| Glucocorticoid-induced osteoporosis(GIOP) is the most common type of secondary osteoporosis. After GCs treatment several weeks, the loss of bone mass occurs and more than 30% bone mass lost in initially 3- 6months, about 6%-12% bone mass lost in the first year of GCs treatment,subsequently, 2%-3% bone mass lost will lost in each year after first year of GCs treatment. GCs have no safe dose, low-dose prednisone can increase the risk of hip and spine fractures in patients, and no gender differences in the fracture risk. Therefore, GIOP prevention has important clinical significance.Pathogenesis of GIOP is different from other type of osteoporosis which induced by age, estrogen deficiency or braking. GCs direct effect of bone marrow mesenchymal stromal cells(BMSCs), induce BMSCs differentiate into fat cells, fat cells are increased bone marrow cavity,causing bone marrow lipid metabolism disorder, and further reduce osteoblast differentiation. Thus, lipid metabolism disorder of bone marrow caused by GCs plays an important role in the pathogenesis of GIOP.Long-term use GCs may also encourage extramedullary adipose tissue mobilization and decomposition of the body increased total cholesterol(TCHO) and triglycerides(TG) levels, leading to hyperlipidemia, and hyperlipidemia may induce the bone osteoporosis occurs. The study suggested that high-fat fed rats can appear osteoporosis, and lipid metabolism disorders can increase the degree of osteoporosis, that indicate lipid metabolism has a close relationship with the happen and development of osteoporosis. Clinically application of lipid-lowering drug therapy can increase bone and reduce the risk of fractures. If the lipid metabolism disorder of bone marrow was effectively inhibited and the body’s dyslipidemia state was corrected, that can play a preventive effect to the incidence of GIOP, the degree of bone osteoporosis and fracture risk.GIOP clinical drug therapy is costly, and induce many side effects, it is necessary to search for a safe, side effects, low cost method of treatment.Pulsed electromagnetic fields(PEMF) therapy as a physical factor means a noninvasive, is widely used in a variety of orthopedic diseases, such as fresh fractures, delayed and nonunion fractures, osteoporosis, diabetes,osteoporosis, osteonecrosis and osteoarthritis. Evidence-based medicine has been proven safe PEMF in the treatment of osteoporosis and effective,PEMF has also been widely used as an adjunct to the prevention and treatment of OP. Clinical and experimental studies have found PEMF in anti-osteoporosis but also can improve the body of dyslipidemia. Toinvestigate whether the effect of PEMF on lipid metabolism could influence bone metabolism, is important for osteoporosis prevention by PEMF.There are no reports about PEMF affect bone metabolism by regulating lipid metabolism pathways in animal models GIOP. In this study,the GIOP rats were employed to study the effect of PEMF on regulating the lipid metabolism and bone metabolism of GIOP rats. To elucidate the anti-osteoporosis mechanism of PEMF has important scientific significance and clinical application.PART ⅠObjective Female SD rats were employed as experimental subjects,and received intramuscular super-physiological dose dexamethasone to build the GIOP rats for investigate the therapeutic mechanism of PEMF on GIOP.Methods 20 healthy female SD rats were random divided into control group and GIOP group. The dexamethasone(2.5 mg / kg, 2 times per week)was injected into right buttocks muscle of rats in GIOP group for 12 weeks.Saline(0.5 ml / kg, 2 times per week) was injected into right buttocks muscle of rats in control group for 12 weeks. After 12 weeks, bone density,Bone histomorphometry analysis, bone metabolism analysis, and lipid metabolism biochemical parameters was conducted to evaluate GIOP ratmodel.Results Compared with normal rats, whole body bone mineral density,bone mineral content and serum ALP levels of GIOP group were decreased significantly and serum TRAP, TG, TCHO and LDL levels were increased significantly. Trabecular bones were severely damaged.Conclusion Intramuscular injection of dexamethasone(2.5mg / kg, 2times / week) 12 weeks can be successfully established rat model of GIOP.PART ⅡObjective To observe the effect of PEMF GIOP intervention on rat BMSCs adipogenesis and lipid metabolism, and further investigate the mechanism.Methods(1) 40 healthy female SD rats were random divided into control group,GIOP group, calcium group and PEMF group. The dexamethasone(2.5 mg/ kg, 2 times per week) was injected into right buttocks muscle of rats in GIOP group, calcium group and PEMF group for 12 weeks. Saline(0.5 ml /kg, 2 times per week) was injected into right buttocks muscle of rats in control group for 12 weeks. After 12 weeks, bone density, bone metabolism,and lipid metabolism biochemical parameters was conducted to evaluate GIOP rat model.(2) PEMF group were given PEMF(50 Hz, 4.0 m T) 12 weeks.calcium group were given calcium gluconate tablets oral treatment and sham PEMF radiation 12 weeks. GIOP normal group and model group were given sham PEMF radiation and normal saline for 12 weeks.(3) Serum lipid metabolism biochemical tests was evaluated.(4) Oil Red O staining and L4 vertebral bone histopathological evaluation were conducted.(5) The m RNA expressions of lipid metabolism related indicators PPARγ, C/EBPα, FABP4 and Axin2 were detected by Real-time PCR assay.(6) The protein expressions of lipid metabolism related indicators PPARγ, C/EBPα, FABP4 and Axin2 were detected by Western blot assay.Results(1) Compared with normal rats, whole body bone mineral density,bone mineral content and serum ALP levels of GIOP group, alcium group and PEMF group were decreased significantly and serum TRAP, TG,TCHO and LDL levels were increased significantly after intramuscular injection of dexamethasone for 12 weeks. GIOP rat model successfully copied.(2) The results of serum lipid metabolism biochemical tests showedthat lipid disorder in GIOP rats were significantly improved after 12 weeks of PEMF intervention.(3) Oil Red O staining and L4 vertebral bone histological observation showed that the fat cells in bone marrow were significantly decreased after12 weeks of PEMF intervention.(4) The results of Real-time PCR assay showed that the m RNA expressions of PPAR-γ, C/EBPα, FABP4 and Axin2 were significantly decreased after 12 weeks of PEMF intervention.(5) The results of Western blot showed that the protein expressions of PPAR-γ, C/EBPα, FABP4 and Axin2 were significantly decreased after 12 weeks of PEMF intervention.Conclusion PEMF can inhibit the adipogenic differentiation of BMSCs in GIOP rats, the fat cells in the bone marrow were decreased,lipid metabolism disorders of bone marrow were improved. PEMF inhibit BMSCs differentiation of GIOP rats partly via decreasing Axin2/PPARγsignaling pathway.PART ⅢObjective In this part of the study, GIOP rats who were conducted in the second part of the study as subjects to observed PEMF on GIOP rat BMSCs osteogenic differentiation and bone metabolism, then further investigate the mechanism of PEMF regulate bone metabolism in GIOP rats.Methods(1) Test of bone mineral density and bone mineral content were conducted.(2) Serum bone metabolism biochemical tests was evaluated.(3) Bone tissue pathology and bone histomorphometry analysis were conducted.(4) The m RNA expressions of Wnt10 b, LRP5, β-catenin, OPG,RANKL, Runx2, Dkk1 and SOST were detected by Real-time PCR assay.(5) The protein expressions of Wnt10 b, LRP5, β-catenin, OPG,RANKL, Runx2, Dkk1 and SOST were detected by Western blot assay.Results(1) The results of bone mineral density and bone mineral The levels of bone mineral density and bone mineral content were significantly increased after 12 weeks of PEMF intervention.(2) The results of serum bone metabolism biochemical tests showed that bone metabolism disorders in GIOP rats were significantly improvedafter 12 weeks of PEMF intervention.(3) The results of bone tissue pathology and bone histomorphometry analysis showed that bone trabecula was improved after 12 weeks of PEMF intervention.(4) The results of Real-time PCR assay showed that the m RNA expressions of Wnt10 b, LRP5, β-catenin and OPG were significantly increased and the m RNA expressions of RANK and Dkk1 were significantly decreased after 12 weeks of PEMF intervention.(5) The results of Western blot showed that the protein expressions of Wnt10 b, LRP5, and Runx2 were significantly increased and the protein expressions of RANK and Dkk1 were significantly decreased after 12 weeks of PEMF intervention.Conclusion PEMF can promote osteogenic differentiation of BMSCs in GIOP rats, bone metabolism disorders were improved, bone loss was inhibited, bone formation was promoted. This part of the study showed that PEMF intervention activation Wnt10b/LRP5/β-catenin signaling pathway partly through inhibiting Axin2/PPARγ signaling pathway and downregulated Dkk1 expressions. PPARγ-Axin2-Wnt signaling axis involved the intervention of PEMF on lipid metabolism and bone metabolism. |
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