Based On Wnt/β-catenin Signaling Pathway To Explore The Mechanism Of Modified Liuwei Dihuang Decoction In Improving Renal Bone Disease

Objective:Under the guidance of the"Kidney Governing Bone"theory in traditional Chinese medicine and based on Wnt/β-catenin signaling pathway,this study was conducted to observe the effects of modified Liuwei Dihuang Decoction on renal osteodystrophy（ROD）,renal function（urea nitrogen,serum creatinine）,serum calcium and phosphorus metabolism,i PTH,1,25（OH）₂D₃,FGF23,RANKL and Osteocalcin,and renal and bone histology in rats,as well as the expression of Wnt4 andβ-catenin protein in bone tissue,from which to explore the protective mechanism of Modified Liuwei Dihuang Decoction on mineral and abnormal bone metabolism of chronic kidney disease,and moreover to provide experimental basis for further application of this recipe.Methods:A total of 130 healthy male SD rats were divided into 5 groups randomly according to body weight discrepancy:normal group（n=10）,high phosphorus group（n=30）,model group（n=30）,traditional Chinese medicine group（n=30）and calcitriol group（n=30）and the rats in each group was divided respectively into 3 time points at 8,10 and 12 weeks randomly.10 rats in each group were randomly killed at each time point expect those in the normal group were killed at one time when the experiment was completed.After adaptive feeding for 1 week,the rats in normal group were fed with normal diet,while those in high phosphorus group were fed with high phosphorus diet（phosphorus content 1.03%）and then received distilled water（1m L/100g.d）by intragastric administration.In the meantime the rats in model group,Chinese medicine group and calcitriol group were fed with adenine and high phosphorus diet（adenine content 0.75%and phosphorus content 1.03%）（the first to fourth week were fed with adenine diet,while the fifth to sixth week were fed with high phosphorus diet）.After the 6 weeks of model establishment,the rats in model group were fed with high phosphorus diet（P content 1.03%）and received distilled water（1m L/100g.d）by intragastric administration while the rats in TCM group received modified Liuwei Dihuang Decoction（1g/100g.d）by intragastric administration and the rats in calcitriol group received calcitriol solution（0.009ug/100g.d）by intragastric administration.The contents of BUN,Scr,calcium and phosphorus in each group were determined by automatic biochemical analyzer,and the contents of i PTH,FGF-23,1,25（OH）₂D₃,RANKL and Osteocalcin in serum of rats in each group were determined by enzyme linked immunosorbent assay（ELISA）.Bone mineral density of femur was measured by bone densitometer.PASM and Masson staining were performed on the kidneys,Goldner’s trichrome staining was performed on the femurs,and histomorphological changes in the renal interstitium,tubules,and femurs were observed by light microscopy.Immunohistochemistry and Western blot were used to detect the expression of Wnt andβ-catenin protein in bone tissue.The expressions of Wnt andβ-catenin in bone tissues were detected by immunohistochemistry and Western blot.Results:1.Compared with the normal group and the high phosphorus group at the same time point at 8,10 and 12 weeks,the semi-quantitative scores of BUN,Scr,blood phosphorus,i PTH,FGF-23,RANKL,Osteocalcin and tubulointerstitial of the rats in model group were significantly increased,while the serum calcium level and 1,25（OH）₂D₃were decreased,with statistical significance（P<0.05 or P<0.01）.Compared with the model group at the same time point at 8,10 and12 weeks,the semi-quantitative scores of BUN,Scr,blood phosphorus,i PTH,FGF-23,RANKL,Osteocalcin and tubulointerstitial of the rats in the TCM group and the calcitriol group were significantly decreased,while the levels of serum calcium and 1,25（OH）₂D₃ were increased,with statistical significance（P<0.05 or P<0.01）.There was no significant difference in the above indexes between the TCM group and the calcitriol group at the same time point（P>0.05）.2.Test results of femoral bone mineral density:compared with the normal group,the femoral bone mineral density of rats in the high phosphorus group was not significantly changed at each time point;nevertheless,compared with both normal group and high phosphorus,the femoral bone mineral density of rats in the model group was significantly decreased at the 8th,10th and 12th week after modeling,and the decrease was more obvious with time extension（P<0.05,or P<0.01）.Compared with model group,there was no significant difference in femoral bone mineral density between TCM group and calcitriol group at week 8（P>0.05）.At week 10 and 12,the density of femoral head in TCM group and calcitriol group was significantly increased（P<0.01）,while there was no statistical significance in the density of femoral head between TCM group and calcitriol group at the same time points（P>0.05）.3.Morphological results showed that:（1）Compared with the normal group,there were no significant changes in renal interstitium and renal tubule morphology at each time point in the renal tissue of rats in the high phosphorus group.However,compared with both normal group and high phosphorus,rats in model group were observed to suffer renal tubule injury（dilatation or atrophy）,glomerular basal membrane was obviously thickened,mesangial membrane was widened,and renal interstitium showed morphological changes such as inflammatory cell infiltration and fibrous proliferation which resulted in the increase of renal tubulointerstitial injury scores and gradually worsened with time（P<0.05）.Compared with model group,renal interstitial fibrous and renal tubule injury scores in TCM group and calcitriol group were significantly decreased at each time point（P<0.05 or P<0.01）,while renal tissue morphological changes in TCM group and calcitriol group were not statistically significant（P>0.05）.（2）Bone histopathology:compared with the normal group,there were no significant changes in osteoblasts,bone trabeculae and bone cells at each time point in the simple high phosphorus group;compared with both normal group and high phosphorus,in the model group,the width of bone trabecular increased,the average osteoid area increased and osteoblasts decreased at 8,10 and 12 weeks after modeling.Compared with the model group,the number of osteoblasts increased and the width of bone trabecular beam and the average osteoid area decreased in the TCM group and the calcitriol group at each time point,while there was no significant difference between the TCM group and the calcitriol group at each time point.4.Results of bone immunohistochemistry:Compared with the normal group,the expression of Wnt4 andβ-catenin protein in the high phosphorus group was not significantly changed at each time point（P>0.05）.However,compared with both normal group and high phosphorus,the expression of Wnt4 andβ-catenin in model group was significantly decreased,and the protein expression of Wnt4 andβ-catenin in model group was gradually decreased with time（P<0.05 or P<0.01）.Compared with model group,the protein expressions of Wnt4 andβcatenin in TCM group and calcitriol group increased,and they increased with the prolonging of intervention time（P<0.05 or P<0.01）,while there was no statistical significance in the protein expression of bone tissue between TCM group and calcitriol group at each time point（P>0.05）.5.Western blot results of bone tissue:Compared with the normal group,the protein expression of Wnt4 andβ-catenin protein in the simple high phosphorus group was not significantly changed at each time point（P>0.05）;However,compared with both normal group and high phosphorus,the protein expression of Wnt4 andβ-catenin in model group was significantly decreased,and was gradually decreased with time（P<0.05 or P<0.01）.Compared with the model group,the protein expressions of Wnt4 andβ-catenin in the TCM group and the calcitriol group increased,and the protein expressions increased with the prolonging of intervention time（P<0.05 or P<0.01）,while there was no statistical significance in the protein expression of bone tissue between the TCM group and the calcitriol group at each time point（P>0.05）.Conclusion:1.Modified Liuwei Dihuang Decoction can reduce BUN and Scr levels and improve renel interstitial fibrosis in CKD-MBD rats.The vivo experiment confirmed the protective effect of modified Liuwei Dihuang Decoction on kidney in CKD-MBD rats.2.Modified Liuwei Di Huang Decoction can increase the levels of blood calcium and 1,25（OH）₂D₃,reduce the levels of blood phosphorus,i PTH,FGF-23,RANKL and Osteocalcin,regulate the disorder of calcium-phosphorus metabolism,improve secondary hyperparathyroidism,and regulate the mineral and bone metabolism levels of CKD.The vivo experiment confirmed the protective effect of modified Liuwei Dihuang Decoction on the bone of CKD-MBD rats,and the effect was similar to that of calcitriol.3.The Wnt4 andβ-catenin proteins were down-regulated in bone tissue of CKD-MBD rats,suggesting that the Wnt/β-catenin signaling pathway was inhibited in bone tissue of CKD-MBD rats.4.Modified Liuwei Dihuang Decoction can up-regulate Wnt4 andβ-catenin proteins in renal and bone tissue,suggesting that the mechanism of Modified Liuwei Dihuang Decoction may be related to the activation of Wnt/β-catenin signaling pathway.