| Chronic Kidney Disease(CKD)is defined as a chronic disease caused by various factors of kidney structure and(or)kidney function long-term abnormalities(lasting more than 3 months),which seriously endangers human health.CKD is characterized by high prevalence rate,low public awareness rate,long course of disease,poor prognosis,high medical cost and heavy burden on patients.It poses a serious threat to human health and social development.In recent years,the prevalence of CKD in adult population is increasing year by year.Chronic kidney disease,Chinese medicine thinks that it belongs to "edema,asthenia,hematuria,guange" and other categories,the disease location involves kidney,spleen,liver,lung,heart,sanjiao etc.,the disease is closely related to the dysfunction of lung,spleen and kidney.Kidney-yang deficiency syndrome is an important syndrome type of chronic kidney disease.Yang deficiency is insufficient to promote gasification,qi,blood and body fluid can not nourish the tissue,causing ischemia and hypoxia.Modern research results show that chronic kidney disease exists a state of ischemia and hypoxia at the end of the tissue,and this unfavorable state aggravates the pathological damage of the kidney tissue,forming a vicious circle.Hypoxia can lead to pyrotopia of tissue cells,leading to tissue damage,and pyrotopia is an important way of programmed cell death,so it is of great significance to study hypoxic pyrotopia in the course of chronic kidney disease.Hypoxia inducible factor-1α(HIF-1α)is an important regulatory factor in the hypoxia process found in current studies,and is involved in many pathological processes related to hypoxia,suggesting that the study on the pyrodeath pathway associated with HIF-1α is of great significance to reveal the pathogenesis of kidney-yang deficiency syndrome in chronic kidney disease.Zhenwu Decoction is a classic Chinese medicine prescription with the effect of warming yang to promoting water.Previous basic studies and clinical studies have shown that Zhenwu Decoction has a good clinical effect on the treatment of kidney-yang deficiency syndrome in chronic kidney,disease.However,no relevant studies have been found on whether Zhenwu Decoction can improve the hypoxia state of kidney in chronic kidney disease,thereby reducing kidney damage and protecting the kidney.In this study,a rat model of chronic kidney disease with kidney-yang deficiency syndrome through combination of disease and syndrome was constructed,and the mechanism of Zhenwu Decoction on kidney-yang deficiency syndrome in chronic kidney disease was studied based on HIF-1α/NLRP3/GSDMD signal pathway.Object:In this study,the animal model of chronic kidney disease with kidney-yang deficiency syndrome was prepared by adriamycin combined with hydrocortisone.Zhenwu Decoction was used as the intervention group,and wenshenyang formula was used as the positive control group.The effects of Zhenwu Decoction on the general situation,body weight,kidney index,testicular index,24h urine protein,body temperature,kidney appearance,testicular appearance,renal function,TC,TG,LDL,NEFA,TXB2,TP,ALB,cAMP,cGMP,cAMP/cGMP,T3,T4,TSH,CORT,T.HE and Mallory staining and transmission electron microscopy were used to observe the pathological changes of renal tissues of rats.The chemical constituents of Zhenwu decoction compound were identified by UHPLC-Q-Exactive-Orbitrap/MS.Transcriptomics was used to detect the differential gene expression in the kidney of rats.The mRNA and protein expression levels of PHD2,HIF-1α,VHL,NLRP3,and GSDMD were detected by in situ hybridization,RealTime PCR and immunohistochemistry.Method:1.Identification of chemical components of Zhenwu decoctionThe chemical constituents of Zhenwu decoction compound were identified by UHPLC-Q-Exactive-Orbitrap/MS.2.Preparation and grouping of rat model of kidney yang deficiency syndrome of chronic kidney disease58 male healthy SPF class SD rats were fed adaptively for 1 week,free drinking water and ordinary rat feed.They were randomly divided into the normal group(n=10),adriamycin group(n=12),adriamycin+hydrocortisone group(n=12),Zhenwu Decoction group(n=12)and wenshenyang formula group(n=12).In the second week of the experiment,except the normal group,the other rats were placed in the rat fixator,after fixed the tail of the rats,the tail was hot packed with warm water and sterilized with 75%alcohol,so that the tail veins of the rats were fully exposed and adriamycin was injected.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 group rats were injected with normal saline(2 ml/kg)through the tail vein.The rats were placed in the metabolic cage to collect urine for 24 hours.The success of CKD model was confirmed by 24 h urinary protein ≥50 mg.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 every morning,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:the gavage doses of Zhenwu Decoction and Wenshenyang formula were converted into 4.76 times according to the conversion of body surface area between adults and animals,the doses were 5.55g·kg-1·d-1 and 2.38g·kg-1·d-1,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 deionized water(equal volume).3.Evaluation of model of kidney yang deficiency syndrome with chronic kidney disease and study on the intervention effect of Zhenwu Decoction3.1 General conditionsThe activity,fur gloss,mental state and diet of rats in each group were observed every day.3.2 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.3.3 24-h urinary proteinAt 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.3.4 Body temperatureInfrared temperature gun detects the temperature of the tail of the rat,once a week.3.5 General morphology of kidney and testisAfter modeling treatment,the materials were taken to observe the general morphology of kidney and testis.3.6 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,BUN,UA,TC,TG,LDL,TP and ALB were detected by automatic biochemical instrument;the contents of NEFA,TXB2 were detected by ELISA.3.7 Detection of energy metabolism indexesSerum cAMP and cGMP were detected by ELISA.3.8 Detection of adrenal axis indexesSerum CORT were detected by ELISA.3.9 Detection of thyroid axis indexesSerum T3,T4,TSH were detected by ELISA.3.10 Detection of gonadal axis indexesSerum T were detected by ELISA.3.11 HE and Mallory stainingThe pathomorphological changes of rat kidney were observed by HE staining and Mallory staining.3.12 TEM observationThe morphological changes of glomerular basement membrane and foot process were observed by transmission electron microscope.3.13 TranscriptomicsTranscriptomics was used to analyze the differential gene expression and pathway between the normal group and the adriamycin+hydrocortisone model group,the adriamycin+hydrocortisone group and the Zhenwu decoction group.3.14 In situ hybridization and RealTime PCRIn situ hybridization and RealTime PCR were used to detect the expression of PHD2,HIF-1α,VHL,NLRP3,and GSDMD mRNA in rat kidney.3.15 Immunohistochemical methodImmunohistochemistry was used to detect the expression of PHD2,HIF-1αVHL,NLRP3,and GSDMD protein in renal tissue of rats in each group.4 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 24.0 software.Result:1.Identification of chemical components of Zhenwu decoctionUHPLC-Q-Exactive-Orbitrap/MS identified 62 compounds,including alkaloids and terpenoids.2.General informationCompared with the normal group,adriamycin+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 Zhenwu Decoction group and wenshenyang formula group was better than that in adriamycin+hydrocortisone group.3.Body mass and organ indexCompared with the normal group,the body weight of the adriamycin+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 adriamycin+hydrocortisone group,the body weight of the Zhenwu Decoction group increased significantly(P<0.01),the renal index decreased(P<0.01),and the testicular index increased.4.24-h urinary proteinIn the first week,there was no significant difference in 24-h 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 the adriamycin+hydrocortisone group,24-h urinary protein in the Zhenwu Decoction group and wenshenyang formula group decreased.5.Body temperatureCompared with the normal group,the body temperature of rats in the adriamycin+hydrocortisone group decreased significantly(P<0.01),and the body temperature of rats in the Zhenwu Decoction group increased compared with the adriamycin+hydrocortisone group.6.General morphology of kidney and testisCompared with the normal group,the kidneys of the adriamycin+hydrocortisone group were grayish white and enlarged in size.The kidney volume of Zhenwu Decoction group was smaller than that of adriamycin+hydrocortisone group,and the color was between brown and grayish 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 Zhenwu Decoction group was slightly larger than that of the model group.7.Renal function Scr,BUN,UACompared with the normal group,Scr in the adriamycin+hydrocortisone group increased significantly(P<0.01),BUN and UA increased;Compared with adriamycin+hydrocortisone group,Scr in Zhenwu Decoction group decreased significantly(P<0.01),and BUN,UA decreased.8.TC,TG,LDL,NEFA,and TXB2Compared with the normal group,the content of TC and TG in the adriamycin+hydrocortisone group increased significantly(P<0.01),LDL increased and NEFA decreased significantly(P<0.01),TXB2 increased significantly(P<0.01);Compared with the adriamycin+hydrocortisone group,TC and LDL decreased and NEFA increased(P<0.05),TXB2 decreased significantly(P<0.01)in Zhenwu Decoction group.9.TP,ALBCompared with the normal group,the content of TP and ALB in the adriamycin+hydrocortisone group decreased significantly(P<0.05 and P<0.01);Compared with the adriamycin+hydrocortisone group,TP and ALB in Zhenwu Decoction group increased(P<0.05).10.Energy metabolism indexes cAMP、cGMP、cAMP/cGMPCompared with the normal group,cAMP in the adriamycin+hydrocortisone group decreased significantly(P<0.01),cGMP increased significantly(P<0.01)and cAMP/cGMP ratio decreased significantly(P<0.01).Compared with the adriamycin+hydrocortisone group,cAMP and cGMP in Zhenwu Decoction group increased significantly(P<0.01).Compared with the Zhenwu Decoction group,cAMP in wenshenyang formula group increased(P<0.05),cGMP decreased(P<0.05)and cAMP/cGMP ratio increased(P<0.05).11.Adrenal axis indexes CORTCompared with the normal group,CORT in the adriamycin+hydrocortisone group decreased significantly(P<0.01),and CORT in Zhenwu Decoction group increased significantly(P<0.01)compared with the adriamycin+hydrocortisone group.12.Thyroid axis indexes T3,T4 and TSHCompared with the normal group,the contents of T3 and T4 in the adriamycin+hydrocortisone group decreased significantly(P<0.01),and the content of TSH increased significantly(P<0.01).13.Gonadal axis indexes TCompared with the normal group,T in the adriamycin+hydrocortisone group decreased,and T in Zhenwu Decoction group increased compared with the adriamycin+hydrocortisone group.14.Kidney pathology changes in CKD ratsHE staining:compared with normal group,the adriamycin+hydrocortisone group rat kidney glomerular mesangial area visible widen and mesangial cell hyperplasia of different level;renal tubular expansion or atrophy;a lot of protein tubules stay in the renal tubular;epithelial cell vacuolar degeneration;renal interstitial has a large number of inflammatory cells infiltration,balloon wall adhesion.Compared with the adriamycin+hydrocortisone group,the proliferation of mesangial cells in Zhenwu Decoction group was reduced,the expansion or atrophy of renal tubules was mild,the protein tubules and the infiltration of inflammatory cells in renal interstitium was reduced.Mallory staining:compared with the normal group,the adriamycin+hydrocortisone group showed a large amount of collagen fiber deposition in the glomerular mesangial area and in the renal interstitium.Compared with the adriamycin+hydrocortisone group,Zhenwu Decoction group had a small amount of collagen fiber deposition in the mesangial area of the glomeruli,and a small amount of collagen fiber deposition in the renal interstitium.TEM results:compared with the normal group,the glomerular basement membrane segment in the adriamycin+hydrocortisone group was segmentally thickened and the foot process was widely fused.Compared with the adriamycin+hydrocortisone group,the glomerular basement membrane segmentally thickened was not obvious and the foot process fusion was reduced in the Zhenwu Decoction group.15.Transcriptomics in renal tissueCompared with the normal group,the expression of regulation of programmed cell death genes in the adriamycin+hydrocortisone group was significantly up-regulated,and the redox-related genes and oxidative phosphorylation pathways were significantly down-regulated.Compared with the adriamycin+hydrocortisone group,the expression of nucleotides and organic nitrogen compound synthesis genes in the Zhenwu Decoction group was significantly up-regulated,genes related to antigen presentation were significantly down-regulated,and the oxidative phosphorylation pathway was significantly up-regulated.16.Expression of PHD2,HIF-1α,VHL,NLRP3,and GSDMD mRNA in renal tissueIn situ hybridization:compared with the normal group,the expressions of HIF-1α,NLRP3 and GSDMD mRNA in the adriamycin+hydrocortisone group increased significantly(P<0.01),and the expression of PHD2 and VHL decreased significantly(P<0.05,P<0.01);Compared with the adriamycin+hydrocortisone group,the expression of HIF-1α,NLRP,and GSDMD mRNA in Zhenwu Decoction group decreased significantly(P<0.01),and the expression of PHD2 and VHL increased(P<0.05).RealTime PCR:compared with normal group,the mRNA expression levels of PHD2 and VHL in the adriamycin+hydrocortisone group were significantly decreased(P<0.01),the mRNA expression levels of HIF-1α and NLRP3 were significantly increased(P<0.01),and the mRNA expression levels of GSDMD were increased(P<0.05).Compared with the adriamycin+hydrocortisone group,PHD2 mRNA in Zhenwu Decoction group had a trend of increase,VHL mRNA expression increased(P<0.05),HIF-1α mRNA decreased(P<0.05),NLRP3 mRNA in Zhenwu decoction group had a trend of decrease,GSDMD mRNA decreased(P<0.05).17.Expression of PHD2,HIF-1α,VHL,NLRP3,and GSDMD protein in renal tissueImmunohistochemical results showed that the expression of HIF-1α,NLRP3,GSDMD in the adriamycin+hydrocortisone group was significantly higher than that in normal group(P<0.01),and the expression of PHD2,VHL decreased significantly(P<0.05,P<0.01);Compared with the adriamycin+hydrocortisone group,HIF-1α,NLRP3,and GSDMD in Zhenwu Decoction group decreased significantly(P<0.05,P<0.01),and the expression of PHD2 increased(P<0.05).Conclusion:1.The establishment standard and evaluation method of animal model of kidney yang deficiency syndrome in CKD were established successfully by adriamycin combined with hydrocortisone.Through the evaluation of three aspects of syndrome,objective indicators and drug counter-evidence in rats,kidney lesions were significant,the symptom of kidney-yang deficiency in CKD was obvious,and the hypothalamic-pituitary-adrenal axis,hypothalamic-pituitary-thyroid axis and hypothalamic-pituitary-gonadal axis had low function,and the energy metabolism was abnormal.2.Zhenwu Decoction can reduce kidney damage,protect the kidney,improve the symptoms of kidney yang deficiency in rats with CKD,and improve the function of energy metabolism,hypothalamic-pituitary-adrenal axis and hypothalamic-pituitary-gonadal axis in rats.3.Zhenwu Decoction may intervene CKD by up-regulating oxidative phosphorylation pathway,nucleotide and organic nitrogen synthesis,and mitochondrial components,down-regulating antigen presentation and other gene expressions.Zhenwu Decoction can improve tissue hypoxia,reduce cell damage,especially mitochondrial damage,and have anti-inflammatory effects;4.Zhenwu Decoction can reduce the expression of HIF-1α/NLRP3/GSDMD mRNA and protein in kidney tissue by regulating the HIF-1α/NLRP3/GSDMD signaling pathway,relieve hypoxia and hypoxic-induced cell pyrogen,reduce kidney damage in rats with kidney-yang deficiency syndrome in CKD,and protect renal function. |
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