Based On AQP-2 To Investigate The Reducing Urination And Norish Kidney-yang Machanisms Of SQW

The formula of Suoquanwan(SQW)was recorded in a Chinese medicine book, named Furenliangfang,which collected hundreds of formulas of Chinese medicine combinations.SQW consists three herb medicines,Radix Linderae,Radix dioscoreae, and alpinia oxyphylla Miq.The function of SQW is to warm kidney,stop polyuria and hold excessive urination.In clinical practice,SQW is used for the treatment of polyuria and excessive urination due to Yang-deficiency of Kidney.In short words,it acts to warm kidney and hold excessive urination,in which the former is the root target and the latter is the emergency target of treatment.Whether each ingredient or the combination of SQW,have the blank in the field of researches for their action of warming kidney on Yang-deficiency of Kidney and holding excessive urination on the pathophysiological changes of the kidney damage.It is ironic that SQW has good clinical efficacy but the basis of its action is absent.The thesis is focused on such therapeutical basis of SQW action in animal model of Yang-deficiency of Kidney induced by adenine which have characteristics of polyuria.The target of investigation involves in the expression of aquaporin 2(AQP-2),which is the archetypal molecular water channel,the concentration of metabolites and ions in urine and the level of aldosterone in serum,which may be the causes of polyuria,and the immunity function changes due to Yang-deficiency of Kidney.At the first stage,the animal model of Yang-deficiency of Kidney induced by adenine was investigated.By repeated experiments,fix-dose drug administration in gavage(i.g.)was improved more stable and repeatable than the normal method by adding adenine into feed.And suitable dosage and regimen were determined to assure that the animal with characteristics of Yang-deficiency of Kidney lives for certain period.So the dosage of 250mg/kg/d adenine i.g.in rats for one month was chosen as ideal experimental condition.The model rats showed some typical symptoms of Yang-deficiency of Kidney,such as polyuria,chill and cold of limbs,huddling-up,low baseal metabolism,weightloss,listlessness,aversion to cold and hypotrichosis.It is a good Kidney of Yang-deficiency and polyuria model.SQW could reduce urine and regelate the syndrome.The pharmacological machanisms of SQW were investigated in following aspects.1.The influence of SQW on urine in kidney of Yang-deficiency model.2.The influence of SQW on expression of AQP-2 mRNA and AVPR-V2 mRNA in kidney of Yang-deficiency model by realtime fluorescence quantitative PCR method was investigated.3.The influence of SQW on the in situ expression of AQP-2 and AVPR-V2 in rats' kidney was observed by immunohistochemistry method.4.The influence of SQW on the expression of AQP-2 and AVPR-V2 was obsersed by Westernblot.5.The pathological changes of kidney in rats were observed with HE dying method before and after SQW administration.6.The serum level of aldosterone and cAMP were detected by radioimmuno analysis.7.The level of BUN,Cr,sodium,potassium,chlorine in urine were detected.8.The level of CD3/CD4/CD8 in blood were investigated by flow cytometry assay.9.The weight and organ indexes were observed in Yang-deficiency model treated with SQW.The results showed that SQW elevated significantly the expression level of AQP-2 mRNA and AVPR-V2 mRNA in kidney-yang deficieny rats with polyuria. SQW also increased the in situ expression of AQP-2 in the membrane and plasma of nephron collecting duct cells and AVPR—V2 expressed in the plasma of the wall cells of glomerulus,collecting duct,renal tubule and Bowman capsule.By Western blot,both AQP-2 and AVPR-V2 protein expressions were increased.Some histological changes were observed in SQW group:the brown crystal granules in cavern of Bowman capsule and renal tubule.And inflammatory infiltration were decreased.The serum cAMP and ALD level increased after SQW administration in model rats.The pathological characteristics of kidney function of model rats were switched:urine potassium excretion elevated and the volume of sodium and water excretion dropped. SQW increased the percent of CD3~+ and CD3~+CD4~+ in blood and decrease the percent of CD3~+CD8~+.SQW increased the weight and thymus gland,adenohypophysis and aderenal indexes.The thesis involves three aspects of the pharmacology of SQW.First,the influence of SQW on the express and positioning of AQP-2 and AVPR-V 2 in kidney were investigated.It was implied that SQW regulates the expression and distribution of AQP-2,which is the main factor in water balance mechanism.Second,SQW acts on several certain pathophysiological changes resulting in polyuria,such as the urine metabolites,concentration of ions,and serum level of aldosterone.Third,the weakened immunity function due to kidney-Yang-deficieny were strengthened by SQW,which showed its has multi-targeting drug action and integral regulation.