| Diabetic kidney disease(DKD)is one of the common complications of diabetes mellitus(DM).About 20%-40%of DM patients progress to DKD within 20-25 years after onset,and DKD has become the leading cause of chronic kidney disease(CKD)in China.Bone metabolic disorder,which is one of the common complications of DKD,is characterized by bone pain,osteoporosis,fracture,and bone deformity.Bone metabolic disorder not only causes bone disease but also leads to disorder of calcium and phosphorus metabolism,which increases the risk of cardiovascular and other high-mortality complications.Shenyuan granules(SYG),which has been proved to possess the function of tonifying spleen and kidney and purging fu and removing turbidity,can improve the renal function,vascular calcification,and bone mineral density reduction of DKD model animals,this study will further explore the role and mechanism of SYG in ameliorating bone metabolism of DKD.Objective:To explore the mechanism of SYG on bone metabolism of db/db mice based on VDR/RXR/FGF23 signaling pathway,and to provide the theoretical basis for prevention and treatment of bone metabolism abnormality of DKD with traditional Chinese medicine.Methods:In vivo,DKD mouse model was established by db/db mice with high phosphorus diet,30 db/db mice were randomly divided into the model group(db/db+distilled water),low dose of Shenyuan granules group(db/db+SYG1.5g/kg,db/db+SYG-L)and high dose of Shenyuan granules group(db/db+SYG 6g/kg,db/db+SYG-H),each group were given 1.2%high phosphorus diet.Another 10 db/m mice were used as the control group(db/m+distilled water)and were fed with 0.2%normal phosphorus diet.The mice in each group were intervened once a day for 12 weeks.The body weight,water intake,food intake,over-all conditions were observed,and recorded the changes in fasting blood glucose(FBG).At the 12th week of drug intervention,24h urine of each group was collected,urine microalbumin(m ALB)and urine creatinine(UCr)were detected,and UACR(urine albumin-to-creatinine ratio)was calculated.After 12 weeks of intervention,blood,kidney and bone samples were collected after 10%chloral hydrate anesthesia.The levels of serum creatinine(SCr),urea nitrogen(BUN),calcium(Ca),phosphorus(Pi),parathyroid hormone,1,25(OH)2D3(1,25D),fibroblast growth factor 23(FGF23),and Klotho were measured.The pathological changes of the kidney of mice were observed by HE,PAS,and Masson staining,and the pathological changes of bone tissue were observed by HE,TRAP,Von Kossa,and Goldner`s trichromatic staining.The changes in bone microstructure and histomorphometry were detected by Micro CT.Real-time PCR,Western Blot,and immunohistochemistry were used to detect the expressions of bone metabolic markers such as ALP,OC,and CTSK,as well as the expressions of FGF23,VDR,and RXR in the bone tissues of mice.The co-expression of VDR/RXR in the bone tissues of mice in each group was detected by double immunofluorescence.In vitro,the effects of high glucose on the viability and FGF23 expression of the osteoblast cell line UMR-106 were studied.The cells were treated with different concentrations of glucose(5.5 mmol/L,10 mmol/L,20 mmol/L,and30 mmol/L),respectively,the cell viability was detected by CCK-8 assay,and the expression of FGF23 was detected by Real-Time PCR and Western Blot.Then,the effect of 1,25D on the expression of FGF23 in UMR-106 cells under high glucose or non-high glucose(30 mmol/L)conditions was observed.0.1nmol,1 nmol,and 10 nmol of 1,25D were added to cells respectively,the expression of FGF23 was detected by Real-Time PCR and Western Blot,and screen the appropriate concentration of 1,25D for subsequent experiments.Finally,the effect of the SYG containing serum on the VDR/RXR/FGF23 signal pathway of UMR-106 cells was observed.The Wistar rats(n=15)were given intragastric administration of 2.72 g/kg Shenyuan granules,and the rats in the control group(n=15)were given the same volume of distilled water.Both rats were processed twice a day for 7 days,after centrifugation,deactivation,filtration,and repacking.In the previous study,epimedium glycoside,astragalus glycosides and emodin were detected in the serum by UPLC-MS/MS.The suitable serum concentration of SYG containing serum was selected by CCK-8assay for follow-up experiments.The expression of VDR,RXR,and FGF23was detected by Real-Time PCR and Western Blot,and the co-expression of VDR/RXR was detected by double immunofluorescence labeling.Results:1.Effect of SYG on general condition?renal function and renal pathomorphism of db/db mice.(1)General condition:compared with the control group,the weight,food intake,water intake,and FBG of the model group were significantly increased(P?0.01),accompanied by obesity,fur loss on the neck,delayed wound healing and decreased activity frequency,unresponsive,etc.Compared with the model group,there were no significant differences in body weight,food intake,water intake and FBG in SYG groups(P?0.05),but no fur loss occurred in SYG group and its response was more sensitive.(2)Renal function:compared with the control group,the levels of SCr,BUN,Pi,PTH,1,25D,and FGF23 levels of the model group were significantly increased(P?0.01),while the levels of serum Ca were decreased(P?0.01).Compared with the model group,the levels of serum SCr,BUN,Pi,PTH,1,25D and FGF23 in SYG-interfered db/db mice were decreased(P?0.05 or P?0.01),and the level of serum Ca was increased(P?0.05).(3)Urine albumin-to-creatinine ratio:compared with the control group,the m ALB content of the model group was significantly increased(P?0.01),while the UCr level was significantly reduced(P?0.01),resulting a significant increase in UACR(P?0.01).After the intervention of SYG,the m ALB content in db/db mice was decreased(P?0.05),and the UACR was also decreased significantly(P?0.05).(4)Renal pathological morphology:compared with the control group,the renal glomerular area,mesangial matrix proliferation,and fibrosis area of the model group were significantly increased(P?0.01),and it was accompanied by renal tubules dilation of the lumen and shedding of the brush border.After the intervention of SYG,the glomerular area,mesangial matrix and fibrosis area of db/db mice decreased in different degrees(P?0.05 or P?0.01),and the injury of renal tubules was also reduced.2.Effect of SYG on bone histomorphology of db/db mice.(1)Bone microstructure:compared with the control group,mice in the model group lost significant bone mass,bone volume fraction(BV/TV),trabecular number(Tb.N),trabecular thickness(Tb.Th),and bone mineral density(BMD)of the femur bone were significantly decreased in the model group(P?0.01),while trabecular resolution(Tb.Sp)and structural model index(SMI)both increased(P?0.01).The intervention of SYG can increase BV/TV,Tb.N,Tb.Th,and BMD of db/db mice(P?0.05 or P?0.01),and reduce their Tb.Sp and SMI(P?0.05 or P?0.01).(2)Bone histomorphology:compared with the control group,the distal metaphysis of the femur bone in the model group decreased significantly in bone volume,the number and thickness of trabecular bone decreased,the distribution of trabecular bone was sparse,the connection was decreased,and the marrow cavity of the model group was filled with a large number of adipocytes,there are erosive cavities in the cortical bone.In the model group,the osteoclast-labeled area and calcium deposition in the bone decreased significantly,and the mineralized function of bone trabecula was impaired.Compared with the model group,the intervention of SYG increased the number,thickness,distribution,and connection of bone trabecula in db/db mice,decreased the number of adipocytes in bone marrow cavity.The marked area of osteoclasts,calcium deposition,and mineralization function was improved in different degrees.3.Effect of SYG on bone metabolism in db/db mice.Compared with the control group,the expression of ALP and OC m RNA and protein was significantly increased in the model group(P?0.05),while the expression of CTSK m RNA and protein was significantly decreased(P?0.05).After the intervention of SYG,the expression of ALP and OC in bone tissue of db/db mice was decreased(P?0.05),while the expression of CTSK was increased(P?0.05),which improved the bone metabolism of db/db mice.4.Effect of SYG on VDR/RXR/FGF23 signal pathway in bone tissue of db/db mice.Compared with the control group,the expression of FGF23,VDR,and RXR in the bone tissue of the model group was significantly up-regulated(P?0.01),and the co-expression of VDR/RXR was also increased by double immunofluorescence labeling.The expression of FGF23,VDR,and RXR in bone tissue of db/db mice was decreased by SYG intervention(P?0.05),and the co-expression of VDR and RXR in bone tissue was also decreased.5.Effects of high glucose on the viability and FGF23 expression of UMR-106 cells.Compared with the control group,the normal concentration of glucose(5.5 mmol/L)did not change the activity of UMR-106 cells and the expression of FGF23,while the high concentration of glucose(30 mmol/L)significantly decreased the activity of cells and inhibited the expression of FGF23(P?0.05).6.Effects of 1,25D on the expression of FGF23 in UMR-106 cells exposed to high glucose.In a non-high glucose condition,the expression of FGF23 was up-regulated in a concentration-dependent manner by 1,25D.10 nmol 1,25D significantly increased the expression of FGF23(P?0.01).Compared with the control blank group,the expression of FGF23 in UMR-106 cells was significantly decreased in a high glucose environment(P?0.05),but similarly10nmol 1,25D also up-regulated the expression of FGF23(P?0.05).7.Effect of SYG containing serum on VDR/RXR/FGF23 signaling pathway of UMR-106 cells.CCK-8 assay test showed that 10%concentration of SYG containing serum had no obvious effect on the viability of UMR-106cells,so this concentration was selected for subsequent experiments.Compared with the control group,the expression of FGF23,VDR,and RXR m RNA and protein in the high glucose group decreased significantly(P?0.05),and the co-expression of VDR/RXR in the cells also decreased.Compared with the control group,there was no significant difference in the expression of FGF23,VDR,and RXR in the hypertonic group.Compared with the high glucose group,the expression of FGF23,VDR,and RXR in the 1,25D group was significantly up-regulated(P?0.05),and the co-expression of VDR/RXR was also significantly increased.Compared with the 1,25D group,the expression of FGF23,VDR,and RXR in the SYG group was all down-regulated(P?0.05),and the co-expression of VDR/RXR was also reduced(P?0.05).Conclusion:1.db/db mice combined with high phosphorus diet showed significantly impaired renal structure and function,disorder of calcium and phosphorus metabolism,and SYG can improve renal function and calcium and phosphorus levels of db/db mice,and reduce renal pathological changes;2.db/db mice showed significant damage in bone microstructure,bone morphometric parameters and osteopathological morphology.SYG ameliorate bone mass loss,bone microstructure destruction and bone remodeling abnormality in db/db mice,and play a role in improve the quality and strength of bones in db/db mice;3.SYG can improve bone metabolic disorder and inhibit the expression of FGF23 in the bone of db/db mice.The mechanism may be related to the regulation of the VDR/RXR/FGF23 signaling pathway in the bone tissue of SYG.4.In this study,the mechanism of bone metabolism disorder in DKD was briefly discussed from kidney-bone axis,which expanded the scientific connotation and application of the kidney governing bones theory to a certain extent. |
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