The Intervention Mechanism Of Heshenluo Ningfeng Granule To The Disorder Of Calcium And Phosphorus Metabolism In Chronic Renal Failure Basing On The Method Of Heluoxiezhuo And Peituningfeng

Object The theoretical hypothesis of shen-luo-yu-zhi,pi-xu-feng-dong in Traditional Chinese Medicine?TCM?was built in the present study,consequently,the intervention mechanism of Heshenluoninfeng Granule based on the method of“Heluoxiezhuo and Peituningfeng”was investigated in rats with calcium and phosphorus metabolism disorder of Chronic Kidney Disease.Method Rats model of chronic renal failure?CRF?with calcium and phosphorus metabolic disorders was prepared by 5/6 nephrectomy and high-phosphorus diet gavage.Sixty-five SPF male Wistar rats were randomly divided into normal control group?n=8?,sham operation group?n=8?and CRF model group?n=49?for four weeks.The remaining60 rats with CKD were randomly divided into the following 7 groups for drug intervention:A)normal control group?n=8?,B)sham operation group?n=8?,C)model group?n=10?,D)Western medicine group?n=8?,E)Chinese medicine low dose group?n=8?,F)Chinese medicine middle dose group?n=9?,G)Chinese medicine high dose group?n=9?.Eight weeks after intervention,the general data including the body weight,mental state,feeding,activity,coat color,urine output and fecal material was recorded.Histological changes in kidney renal tissues were observed by HE and PAS staining respectively.Blood urea nitrogen?BUN?,serum creatinine?Scr?,serum calcium(Ca²⁺),phosphorus(P³⁺),Alkaline phosphatase?ALP?,Parathyroid hormone?PTH?etc and 24-hour urine protein were detected.Serum N-terminal propeptide of type I precollagen?PINP?value was detected using Enzyme-linkedimmunosorbent assay?ELISA?.The renal tissue of each group was observed by light microscope.The expressions of Klotho protein,Wnt4,?-catenin,Bone Morphogenetic Protein-7?BMP-7?,Osteocalcin?OC?and PINP protein in rat kidneys were demonstrated by immunohistochemistry.In addition,The protein expression of Klotho protein,BMP-7,Fibroblast Growth Factor-23?FGF23?and OC were showed using of Western blot.The mRNA of Klotho,BMP-7,Wnt4 and?-catenin in rat kidneys were detected by RT-PCR.Result 1.Pathological changes of disordered glomerular structure,Hyperplastic mesangial cells,Partial glomerulosclerosis,tubular ectasia,inflammatory cell infiltration and focal fibrous tissue hyperplasia in renal interstitium were observed in the model group.The degree of glomerular hyperplasia and interstitial inflammatory cell infiltration in Chinese medicine middle and high dose group were reduced than that in model group.2.Serum Scr,BUN in Chinese medicine middle and high dose group were significantly reduced while serum PTH,ALP were lower and serum Ca²⁺,weight higher than those in model group?P<0.01?.There was a statistically significant difference in Scr,BUN,and P³⁺between Chinese medicine group and western medicine group?P<0.05?,Both of which have the same effect in improving Ca²⁺and lowering PTH,ALP or PINP levels?P>0.05?.3.Protein Expressions of Wnt,?-catenin in Chinese medicine group obviously declined and mRNA expressions significantly down-regulated compared with the model group?P<0.01?and western medicine group?P<0.05?.4.Both of the protein and mRNA expressions of Klotho BMP-7 in Chinese medicine middle and high dose group up-regulated,while protein expressions of FGF23,OC and PINP down-regulated compared with the model group?P<0.05?.Protein expression of FGF23 significantly decreased?P<0.01?,protein and mRNA expressions of BMP-7 up-regulated in Chinese medicine group,compared with the western medicine group?P<0.05?.Above the two groups have the same effect on up-regulating protein and mRNA expression of Klotho,with the similar inhibitory effect on the protein expressions of OC and PINP?P>0.05?.Conclusion HeluoNingfeng Granules can alleviate pathological damage such as glomerular sclerosis in rats with calcium and phosphorus metabolism disorder in chronic renal failure and improve physical condition.It also can effectively reduce serum creatinine,urea nitrogen,increase blood calcium levels,control of phosphorus,PTH and ALP.Heshenluo Ningfeng Granule could inhibit the high protein expression of FGF23,up-regulate Klotho protein and mRNA expression,and further regulate the dynamic balance of Klotho/FGF23 axis.It can affect the activity of Wnt/?-catenin signal transduction pathway,down-regulate?-catenin,hinder the transcription of?-catenin target gene and effectively cut off the conduction pathway by the way of further reducing the levels of serum P³⁺and PTH,inhibiting SHPT,promoting protein and gene expression of BMP-7.Furthermore,HeluoNingfeng Granules could control the activity of ALP,reduce the abnormal expression of OC and PINP together with the serum PINP.Therefore,it is effective in inhibiting the abnormal differentiation and bone remodeling of osteoblasts,and regulateing bone metabolism which migh exert biological effect.In short,HeshenluoNing-feng Granule could play an important role in alleviating calcium and phosphorus metabolism in chronic renal failure and renal osteodystrophy,delaying the progression of the disease,by means ofthe regulation of Klotho/FGF23-axis.