| Purpose:In this study, the pathway of Ca2+ transport was mainly investigated. To observe the gene and protein expression of Ca2+ transport associated protein TRPV5, CaBP-D28k,NCX1 and PMCA1b in nephridial tissue and bone tissue of rats which had kidney-deficiency osteoporosis induced by dexamethasone, then to reveal the molecular mechanism of dexamethasone-induced kidney-deficiency osteoporosis, and to explain the curative mechanism of tonifying-kidney herbs.Materials and methods:In this study, we used dexamethasone 2.5mg/kg intramuscular injection twice in a week in rats to induce kidney-deficiency osteoporosis, at the same time used tonifying-kidney herbs to prevent and treat. The normal rats were as standard control, model rats were as blank control and positive control were GUSHUKANG granules, and to compare the therapeutic effect of invigorate-spleen herbs and promoting-blood flow herbs. Above process were 9 weeks. After 9 weeks, killed the rats to collect serum,femoral bone and nephridial tissue to detect. Using dual-energy X-ray absorptiometry (DEXA) to detect BMD of femoral bone upper 1/3. Detected serum Ca, P and ALP levels by biochemistry method. Enzyme linked immunosorbent assay (ELISA) were used to detect the serum TRAP level. Real-Time Quantitative PCR and Western Blotting were used to detect the gene and protein expressions of TRPV5,CaBP-D28k,NCX1,PMCA1b in nephridial tissue and bone tissue of rats.Results:1. Results of femoral bone upper 1/3 BMD of rats: Compared with normal group, BMD of blank group were obviously decreased(P<0.01). Compared with bland group, in each medication groups, femoral bone upper 1/3 BMD were all increased, and tonifying-kidney group were most obviously, statistical significance were predominant(P<0.01).2. Results of bone metabolism markers: Compared with normal group, TRAP of blank group were obviously increased(P<0.01). Compared with bland group, TRAP were all decreased in each medication groups(P<0.01), and tonifying-kidney group and GUSHUKANG granules were most obviously, to approach to the level of normal group. Compared with normal group, blood calcium level had no obviously variation. Compared with blank group, blood calcium level of each medication groups were increased(P<0.05). Blood phosphonium level had no obviously variation. Compared with blank group, only GUSHUKANG granules group increased(P<0.01).3.TRPV5 mRNA and protein expression of nephridial tissue: Compared with normal group, TRPV5 mRNA and protein expression of blank group were obviously decreased(P<0.01). Compared with blank group, TRPV5 mRNA and protein expression of each medication groups were all up-regulated in different degree, and tonifying-kidney group were most obviously(P<0.01).4. CaBP-D28k protein expression of nephridial tissue: CaBP-D28k protein was expressed in nephridial tissue of all groups. Compared with normal group, CaBP-D28k protein expression of blank group were obviously decreased(P<0.01). Compared with blank group, CaBP-D28k protein expression of each medication groups were all up-regulated in different degree, and tonifying-kidney group were most obviously(P<0.01).5. NCX1,PMCA1b mRNA and protein expression of nephridial tissue: Compared with normal group, NCX1 mRNA and protein expression of blank group were obviously decreased(P<0.01). Compared with blank group, NCX1 mRNA and protein expression of each medication groups were all up-regulated in different degree, and tonifying-kidney group were most obviously(P<0.01). PMCA1b protein was expressed in nephridial tissue of all groups. Compared with normal group, PMCA1b protein expression of blank group were obviously decreased(P<0.01). Compared with blank group, PMCA1b protein expression of each medication groups were all up-regulated in different degree, and tonifying-kidney group were most obviously(P<0.01).6. TRPV5 mRNA and protein expression of bone tissue: Compared with normal group, TRPV5 mRNA and protein expression of blank group were obviously increased(P<0.01). Compared with blank group, TRPV5 mRNA and protein expression of each medication groups were all down-regulated in different degree, and tonifying-kidney group were most obviously(P<0.01).7.CaBP-D28k protein expression of bone tissue: CaBP-D28k protein was expressed in bone tissue of all groups'rats. Compared with normal group, CaBP-D28k protein expression of blank group were obviously increased(P<0.01). Compared with blank group, CaBP-D28k protein expression of each medication groups were all down-regulated in different degree, and tonifying-kidney group were most obviously(P<0.01).8. NCX1 mRNA and protein expression of bone tissue: Compared with normal group, NCX1 mRNA and protein expression of blank group were obviously increased(P<0.01). Compared with blank group, NCX1 mRNA and protein expression of each medication groups were all down-regulated in different degree, and tonifying-kidney group were most obviously(P<0.01).Conclusions:1. Used dexamethasone 2.5mg/kg intramuscular injection twice in a week for continuous 9 weeks, kidney-deficiency osteoporosis rat model can be induced.2. TRPV5 and NCX1 are expressed in nephridial tissue of normal rats in gene and protein level, CaBP-D28k and PMCA1b are expressed in protein level, indicate that renal tubule of normal rats exist calcium transport pathway mediated by TRPV5, which can contribute to maintain the stability of calcium metabolism in the body.3. TRPV5 and NCX1 are expressed in bone tissue of normal rats in gene and protein level, CaBP-D28k are expressed in protein level, indicate that osteoclast of normal rats exist calcium transport pathway mediated by TRPV5, which can contribute to bone metabolism and maintain the stability of calcium metabolism in the body.4.The down-regulation of mRNA and protein expression of TRPV5,CaBP-D28k,NCX1 and PMCA1b in nephridial tissue of blank group indicate the function inhibition of calcium transport pathway associated proteins in kidney, and malabsorption of calcium in renal tubule epithelium, which maybe the one of pathomechanism of kidney-deficiency osteoporosis induced by glucocorticoid.5. The up-regulation of mRNA and protein expression of TRPV5,CaBP-D28k,NCX1 in bone tissue of blank group indicate the active function of calcium transport pathway associated proteins in bone, so result in enhancement of bone resorption and decreased BMD, which maybe the one of pathomechanism of kidney-deficiency osteoporosis induced by glucocorticoid.6. Tonifying-kidney herbs can boost the expression of calcium transport pathway associated proteins in kidney, inhibit expression of calcium transport pathway associated proteins in bone resorption process, from which to reinforce calcium absorption in kidney and inhibit bone resorption to reduce the loss of bone content,thereby to prevention and cure osteoporosis.
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