Relationship Between Kidney-deficiency Osteoporosis Mice And EPO And Improvement Effect Of You-gui-yin On Them

BackgroundThe understanding of"kidney governing bones"is different but connected between traditional Chinese medicine and modern western medicine.The modern western medicine discovers that partial or total loss of renal function will contribute to the bone metabolism disorder,such as osteoporosis,bone sclerosis.The theory of traditional Chinese medicine is that“kidney governing bones”,which means“kidney essence”can nourish bones.EPO is an erythropoietin producing hormone which can promote hematopoiesis and protect brain.And EPO is supposed as one of the biological substances of“kidney essence”.How EPO levels are changed in kidney-deficiency osteoporosis mice,and the effects of rhEPO and the kidney-tonifying traditional Chinese medicine on the kidney-deficiency osteoporosis mice and its correlation with EPO still have not been reported and need to be clarified..This study is supported by the National Natural Science Foundation of China（81473549）and the Ministry of Education Central University Fundamental Research Funding Project（XDJK2017D158）.PurposeTo observe the correlation between kidney-deficiency osteoporosis and EPO,and the improvement of You-gui-yin（YGY）on them.To verify the hypothesis that“EPO is one of the biological substances of’Kidney essence’”so that finding out one corresponding biological substance for’kidney essence’in traditional Chinese medicine theory.Methods1.The establishment of kidney-deficiency osteoporosis mouse model and the criteria for the model（1）Male C57BL/6J mice weighing from 22 to 26 g were fed with 0.2%adenine model diet for 5 weeks and drunk water freely.Body weight and temperature were measured every week.Other indicators were tested after the model was established.（2）Body weight,temperature,testosterone,CORT,alkaline phosphatase,bone mineral density and trabecular number were significantly lower in kidney-deficiency osteoporosis model mice than that in normal mice（all P<0.05）;creatinine,urea,calcium and phosphorus were significantly higher in kidney-deficiency osteoporosis model mice than that in than normal mice（all P<0.05）.（3）The test showed that the success rate of the modelling method described above is80%⁹0%,which is stable and feasible.2.Groups and drug administration（1）After being fed with 0.2%adenine model diet for 2 weeks,the mice were randomly divided into model group and EPO 5000 U·kg^-1group,each group contains 15 mice,ensuring that there was no significant difference between the body temperature,weight,renal function,endocine,bone metabolism and bone microscopic parameters in all modeling groups（all P>0.05）.Being fed with 0.2%adenine diet continuously,EPO group were subcutaneously injected with 5000 U·kg^-1EPO,normal and model group were subcutaneously injected with equal volume of saline.All mice were injected for 3 weeks and 3 times a week.（2）After being fed with0.2%adenine diet for 2 weeks,the model mice were randomly divided into model group,YGY 10 g·kg^-1 group,20 g·kg^-1 group and 40 g·kg^-1 group using random number table,each group contains 20 mice,ensuring that there was no significant difference between the body temperature,weight,renal function,endocine,bone metabolism and bone microscopic parameters in all modeling groups（all P>0.05）.Fed 0.2%adenine dietcontinuously,the YGY groups were intragastricly administered with corresponding concentrations of YGY decoction and the normal and model control groups were given the same volume of saline.All mice were administered for 3 weeks,and 1 times per day.3.Measurement of indexes for kidney-deficiency osteoporosis（1）The posture of the mice and the color of hair were observed.The weight and temperature of each group were measured each week.（2）The general morphology and histomorphology of kidney in each group were observed.（3）The blood was taken from the heart,serum was collected by centrifugation,and the contents of creatinine and urea were measured by automatic blood biochemical analyzer.（4）The contents of CORT and T in serum were measured by ELISA.（5）After decalcification of the femur,HE staining was performed on paraffin sections to observe its histomorphology.（6）The bone mineral density and microscopic of femur were measured by micro-CT.（7）The contents of Ca,P and ALP were measured by automatic blood biochemical analyzer.4.Determination of serum and bone EPO and signaling pathway proteins（1）The level of EPO in serum was measured by ELISA.（2）The expression of HIF-1α,HIF-2α,EPO and EPOR were determined by western blot.5.Quality controlof YGY（1）Establishment of the fingerprint of YGY（1）Establishing the fingerprint of You-gui-yin by HPLC.Determination conditions:Separation using the Pursuit XRs 5 C18 column（4.6 mm x 250 mm,5μm）.The mobile phase is acetonitrile（A）-0.1%phosphoric acid solution（B）;gradient elution:1%A→10%A（0³0 min）,10%A→17%A（30⁸0 min）,17%A→30%A（80¹05min）,30%A→55%A（105¹25 min）;flow rate is 1.0 mL min^-1;and column temperature is 30℃.（2）The fingerprint of YGY were established by HPLC,and the similarity of the 10 batches of fingerprints and the control fingerprints was greater than0.904.（2）Determination of the main components of YGY:（1）The determination conditions were the same as above.（2）The HPLC chromatographic conditions,linearity,precision,stability,repeatability,and sample recovery were well established.The contents of geniposide,morroniside,chlorogenic acid,geniposide,logicoside,pinolediol diglucoside,glycyrrhizin,rutin and glycyrrhizic acid all meet the requirements of the 2015^th edition of Pharmacopoeia.Results1.Decreased EPO levels in serum and bone of kidney-deficiency osteoporosis mice.Compared with the normal group,the expression of serum and femur EPO in model group were significantly reduced;the expression of femur HIF-1α,HIF-2αand EPO in model group were significantly reduced,and the expression of femur EPOR in model group were significantly elevated（P<0.05）.2.rhEPO significantly improved the physique of kidney-deficiency osteoporosis mice.Compared with the model group,gloss,contraction and arching of the hair of EPO group were improved;body weight and body temperature of EPO group were increased significantly;the"Great White Kidney"symptoms and morphology of the renal corpuscles and renal tubules of EPO group were improved significantly;the number of nephrons of EPO group was increased significantly;purple blue adenine crystals of EPO group was reduced significantly;serum creatinine and urea levels were reduced significantly;T and CORT levels in serum increased significantly;the number of trabecular bone and bone mineral density was increased,while the trabecular bone connection interruption point was reduced significantly in EPO group.The serum calcium content of EPO group is significantly reduced（P<0.05）.Conclusilvely,rhEPO can significantly improve the physique of kidney-deficiency osteoporosis mice.3.rhEPO significantly elevated the expression of HIF-1αand HIF-2αin the femur,and reduce the expression of EPOR Compared with model group,the expression of HIF-1α,HIF-2αand EPO in the femur of EPO group were significantly elevated,and the contents of EPOR were significantly reduced（P<0.05）.4.YGY significantly improved the physique of kidney-deficiency osteoporosis mice Compared with the model group,gloss,contraction and arching of the hair of YGY 20 g·kg^-1,40 g·kg^-1 groups were improved;body weight and body temperature were increased significantly.Compared with the model,the"Great White Kidney"symptoms of YGY 10 g·kg^-1,20 g·kg^-1,40 g·kg^-1 groups were improved,the number of nephrons was increased significantly;the morphology of the renal corpuscles and renal tubules improved significantly;and the purple blue adenine crystals reduced significantly.Compared with the model group,the number of nephrons of YGY 20g·kg^-1 and 40 g·kg^-1 groups was increased significantly;the morphology of the renal corpuscles and renal tubules improved significantly;and the purple blue adenine crystals reduced significantly.Compared with the model group,serum creatinine and urea levels of YGY 20 g·kg^-11 and 40 g·kg^-1 groups were reduced significantly;the number of trabecular bone and bone mineral density was increased;the trabecular bone connection interruption point was also reduced significantly;the serum calcium content was significantly reduced;and osteoporosis symptoms were significantly reduced（P<0.05）.5.YGY significantly increased the EPO level in the blood and the bone of kidney-deficiency osteoporosis mice and regulated the EPO signaling pathway（1）Compared with model group,the EPO level in serum and femur of YGY 10 g·kg^-11 and20 g·kg^-1 groups were significantly elevated（P<0.05）.（2）Compared with model group,the expression of HIF-1α,HIF-2αand EPO in the femur of YGY 10 g·kg^-1 and20 g·kg^-1 groups were significantly elevated,and the contents of EPOR were significantly reduced（P<0.05）.Conclusions1.After fed with 0.2%adenine diet for 5 weeks,a kidney-deficiency osteoporosis model was successfully established.The model not only showed the symptoms of kidney-deficiency and osteoporosis,but also had reduced levels of EPO in serum and femurs and their signaling pathway-related proteins HIF-1α,HIF-2αand EPO,and had increased expressions of EPOR.2.Exogenous EPO significantly improved the physique of kidney-deficiency osteoporosis mice,which might be related to the regulation of related protein expressions in HIF-EPO signaling pathway.The expression of EPO in serum and femur,and HIF-1α,HIF-2α,and EPO in femur were elevated.The expression of EPOR content in femur were reduced.3.YGY significantly improved the physique of kidney-deficiency osteoporosis mice,owing to regulating the expression of related proteins in HIF-EPO signaling pathway.The expressions of EPO in serum and femur,and HIF-1α,HIF-2α,and EPO in femur were elevated.The expressions of EPOR in femur were reduced.The fingerprints of YGY and the determination of 9 components provided the basic guarantee for the credibility and repeatability of the results.4.Kidney-deficiency osteoporosis had a significant correlation with decreased EPO content.Exogenous EPO significantly improved kidney-deficiency and osteoporosis.YGY significantly improved kidney-deficiency osteoporosis and increased the EPO content at the same time.All of these above results supported the hypothesis that"EPO may be one of the biological substance of’renal essence’"...