Experimental Study On The Effect Of Wuling Powder On Water And Liquid Metabolism Of Kidney-yang Deficiency Edema Model Rats Based On AQP3

OBJECTIVE: In this study,we prepared the rat model of kidney-yang deficiency edema,observed the effect of Wuling powder on the water metabolism of rats with kidney-yang deficiency edema,and detected the expression of AQP3 by immunohistochemistry,RT-PCR and Western Blot,in order to explore the correlation between the regulation of water metabolism and AQP3 in rats with kidney-yang deficiency edema by Wuling powder.The aim of this study is to reveal the regulatory mechanism of classical prescription Wuling Powder on renal water metabolism from the level of aquaporin,and to provide theoretical and experimental basis for clinical use of Wuling Powder in the treatment of water metabolism-related diseases.METHODS:1.The rat model of renal Yang deficiency and edema was prepared by injecting hydrocortisone into the arm muscle and doxorubicin hydrochloride solution into the tail vein;2.The behavior of rats was observed.The body weight,body temperature,urine volume and 24-hour urinary protein were measured.The contents of serum cAMP and cGMP were measured.3.50 male rats were randomly selected and randomly divided into 5groups,namely model group(MG group),low-dose wuling powder group(wls-l group),medium-dose wuling powder group(wls-m grou),highdose wuling powder group(wls-h group)and fuxime group(F group),with 10 rats in each group.Ten unmodeled normal rats were randomly selected as the blank group(NG).4.Each group was given the same volume of different drugs for 7 days,and the urine volume of each group(1h,2h,4h,24h)was collected and measured with the metabolic cage device.5.After the experiment,blood was collected from the abdominal aorta of rats in each group,and the rats were sacrificed and their kidneys were extracted.6.Renal tissue injury was observed by HE staining;7.Immunohistochemistry,rt-pcr and Western Blot were used to detect and analyze the expressions of AQP3 mRNA and AQP3 protein in the kidney of rats.Results:1.The results of behavioral observation,body weight,body temperature,urine volume and 24-hour urinary protein test showed that compared with NG group,the weight and body temperature of kidney-yang deficiency edema model group decreased significantly,urine volume decreased,and 24-hour urinary protein content increased.The serum levels of cAMP and cGMP in rats were detected by ELISA method.Compared with NG group,the content of cAMP in each group of kidney-yang deficiency edema model decreased,and the content of cGMP was increased.The value of cAMP/cGMP decreased significantly with the increase of cAMP.In conclusion,it is suggested that the model of kidney-yang deficiency edema obtained in this experiment is stable and reliable.2.By measuring the 24-hour total urine volume of rats,it was found that the 24-hour urine volume of rats in MG group decreased significantly compared with that in NG group(p<0.01).Compared with MG group,the24-hour urine volume of rats in WLS-L group and WLS-H group increased significantly(p<0.05),while the 24-hour urine volume of rats in WLS-M group and F group increased significantly(p<0.01).3.By analyzing the urine volume of rats in each group at the corresponding time points(1h,2h,4h)after administration,the urine volume of each experimental group increased with the increase of time,but the increase degree of each group was different.Compared with NG group,the urine volume of MG at each time point at 1h,2h and 4h after administration had significant difference(p<0.01);compared with MG group,the urine volume of WLS-L group,WLS-M group,WLS-H group and F group at each time point at 1h,2h and 4h after administration hadsignificant difference(p<0.01).4.By using HE staining,immunohistochemistry,RT-PCR and Western Blot techniques,AQP3 gene and AQP3 protein were detected in rat kidneys.The results showed that AQP3 was mainly distributed in the basolateral membrane of renal collecting duct epithelial cells.Gray value of density in each area was analyzed by image software.Compared with NG group,the expression of AQP3 gene and AQP3 protein in MG group increased significantly(p<0.01).Compared with MG group,the expression of AQP3 and AQP3 in WLS-L group,WLS-M group,WLS-H group and F group decreased significantly(p<0.01).Conclusion:1.Wuling powder has diuretic effect on kidney Yang deficiency and edema model rats,and its diuretic intensity is related to drug concentration.Wuling powder with medium dose has the most significant diuretic effect.2.The diuretic effect of Wuling powder on kidney-yang deficiency edema rats is related to the regulation of renal AQP3.The diuretic effect may be achieved by down-regulating the expression of AQP3 mRNA and AQP3 in kidney.