| Aim: 1. To explore the feasibility Wistar inbred rats to analysis glucocorticoid genetic susceptibility,Two strains of rats(Wistar inbred rats and Wistar closed rats) were injected corticosteroids. 2. To elucidate the effect of glucocorticoid on bone metabolism, biomechanical properties of bones and formation and bone marrow cell differentiation into adipocytes and osteoclast in adult rats at different frequencies. 3. To study the influence of Sodium ferulate on bone metabolism in glucocorticoid–induced osteoporosis rats.Methods: 1. Experimental animal grouping:(1)GIOP modeles:32 female Wistar inbred Rats and Wistar closed Rats(3-month-old) were divided into four groups by randomized block design, which were control group(C), low-frequency group(L), middle-frequency group(M), high-frequency group(H).(2)Experiments of Sodium ferulate for the treatment 30 female Wistar Rats(3-month-old) were divided into five groups by randomized block design, which were control group(C), model group(A-G), low-dose Sodium ferulate group(A-L), middle-dose Sodium ferulate group(A-M), high-dose Sodium ferulate group(A-G). 2. Regimen:(1)GIOP Modules:Group C was intramuscular injection of 0.9% Na Cl; Groups L/M/H were intramuscular injection of dexamethasone(Dex) 1mg/kg/time: Group L 1 times a week, middle-frequency Group M 3 times a week, high-frequency Group H 5 times a week. Each rat were sacrificed after 90 days in the administration,(2) Experiments of Sodium ferulate(SF)for the treatment: Group A-C was intramuscular injection of 0.9% Na Cl; Stop medication for 10 days after Groups A-G/ A-L/ A-M/ A-H were intramuscular injection of dexamethasone(Dex) 1mg/kg/time, 5times a week for 90 days. Group A-G was intramuscular injection of 0.9% Na Cl 3 time/week. Group A-L/ A-M/ A-H were intramuscular injection of SF 20/ 40/ 80mg/kg/day for 20 days. Double fluorochrome labeling with calcein was performed before necropsy. 3. Bone histomorphometry:The longitudinal proximal tibial metaphyseal was used to analysis the bone histomorphometry. 4. Biomechanical characters: The femur was conducted in the three-Point bending test. The specimen is the whole length of femur. the maximum load and Load-deformation curve was recorded and absorption capability index were obtained. Biomechanical characters of fifth lumbar vertebral trabecular bone. 5. Bone marrow cell culture: Induce differentiation of bone marrow–derived macrophages(BMMs) towards osteoclast. Osteoclast cells were evaluated by tartrate-resistant acid phosphatase(TRAP) staining and these cells formed resorption pins on bone slices. Oil red O staining was assessed to identify and count adipocytes.Results: 1 GIOP modeling of Wistar inbred rats(1)The body weight of Wistar inbred Rats were more steady and changed little. The control and the low group rats weigh positive increase, while, the middle and the high weigh decrease.(2)About the static parameters: The Wistar inbred rats proximal tibia cancellous bone volume(%Tb.AT), the thickness of bone trabaculae(Tb.Th), the number of bone trabaculae(Tb.N), the separate degree of bone trabaculae(Tb.Sp) in J-Low, J-Mid and J-Hig group changed little compared with J-Con group.(3)The development parameter: Wistar inbred rats bone mineral deposition rate, mark perimeter rate and the bone formation rate decrease in F-H group compared with the F-C. MAR,%L.Pm, BFR/TV, BFR/BV and BFR/BS reduced in F-H group compared with the F-L and the F-M..(4)Osteoblast and osteoclast: Compared with the J-C, Wistar inbred rats bone osteoblast number per mm, percent osteoblast surface perimeter and percent osteoclast surface perimeter decrease in J-H. Osteoclast number per mm and %Oc.S/BS were decreased in J-M group. In J-L group %Oc.S/BS decreased. Compared with J-L group, Ob.N/ BS and %Ob.S/BS in J-H group decreasesignificantly; %Ob.S/BS and Ob.N/BS decreased in the J-M. Ob.N/BS and %Ob.S/BS decreased in J-H group compared with the J-M.(5)In growth-plate, the thickness of growth-plate(G.P.Wi) in J-M and J-H group were decreased compared with control group and the J-L. In contract, Height of Degenerative Cell(D.C.H) in J-Low group increase compared with group J-C.(6)Three-point bending test: In Wistar inbred rats, compared with the control group, the maximum load in J-L, J-Mid and J-H group was significantly reduced. Maximum stress in J-H group significantly reduced compared with the control group. elastic load was reduced significantly in J-L, J-M and J-H group compared with the control group. The femur length was reduced in J-M and J-H group compared with the control group. Compared with group J-L, maximum load, maximum stress, elastic load, fracture energy and femur length was significantly reduced in group J-H, while, femur length was significantly reduced in J-M group.(7)Compression Fracture of Lumbar Vertebra: Wistar inbred rats bone toughness and fracture energy decreased in group J-H compared with the control group. Compared with group J-L, fracture energy was reduced in group J-H. 2 GIOP modeling of Wistar closed Rats:(1)The rat body weight change: The group C/L and M rats weighing positive increase, while, the group H weighing indicate negative growth.(2)About the static parameters: Compared with the F-C, the Wistar closed Rats proximal tibia cancellous %Tb.AT, Tb.Th and Tb.N decreased in F-H group, and Tb.N in the F-M decreased significantly. Compared with the F-L, the Wistar closed Rats proximal tibia cancellous %Tb.AT, Tb.N and Tb.Th decreased in F-H group, while, Tb.Sp increased. %Tb.AT and Tb.N decreased in F-M group, while, Tb.Sp increased. The Wistar closed Rats proximal tibia cancellous Tb.Th decreased in the F-H compared with the F-M.(3)The development parameter: Wistar closed Rats compared with the F-C MAR, BFR/BS, and %L.Pm, BFR/TV and BFR/BV reduce in F-H group. Compared with F-L, MAR,%L.Pm, BFR/TV, BFR/BV and BFR/BS decrease in F-H group, while, MAR, BFR/TV, BFR/BV and BFR/BS decrease in F-M group. MAR, %L.Pm and BFR/BS reduce in F-H group compared with the F-M.(4)Bone marrow cell culture:Compared with the F-C, Wistar closed rats bone Ob.N/ BS and %Ob.S/ BS decrease in F-M group and F-H group; while,Oc.N/BS and %Oc.S/BS increased. Compared with the F-L, Wistar closed rats bone Ob.N/ BS and %Ob.S/ BS decrease in F-H group; while, Oc.N/BS and %Oc.S/BS increased. The Ob.N/ BS and %Ob.S/ BS decrease in F-M group; while in contrast, Oc.N/BS and %Oc.S/BS increased.(5)In growth-plate,In Wistar closed Rats, G.P.Wi was decreased in F-M and F-H group compared with control group and the F-L, while, compared with group F-M G.P.Wi was decreased in the F-H. D.C.H was reduced in the F-M and F-H group compared with control group. Compared with F-L group, the F-M and F-H group was decreased. D.C.H was reduced in F-H group compared with the F-M.(6) Three-point bending test: In Wistar closed rats, compared with the control group, maximum load, maximum stress, elastic load, fracture energy and femur length was significantly reduced in group J-H, while, maximum load, elastic load and femur length was reduced in group J-H. Compared with group J-L, fracture energy and femur length was significantly reduced in J-M group. Fracture energy, elastic load and femur length was significantly reduced in J-M group compared with group J-L.(7)Compression Fracture of Lumbar Vertebra:Wistar closed Rats lumbar, compared with the control group, maximum load, maximum stress, elastic load, fracture energy and bone toughness decreased in group F-M and F-H, while, maximum load, maximum stress, elastic load, fracture energy and bone toughness was reduced in group F-L. Compared with group F-L and F-M, maximum load was decreased in F-H group. 4.Bone marrow cell culture and differentiation:(1)Fat cell experiment: Compared with the control group, the number of fat cell in M and H roup was increased significantly. Compared with group L, the number of fat cell in M and H group was increased significantly. There was no fat cell in group L.(2)Osteoclast number: compared with group C and L, the number of osteoclast was decreased in H group.(3)The number of Oc resorption pit: The number of OC resorption pit staining was significantly increased in group H compared with group L and C.(4)The area of Oc resorption pit: The area of Oc resorption pit was increased significantly in group H compared with group L and C. 4. Effect of Sodium Ferulate on glucocorticoid-induced osteoporosis :Bone histomorphometry studies:(1) The proximal tibia cancellous bone trabecula thickness,area and area percentage were decreased in model group compared with control group. The proximal tibia cancellous bone trabecula thickness,area and area percentage were increased in middle-dose Sodium ferulate group and high-dose Sodium ferulate group compared with model group.(2)The calculation parameters of cancellous bone mark perimeter rate(%L.Pm) and the bone formation rate(BFR/BV, and BFR/BS) was increased in model group compared with control group. The bone formation rate was increased in middle-dose Sodium ferulate group and high-dose Sodium ferulate group compared with model group.(3)Osteoclast number per mm/osteoblast number per mm/percent osteoblast surface perimeter and percent osteoclast surface perimeter were increased in model group compared with control group.(4)In growth-plate,the thickness of growth-plate was increased in model group compared with control group. ConclusionConclusion:1.Effects of glucocorticoid on the closed rats 1mg/kgã€5 times/w for 90 days,is bone loss and bone structure destroyed, levels of osteogenesis decreased and role of osteoclasts increased, bone formation reduced and bone resorption increased, cartilage bone reduced and mechanical properties of bone deterioration. The effect on the inbred is osteogenesis and role of osteoclasts reduced, bone formation decreased and subchondral bone formation increased; measurements showed no reduction in bone mass, the bone structure destroyed as it is showed in bone biopsy and mechanical properties of bone deterioration. 2. It is indicated by the results GC induces changes of bone metabolism due to Influence of MSC differentiation into adipocytes instead of OB and BMMs differentiation into OC in adult rats, inhibit periosteal bone formation and reduce the stiffness and flexibility of skeleton. 3. This study demonstrates SF can effectively treat GIOP in rats. Its mechanism of action is mainly achieved by the reduction of bone loss in osteoporotic model. |
