Roles of TNF-α, GSK-3β and RANKL in the occurrence and development of diabetic osteoporosis

Abstract

Objective: To investigate the roles of TNF-α, GSK-3β and RANKL in the occurrence and development of diabetic osteoporosis.

Methods: Diabetic rat model was established; tissue section technology was used to observe the situation of osteoporosis in diabetic rats; rat serum levels of OC, RANKL, GSK-3β, P38mapk, TNF-α and INS were detected by Elisa assay; osteoblasts and osteoclasts were primarily cultured and identified by immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) staining respectively. The effects of GSK-3β inhibitors, lithium chloride, TNF-α antagonists and RANKL antagonists on the proliferation of osteoblasts and osteoclasts were evaluated; quantitative PCR was used to assess the effects of GSK-3β inhibitors, lithium chloride, 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: Diabetic rat model was successfully established; osteoblasts and osteoclasts were successfully isolated and cultured. Elisa experiments showed that in diabetic model group, the levels of RANKL, GSK-3β, P38mapk and TNF-α were significantly increased, while the levels of osteocalcin (OC) and insulin (INS) were significantly reduced; MTT results showed that osteoclast proliferation in GSK-3β inhibitor and lithium chloride 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 lithium chloride 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. In GSK-3β inhibitor and lithium oxide groups, TNF-α and 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-3β gene expression level in diabetic group was lower than the control group.

Conclusion: 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-α and 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.

Keywords: GSK-3β; RANKL; TNF-α; diabetes; osteoporosis.

Figure 1

Bone tissue observation in diabetic rats and normal rats. A. The left femur of control group; B. The left femur of diabetic model group; C. L6 vertebrae of control group; D. L6 vertebrae of diabetic model group.

Figure 2

Comparison of the serum concentration of testing items.

Figure 3

Primarily cultured osteoblasts and osteoclasts. A. Normal osteoblasts (100 ×); B. Diabetic osteoblasts (100 ×); C. Normal osteoclasts (100 ×); D. Diabetic osteoclasts (100 ×).

Figure 4

Identification of osteoblasts. A. Normal osteoblasts (200 ×); B. Diabetic osteoblasts (200 ×).

Figure 5

Identification of osteoclasts. A. Normal osteoclasts (400 ×); B. Diabetic osteoclasts (400 ×).

Figure 6

MTT results of osteoclasts: 1. Negative control group; 2. GSK-3β inhibitor group (Final concentration was 5 mM); 3. Lithium chloride group; (Final concentration was 5 mM); 4. TNF-α antagonist group; (Final concentration was 10 μg/ml); 5. RANKL antagonist group (Final concentration was 10 ug/ml).

Figure 7

MTT results of osteoblasts: 1. Negative control group; 2. GSK-3β inhibitor group (Final concentration was 5 mM); 3. Lithium chloride group (Final concentration was 5 mM); 4. TNF-α antagonist group (Final concentration was 10 ug/ml); 5. RANKL antagonist group (Final concentration was 10 ug/ml).

Figure 8

Effects of GSK-3β inhibitor and lithium chloride on TNF-α (A) and RANKL (B) gene expression in osteoblasts and TNF-α (C) and RANKL (D) gene expression in osteoclasts: 1, the control group (control rats); 2, GSK-3β inhibitor group (control rats); 3, lithium chloride group (control rats); 4, the control group (diabetic rats); 5, GSK-3β inhibitor group (diabetic rats); 6, lithium chloride group (diabetic rats).

Figure 9

Effect of TNF-α and RANKL antagonists on gene expression of GSK-3β in osteoblasts (A) and osteoclasts (B): 1, the control group (control rats); 2, TNF-α antagonist group (control rats); 3, RANKL antagonist group (control rats); 4, the control group (diabetic rats); 5, TNF-α antagonist group (diabetic rats); 6, RANKL antagonist group (diabetic rats).