Zoledronic acid inhibit the differentiation of osteoclast to reduce the loss of bone mass in mice after ionizing radiation

Field: Pharmacology, Toxicology and Pharmaceutics (Life Sciences)

Objective
To study the mechanism of how zoledronic acid (ZA) inhibit the bone loss in mice which were suffered total ionizing radiation (IR), we would discuss through three aspects including osteoclast (OC) proliferation, OC differentiation and the bone marrow mirco-environment.

Methods
50 male BALB/c mice suffered one-time 6.0 dose radiation were randomly divided into 5 groups, 10 in radiation group, 30 in low, middle, high treatment group, 10 in bone marrow group. Low, middle, high treatment groups were each injected with 0.000 25, 0.000 50, 0.001 00 mg/g ZA through caudal vein. Bone marrow group were injected with 2×106 allograft bone marrow cells in 4 hours. 4 weeks later, detect bone mineral density by small animal imaging measurement. Count the percentage of osteoclast primitive cells (CD34+ CD115+ ), osteoclast precursor cells [CD34+ CD115+ receptor activator of nuclear factor κB ligand (RANKL)+ ] and its activate cells [CD34+ CD115+ RANKL+ CXC chemokine receptor 4 (CXCR4)+ ] in bone marrow by flow cytometry. Analyzed bone trabecular and osteoclast precursor cells by bone marrow pathology. Detected the percentage and absolute value of blood cells and osteoclast precursor cells by mall animals 5 classification blood cell analyzer. Assess the level of cytokines in serum related with OC activation by cytometric beads array (CBA) and enzyme-linked immuno sorbent assay (ELISA) kits.

Results
The bone mineral density of mice in radiation group, low treatment group, middle treatment group, high treatment group and bone marrow group were correspondingly (0.51±0.06), (1.26±0.17), (2.24±0.29), (1.27±0.20), (2.52±0.23) g/cm3. The bone mineral density of mice in radiation group were lower than others in four groups (P=0.000). The bone mineral density of mice in middle treatment group were higher than low treatment group and high treatment group (P=0.000, P=0.942) and got close to bone marrow group (P=0.004). The present of cells with CD34+ CD115+ , CD34+ CD115+ RANKL+ , CD34+ CD115+ RANKL+ CXCR4+ were (4.09±0.97)%, (4.11±1.64)%, (18.71±6.23)% in middle treatment group, which are absolutely lower than radiation group (7.19±1.11)%, (9.01±4.06)%, (40.16±10.31)% (P=0.000, P=0.000, P=0.000) and moved towards to bone marrow group (10.14±2.13)%, (2.86±0.82)%, (11.86±3.39)% (P=0.000, P=0.241, P=0.056). Bone trabecular in the pathology of mice in radiation group became tenuous, discontinuous and messy. Limited bone marrow primitive cell were observed, which were instead of much necrotic tissue and adipose tissue. We could observe hyperaemia in bone marrow stromal mostly and polykaryocyte locally. After treatment of ZA, bone trabecular became stronger and more continuous and primitive cells were increased in the marrow cavity and degenerative tissues were decreased, especially in middle treatment group. Bone trabecular of mice in bone marrow group become compact, fullness and well-ordered. The percentage of primitive cell was obviously increased. Hemopoietic tissues were integrity.

Conclusion
ZA can restore bone marrow mirco-environment, accelerate self-rehabilitation. The number of marrow blast was increased and inhibit differentiation of OC. So we could conclude that ZA could prevent the bone mass loss after radiation.

Key words:
Zoledronic acid; γ-ray radiation; Bone marrow; Osteoclast; Bone mineral density