Effects Of Low-dose Zoledronic Acid On Osteoclasts In Ovariectomized Rats And Its Mechanism

Objective: To observe the effects of low-dose zoledronic acid(ZOL)on mandibular bone resorptionin the ovariectomized rats and explore the mechanism of nuclear factor kappa-B(NF-?B)signal system in the inhibition of osteoclast formation by ZOL.Methods: 72 female SD rats were randomly divided into three groups(n=24/group): sham operation control group(Sham),ovariectomized model group(OVX)and ovariectomized ZOL treatment group(ZOL-20).The latter two groups were ovariectomized to establish the model of osteoporosis,and the sham operation control group was performed to remove the fat around the ovary of the same quality.Three months after ovariectomy,the treatment group was subcutaneously injected with ZOL 20?g/kg,the Sham group and the OVX group were subcutaneously injected with the same dose of normal saline.At the same time,the left mandibular molars of all rats were extracted under anesthesia,then half the rats(n=12/group)were killed at 4 and 12 weeks after tooth extraction,respectively and the mandible tissue on the side of tooth extraction was taken out for the related-detection.The residual alveolar bone in the extraction fossa of rats was observed by imaging X-ray.The pathological changes of mandibular alveolar bone were detected by hematoxylin-eosin staining(HE)and Masson trichrome staining(Masson).The number of apoptosis of osteoblasts was detected by terminal-deoxynucleotidyl transferase mediated nick labeling(TUNEL)apoptotic assay.The changes of bone resorption marker CTX-I(Cross-linked Cterminal telopeptide of type I collagen)and bone formation marker PINP(Procollagen type IN-terminal propeptide)in serum were detected by Enzyme linked immunosorbent assay(ELISA).Real-Time quantitative PCR(RT-q PCR)was used to detect the expression of osteoclast-related genes : NF-? B?receptor activator of nuclear kappaB(RANK)and nuclear factor of activated T cells c1(NFATc1).Immunohistochemical technique was used to detect the expression of RANKL and NF-? B in mandibular alveolar bone;The expression levels of osteoclast related proteins(RANKL,I?B? and p I?B?,NF-?Bp65 and p-p65)were detected by Western blot(WB).Results: 1.Imaging X-ray: The alveolar bone density decreased in all groups,but the alveolar bone mineral density in the ZOL treatment groups were higher than that in the OVX model groups at 4 weeks and 12 weeks.2.Histopathology: HE staining showed that the bone of each group was looser at 4 weeks after operation than that at 12 weeks,and osteogenesis was active in ZOL treatment group compared with OVX model group,bone trabeculae were closely arranged and the structure was relatively complete;Masson staining showed that there was mainly light blue immature braided bone at 4 weeks and light red mature bone tissue at 12 weeks.The number of collagen fibers in ZOL treatment group was significantly lower than that in OVX model group(P < 0.05).3.TUNEL detection showed that compared with 4 weeks,the number of osteoblasts apoptosis in each group decreased in varying degrees at 12 weeks,especially in the ZOL treatment group was significantly less than that in the OVX model group(P < 0.05).4.ELISA serological testing showed that both CTX-I and PINP in the OVX model group increased in varying degrees,bone resorption marker CTX-I and bone formation marker PINP which decreased after ZOL treatment(P < 0.05).5.The results of RT-PCR showed that compared with the OVX model group,the expression levels of NF-?B,RANK and NFATc1 genes in ZOL treatment group decreased in different degrees at 4 weeks and 12 weeks,especially RANK and NFATc1(P<0.05).6.The results of immunohistochemistry showed that compared with the OVX model group,the expression of RANKL and NF-?B protein decreased after treatment with ZOL(P < 0.05).There was no significant difference in the expression of RANKL protein at 4 weeks and 12 weeks,but the expression level of NF-kappa B protein in the OVX model group was slightly lower at 12 weeks(P < 0.05).7.Western blot results showed that compared with the OVX model group,the expression levels of proteins(RANKL?pI?B?and p-p65)at 4 and 12 weeks decreased significantly after ZOL treatment(P <0.05),but there was no significant difference in the expression of total proteins(I?B? and p65)in each group.Conclusion: ZOL inhibit mandibular resorption in a certain extent and NF-? B signal pathway might be involved in the inhibition of osteoclast differentiation and maturation,and ZOL regulate the apoptosis of mandibular osteoblasts at the same time.