Roles Of TNF-α, GSK-3β And RankL In The Occurrence And Development Of Diabetic Osteoporosis

Objective: To investigate the roles of TNF- α, GSK-3β and RANKL in the occurrence and development of diabetic osteoporosis.Methods:(1)Diabetic rat model was established: female SD rats were randomly served as control group and diabetic group,Type diabetes was established by high fat and high-sugar feeding for eight weeks combined with one injection of small-dose(50 mg/kg body weight) of streptozotocin(STZ).The diabetes was confirmed with random blood glucose levels>7.8mmo L /L one week after the injection.Rats in control group were only injected the same volume of citric acid buffer. One week after dibetes group was set, rats were underwent bilateral ovariectomy surgery after anesthesia, and sham operation was performed in rats of control group.Tissue section technology was used to observe the situation of osteoporosis in diabetic rats; rat serum levels of OC, TNF-α, GSK-3β, P38-mapk,RANKL and INS were detected by elisa assay. The femur and the sixth lumbar vertebra were harvested and freed of soft tissue attachments carefully. Histomorphometry analysis was performed with an analyze software for multimedia pathological image.(2)Osteoblasts and osteoclasts were isolated and cultured. Morphological changes were observed under inverted microscop and identified by immunohistochemistry and tartrate resistant acid phosphatase(TRAP) staining respectively. The effects of GSK-3β inhibitors, TNF-αantagonists and RANKL antagonists on the proliferation of osteoblasts and osteoclasts were evaluated; quantitative PCR was used to assess the effects of GSK-3β inhibitors, on TNF-α and RANKL gene expression in osteoblasts and osteoclasts, and the effects of TNF-α and RANKL antagonists on GSK-3β gene expression in osteoblasts and osteoclasts.Results:(1) Diabetic rat model was successfully established; osteoblasts and osteoclasts were successfully isolated and cultured. BMD was significantly decreased at 8 and 12 weeks, when diabetic model group compared with control group.Elisa experiments showed that in diabetic model group, the levels of RANKL, GSK-3β,P38 mapk and TNF-αwere significantly increased, while the levels of osteocalcin(OC) and insulin(INS) were significantly reduced; MTT results showed that osteoclast proliferation in GSK-3 β inhibitor and negative groups were weaker than the untreated group, while osteoclast proliferation in TNF-α antagonist group and RANKL antagonist group was very close to the untreated group. Osteoblast proliferation in GSK-3β inhibitor and negative groups were weaker than the untreated group, while osteoblast proliferation in TNF- α antagonist group and RANKL antagonist group was higher than the untreated group. In all of the corresponding groups, cell proliferation in the diabetic group was stronger than the untreated group.Flow cytometry showed that in the GSK-3 inhibitor group,the diabetic group osteoclasts apoptosis rate was significantly lower than that of the control group;while in TNF-αantagonist and RANKL antagonist group the diabetic group osteoclasts apoptosis rate was significantly higher than that of the control group. In GSK-3β inhibitor group and TNF-αantagonist group, the osteoblasts apoptosis rate of the diabetic group, was significantly lower than that of the control group; in RANKL antagonist group. In the diabetic group, the osteoblasts apoptosis rate showed no significant difference to the control group rats. In GSK-3βinhibitor and negative groups, TNF-αand RANKL gene expression levels were elevated, but TNF-α and RANKL gene expression levels in the diabetic group were slightly lower than the control group. GSK-3β gene expression level in TNF-α antagonist group and RANKL antagonist group was reduced; GSK-3 β gene expression level in diabetic group was lower than the control group.Conclusion:(1)Diabetes rat model is successfully set up by ovariectomizing rats after three weeks.(2)In diabetic rats,TNF-α,GSK-3β and RANKL levels were elevated;GSK-3β could promote the proliferation of osteoblasts and osteoclasts, and inhibit the expression of TNF-α and RANKL; TNF-αand RANKL can suppress the proliferation of osteoblasts while had little effect on osteoclast proliferation; they also can promote the GSK-3β gene expression; interactions between the three broke the balance between osteoblasts and osteoclasts, leading to osteoporosis.