| Objective Long term treatment with glucocorticoids (GC) can induce osteoporosis (GIO), the incidence of GIO is only behind the postmenopausal osteoporosis. It occurs disability and carries financial and psychosocial consequences for affected individuals, their families and their communities. Owing to the catabolic activity of GC on bone and the multiplicate etiology, the ideal and specific agents for GIO have not been discovered. Danshen {Radix Salvia Miltiorrhiza), a traditional Chinese medicine, has been widely used in clinical practice in China and other Asia countries for the managements of cardiovascular diseases, arthritis and other inflammation-related diseases based on its pharmacological actions of anti-peroxidation and improvement of microcirculation system, we assume that these actions of danshen would antagonize the adverse effects of GC and remain benefit effects to the underling diseases of GIO. The purpose of this study was to investigate 1) the effects of water extract of danshen (DS) on experimental GIO by applying bone histomorphometry and bone biomechanical properties tested in vivo; 2) the cells and biomolecular mechanism of bioactive compound danshensu (DSU) on rats calvarial osteoblast (rOB) and rats bone marrow stromal cells (rMSCs) in vitro, focus on studying anabolic effect of DSU at rOB in vitro and the abilities of osteogenesis and adipogenesis of DSU at rMSCs in vivo and in vitro. The in vitrostudy will illustrate the dose and time response of DSU on cells. Methods(In vivo study): Experimental design 1 for growing rats: Thirty-two 3-month-old SD male rats weighing 228?0g were divided into four groups and treated for 12 weeks as follows: (1) intact rats was given with vehicle as control (Cont.); (2) Prednisone was given orally to the rats at dose of 2.7mg/kg/d as the model group (GC); (3) DS at dose of 5.0g/kg/d were plus to the prednisone treated rats (GC+DS) and ?Integrated medicine (Calcium 0.5/kg/d + stanozolol 0.5 mg/kg/d + VitD3 25 IU/kg/d ) were plus to the the prednisone treated rats (GC+Co.). Experimental design 2 for age rats: Eighty-six 6-month-old male rats weighing 468?0g were used. The first six rats were killed at first day of study as basal. The remain rats were divided into ten groups and treated for 6 weeks and 12 weeks respectively as follows: group (1) and (2) were short-term control (6w-Cont, distalled water p.o) and GC treatment model group (6w-GC, Prednisone at dose of 3.0mg/kg/d p.o for 6 weeks); Group (3) and (4) were long-term control (12w-Cont.) and GC treatment model group (12w-GC, prednisone at dose of 3.0mg/kg/d p.o for 12 weeks); Group (5) was on prednisone for 6 weeks and off for 6 weeks (GC/off); Group (6) and (7) were carried CG/off then treated with DS at dose of 5.0g/kg/d for 6 weeks (CG/off+DS/on6w) and for 12 weeks (CG/off+DS/onl2w); Group (8), (9) and (10) were GC-treated rats plus DS at low dose 2.5g/kg/d (GC + DS-L), mid dose 5.0g/kg/d (GC + DS-M) and high dose 10.0g/kg/d (CG + DS-H) for 12 weeks. Double in vivo fluorochrome labeling was performed before killing the rats. The blood serum was separated for testing biochemistry indices; The selected soft tissue (testicles, adrenal glands, thymus, spleen and liver) were removed and weighed separately. The longitudinal proximal tibial metaphyseal (PTM), forth lumber vertebral body (LV4) and tibiae shaft (TX) sections were performed undecalcifiedly and used to the bone histomorphometric analysis. Thefemur and fifth lumber vertebral body (LV5) were conducted three-point bending test and reduced-platen compression test separately; The bone calcium content and bone hydroxyproline content of right ulna were also tested.(In vitro study): New born SD rat calvarial osteoblasts (rOB) were cultured, and the rats bone marrow stromal cells (rMSCs) were isolated by density-gradient centrifugation and cultured. Biochemical measurement of alkaline phosphatase (ALP) activity, Reverse transcription-Polymerase chain reaction (RT-PCR), and gene reporter assays were used to investigate the anabolic effects of DSU on... |
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