| Chronic Kidney Disease(CKD)refers to the progressive damage of Kidney structure and function caused by various reasons over a course of 3 months.According to relevant statistics,by 2017,there were 697.5 million patients with chronic kidney disease worldwide and 132.3 million in China,which seriously harms people’s health.Chronic kidney disease is insidious and develops slowly,leading to renal interstitial fibrosis and glomerulosclerosis,and eventually to chronic kidney failure.Chronic kidney disease(CKD)is known in traditional Chinese medicine(TCM)on "edema,dysuria,dysuria,dysuria"(URination).Chronic kidney disease(CKD)has a complex pathogenesis,which is characterized by deficiency of the root and deficiency of the kidney.Congenital deficiency,deficiency of spleen and kidney,or invasion of external evil,excessive sexual affairs,disorder of emotions,loss of treatment,can lead to kidney Yang deficiency syndrome,then the function of the essence of the kidney transpiration and vaporization dereliction of responsibility,water flow is not smooth,qi and blood block,damp,poison,blood stasis connotation,deficiency and solid inclusion,hair and disease.The inflammatory state can be classified as "turbid poison","damp heat" and "blood stasis" in TCM.In recent years,an important inflammatory pathway,TLR4/MyD88/NF-κB signaling pathway,has been found to be closely associated with chronic kidney disease.At present,western medicine treatment of chronic kidney disease is mainly symptomatic treatment,disease control is insufficient,there are certain limitations.Traditional Chinese medicine has unique advantages in preventing chronic kidney disease,delaying disease progression,alleviating clinical symptoms and protecting residual renal function in the body.Kidney Yang deficiency syndrome is an important syndrome type of chronic kidney disease.Kidney is the root of congenital disease,and the main treatment principle is to warm kidney Yang.The recipe for Warming Shenyang is composed of three herbs,cistanche deserticola,Herba icarii and Guminbu.Previous studies have confirmed that the recipe for warming Shenyang can significantly improve the deficiency syndrome of kidney-yang by warming and tonifying kidney-yang.Therefore,by establishing the "disease syndrome" combined animal model of chronic kidney disease with kidney-yang deficiency syndrome,this study explored the effects of Wenshenyang prescription on renal protection and TLR4/MyD88/NF-κB signaling pathway in rats with chronic kidney disease with kidney-yang deficiency syndrome,and explained the mechanism of wenshenyang prescription,so as to provide experimental basis for the treatment of kidney-yang deficiency syndrome with Wenshenyang prescription.Objective:In this study,intravenous injection of adriamycin combined with intramuscular injection of hydrocortisone was used to construct the animal model of "disease and syndrome"combined with kidney Yang deficiency syndrome of chronic kidney disease.Wenshenyang Prescription was given for intervention,and Zhenwu Decoction was used for control.The effects of Wenshenyang prescription on general condition,body weight,organ index,24 h urinary protein,body temperature,blood biochemical indexes and pathological changes of kidney tissue were observed.The mRNA expression of TLR4,MyD88 and NF-κB were detected by in situ hybridization and RT-PCR.The expression of TLR4,MyD88 and NF-κB in rat kidney were detected by immunohistochemistry.Method:1.Preparation,grouping and administration of rat model of chronic kidney disease with kidney-yang deficiency syndromeA total of 58 healthy SPF MALE SD rats were accommodated for 1 week and randomly divided into normal group(n=10)and model group(n=48).In the second week of the experiment,except for the normal group,the model group was injected with adriamycin by tail vein at 4 mg/kg for the first time.In the third week of the experiment,the model group was injected with adriamycin by tail vein at 4 mg/kg for the second time in the same way,and the normal group was injected with normal saline by tail vein at 2 mL/kg.The criteria for successful construction of the chronic kidney disease rat model were 24 h urinary protein quantification ≥50 mg,and the rats were grouped by stratified random method based on body weight and 24 h urinary protein quantification:Rats in adriamycin group(12),hydrocortisone group(12),Zhenwu Decoction group(12)and Wenshenyang Prescription group(12)were injected with hydrocortisone 10 mg/kg per day intramuscular in hind limbs at the beginning of the 6th week,and rats in Zhenwu Decoction group and Wenshenyang Prescription group were given free water and diet for 8 consecutive weeks.At the same time,wenshenyang prescription group was given Wenshenyang prescription for intervention treatment,and Zhenwu Decoction group was given Zhenwu Decoction for control.According to the body surface area of adults and animals,the dosage of Wenshenyang Prescription group and Zhenwu Decoction group was 4.76 times,and the dose was 2.38g·kg-1·d-1 and 5.55g·kg-1·d-1,respectively.The volume was 1 mL per 100 g body weight of rats.The drug was administered intragastric every afternoon;Normal group and doxorubicin group were given equal volume of deionized water intragastually for 8 weeks.2.Evaluation of rat model of chronic kidney disease with kidney-yang deficiency syndrome and study on the intervention effect of Warming Kidney-yang prescription2.1 General InformationThe mental condition,activity,diet and hair of the rats were observed and recorded daily.2.2 Body mass and renal and testicular indicesThe rats were weighed and recorded weekly.Rats were anesthetized with 1%Pentobarbital Sodium at the beginning of the 14th week.After blood was collected from femoral artery,kidney and testis were dissected and organ index was calculated.2.3 Urine protein quantification at 24 hMetabolic cages were used to collect 24 h urine of rats at week 1,5,7 and 9 of the experiment,respectively,and the 24 h urine protein quantity of rats was calculated.2.4 temperatureInfrared temperature measuring gun was used to measure the temperature of rat tail root every week,and the temperature was recorded.2.5 Gross morphology of kidney and testisAt the 14th week,the rats were sampled,and the gross morphology of kidney and testis in each group was observed.2.6 Blood biochemical index detectionAt the beginning of the 14th week,the levels of Scr,UA,BUN,TC,TG,LDL,TP and ALB were detected by automatic biochemical instrument.The contents of NEFA,cAMP,cGMP,CORT,T3,T4,TSH and T were detected by enzyme-linked immunoassay.2.7 HE and Mallory stainingThe pathological changes of renal tissue in each group were observed.2.8 Transmission electron MicroscopeThe structure of glomerular basement membrane and foot process were observed in each group.2.9 In situ hybridization and RT-PCR experimentsThe mRNA expression levels of TLR4,MyD88 and NF-κB were detected.2.10 Immunohistochemical testsThe protein expression levels of TLR4,MyD88 and NF-κB were detected.3.Image analysis and data statisticsImage analysis software image-J was used to analyze the experimental results,and SPSS 23.0 software was used to make statistics on the data.Result:1.GeneralAt the 4th week of the experiment,the doxorubicin model group gradually developed dark hair color,lower limb edema and weight loss.At the 8th week of the experiment,the hydrocortisone model group showed symptoms of kidney-yang deficiency,such as emaciation,lower limb edema,low hair luster,arched back,lazy movement,crouching and listlessness.Compared with hydrocortisone group,the general state of rats in Wenshenyang prescription group and Zhenwu Decoction group was improved.2.Body weight and organ indexCompared with normal group,the body weight of rats in hydrocortisone group was significantly decreased(P<0.01),the renal index was significantly increased(P<0.01),and the testicular index was significantly decreased(P<0.01).Compared with hydrocortisone group,body weight of rats in Wenshenyang formula group was significantly increased(P<0.01),kidney index was significantly decreased(P<0.05),and testicular index had no significant difference.3.24 h urine protein quantificationCompared with normal group,24 h urinary protein in hydrocortisone group was significantly increased(P<0.01);Compared with hydrocortisone group,the 24 h urinary protein of rats in Wenshenyang prescription group had a decreasing trend.4.The temperatureCompared with normal group,the body temperature of hydrocortisone group was significantly decreased(P<0.01).Compared with hydrocortisone group,the body temperature of rats in wenshenyang prescription group was increased.5.Appearance of rat kidney and gross morphology of testisCompared with the normal group,the renal volume of hydrocortisone group was enlarged and the color was gray.Compared with the hydrocortisone group,the size of the kidney of the rats in the wenshenyang prescription group decreased and the degree of gray color decreased.Compared with normal group,the testis of hydrocortisone group,Zhenwu Decoction group and Wenshenyang prescription group decreased significantly,and the color of testis had no significant change.6.Blood biochemical indexes6.1 Renal function:Compared with normal group,Scr and UA in hydrocortisone group were significantly increased(P<0.05),and BUN showed an increasing trend;Compared with hydrocortisone group,Scr of rats in Wenshenyang prescription group was significantly decreased(P<0.01),and BUN showed a decreasing trend.6.2 Blood lipid:Compared with normal group,TC and TG in hydrocortisone group were significantly increased(P<0.01),LDL was significantly increased(P<0.05),and NEFA was significantly decreased(P<0.01);Compared with hydrocortisone group,TC,TG and LDL in Wenshenyangfang group were decreased,while NEFA was significantly increased(P<0.01).6.3 Protein:Compared with normal group,TP and ALB in hydrocortisone group were significantly decreased(P<0.01);Compared with hydrocortisone group,TP and ALB in Wenshenyang formula group were significantly increased(P<0.05).6.4 cAMP,cGMP and cAMP/cGMP:Compared with normal group,cAMP and cAMP/cGMP in hydrocortisone group were significantly decreased(P<0.01),while cGMP was significantly increased(P<0.01).Compared with hydrocortisone group,cAMP and cAMP/cGMP in Wenshenyang formula group were significantly increased(P<0.01).6.5 Adrenal axis index:Compared with normal group,CORT in hydrocortisone group was significantly decreased(P<0.01);Compared with hydrocortisone group,CORT of rats in Wenshenyang formula group was significantly increased(P<0.01).6.6 Thyroid axis index:Compared with normal group,T3 and T4 in hydrocortisone group were significantly decreased(P<0.01),and TSH was significantly increased(P<0.01).6.7 Gonad axis index:Compared with normal group,T in hydrocortisone group showed a decreasing trend;Compared with hydrocortisone group,T in wenshenyang formula group showed an increasing trend.7.Pathological changes of kidney in ratsHE staining:Compared with the normal group,the renal tubules in hydrocortisone group showed protein tubules,widening of mesangial area,proliferation of mesangial cells and podiocytes,vacuolar change of renal tubule epithelial cells,and a large number of inflammatory cells infiltrated in renal interstitium.The tubular type of tubulin decreased,mesangial cell proliferation was mild,and inflammatory cells infiltrated in renal interstitium.Mallory’s staining:Compared with the normal group,the hydrocortisone group showed significant deposition of collagen fibers in the glomerular mesangial region.A small amount of collagen fibers were deposited in the mesangial region of glomerulus in wenshenyang prescription group.Transmission electron microscopy(TEM)results:The structures of glomerular basement membrane and foot process were normal in normal group.Compared with the normal group,the glomerular basement membrane segment was thickened and the foot process was extensively fused in the adriamycin and hydrocortisone groups.Compared with model group,the thickness of glomerular basement membrane segment in zhenwu Decoction group and Wenshenyang prescription group was not obvious,and the fusion degree of foot process was reduced.8.Effects of Wenshenyang Prescription on TLR4/MyD88/NF-κB pathway gene expression in kidney of kidney Yang deficiency syndrome rats with chronic kidney diseaseCompared with normal group,the mRNA expression levels of TLR4,MyD88 and NF-κB in renal tissue of other groups were significantly increased(P<0.05,P<0.01);Compared with hydrocortisone group,the mRNA expressions of TLR4,MyD88 and NF-κB in renal tissue of rats in Wenshenyang formula group were significantly decreased(P<0.05,P<0.01).9.Effects of Wenshenyang Prescription on protein expression of RENAL TLR4/M yD88/NF-κB pathway in rats with kidney Yang deficiency syndrome of chronic kidney diseaseCompared with normal group,the protein expressions of TLR4,MyD88 and NF-κB in kidney tissues of other groups were significantly increased(P<0.01);Compared with hydrocortisone group,the protein expressions of TLR4,MyD88 and NF-κB in renal tissue of rats in Wenshenyang formula group were significantly decreased(P<0.01).Conclusion:1.The rats were injected with adriamycin twice through caudal vein,and the 24 h urinary protein,renal function,blood lipid,protein indexes and renal pathology were used as evaluation criteria to successfully construct the rat model of chronic kidney disease.2.The rats were injected with hydrocortisone and treated with Warming Shenyang prescription for intervention.The model was evaluated comprehensively from three aspects:physical signs,objective indicators and drug evidence,with the signs of kidney-yang deficiency,hypothalamic-pituitary-adrenal axis,thyroid axis,gonad axis and energy metabolism indicators as the evaluation criteria.The rat model of chronic kidney disease with kidney-yang deficiency syndrome combined with "disease syndrome" was successfully constructed.3.Warming Kidney Yang recipe can alleviate kidney damage of kidney-yang defic iency syndrome rats of chronic kidney disease to a certain extent,thus playing the rol e of kidney protection,and improve the symptoms of kidney-yang deficiency,hypothal amic-pituitary-adrenal axis,hypothalamic-pituitary-gonad axis function and abnormal en ergy metabolism.4.Warming Shenyang recipe can affect the functions of hypothalamic-pituitary-adrenal axis and hypothalamic-pituitary-gonad axis by intervening the protein and mRNA expression of renal TLR4/MyD88/NF-κB signaling pathway in rats with kidney-yang deficiency syndrome of chronic kidney disease,and play a good role in improving and treating kidney-yang deficiency syndrome of chronic kidney disease. |
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