| BackgroundOsteoporosis(OP) is considered as dysostosis disorder due to the damage of microstructure of bony tissues, the persistent decreasing of bony matrixes and mineral bones,thinness of sclerotin, the decrease of the number of trabecular bones, and the increase of bony fragility and the risk of fracture. Glucocorticoids(GC) are valuable and indispensable therapeutic agents in the management of many diseases such as asthma, rheumatic diseases, and connective tissue diseases.GC impair the proliferation, differentiation, and function of osteoblasts and induce the apoptosis of mature osteoblasts and osteocytes, also favor in osteoclastogenesis and as a consequence increase bone resorption. Half a percent of the population takes oral GC, making GC-induced osteoporosis(GIOP) is the most common and serious side effect for patients receiving GC.GIOP is characterized by disturbed bone remodelling associated with reduced bone mass and abnormally low quality of the bone tissue, resulting in loss of both cortical and cancellous bone, thus increasing the risk of fracture.Receptor activator of nuclear factor-KB(RANK),receptor activator of nuclear factor-KB ligand(RANKL) and osteoprotegerin(OPG) are the cytokines which regulate the differentiation and activation of osteoclast. RANKL/RANK/OPG system plays a vital role in bone metabolism. RANKL binds to its receptor, RANK, can induce differentiation and activation of osteoclast and inhibit steoclast apoptosis. This process can be blocked by OPG, which can decrease bone destruction. The RANKL/RANK/OPG system is implicated in many bone loss diseases.This system could be performed as a therapeutic target.Bisphosphonates are considered to be the pharmacologic gold standard of care for the prevention and treatment of GIOP. Aln is a member of the third generation of modified pyrophosphates, which is a potent antiresorptive agents; both have been approved and are in use for treatment of various disorders characterized by increased osteoclast-mediated bone resorption. GIOP and male osteoporosis are also treated with Aln successfully. SrR is a novel anti-osteoporotic treatment with a dual mode of action, both increasing bone formation and decreasing bone resorption, which rebalances bone turnover in favor of bone formation and increases bone strength. Therefore, SrR might be applicable as a bone therapeutic agent to treat secondary osteoporosis in the clinic.ObjectiveTo study the effects of administration Strontium ranelate(SrR) on the cancellous and cortical bone in glucocorticoid(GC)-treated rats by means of bone micromorphology, DEXA, biomechanics and histomorphometry, bone turnover marker, and observe the expression of OPG/RANKL mRNA and protein activity in the left femur, as well as to provide futher experimental evidence for exploring the mechanism of SrR.MethodsIn successful model experiment foundation, thirty-two 3.5-month male Sprague-Dawley rats were randomized into four groups:age-matched normal control(Nrm), methylprednisolone(Met) (5.0mg/kg, sc, per day for 5 days/week), Met plus Aln orally(1.0 mg/kg/d) and Met plus SrR orally(900 mg/kg/d).The study period was 9 weeks. Blood samples were obtained before sacrifice to determine the serum ALP, OC and TRACP.DEXA was evaluated in the right femoral diaphysis and fifth lumbar vertebrae(L5), after measuring BMD, the right femur was prepared for shaft three-point bending test, L4 and L5 were prepared for indentation and compression test. Histomorphometry was performed in the right proximal tibial metaphysic and tibial diaphysis, RT-PCR and Western-blot were detected in the left femur, SEM was performed in the left tibia.Statistical methodsAll data were reported as mean±standard error, SPSS13.0 software was used for these analyses.Group Nrm and group Met comparison were run using independent-samples T test, and when a significant difference was revealed(P<0.05);group Aln and group SrR assays performed were analyzed by one-way ANOVA,when significance was revealed(P<0.05).Results1.Osteoporosis model established by simulated weightlessness:All rats gained substantial body weight, significant differences were found in two groups(P<0.001). Serum ALP and OC were significantly lower in Met than in Nrm(P<0.01),serum TRACP was significantly higher in Met than in Nrm(P<0.01);the BMD of the right femur and L5 were significantly lower in Met than in Nrm(P<0.001);the max load and rigidity were significantly lower in Met than in Nrm(P<0.05).Quantitative histomorphometry,%Tb.Ar, Tb.Th and Tb.N were significantly lower in Met than in Nrm(P<0.01).Tb.Sp was significantly higher in Met than in Nrm(P<0.01). %L.Pm,MAR,BFRs and %O.Pm were significantly lower in Met than in Nrm(P< 0.01);%E.Pm was significantly higher in Met than in Nrm(P<0.01).2.Effects of SrR on osteoporosis in GC-treated rats:(1)In the right femur and L5 BMD were found significant differences in all groups(P <0.05).Met was significantly lower than Nrm group(P<0.001);Aln and SrR were significantly higher than Met group(P<0.001).It should be noted that SrR led to significantly higher in the right femur and L5 BMD compared with the Aln group(P <0.05).(2) The cortical bone biomechanical property such as the max load and rigidity of femoral shaft three-point bending test, four groups were found significant differences (P<0.05).Met was significantly lower than in Nrm group(P<0.001);there were no significant difference between Aln and Met groups(P>0.05);SrR was significantly higher than in Met group(P<0.001).It should be noted that SrR led to significantly higher compared with Aln group(P<0.05).The cancellous bone biomechanical property like the max load and rigidity of vertebral body(L4) indentation were found significant differences in four groups(P<0.05).Met was significantly lower than in Nrm group(P<0.01);Aln was significantly higher than in Met group(P<0.05);SrR was significantly higher than in Met group(P<0.05);there were no significant difference between Aln and SrR groups(P>0.05).The max load and rigidity of vertebral body(L5) in the compression test were found significant differences in four groups(P<0.05).Met was significantly lower than in Nrm(P<0.01);Aln was significantly higher than in Met group(P<0.05);SrR was significantly higher than in Met group(P<0.05); there were no significant difference between Aln and SrR groups(P>0.05).(3)Bone turnover marker:Serum ALP, OC and TRACP,significant differences were found in four groups(P<0.05).Serum OC and ALP were significantly lower in Met than in Nrm group(P<0.001),serum TRACP was significantly higher in Met than in Nrm group(P<0.001);there were no significant difference between Aln and Met groups(P>0.05); SrR was significantly higher than in Met group(P<0.05),but lower in Nrm group(P<0.05);serum TRACP was significantly lower in Aln and SrR than in Met group(P<0.05), but higher in Nrm group(P<0.05);there were no significant difference between Aln and SrR groups(P>0.05).(4) In SEM observation, trabecular bone of the left tibia was found with normal network structure and collagenous fibers with regular arrangement in Nrm group; trabecular bone of the left tibia was manifested with fewness, fragility, microcrack and collagen fibers displaced by absorption lacuna in Met group;trabecular bone getting more, network structure getting better and collagenous fiber were much more regular in Aln and SrR groups.(5) Quantitative histomorphometry①Histomorphometric indices in the proximal tibia metaphysic: Static histomorphometric indices(%Tb.Ar,Tb.Sp,Tb.N and Tb.Sp) in cancellous bone, differences were found in four groups(P<0.05).Met significantly decreased %Tb.Ar,Tb.Sp and Tb.N compared with the Nrm group(P<0.01).Aln was significantly higher than in Met group(P<0.05);SrR was significantly higher than in Met group(P<0.05);there were no significant differences between Aln and SrR groups(P>0.05).Tb.Sp, Met was significantly higher than in Nrm group(P<0.001), Aln was significantly lower than in Met group(P<0.01);SrR was significantly lower than in Met groups<0.01);there were no significant difference between Aln and SrR groups(P>0.05).Dynamic histomorphometric indices(MS/BS,MAR,BFRs and ES/BS),differences were found in four groups(P<0.05).Met significantly decreased MS/BS,MAR and BFR/BS compared with the Nrm group(P<0.001).there were no significant differences between Aln and Met groups(P>0.05);SrR was significantly higher than in Met group(P<0.05).It should be noted that SrR led to higher in MS/BS compared with the Aln group(P<0.05).ES/BS,Met was significantly higher than in Nrm group(P<0.001),Aln was significantly lower than in Met group(P< 0.01);SrR was significantly lower than in Met group(P<0.01);there were no significant difference between Aln and SrR groups(P>0.05).②Histomorphometric indices in the tibial diaphysis: Static histomorphometric indices(%Ct.Ar and%Ma.Ar) in cortical bone and (MS/BS, MAR and BFRs)in periosteum, differences were found in four groups(P<0.05).Met significantly decreased Ct.Ar,MS/BS,MAR and BFRs compared with the Nrm group(P<0.01).there were no significant difference between Aln and Met groups(P >0.05); SrR was significantly higher than in Met group(P<0.05); there were no significant differences between Aln and SrR groups(P>0.05).%Ma.Ar, Met was significantly higher than in Nrm groups<0.01);there were no significant difference between Aln and Met groups(P>0.05), and no significant difference between SrR and Met groups(P>0.05), and no significant difference between SrR and Aln groups(P>0.05).Dynamic histomorphometric indices (MS/BS,MAR, BFRs and ES/BS)in periosteum, significant differences were found in four groups(P<0.05). Dynamic histomorphometric indices (MS/BS,MAR and BFRs), there were no significant differences between Met and Nrm groups(P>0.05).For MAR and BFRs, Aln was lower than in Met group(P<0.05); for MAR and MS/BS,SrR was higher than in Met group(P<0.05).Furthermore, SrR led to a significantly higher in MS/BS, MAR, BFR/BS compared with the Aln group group(P<0.01).ES/BS,Met was significantly higher than in Nrm group(P<0.01),Aln was significantly lower than in Met group(P<0.01);SrR was significantly lower than in Met group(P<0.01);there were no significant differences between Aln and SrR groups(P>0.05).(6) Effects of Strontium ranelate on OPG/RANKL related changes in protein activity in GC-treated rats①RT-PCR assay:the expression levels of OPG mRNA and RANKL mRNA, differences were found in three groups(P<0.001),Met significantly decreased OPG mRNA and RANKL mRNA compared with the Nrm group(P<0.001).SrR was significantly higher than in Met group(P<0.001).②Western blot detection:OPG and RANKL protein expression, differences were found in three groups(P<0.001),Met significantly decreased OPG and RANKL protein expression compared with the Nrm group(P<0.001).SrR was significantly higher than in Met group(P<0.001). Conclusion1.The effect of long-term(9 weeks) GC administration, employing a dose equivalent to 5.0mg/kg, sc, per day for 5 days/week, the model of GC-induced osteoporosis characterized by a severe increasing bone formation and decreasing bone resorption, a decrease in BMD, and bone strength. The model was established successfully.2.SrR at the dose of 900mg/kg/d has more effective than Aln 1.0mg/kg/d in stimulate an anabolic bone response, and improve the resistance of bone over load to fracture in GC-treated rats.SrR has mildly effective in stimulate an anabolic bone response than Aln in prevention GC-induced osteopenia, suggesting the possibility that the SrR may be applicable to use as a bone therapeutic agent to treat secondary osteoporosis in clinic.3.The level of gene and protein expression of OPG/RANKL in the normal rat bone. The GC administration decreased bone OPG gene and protein expression, increased RANKL gene and protein expression; SrR increased bone OPG gene and protein expression as well as decreased RANKL gene and protein expression, one of the mechanism maybe that the SrR could regulate OPG and RANKL in GC-induced osteoporosis.
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