通过5/6肾切除法和腺嘌呤法,配合4%酪蛋白饲料喂养制备了两种CRF营养不良大鼠模型。本实验中观察到：造模8周末,各造模大鼠与正常组相比,体重显著减轻,ALB显著下降,24hUpro、BUN、Scr显著升高,提示已符合CRF营养不良模型。在干预过程中,两种模型均可观察到：与正常组比较,模型对照组大鼠一般状况明显较差,体重显著下降,24hUpro、BUN、Scr水平持续升高,RBC计数、Hb、Hct、ALB、PA、IGF-1、EPO、Tf等营养指标显著降低,血脂代谢及电解质紊乱,存在低钙、高磷及高PTH血症,肾脏病理可见肾小球硬化、坏死,说明CRF大鼠存在着：营养物质摄入减少、继发性甲旁亢、CRF贫血以及因尿毒症毒素、代谢性酸中毒、内分泌紊乱、炎症、脂质代谢紊乱等引起的蛋白质分解增加与合成减少,上述各种因素相互作用导致了CRF营养不良的发生。而各组干预后,发现SSYZ可显著改善大鼠营养状况,降低24hUpro、BUN、Scr水平,升高RBC计数、Hb、Hct、ALB、PA、IGF-1、EPO、Tf水平,降低血清K、Na、P及PTH水平,升高血Ca,改善血脂水平,减轻肾脏组织病理损害,同时与开同作对照,结果说明了肾衰养真颗粒对CRF营养不良的疗效确切,其作用机制可能通过多途径、多靶点起作用的,疗效更优于原汤剂剂型,所以通过对SSYZ的进一步开发利用,有望填补国内治疗CRF营养不良的中药新药空白。
General Introduction
Malnutrition is a major complication of chronic renal failure (CRF). Although renal replacement therapy has been developed, the mortality of patients with end-stage renal disease (ESRD) is still very high. Malnutrition plays an important role in a number of causes of death. According to statistics, in the patients with ESRD, there were 20%-50% patients complicated with malnutrition Malnutrition can lead to immune dysfunction, anemia, frequent infections, even lead to multiple organ failure, which not only affects the prognosis of patients with CRF, but also affected its mortality rate. There are many reasons which can cause malnutrition in chronic renal failure, which mainly include the following aspects:1. Reduced nutrient intake. 2. Metabolic and endocrine disorders:an increase in protein synthesis and a decrease in protein degradation which is caused by uremic toxins, metabolic acidosis, endocrine disorders, inflammation, lipid metabolism disorders, etc.3.CRF anemia.4. protein loss in the process of hemodialysis. With the improvement of dialysis technology, patients with maintenance dialysis increased, while the incidence of malnutrition in CRF was much higher. Nowadays, a-keto acid (brand name: Ketosteril) is the main medicinal treatment on malnutrition in CRF. But the price is high and most patient can't afford it. The efficacy of other treatment, such as intravenous albumin, energy mixture, water-soluble vitamins, recombinant growth hormone, is short-term and limited. So their adverse reactions unavoidably limit their clinical application. Traditional Chinese herbal compound has better effects on malnutrition of CRF patients, but it is inconvenient to take and the effects are not uniform and the quality is difficult to control. Therefore, there is urgent need to seek for alternative remedies for Crohn's diseases and the Chinese drugs preparation may be the best choice.
Granule of Shengshuai Yangzhen (SSYZ) was empirical prescription based on "Renshen Yangrongtang", by which WeiLian-bo professor treated malnutrition in chronic renal failure for many years. It was composed of Milkvetch Root, Largehead Atractylodes Rhizome and so on. Our pre-clinical observation showed that total effective rate of therapeutic effect on malnutrition of CRF patients was 81.7%, which was more effective than Ketosteril control group (effective rate 63.2%, P<0.01) and could effectively relieve the state of anemia in CRF. Therefore, it has important social significance and economic value to explore it.
In this study we established two types of CRF rat model with malnutrition to investigate the protective effects and improvement of Granule of SSYZ on renal of rats and nutritional status and complete the main pharmacodynamics research of Granule of SSYZ treat CRF rats with malnutrition according to Chinese medicine as 6(1) catalog required.
Objective
1.To establish CRF rat modle with malnutrition by 5/6 nephrectomy or by adenine and to investigate the onset time of malnutrition and its malnutrition status dynamically.2. To detected the blood parameters including serum creatinine(Scr), blood urea nitrogen (BUN),24-hour urinary protein (24h Upro), albumin(ALB) and hemoglobin (Hb) and the level of IGF-1, EPO, Tf and PTH of animals and to investigate the effects of SSYZ on these indicatrixes above and to explore its possible mechanism to complete its main pharmacodynamics research according to Chinese medicine as 6 (1) catalog required.
Methods
To establish two types of CRF rats with malnutrition. Model I:Rats were received 5/6 nephrectomy for preparation of CRF models, and fed wiht 4% casein at the same time to make malnutrition of CRF rats. Model II:Rats were fed with 0.5% adenine and 4% caseinum to make malnutrition of CRF rats. The onset time of malnutrition and correlated parameters were dynamically observed in two types of model. Those contented with malnutrition of CRF were randomized into normal control group, model control group, Ketosteril group, broth of SSYZ group and Granule of SSYZ high, middle, low group. All rats were treated with corresponding drugs for 8 weeks. The general status of rats, food-intake, body weight were observed during the experiment. During and after treatment, the blood parameters, such as ALB, BUN, Scr,24hUpro, Hb, PA, IGF-1, EPO, Tf, PTH, K, Na, Ca, P and so on, were observed, and the macroscopic appearance changes and pathological change of renal tissue were observed, too.
Results
1. Preparation of the two models:Model I:Malnutrition status in CRF rats by 5/6 nephrectomy began at the end of 8th week after model establishment.50 rats were consistent with CRF malnutrition model. The correlated parameters were detected as following:Scr:(123.43±18.00)μmol/L, BUN:(23.68±5.65)mmol/L, ALB:(30.02±1.80) g/L,24hUpro:(62.91±5.12)mg/24h, when weigh reduced 20% contrast to normal group (327.28±35.39 vs 419.63±29.91)g.29 rats were dead in the process of modeling. The survival rate was 61.84%. ModelⅡ:Malnutrition status in CRF rats fed with 0.5% adenine began at the end of 8 week. The correlated parameters were detected as following:Scr:(113.02±17.27)μmol/L, BUN:(28.30±4.34)mmol/L, ALB:(29.38±2.74)g/L,24hUpro:(68.65±5.79)mg/24h, when weigh reduced 20% contrasted to normal group (276.45±32.26vs 419.63±29.91)g.14 rats were dead in the process of modeling. The survival rate was 81.58%. During the preparation of the two models, normal rats were lively, responsive, their coats were moist. They had good appetite, normal urine, and their body weight were gradually increased. While CRF rats with malnutrition had bad spirit and poor apathetic and were unresponsive and their urine outputs were decreased and weight lost and there was significant difference between the normal group and the model (P<0.05).
2. After 8-weeks treatment, the experimental results of the two types of models were similar. Compared to model control group, the general status of the treatment groups was obviously relieved. The weight was significantly increased (P<0.05).
3. After 8-weeks treatment, the levels of 24hUPro, Scr and BUN in treatment groups were significantly lower than the model group (P<0.05). In malnutrition rats with CRF by 5/6 nephrectomy:the levels of 24hUPro and BUN had no difference among Ketosteril group, broth of SSYZ group and Granule of SSYZ high, middle, low group(P>0.05), while the level of Scr in SSYZ group was significantly reduced compared with Ketosteril group(P<0.05). In malnutrition rats with chronic renal failure by adenine:the levels of 24hUPro and Scr in SSYZ group were significantly lower than Ketosteril group(P<0.05) and there was no difference in other parameters among Ketosteril group, broth of SSYZ group and Granule of SSYZ high, middle, low group. The expression of BUN was significantly higher than Ketosteril group (P<0.05) and there was no difference among other groups.
4. The status of malnutrition of rats after 8-weeks treatment.
In CRF rats with malnutrition by 5/6 nephrectomy:the level of RBC count of SSYZ group was markedly increased compared with the model group(P<0.05), while there was increase in Ketosteril group. In contrast with the model group, the levels of Hb, Hct, ALB, PA, IGF-1, EPO and Tf in serum were all elevated in treatment groups, while the expression of PTH in model group was significantly higer than the normal group and was decreased after given corresponding drugs. SSYZ significantly alleviated the levels of blood lipid and electrolyte in model group. In CRF rats with malnutrition by adenine:the levels of RBC count, Hb, Hct, ALB, PA, IGF-1, EPO and Tf in all treatment groups were markedly increased compared with the model group(P<0.05). The expression of PTH in model group was significantly higer than the normal group and was decreased after given corresponding drugs. SSYZ significantly alleviated the levels of blood lipid and electrolyte in model group.
5. The comparation of pathological change of renal tissue
In CRF rats with malnutrition by 5/6 nephrectomy:In control group, the glomeruli structure, regular arrayed renal cells, complete basal laminal, no inflammatory cell infiltration and fibroplasias can be clearly detected, while in the model group, obviously compensatory hypertrophy of glomeruli, wider mesangial region, proliferation of rat mesangial cells can be observered; in parts of renal tissue, hypertrophy of glomeruli, degeneration, necrosis, abscission and groundmass increase, thickening vessel wall even can be detected. Lymphocyte infiltrated in renal interstitium. However, histological analysis of renal tissue from drug-treated rats showed greatly reduced inflammatory cell infiltration, hypertrophy of glomeruli and hypertrophy of glomeruli. The effects of Granule of SSYZ high and middle groups on the pathological changes were better than that of Granule of SSYZ low and broth of SSYZ group and the effect of Ketosteril group is inferior to Granule of SSYZ middle group.
In CRF rats with malnutrition by adenine:In normal group, normal glomeruli structure, regular arrayed renal cells, nocrystal deposition in renal tubule were observed. In model group, the numbers of glomeruli was significantly reduced. Interstitial fibrosis, tubular atrophy, glomerular sclerosis and the extension of capsular space were detected. The atrophy, necrosis of the nephric tubule, the enlargement of capsular space could also be detected. There were an amount of some black needle crystal in the nephric tubule, which showed protein cast, cellular cast, serious interstitial fibrosis and inflammatory cell infiltration. Granule of SSYZ groups and Ketosteril group obviously reduced the pathological changes of the renal tissue from CRF with malnutrition. Mild proliferation of mesangial cells and mild expansion of tubular were obsevered. There were only a few of inaurate crystal in the lumina and a few inflammatory cell infiltration in the renal interstitium.
Conclusions
In this study we established two types of CRF rats with malnutrition by 5/6 nephrectomy or Adenine simultaneously 4% casein fed. At the end of 8th week after model establishment, the weigh and the level of ALB in model group were significantly reduced, while 24hUpro, BUN and Scr were obviously increased compared with normal group, which indicated that the status of malnutrition had already been existed. During the process of intervention, the general status of CRF rats with malnutrition without treatment were bad, their weight was obviously declined and the levels of RBC, Hb, Hct, ALB, PA, IGF-1, EPO and Tf was decreased, while BUN, Scr and 24hUpro were obviously increased, contrast to normal group. It also had lipid metabolism and electrolyte disorders, low calcium, high phosphorus and high PTH hyperlipidemia and the observation of renal pathology showed glomerular sclerosis and necrosis. All the evidence showed that CRF rats with malnutrition had an increase in protein degradation and a decrease in protein synthesis which was caused by uremic toxins, metabolic acidosis, endocrine disorders, inflammation, lipid metabolism disorders, etc. The mutual effects of these reasons finally led to the genesis of CRF with malnutrition. After administratment of corresponding drugs, the malnutrition status of rats were relieved, the expression of RBC, Hb, Hct, ALB, PA, IGF-1, EPO and Tf were significantly elevated and 24hUpro, BUN, Scr and PTH were reduced. The disorders of electrolytes and lipid metabolism were effectively corrected. All these data suggested that Granule of SSYZ could effectively alleviate the symptoms of the malnutrition of CRF and relieved the sclerosis and necrosis of glomerular, whic might work through multi-channel and multi-target. There was no difference in the efficacy of Granule of SSYZ on CRF malnutrition in comparison with Ketosteril but superior to broth of SSYZ group. The results indicated that it was worthy of exploring Granule of SSYZ and it would filled the Chinese Medicines blank treated CRF malnutrition.
营养不良是慢性肾衰竭(chronic renal failure, CRF)临床常见的重要并发症,尽管肾脏替代治疗在不断发展,但终末期肾脏疾病(ESRD)患者的死亡率仍然很高,而且在众多死亡因素中,营养不良占重要地位。据统计,在ESRD患者群中,营养不良发生率约为20%-50%。营养不良可以导致患者免疫功能低下、贫血、频发感染,甚至导致多脏器衰竭,其发生不仅影响CRF患者的预后,而且与其病死率增高有关。引起CRF营养不良的原因很多,主要包括以下几个方面：1.营养物质摄入减少；2.代谢及内分泌紊乱：因尿毒症毒素、代谢性酸中毒、内分泌紊乱、炎症、脂质代谢紊乱等引起的蛋白质分解增加与合成减少；3.CRF贫血；4.透析过程中丢失蛋白质和氨基酸。随着透析技术的日益改进和完善,维持性透析病人增加,CRF营养不良的发生率进一步提高。目前临床上常使用α-酮酸(商品名：开同)进行治疗,但其价格昂贵,难以普遍应用。其它治疗如静滴白蛋白、能量合剂、水溶性维生素,注射重组生长激素等,疗效短暂且有限,并不能从本质上改善CRF患者的蛋白质合成,使用受限。传统的中药复方汤剂有不同程度的改善CRF营养不良的作用,但服用不方便,疗效不统一,质量难以控制。而且至今为止,国内尚无治疗CRF营养不良的中药新药问世。因此,开发有效的防治CRF营养不良的药物,具有十分重要的社会价值和意义,其潜在的市场和经济效益也相当可观。
祖国医学认为,CRF营养不良其病机属于脾肾衰败,气血亏虚,浊毒内阻。在治疗上若单纯益气养血,则有助邪碍脾,关门留寇之弊,若单纯逐瘀降浊,则又耗伤正气,损伤脾胃,故须降浊排毒与益气养血并用,攻补兼施,方奏气血双补、通腑降浊之功。在此原则指导下,导师魏连波教授据多年临床经验在人参养荣汤基础上筛选药物组方,研制出治疗CRF营养不良的中药新药—肾衰养真颗粒,方由黄芪、白术、当归等六味中药组成,有补益气血、通腑降浊之用。我们前期临床观察已证明肾衰养真颗粒纠正CRF营养不良的总有效率达81.7%,显著优于开同对照组(有效率63.2%,P<0.01),并能很好的改善CRF贫血。因此,对其进一步开发利用具有重要的社会意义和经济价值。
本课题拟按照中药新药6(1)类申报要求,通过建立两种CRF营养不良大鼠模型,研究肾衰养真颗粒对模型大鼠的肾脏保护作用及营养状态的改善作用,从而完成肾衰养真颗粒治疗两种CRF营养不良模型大鼠的主要药效学研究。
目的
1.建立5/6肾切除CRF营养不良大鼠模型及腺嘌呤CRF营养不良大鼠模型,动态观察两种模型CRF营养不良的发生时间和营养状况；
2.观察CRF营养不良模型大鼠尿素氮(BUN)、血肌酐(Scr)、24h尿蛋白(24hUpro)等肾功能指标及血红蛋白(Hb)、白蛋白(ALB)、前白蛋白(PA)、胰岛素样生长因子1(IGF-1)、促红细胞生成素(EPO)、转铁蛋白(Tf)、甲状旁腺激素(PTH)等相关营养指标的变化,以及肾衰养真颗粒对上述指标的影响,探讨其改善CRF营养不良的作用机制,从而按照中药新药分类6.1的技术要求,完成其主要药效学研究。
方法
制备两种CRF营养不良大鼠模型。模型一：采用5/6肾切除联合4%酪蛋白饲料喂养制作5/6肾切除CRF营养不良大鼠模型；模型二：用含0.5%腺嘌呤加4%酪蛋白的饲料喂养制作腺嘌呤CRF营养不良大鼠模型。动态观察两种模型CRF营养不良的发生时间和相关指标的变化,符合CRF营养不良模型条件的大鼠随机分为正常对照组、模型对照组、开同(KT)阳性对照组、肾衰养真汤剂对照组和肾衰养真颗粒(SSYZ)高、中、低剂量组。给药治疗8周,观察大鼠的一般状况、体重、摄食,检测红细胞(RBC)计数、Hb、红细胞比积(Hct)、24hUPro、Scr、BUN、ALB、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、甘油三脂(TG)、PA、IGF-1、EPO、Tf、PTH及血钾(K)、钠(Na)、钙(Ca)、磷(P)的含量,留取大鼠肾脏,观察大鼠肾脏外观,光镜(HE染色)观察肾组织病理学改变。
结果
1.两种模型制备情况5/6肾切除CRF营养不良大鼠模型造模8周末,47只大鼠符合CRF营养不良模型,检测Scr: (123.43±18.00)μmol/L, BUN: (23.68±5.65)mmol/L,24Upro:(62.91±5.12)mg/24h, ALB:(30.02±1.79)g/L,体重：(328.04±35.62)g,与正常组体重(418.50±31.97)g比较平均减少20%以上。造模过程中共死亡29只,存活率为61.84%。腺嘌呤致CRF营养不良大鼠模型造模8周末,62只大鼠符合CRF营养不良模型,检测Scr：(113.02±17.27)μmol/L, BUN: (28.30±4.34)mmol/L,24Upro:(68.65±5.79)mg/24h, ALB:(29.38±2.74)g/L,体重：(276.45±32.26)g,与正常组体重(418.50±31.97)g比较平均减少20%以上。造模过程中共死亡14只,存活率为81.58%。两种模型制备过程中,正常组大鼠精神状况良好,活泼好动,反应灵敏,毛色润泽,食欲良好,尿量正常,体重逐渐增加。CRF营养不良模型大鼠精神萎靡,反应迟钝,毛竖枯槁无光泽,动作迟缓,弓背蜷体震颤,食欲减退,尿量减少,体重增加缓慢,与正常组之间差异显著(P<0.05)。
2.干预后各组一般状况的比较干预8周末,两种模型均见：与模型组比较,各治疗组大鼠一般状况显著改善,体重亦显著增加(P<0.05)。
3.各组肾功能指标的比较两种模型均见：干预8周末,与模型组相比,各治疗组大鼠24hUPro、Scr、BUN均显著降低(P<0.05)；在5/6肾切除致CRF营养不良大鼠模型中：与KT组相比,SSYZ各剂量组及汤剂组大鼠24hUPro、BUN水平无显著差异(P>0.05),而Scr水平显著低于KT组(P<0.05)；在腺嘌呤致CRF营养不良大鼠模型中：与KT组相比,SSYZ中剂量组大鼠24hUPro、Scr水平显著低于KT组(P<0.05), SSYZ高剂量组Scr水平亦显著低于KT组,其余各组之间无统计学差异。SSYZ低剂量组与汤剂组大鼠BUN水平显著高于KT组(P<0.05),余各组间无统计学差异。
4.各组营养指标的比较
在5/6肾切除CRF营养不良大鼠模型中：与模型组相比,SSYZ各剂量组与汤剂组RBC计数均显著增加(P<0.05),而KT组未见显著增加；各治疗组大鼠Hb、Hct、ALB、PA、IGF-1、EPO、Tf水平均显著高于模型组(P<0.05), PTH显著降低(P<0.05),各治疗组大鼠血脂水平及电解质水平亦较模型组显著改善。
在腺嘌呤CRF营养不良大鼠模型中：与模型组相比,各治疗组大鼠血RBC计数、Hb、Hct、ALB、PA、IGF-1、EPO、Tf;水平均显著优于模型组(P<0.05),PTH显著降低,各治疗组大鼠血脂水平及电解质水平亦较模型组改善显著。
5.各组肾脏病理变化的比较
在5/6肾切除CRF营养不良大鼠模型中：正常组肾小球、肾小管及间质结构清晰,排列分布及形态正常。模型组大鼠肾小球代偿性肥大,可见较多肾小球呈节段性硬化,系膜增宽；部分肾小管扩张,变性,坏死,脱落；间质呈慢性淋巴细胞浸润。各治疗组也见部分肾小球肥大,肾小球基底膜(glomerular basement membrance, GBM)增厚,肾小球系膜增宽,但病变范围小且较轻,部分肾小球局灶性硬化,肾小管上皮细胞部分脱落,间质见少量淋巴细胞浸润,肾小管损害明显减轻。各治疗组中SSYZ高中剂量效果较SSYZ低剂量及汤剂组为好,KT组效果不及SSYZ中剂量组。
在腺嘌呤CRF营养不良大鼠模型中：正常组大鼠肾脏组织结构如常,肾小球、肾小管间质无病理改变,管腔内无结晶物沉积。模型组大鼠肾小球数目明显减少,肾小球萎缩,囊腔扩张,肾小球系膜细胞中度增生,肾小管萎缩、坏死、管腔明显扩大,管腔内有大量棕黑色针状结晶,可见蛋白管型、细胞管型,肾间质重度纤维化,成纤维细胞明显增多,大量炎性细胞浸润。各治疗组病理改变较模型组为轻：SSYZ颗粒组肾小球系膜细胞轻度增生,肾小管上皮细胞比较完整,肾小管轻度扩张,管腔内有少量针状结晶,肾间质轻度炎性细胞浸润,轻度纤维化。KT组肾小球系膜细胞轻度增生,少量肾小管上皮细胞脱落,肾小管中度扩张,肾小管中有少量针状结晶,肾间质纤维化较轻。
结论
本课题通过5/6肾切除法和腺嘌呤法,配合4%酪蛋白饲料喂养制备了两种CRF营养不良大鼠模型。本实验中观察到：造模8周末,各造模大鼠与正常组相比,体重显著减轻,ALB显著下降,24hUpro、BUN、Scr显著升高,提示已符合CRF营养不良模型。在干预过程中,两种模型均可观察到：与正常组比较,模型对照组大鼠一般状况明显较差,体重显著下降,24hUpro、BUN、Scr水平持续升高,RBC计数、Hb、Hct、ALB、PA、IGF-1、EPO、Tf等营养指标显著降低,血脂代谢及电解质紊乱,存在低钙、高磷及高PTH血症,肾脏病理可见肾小球硬化、坏死,说明CRF大鼠存在着：营养物质摄入减少、继发性甲旁亢、CRF贫血以及因尿毒症毒素、代谢性酸中毒、内分泌紊乱、炎症、脂质代谢紊乱等引起的蛋白质分解增加与合成减少,上述各种因素相互作用导致了CRF营养不良的发生。而各组干预后,发现SSYZ可显著改善大鼠营养状况,降低24hUpro、BUN、Scr水平,升高RBC计数、Hb、Hct、ALB、PA、IGF-1、EPO、Tf水平,降低血清K、Na、P及PTH水平,升高血Ca,改善血脂水平,减轻肾脏组织病理损害,同时与开同作对照,结果说明了肾衰养真颗粒对CRF营养不良的疗效确切,其作用机制可能通过多途径、多靶点起作用的,疗效更优于原汤剂剂型,所以通过对SSYZ的进一步开发利用,有望填补国内治疗CRF营养不良的中药新药空白。
General Introduction
Malnutrition is a major complication of chronic renal failure (CRF). Although renal replacement therapy has been developed, the mortality of patients with end-stage renal disease (ESRD) is still very high. Malnutrition plays an important role in a number of causes of death. According to statistics, in the patients with ESRD, there were 20%-50% patients complicated with malnutrition Malnutrition can lead to immune dysfunction, anemia, frequent infections, even lead to multiple organ failure, which not only affects the prognosis of patients with CRF, but also affected its mortality rate. There are many reasons which can cause malnutrition in chronic renal failure, which mainly include the following aspects:1. Reduced nutrient intake. 2. Metabolic and endocrine disorders:an increase in protein synthesis and a decrease in protein degradation which is caused by uremic toxins, metabolic acidosis, endocrine disorders, inflammation, lipid metabolism disorders, etc.3.CRF anemia.4. protein loss in the process of hemodialysis. With the improvement of dialysis technology, patients with maintenance dialysis increased, while the incidence of malnutrition in CRF was much higher. Nowadays, a-keto acid (brand name: Ketosteril) is the main medicinal treatment on malnutrition in CRF. But the price is high and most patient can't afford it. The efficacy of other treatment, such as intravenous albumin, energy mixture, water-soluble vitamins, recombinant growth hormone, is short-term and limited. So their adverse reactions unavoidably limit their clinical application. Traditional Chinese herbal compound has better effects on malnutrition of CRF patients, but it is inconvenient to take and the effects are not uniform and the quality is difficult to control. Therefore, there is urgent need to seek for alternative remedies for Crohn's diseases and the Chinese drugs preparation may be the best choice.
Granule of Shengshuai Yangzhen (SSYZ) was empirical prescription based on "Renshen Yangrongtang", by which WeiLian-bo professor treated malnutrition in chronic renal failure for many years. It was composed of Milkvetch Root, Largehead Atractylodes Rhizome and so on. Our pre-clinical observation showed that total effective rate of therapeutic effect on malnutrition of CRF patients was 81.7%, which was more effective than Ketosteril control group (effective rate 63.2%, P<0.01) and could effectively relieve the state of anemia in CRF. Therefore, it has important social significance and economic value to explore it.
In this study we established two types of CRF rat model with malnutrition to investigate the protective effects and improvement of Granule of SSYZ on renal of rats and nutritional status and complete the main pharmacodynamics research of Granule of SSYZ treat CRF rats with malnutrition according to Chinese medicine as 6(1) catalog required.
Objective
1.To establish CRF rat modle with malnutrition by 5/6 nephrectomy or by adenine and to investigate the onset time of malnutrition and its malnutrition status dynamically.2. To detected the blood parameters including serum creatinine(Scr), blood urea nitrogen (BUN),24-hour urinary protein (24h Upro), albumin(ALB) and hemoglobin (Hb) and the level of IGF-1, EPO, Tf and PTH of animals and to investigate the effects of SSYZ on these indicatrixes above and to explore its possible mechanism to complete its main pharmacodynamics research according to Chinese medicine as 6 (1) catalog required.
Methods
To establish two types of CRF rats with malnutrition. Model I:Rats were received 5/6 nephrectomy for preparation of CRF models, and fed wiht 4% casein at the same time to make malnutrition of CRF rats. Model II:Rats were fed with 0.5% adenine and 4% caseinum to make malnutrition of CRF rats. The onset time of malnutrition and correlated parameters were dynamically observed in two types of model. Those contented with malnutrition of CRF were randomized into normal control group, model control group, Ketosteril group, broth of SSYZ group and Granule of SSYZ high, middle, low group. All rats were treated with corresponding drugs for 8 weeks. The general status of rats, food-intake, body weight were observed during the experiment. During and after treatment, the blood parameters, such as ALB, BUN, Scr,24hUpro, Hb, PA, IGF-1, EPO, Tf, PTH, K, Na, Ca, P and so on, were observed, and the macroscopic appearance changes and pathological change of renal tissue were observed, too.
Results
1. Preparation of the two models:Model I:Malnutrition status in CRF rats by 5/6 nephrectomy began at the end of 8th week after model establishment.50 rats were consistent with CRF malnutrition model. The correlated parameters were detected as following:Scr:(123.43±18.00)μmol/L, BUN:(23.68±5.65)mmol/L, ALB:(30.02±1.80) g/L,24hUpro:(62.91±5.12)mg/24h, when weigh reduced 20% contrast to normal group (327.28±35.39 vs 419.63±29.91)g.29 rats were dead in the process of modeling. The survival rate was 61.84%. ModelⅡ:Malnutrition status in CRF rats fed with 0.5% adenine began at the end of 8 week. The correlated parameters were detected as following:Scr:(113.02±17.27)μmol/L, BUN:(28.30±4.34)mmol/L, ALB:(29.38±2.74)g/L,24hUpro:(68.65±5.79)mg/24h, when weigh reduced 20% contrasted to normal group (276.45±32.26vs 419.63±29.91)g.14 rats were dead in the process of modeling. The survival rate was 81.58%. During the preparation of the two models, normal rats were lively, responsive, their coats were moist. They had good appetite, normal urine, and their body weight were gradually increased. While CRF rats with malnutrition had bad spirit and poor apathetic and were unresponsive and their urine outputs were decreased and weight lost and there was significant difference between the normal group and the model (P<0.05).
2. After 8-weeks treatment, the experimental results of the two types of models were similar. Compared to model control group, the general status of the treatment groups was obviously relieved. The weight was significantly increased (P<0.05).
3. After 8-weeks treatment, the levels of 24hUPro, Scr and BUN in treatment groups were significantly lower than the model group (P<0.05). In malnutrition rats with CRF by 5/6 nephrectomy:the levels of 24hUPro and BUN had no difference among Ketosteril group, broth of SSYZ group and Granule of SSYZ high, middle, low group(P>0.05), while the level of Scr in SSYZ group was significantly reduced compared with Ketosteril group(P<0.05). In malnutrition rats with chronic renal failure by adenine:the levels of 24hUPro and Scr in SSYZ group were significantly lower than Ketosteril group(P<0.05) and there was no difference in other parameters among Ketosteril group, broth of SSYZ group and Granule of SSYZ high, middle, low group. The expression of BUN was significantly higher than Ketosteril group (P<0.05) and there was no difference among other groups.
4. The status of malnutrition of rats after 8-weeks treatment.
In CRF rats with malnutrition by 5/6 nephrectomy:the level of RBC count of SSYZ group was markedly increased compared with the model group(P<0.05), while there was increase in Ketosteril group. In contrast with the model group, the levels of Hb, Hct, ALB, PA, IGF-1, EPO and Tf in serum were all elevated in treatment groups, while the expression of PTH in model group was significantly higer than the normal group and was decreased after given corresponding drugs. SSYZ significantly alleviated the levels of blood lipid and electrolyte in model group. In CRF rats with malnutrition by adenine:the levels of RBC count, Hb, Hct, ALB, PA, IGF-1, EPO and Tf in all treatment groups were markedly increased compared with the model group(P<0.05). The expression of PTH in model group was significantly higer than the normal group and was decreased after given corresponding drugs. SSYZ significantly alleviated the levels of blood lipid and electrolyte in model group.
5. The comparation of pathological change of renal tissue
In CRF rats with malnutrition by 5/6 nephrectomy:In control group, the glomeruli structure, regular arrayed renal cells, complete basal laminal, no inflammatory cell infiltration and fibroplasias can be clearly detected, while in the model group, obviously compensatory hypertrophy of glomeruli, wider mesangial region, proliferation of rat mesangial cells can be observered; in parts of renal tissue, hypertrophy of glomeruli, degeneration, necrosis, abscission and groundmass increase, thickening vessel wall even can be detected. Lymphocyte infiltrated in renal interstitium. However, histological analysis of renal tissue from drug-treated rats showed greatly reduced inflammatory cell infiltration, hypertrophy of glomeruli and hypertrophy of glomeruli. The effects of Granule of SSYZ high and middle groups on the pathological changes were better than that of Granule of SSYZ low and broth of SSYZ group and the effect of Ketosteril group is inferior to Granule of SSYZ middle group.
In CRF rats with malnutrition by adenine:In normal group, normal glomeruli structure, regular arrayed renal cells, nocrystal deposition in renal tubule were observed. In model group, the numbers of glomeruli was significantly reduced. Interstitial fibrosis, tubular atrophy, glomerular sclerosis and the extension of capsular space were detected. The atrophy, necrosis of the nephric tubule, the enlargement of capsular space could also be detected. There were an amount of some black needle crystal in the nephric tubule, which showed protein cast, cellular cast, serious interstitial fibrosis and inflammatory cell infiltration. Granule of SSYZ groups and Ketosteril group obviously reduced the pathological changes of the renal tissue from CRF with malnutrition. Mild proliferation of mesangial cells and mild expansion of tubular were obsevered. There were only a few of inaurate crystal in the lumina and a few inflammatory cell infiltration in the renal interstitium.
Conclusions
In this study we established two types of CRF rats with malnutrition by 5/6 nephrectomy or Adenine simultaneously 4% casein fed. At the end of 8th week after model establishment, the weigh and the level of ALB in model group were significantly reduced, while 24hUpro, BUN and Scr were obviously increased compared with normal group, which indicated that the status of malnutrition had already been existed. During the process of intervention, the general status of CRF rats with malnutrition without treatment were bad, their weight was obviously declined and the levels of RBC, Hb, Hct, ALB, PA, IGF-1, EPO and Tf was decreased, while BUN, Scr and 24hUpro were obviously increased, contrast to normal group. It also had lipid metabolism and electrolyte disorders, low calcium, high phosphorus and high PTH hyperlipidemia and the observation of renal pathology showed glomerular sclerosis and necrosis. All the evidence showed that CRF rats with malnutrition had an increase in protein degradation and a decrease in protein synthesis which was caused by uremic toxins, metabolic acidosis, endocrine disorders, inflammation, lipid metabolism disorders, etc. The mutual effects of these reasons finally led to the genesis of CRF with malnutrition. After administratment of corresponding drugs, the malnutrition status of rats were relieved, the expression of RBC, Hb, Hct, ALB, PA, IGF-1, EPO and Tf were significantly elevated and 24hUpro, BUN, Scr and PTH were reduced. The disorders of electrolytes and lipid metabolism were effectively corrected. All these data suggested that Granule of SSYZ could effectively alleviate the symptoms of the malnutrition of CRF and relieved the sclerosis and necrosis of glomerular, whic might work through multi-channel and multi-target. There was no difference in the efficacy of Granule of SSYZ on CRF malnutrition in comparison with Ketosteril but superior to broth of SSYZ group. The results indicated that it was worthy of exploring Granule of SSYZ and it would filled the Chinese Medicines blank treated CRF malnutrition.
引文
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[13]. Kopple JD.Pathophysiology of protein-energy wasting in chronic renal failure.J Nutr,1999,129(1S Suppl):247s-251s.
[14]. Kumar VA, Ledezma ML, Rasgon SA. Daily home hemodialysis at a health maintenance organization:three-year experience[J]. Hemodial Int,2007,11(2): 225-230.
[15]. Movilli E, Zani R, Carli O,et al. Correction of metabolic acidosis increases serum albumin concentrations and decreases kinetically evaluated protein intake in hemodialysis patients:a prospective study [J]. Nephrol Dial Transplant, 1998,13(7):1719-1722.
[16].湛贻璞.慢性肾脏病蛋白营养治疗专家共识[J].中华肾脏病杂志,2005,21(7)：421-424.
[17]. Prakash A, Pande DP, Sharma S, et al. Randomized, double-blind, placebo-controlled trial to evaluate efficacy of ketodiet in predialytic chronic renal failure[J].J Ren Nutr,2004,14(2):89-96.
[18].于俊生.从健脾补肾和解泄浊论治慢性肾衰竭营养不良[J].山东中医药大学学报,2005,29(2)：134-137.
[19].周小梅.健脾益肾方对慢性肾功能衰竭大鼠ob基因表达的影响[J].湖南中医学院学报,2003,23(6)：22-24.
[20].魏连波,邓聪,等.肾衰养真胶囊对5/6肾切除慢性肾衰营养不良大鼠营养状态的影响[J].中药材,2008,31(9)：1391-1395.
[21].邓聪,魏连波,等.肾衰养真胶囊对腺嘌呤致CRF营养不良模型大鼠营养状态的影响[J].中成药,2008,30(3)：333-337.
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[2]魏连波,刘冠贤,叶任高.肾脏病临床备要[M].第一版.北京：人民卫生出版社,1997：339-340.
[3]刘介眉,严庆汉,路英杰.病理组织染色的理论方法和应用[M].第一版.北京：人民卫生出版社,1983：21-6.
[4]杜卉莲,傅晓晴.慢性肾衰动物的模型制备及临床应用[J].中医研究,2005,18(12)：53-57.
[5]李月婷,黄荣桂.慢性肾衰竭实验动物方法研究[J].中国中西医结合肾病杂志,2002,3(1)：62-64.
[6]Yekezawa T, Zheng P, Onra H, et al. Animal model of adenine-induce chronic renal failure in rats [J].Nephron,1986,44:230-234.
[7]郑平东,朱燕俐,丁名城,等.用腺嘌呤制作慢性肾功能不全动物模型[J].中华肾脏病杂志,1989,5(6)：342-343.
[8]周小舟,张盛光,阳晓,等.腺嘌呤所致大鼠慢性肾功能衰竭的机理研究[J].基础医学与临床,1997,17(1)：54-57.
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