【作者】 于化新；

【导师】 王德山；

【作者基本信息】 辽宁中医药大学 ， 中西医结合基础， 2009， 博士

【摘要】 目的水代谢平衡紊乱是慢性肾功能衰竭(CRF)最常见的临床表现之一,往往在CRF早期就出现,是能够导致肾功能急剧恶化的重要危险因素。因此及时纠正CRF早期水代谢平衡紊乱,对改善肾功能,延缓CRF病程进展,提高患者生存质量,具有重要意义。中医认为CRF基本病机属于肾阳虚,临床上采用温补肾阳方法治疗CRF也取得了较好的效果,但机理尚未阐明。本研究以分子生物学、形态学等检测技术,观察了温补肾阳传统名方右归丸对肾阳虚型慢性肾衰模型大鼠肾组织AQP2表达的影响,以及对血中AVP水平、肾组织中血管紧张素Ⅱ(AⅡ)水平、环磷酸腺苷(cAMP)水平、肾组织中血管紧张素ⅡAT1受体(AT1R)表达、环氧合酶2(COX-2)表达、蛋白激酶A(PKA)表达的影响,旨在从分子水平探讨右归丸治疗慢性肾衰水液代谢平衡紊乱的药理作用机制,并为肾阳虚证“气化不利”的病理生理学机制提供实验依据。方法1.动物造模及给药健康雄性Wistar大鼠随机分为对照组、模型组、右归丸组及替米沙坦组。模型组、右归丸组及替米沙坦组大鼠每日上午按200mg/kg/d给予2%的腺嘌呤混悬液灌服,建立肾阳虚型慢性肾衰大鼠模型,对照组大鼠给予相应量蒸馏水灌胃,连续21天。从造模的第3周开始,右归丸组及替米沙坦组大鼠每日上午灌服腺嘌呤的同时,开始每日下午分别灌服右归丸(按3.6 g/kg/d)及替米沙坦(按5.4mg/Kg/d),对照组及模型组大鼠给予相应量蒸馏水灌胃,连续21天。2.指标观察实验过程中,观察大鼠的一般状态、体重、尿量、尿比重、血Scr、BUN含量及肾组织病理形态的变化;采用免疫组化及Western blot方法测定大鼠肾组织AQP2表达;采用放射性免疫方法检测大鼠血AVP含量、肾组织AⅡ及cAMP含量;采用免疫组化方法检测肾组织PKA表达;利用Western blot方法测定大鼠肾组织AT1受体、COX-2蛋白表达,3.统计学处理计量资料用(?)±SD表示,采用SPSS10.0统计学软件进行ANOVA(完全随机设计的单因素方差分析)统计方法处理,以P＜0.05为有显著性差异。结果1.右归丸对肾阳虚型慢性肾衰大鼠肾功能影响结果显示,模型组大鼠出现畏寒肢冷、食量减少、体重下降、活动减少;与对照组比较,尿量明显增加,尿比重下降,血Scr及BUN含量显著增高(P＜0.05),肾组织光镜观察可见肾小球囊腔扩大,间质纤维组织增生及大量炎细胞浸润,肾小管呈代偿性扩张。与模型组比较,右归丸组大鼠摄食较好、活动力较强、被毛较光泽、反应灵敏、体重均有不同程度增加,尿量减少、尿比重增加、血Scr及BUN含量下降(P＜0.05)、肾组织光镜观察可见病理损伤明显减轻。2.右归丸对肾阳虚型慢性肾衰大鼠肾组织AQP2表达影响免疫组化方法检测表明:模型组大鼠肾脏集合管上皮细胞胞膜胞浆AQP2的表达较对照组明显减弱,表现为细胞胞膜和胞浆棕黄色颗粒明显减少,颜色明显变浅,图像分析表明:模型组大鼠肾脏集合管上皮细胞AQP2阳性表达累积光密度值明显低于对照组(P＜0.05);右归丸组大鼠肾脏集合管上皮细胞胞膜胞浆AQP2的表达较模型组明显增强,表现为细胞胞膜胞浆棕黄色颗粒明显增多,颜色明显加深,图像分析结果显示:右归丸组大鼠肾脏集合管上皮细胞AQP2阳性表达累积光密度值明显高于模型组(P＜0.05)。western blot方法检测显示:模型组大鼠肾组织AQP2表达下降,表现为条带窄,密度小,颜色浅。图像分析结果显示:模型组大鼠AQP2表达条带灰度值显著低于对照组;右归丸组大鼠肾组织AQP2蛋白表达增加,表现为条带宽,密度大,颜色深,图像分析结果显示:右归丸组大鼠。肾组织AQP2表达条带灰度值显著高于模型组(P＜0.05)。3.右归丸对肾阳虚型慢性肾衰大鼠血中AVP含量、肾组织中AⅡ、cAMP含量、AT1R表达等影响。结果表明,三组大鼠血中AVP含量无显著性差异(P＞0.05)。与对照组比较,模型组大鼠肾组织AⅡ含量、AT!R及COX-2表达明显升高、cAMP含量及PKA表达显著下降(P＜0.05);与模型组比较,右归丸组大鼠肾组织中AⅡ含量、AT!R及COX-2表达明显降低、cAMP含量及PKA表达增加(P＜0.05)。结果显示,慢性肾衰发生水代谢平衡紊乱的可能机制之一为肾脏集合管AQP2表达下降。中医理论的肾主水功能,主要体现在依靠肾的气化作用“升清降浊”以维持机体水液的动态平衡,当肾阳虚气化功能减退时则出现水液代谢障碍。由此推测:肾脏的气化功能减退的实质之一,可能通过AQP2表达的下调,影响了肾脏的重吸收功能。而肾组织AQP2表达下降的可能机制为,通过肾组织中AⅡ含量升高、AT1R及COX-2表达增加,导致肾组织中cAMP含量及PKA表达降低,使AQP2在肾脏的表达下降;采用右归丸治疗,通过温补肾阳,使肾组织中AⅡ含量、AT1R及COX-2表达降低,增加肾组织中cAMP含量及PKA表达,导致肾脏AQP2的表达增加,改善慢性肾衰水代谢平衡紊乱,保护肾功能,延缓慢性肾衰进展。结论1.肾阳虚型慢性肾衰模型大鼠尿量增加,尿比重降低;血Scr及BUN水平增加;肾组织病理可见明显损伤;右归丸治疗能够明显改善上述病理变化。2.肾阳虚型慢性肾衰模型大鼠肾组织AQP2表达下调;右归丸治疗可使AQP2表达上调。3.右归丸对肾阳虚型慢性肾衰模型大鼠血AVP含量无显著影响。4.肾阳虚型慢性肾衰大鼠模型肾组织中AⅡ含量升高、AT1受体及COX-2表达上调、cAMP含量下降、PKA表达下调。5.右归丸使肾组织中AⅡ含量下降、AT1受体及COX-2表达下调、cAMP含量升高、PKA表达上调。更多还原

【Abstract】 PurposeWater metabolism balance disorder is one of the most common clinical Symptoms of chronic renal failure,often occur at an early stage of CRF.As one of important risk factors,it can cause rapid deterioration of renal function.To rectify timely water metabolism balance disorder,can improve renal function and delay progression of CRF.It has great significance to improve quality of life in patients with CRF.At present,the basic pathogenesis of CRF according to Traditional Chinese Medicine theories was considered to be kidney-yang deficiency.At Early and medium-term of CRF,warm and tonify kidney-yang therapy acquired a better effect in CRF treatment.In this study,we set up the rats model of chronic renal failure with kidney-yang deficiency,to observe renal AQP2 expression changes, changes of angiotensinⅡand cyclic adenosine monophosphate contents in rat renal tissue, and expression changes of angiotensinⅡtype 1 receptor,cyclooxygenase-2 and protein kinase A,by using biology and morphology technique.Order to explore the Pharmacological treatment mechanism of yougui pill in disorder of water metabolism of CRF,and to provide experimental evidence for pathophysiological mechanisms of "obstruction of qi activities" of Kidney-Yang Deficiency.Methods1.Setting up Model and AdministrationThirty Healthy male wistar rats were randomized into normal group,model group,yougui pill group and telmisartan group.From frist day of experiment,rats of model group,yougui pill group and telmisartan group were given by 200mg/Kg/d adenine ig daily morning,the normal control group rats were intragastrically administrated with distilled water of corresponding volume,totally for 21 days.After two weeks adenine ig,rats of yougui pill group and telmisartan group were given with yougui pill(at 3.6g/kg/d) and telmisartan(at 5.4mg/Kg/d) Separately by gastric irrigation daily afternoon,rats of normal control group and model group were intragastrically administrated with distilled water of corresponding volume,,totally for 21 days.2.Indexes were detected.:In experimental process,to observe changes of behavior,average weight,Urine volumes and urine specific gravity,blood Scr and BUN content,renal tissue pathological of rats;Expression of renal tissue AQP2 was detected by immtmohistochemistry method and Western blot analysis;Blood AVP content,renal tissue angiotensinⅡand cyclic adenosine monophosphate content were determined by radioimmunoassay;Renal tissue Protein kinase A expression was detected by immunohistochemistry method;Renal tissue angiotensinⅡtype 1 receptor and cyclooxygenase-2 were determined by Western blot analysis.3.Statistical data analysisThe data were expressed as(?)±SD.Completely randomized design one-way ANOVA was used to deal with data by SPSS10.0 statistical software,P＜0.05 for significantly difference.Results1.Impact of Yougui Pill on renal function of chronic renal failure rats with kidney-yang deficiency.The experimental results show that model group rats appeared intolerance to cold,cold four limbs,loss of appetite,weight loss,less activity.Compared with the normal group,model group rats show a marked increase in urine volume,decreased urine specific gravity,increase of blood Scr and BUN content significantly(P＜0.05) and Renal tissue by light microscopy observation shows glomerular expand cysts,interstitial fibrosis and a large amount of inflammatory cell infiltration,tubular compensatory enlargement.Compared with model group,Yougui Pill group rats show better feeding,stronger activity,more shiny hair,pretty sencitive reaction,body weight increasing,urine volume decreased,urine specific gravity increased,Scr and BUN content in blood decreased(P＜0.05),.Renal tissue pathological injury relieved2.Influence of yougui pill on the expression of renal tissue AQP2 of rats of chronic renal failure with kidney-yang deficiency.In immunohistochemical detection,Compared with the normal group,AQP2 expression of model group rats in principal cell membrane and cytoplasm of kidney connecting duct decreased.It manifested as brown particles reducing significantly and slight loss of color in principal cell membrane and cytoplasm.Imaging analysis showed integrated optical density(IOD) of AQP2 immunoreactivity positive in renal collecting tubular epithelial cells was significantly lower than that in normal group rats(P＜0.05);Compared with model group, AQP2 expression of yougui pill group rats in principal cell membrane and cytoplasm of kidney connecting duct increased.It presented as brown particles increasing significantly and darker in color in principal cell membrane and cytoplasm.Imaging analysis showed integrated optical density(IOD) of AQP2 immunoreactivity positive in renal collecting tubular epithelial cells was significantly higher than that in model group rats(P＜0.05 ).Western blot proved that AQP2 expression of renal tissue decreased obviously in model group rats.Gray scales was determined with image analysis system.Compared with normal group,gray scales of renal tissue AQP2 expression decreased in model group rats(P＜0.05).Compared with model group,AQP2 expression of renal tissue increased obviously in yougui pill group rats with western blot,gray scales of renal tissue AQP2 expression was higher in yougui pill group rats than that in model group(P＜0.05).3.Influence of yougui pill on Contents of blood AVP,angiotensinⅡand cyclic adenosine monophosphate in renal tissue,expression of renal tissue angiotensinⅡtype 1 receptor etc of chronic renal failure rats with kidney-yang deficiency.The results show that blood AVP content of three groups rats was no significant difference(P＞0.05).Compared with the normal group,content of angiotensinⅡ,the expression of angiotensinⅡtype 1 receptor and cyclooxygenase-2 in renal tissue were elevated significantly.cyclic adenosine monophosphate content and expression of protein kinase A of renal tissue were significantly decreased in model group rats(P＜0.05);Compared with the model group,Content of angiotensinⅡ,the expression of angiotensinⅡtype 1 receptor and cyclooxygenase-2 in renal tissue were decreased significantly,cyclic adenosine monophosphate content and expression of protein kinase A of renal tissue were significantly increased in yougui pill group rats(P＜0.05).The results showed that one of the mechanisms of water metabolism balance disorder of chronic renal failure is probably reduced AQP2 expression of renal collecting duct.The theory of Chinese medical considered kidney goveming water..It mainly manifested in maintaining the balance of the body water metabolism.During kidney yang deficiency,it will occur obstruction of qi activities and result in disturbance of water metabolism.The experimental results indicate that one of the essence of obstruction of qi activities is AQP2 expression decreased.The possible mechanism of AQP2 expression decreased in renal tissue is due to that increased angiotensinⅡcontent in renal tissue,increased angiotensinⅡtype 1 receptor and cyclooxygenase-2 expression,This results in renal tissue content of cyclic adenosine monophosphate and protein kinase A expression decreased,making the expression of AQP2 in the kidney decreased.After Warmly Nourishing Kidney-yang treatment with Yougui Pill,angiotensinⅡcontent in renal tissue was decreased,expression of angiotensinⅡtype 1 receptor and cyclooxygenase-2 in renal tissue was decreased and cyclic adenosine monophosphate content and expression of protein kinase A increased.It results in increased renal expression of AQP2,which corrects water metabolic disorder in chronic renal failure, improves renal function,delay the progress of chronic renal failure..Conclusion:1.Chronic renal failure rats with kidney-yang deficiency have a marked increase in urine volume,decreased specific gravity,increase of blood Scr and BUN content and renal tissue pathological injury significantly,treatment with Yougui Pill can improve above facts apparently.2.AQP2 expression of renal tissue of Chronic renal failure rats with kidney-yang deficiency decreased.After treatment with Yougui Pill,AQP2 expression of renal tissue increased.3.Yougui pill have no apparent effects on blood AVP content.of Chronic renal failure rats with kidney-yang deficiency4.Chronic renal failure rats with kidney-yang deficiency show that AngiotensinⅡcontent and expression of angiotensinⅡtype 1 receptor and cyclooxygenase-2 in renal tissue were increased,cyclic adenosine monophosphate content and expression of protein kinase A decreased.5.After treatment with Yougui Pill,AngiotensinⅡcontent and expression of angiotensinⅡtype 1 receptor and cyclooxygenase-2 in renal tissue were decreased,cyclic adenosine monophosphate content and expression of protein kinase A increased.更多还原