| Chronic kidney disease(CKD)is a clinical syndrome with hidden onset.It is chronic and progressive renal damage caused by various primary and secondary kidney diseases.Its initial symptoms are not obvious,the patients have poor awareness of it,the condition is easy to repeat and difficult to heal,and the prognosis is poor in the later stage,which poses a serious threat to people’s health.The prevalence of chronic kidney disease in adult population in China is increasing year by year,which has attracted more and more attention.Traditional Chinese medicine believes that it mostly belongs to the categories of "edema,asthenia,low back pain,hematuria and Guange".The location of the disease involves the viscera such as kidney,liver,spleen,lung and heart.The disease is mainly closely related to the three viscera of lung,spleen and kidney.The kidney is the foundation of the gasification movement,and the kidney yang is the Yuan Yang of the whole body.The deficiency of the kidney Yang has no right to gasification,and the water metabolism is abnormal,resulting in edema.Kidney yang deficiency syndrome is an important syndrome type of chronic kidney disease.Edema is one of the main clinical symptoms of kidney yang deficiency syndrome in chronic kidney disease,which is closely related to kidney yang deficiency and Qi loss.Modern clinical research results show that the edema of renal diseases is related to antidiuretic hormone AVP.For example,the AVP of edema patients with nephrotic syndrome is higher than that of normal control group.The combination of AVP and AQP2 enhances the reabsorption of water and fluid in the middle and distal convoluted tubules and collecting ducts of the kidney,that is,it plays an antidiuretic role.It is suggested that the study of AQP2 related pathways is of great significance to reveal the mechanism of edema in kidney yang deficiency syndrome of chronic kidney disease.Previous clinical studies and basic studies have shown that wenshenyang granule can significantly improve the symptoms of kidney yang deficiency.Wenshenyang formula is an integrated formula of its key drugs Cistanche deserticola,epimedium and Rhizoma Drynariae.Whether wenshenyang formula can increase the gasification of water by warming and Tonifying Kidney Yang,so as to improve water metabolism and protect the kidney remains to be studied.Therefore,this experiment constructs the rat model of kidney yang deficiency syndrome combined with disease and syndrome of chronic kidney disease,and studies the mechanism of wenshenyang Recipe on kidney yang deficiency syndrome of chronic kidney disease based on AVP-V2R-AQP2 pathway.Object:In this study,the animal model of kidney yang deficiency syndrome of chronic kidney disease was prepared by tail vein injection of adriamycin and intramuscular injection of hydrocortisone.Zhenwu decoction was used as the experimental control group,and wenshenyang formula was used to intervene.The effects of wenshenyang formula on the general situation,body mass,kidney index,testicular index,24h urinary protein and urine volume,body temperature,kidney appearance Testicular appearance,renal function,TC,TG,LDL,NEFA,TP,ALB,cAMP,cGMP,cAMP/cGMP,T3,T4,TSH,CORT,T;The pathological changes of rat kidney were observed by he,Mallory staining and transmission electron microscope;The level of plasma AVP in rats with kidney yang deficiency syndrome of chronic kidney disease was detected by ELISA;The expression levels of V2R and AQP2 mRNA in kidney of rats with kidney yang deficiency syndrome of chronic kidney disease were detected by in situ hybridization and RT-PCR;The expression levels of V2R and AQP2 protein in rats with kidney yang deficiency syndrome of chronic kidney disease were detected by immunohistochemistry,and the expression level of V2R protein in rats with kidney yang deficiency syndrome of chronic kidney disease was detected by Western blot.Method:1.Preparation and grouping of rat model of kidney yang deficiency syndrome of chronic kidney disease58 male healthy SPF SD rats were fed adaptively for 1 week,free drinking water and ordinary rat feed.They were randomly divided into 10 rats in the normal group and 48 rats in the model group.In the second week of the experiment,except the normal group,the other rats were placed in the rat fixator,the tail was hot packed with warm water,and then the tail was disinfected with 75%alcohol to fully expose the tail vein of the rats.The syringe was inserted at an angle of 30～45 degrees with the tail.Adriamycin was injected into the tail vein at the dose of 4 mg/kg for the first time.At the third week of the experiment,adriamycin was injected at the dose of 4 mg/kg in the same way.Normal rats were injected with normal saline(2 ml/kg)through the tail vein.The success of CKD model was confirmed by 24 h urinary protein≥50 mg.The rats with successful CKD model were randomly divided into adriamycin group,hydrocortisone group,Zhenwu Decoction group and wenshenyang formula group according to body weight and urinary protein,with 12 rats in each group.At the beginning of the 6th week,hydrocortisone group,Zhenwu Decoction group and wenshenyang formula group were continuously intramuscularly injected with 10 mg/kg in the upper part of their hind legs,free drinking water and diet,For 8 consecutive weeks.The rats in Zhenwu Decoction group and wenshenyang formula group were treated with corresponding drugs while making hydrocortisone model:Zhenwu Decoction and wenshenyang formula were converted into 4.76 times by gavage according to the body surface area of adults and animals,the doses were 5.55g·kg-1·d-1 and 2.38g·kg-1·d-1 respectively,and the Administration volume was 1 ml per 100 g body weight of rats.Intragastric administration was given once a day in the afternoon for 8 weeks;Rats in normal group and adriamycin group were perfused with ultrapure water(equal volume).2.Evaluation of kidney yang deficiency syndrome model of chronic kidney disease and Study on the intervention effect of wenshenyang formula2.1 general conditionsThe activity,fur gloss,mental state and diet of rats in each group were observed every day.2.2 determination of body mass and indexes of kidney and testis of ratsWeigh the body mass of rats every week,take materials after modeling treatment,weigh the kidneys and testicles of rats,and calculate the indexes of kidneys and testicles.2.3 24h urinary protein and urine volumeAt the 1st,5th,7th and 9th weeks of the experiment,24-h urine was collected with rat metabolic cage for 24-h urinary protein detection.2.4 body temperatureInfrared temperature gun detects the temperature of the tail of the rat,once a week.2.5 general morphology of kidney and testisAfter modeling treatment,the materials were taken to observe the general morphology of kidney and testis.2.6 detection of blood biochemical indexesAt the beginning of the 14th week,the femoral artery blood of rats in each group was taken,and the contents of Scr,UA,BUN,TP,ALB,TC,TG and LDL were detected by automatic biochemical instrument;The contents of NEFA,CORT,T3,T4,TSH,cAMP,cGMP and T were detected by enzyme-linked immunosorbent assay.2.7 HE and Mallory stainingThe pathomorphological changes of rat kidney were observed by HE staining and Mallory staining.2.8 TEM observationThe morphological changes of glomerular basement membrane and foot process were observed by transmission electron microscope.2.9 ELISAAt the beginning of the 14th week,the femoral artery blood of rats in each group was taken,and the plasma AVP level was detected by ELISA.2.10 in situ hybridization and RT-PCRIn situ hybridization and RT-PCR were used to detect the expression of V2R and AQP2 mRNA in rat kidney.2.11 immunohistochemical methodImmunohistochemistry was used to detect the expression of V2R and AQP2 protein in renal tissue of rats in each group;The expression level of V2R protein in renal tissue of rats in each group was detected by Western blot.3 image analysis and data statisticsImage analysis was performed by image-j image analysis software.Six visual fields were randomly selected in each immunohistochemical group to measure their OD values,and the data were statistically analyzed by SPSS 23.0 software.Result:1.General informationCompared with the normal group,hydrocortisone rats showed symptoms of kidney yang deficiency,such as withered hair,arched back,laziness,curling,mental depression,thin body,slow movement and edema of lower limbs;The general state of rats in wenshenyang formula group and Zhenwu Decoction group was better than that in hydrocortisone group.2.Body mass and organ indexCompared with the normal group,the body weight of hydrocortisone rats decreased significantly(P<0.01),the renal index increased significantly(P<0.01),and the testicular index decreased significantly(P<0.01).Compared with the hydrocortisone group,the body weight of wenshenyang formula group increased significantly(P<0.01),the renal index decreased(P<0.01),and the testicular index had no significant difference.3.24h urinary protein and urine volumeIn the first week,there was no significant difference in 24h urinary protein between groups;Compared with the normal group,the 24h urinary protein of rats in other groups increased significantly at week 5,7 and 9(P<0.01);At the 9th week,compared with hydrocortisone group,24h urinary protein in Zhenwu Decoction group and wenshenyang formula group decreased.4.Body temperatureCompared with the normal group,the body temperature of rats in hydrocortisone group decreased significantly(P<0.01),and the body temperature of rats in wenshenyang formula group increased compared with hydrocortisone group.5.General morphology of kidney and testisCompared with the normal group,the kidneys of hydrocortisone group were gray white and increased in volume.The kidney volume of wenshenyang formula group was smaller than that of hydrocortisone group,and the color was between brown and gray white.Compared with the normal group,the testis of other groups were significantly smaller than that of the normal group,and the color had no significant change.The testis of wenshenyang formula group was slightly larger than that of the model group.6.Renal functionCompared with the normal group,Scr in hydrocortisone group increased significantly(P<0.01),bun and UA increased;Compared with hydrocortisone group,Scr in wenshenyang formula group decreased significantly(P<0.01),and BUN decreased.7.TC、TG、LDL、NEFACompared with the normal group,the content of TC in hydrocortisone group increased significantly(P<0.01),TG increased(P<0.05),LDL increased and NEFA decreased significantly(P<0.01);Compared with hydrocortisone group,TC,TG and LDL decreased and NEFA increased in wenshenyang formula group.8.TP、ALBCompared with the normal group,the content of TP and ALB in hydrocortisone group decreased significantly(P<0.05 and P<0.01);Compared with hydrocortisone group,TP and ALB in wenshenyang formula group increased.9.cAMP、cGMP、cAMP/cGMPCompared with the normal group,cAMP in hydrocortisone group decreased significantly(P<0.01),cGMP increased significantly(P<0.01)and cAMP/cGMP ratio decreased significantly(P<0.01).Compared with hydrocortisone group,cAMP and cAMP/cGMP ratio in wenshenyang formula group increased significantly(P<0.01)and cGMP decreased significantly(P<0.01)compared with Zhenwu Decoction group,cAMP/cGMP ratio increased significantly(P<0.01).10.Adrenal axis index CORTCompared with the normal group,CORT in hydrocortisone group decreased significantly(P<0.01),and CORT in wenshenyang formula group increased significantly(P<0.01)compared with hydrocortisone group.11.Thyroid axis indexCompared with the normal group,the contents of T3 and T4 in hydrocortisone group decreased significantly(P<0.01),and the content of TSH increased(P<0.05).12.Gonadal axis indexCompared with the normal group,T in hydrocortisone group decreased,and T in wenshenyang formula group increased compared with hydrocortisone group.13.He and Mallory staining resultsThe results of HE staining showed that compared with the normal group,the glomerular mesangial area of hydrocortisone group was widened,mesangial cells showed different degrees of proliferation,renal tubules dilated or atrophied,protein tube type,a large number of inflammatory cell infiltration in renal interstitium and balloon wall adhesion.Compared with hydrocortisone group,the proliferation of glomerular mesangial cells,the infiltration of renal interstitial inflammatory cells and the type of protein tube in wenshenyang formula group were reduced.Mallory staining results showed that compared with the normal group,a large number of collagen fibers were deposited in the glomerular mesangial area of the renal tissue of the hydrocortisone group,and a small amount of collagen fibers were deposited in the glomerular mesangial area of the wenshenyang formula group compared with the hydrocortisone group.14.TEM resultsCompared with the normal group,the glomerular basement membrane segment in the hydrocortisone group was thickened and the foot process was widely fused.Compared with the hydrocortisone group,the glomerular basement membrane segment thickening was not obvious and the foot process fusion was reduced in the wenshenyang formula group.15.Plasma AVPCompared with the normal group,the AVP values of other groups increased(P<0.01,P<0.05),and the AVP of wenshenyang formula group decreased(P<0.05)compared with hydrocortisone group.16.Expression of V2R and AQP2 mRNA in renal tissueThe results of in situ hybridization showed that compared with the normal group,the expressions of V2R mRNA and AQP2 mRNA in hydrocortisone group increased significantly(P<0.01).Compared with hydrocortisone group,the expression of V2R mRNA and AQP2 mRNA in wenshenyang formula group decreased significantly(P<0.01).The results of RT-PCR showed that the expressions of V2R mRNA and AQP2 mRNA in hydrocortisone group were significantly higher than those in normal group(P<0.01);Compared with hydrocortisone group,the expression of V2R mRNA and AQP2 mRNA in wenshenyang formula group decreased significantly(P<0.01).17.Expression of V2R and AQP2 protein in renal tissueImmunohistochemical results showed that the expression of V2R in hydrocortisone group was significantly higher than that in normal group(P<0.01);Compared with hydrocortisone group,V2R in wenshenyang formula group decreased significantly(P<0.01),and the expression of AQP2 decreased..Western blot showed that the expression of V2R in hydrocortisone group increased compared with normal group;Compared with hydrocortisone group,V2R in wenshenyang formula group decreased(P<0.05).Conclusion:1.The rat model of chronic kidney disease can be successfully established by fractional tail vein injection of adriamycin.2.The animal model of kidney yang deficiency syndrome of chronic kidney disease can be successfully induced by repeated tail vein injection of adriamycin and hindlimb injection of hydrocortisone.The model is evaluated mainly from three aspects:obvious symptoms of kidney yang deficiency,low function of hypothalamus pituitary adrenal axis,thyroid axis and gonadal axis in the model rats with disease syndrome combination Abnormal energy metabolism indexes and the improvement effect of wenshenyang Recipe on kidney yang deficiency syndrome in rats with this disease syndrome combination model.3.Wenshenyang recipe can reduce kidney damage to a certain extent,protect the kidney,improve the symptoms of kidney yang deficiency in rats with disease syndrome combination model,and improve the functions of hypothalamus pituitary adrenal axis,gonad axis and energy metabolism.4.Wenshenyang recipe can reduce the content of plasma AVP,the expression of V2R and AQP2 mRNA and protein in renal tissue,regulate water metabolism and reduce edema in rats with kidney yang deficiency of chronic kidney disease by regulating AVP-V2R-AQP2 signal pathway. |
PDF Full Text Request