The Effects Of Cyclosporine A And Insulin On Bone Turnover And Its Related Molecular Mechanism In STZ-induced Diabetic Rats

Part 1: The effects of cyclosporine A and insulin on bone turnover inSTZ-induced diabetic ratsObjective: Diabetes mellitus (DM) is a metabolic syndrome characterized byhyperglycemia due to an absolute or relative deficiency in insulin secretion. Thedecrease in bone mass is believed to be one of the chronical complications in the DMpatients. Many studies have indicated that the decreased bone formation was themajor cause for diabetic bone loss while bone resorption activity showed eitherdecreased or unchanged. But many other studies demonstrated that AGEs, which iselevated in diabetes, can strongly stimulate bone resorption while not affect boneformation process. So the definite mechanism underlying the development of diabeticosteopenia has not hitherto been elucidated. Cyclosporine A, an immunosuppressiveagent, widely used in organ transplantation, was used in the present study. So the aimof our study was to investigate the bone loss and bone turnover in STZ-induceddiabetic rats and the effects of insulin and CsA on the bone turnover. At the same time,the effects of CsA on normal rat skeleton was also investigated.Materials and methods: Of 62 male SD rats studied, 32 were made diabetic byintravenous injection of streptozocin(50mg/kg) on day-7. Thirty nondiabetic rats wererandomly divided into 3 groups as groupCON, groupNL and groupNH. Thirty twodiabetic rats were also randomly divided into 4 groups as groupDM, groupINS andgroup DML, group DMH. From day 0,group NL and DML received a small dose ofCsA(lmg/k/d) by daily subcutaneous injection while group NH and DMH received arelatively large dose of CsA(Smg/kg/d) and group INS were injected subcutaneouslywith insulin (6U/kg/d).After the sacrifice on the day 56, blood were collected and serum were separated for the measurement of Ca, P, ALP and OC. The left tibia wasdissected for bone histomorphometry analysis. Right femur and lumbarvertebrae(L1-L4) were reserved for both BMD and bone biomechanical test.Results: No significant difference was found in serum Ca, P and ALP levelbetween 7 groups of rats studied. STZ-induced diabetic rats were characterized byextreme hyperglycemia, marked weight loss, polyuria and hypercalciuria. Alow-turnover osteopenia was evidenced in this group by decreased BMD (both femurand lumbar vertebrae), impaired bone biomechanics, reduced serum OC level (amarker of formation), decreased trabecular volume and thickness and reduced bonelabel surface and bone formation rate by bone dynamic study. All these abnormalities,except bone dynamic parameters, were partly normalized by insulin treatment. Thedecreased bone label surface and bone formation rate in diabetic rats were completelycorrected by insulin treatment. The intervention of low dose CsA didn't affect bonemass, bone turnover rate and biomechanics in both control rats and STZ-induced DMrats. In both control and STZ-induced DM rats treated with high dose CsA, ahigh-turnover osteopenia was noted by decreased BMD, impaired bone biomechanics,decreased trabecular volume, relatively increased serum OC level and relativelyhigher bone activation frequency by bone dynamic study.Conclusion: STZ-induced diabetic rats showed decreased bone mass andbiomechanics due to reduced bone formation rate. The abnormalities of bone inSTZ-induced DM rat was partially corrected by the injection of insulin. Theintervention of low dose CsA didn't affect bone mass and biomechanics in bothcontrol and STZ-induced DM rats. A high-turnover osteopenia was found with highdose CsA in the control rats and the diabetic osteopenia was also exacerbated bysimilar mechanism. Part 2: The effects of cyclosporine A and insulin on molecularmechanism of bone turnover in STZ-induced diabetic ratsObjective: Decreased bone mass is an important skeletal complications in patientswith T1DM. The major reason for diabetic bone loss is due to decreased boneformation rate, while the change of bone resorption activity in diabetic state needs tobe further investigated. In the present study, our investigation aimed at testing themRNA level of several cytokines and transcription factors related to bone turnover inbone tissues of STZ-induced diabetic rats, insulin-treated diabetic rats andCsA-treated diabetic rats or normal rats. The cytokines and transcription factorsinclude RANKL(receptor activator of nuclear factorκB ligand), OPG(osteoprotegerin),Cbfal(core binding factor 1), OSX(osterix), OC(osteocalcin). Theratio of RANKL/OPG is essential for the bone resorption process. OC is a lateosteoblast marker and the most cell-specific one.Cbfal and OSX are two importanttranscription factors in osteoblast differentiation. In addition to this, one recent studyshowed increased adipocity in bone marrow of STZ-induced diabetic mouse.We alsotry to demonstrate if it is a possible mechanism involved in diabetic osteopenia bydetecting mRNA level of peroxisome proliferators-activated receptor (PPAR)γ2 inbone tissue which can induce adipogenesis over oseoblastogenesis in pluripotent cellsand by counting adipocyte number in femur marrow of rats studied.Materials and methods: The modeling and grouping of the experimentalanimals are described as above. The study of bone tissue mRNA was performed byreal-time quantitative reverse transcription polymerase chain reaction assays.Results: The RANKL/OPG, OSX, Cbfal and OC mRNA expressions weredeclined in the bone tissue of the tibia in the STZ-induced DM rats,compared withthe control. The gene expression of PPARγ2 mRNA, a marker for adipocytes,in thebone tissue of rat tibia showed a remarkable increase in the diabetic rats,comparedwith control. As RNA analysis, histological sections of diabetic femur demonstrated a similar increase in marrow adiposity in comparison with controls. In contrast,however, diabetic rats showed a reduction in adiposity both of liver and peripheraltissues. OSX,Cbfal and OC mRNA level were reversed by the treatment of insulin indiabetic tibia while there was no difference of RANKL/OPG mRNA between theinsulin-treated and those without insulin treatment. An decrease in PPARγ2 mRNAlevels was found in the tibia of insulin-treated rats with reduction in marrow adipocityof femur compared with diabetic rats. The intervention of low dose CsA didn't haveany effects on the mRNA expression of the cytokines or transcription factors studiedand the number of adipocytes in femur marrow. The RANKL/OPG, OSX, Cbfal andOC mRNA expressions were increased by the intervention with high dose CsA in bothnormal and diabetic tibia while PPARγ2 mRNA expression and the number ofadipocytes showed unchanged.Conclusion: A low-turnover osteopenia was evidenced in STZ-induced diabeticrats by significant decrease of both osteoclastic and osteoblastic marker mRNA levelin tibia. We also proposed another pathway that contribute to diabetic bone loss whichinvolves the selection of of adipogenesis over osteoblastogenesis in diabetic bonetissue. The treatment of insulin can stimulate bone formation dramatically byincreasing mRNA level of osteoblastic marker and inhibiting the adipogenesistendency in diabetic bone tissue, but not influence osteoclastic activity of diabetic rats.The use of low dose CsA doesn't affect bone- turnover condition in both intact ratsand STZ rats. High dose CsA can induce a high-turnover osteopenia in intact anddiabetic rats by increasing both osteoblastic and osteoclastic activity, but not affect theadipogenesis process in normal and diabetic bone.