| Background and ObjectionZoledronic acid is the most effective bisphosphonate in clinical application and has been used as an anti-resorptive agent to treat some metabolic bone diseases such as osteoporosis,Paget’s disease,osteolysis,hypercalcemia of malignancy,and cancer-related bone destruction.Zoledronic acid mainly regulates the endogenous bone balance by inhibiting osteoclastic activity,and effectively reduces bone loss and bone turnover.However,the long-term use of zoledronic acid can lead to mandibular necrosis.The incidence of this complication is about from 4.3%to 18.6%(low incidence of complication caused through oral administration and high incidence of complication through intravenous administration).It may be that zoledronic acid induces cytotoxicity towards osteoblasts within bone tissue and reduces osteoblastic activity and osteogenic gene expressions,thus inhibits bone formation.Zoledronic acid reduces bone loss by inhibiting osteoclast production and promoting osteoclast apoptosis.In recent years,zoledronic acid has been found to regulate the activity and function of osteoblasts,thereby regulating bone metabolism and participating in the occurrence and development of bone metabolic diseases.Recent studies have shown that low concentrations of zoledronic acid reduce the expression of BMP-2 in osteoblasts and prevent their differentiation and maturation.Some studies have also shown that low concentrations of zoledronic acid promote osteogenic differentiation and mineralization,which is beneficial to bone formation and repair,while high concentrations of zoledronic acid display toxic effect on osteoblasts,and increase apoptosis and necrosis.Therefore,the regulatory effect of zoledronic acid on osteoblasts is controversial and needs further study.It has been proved that rapamycin target protein-1(mTORC1)can integrate various kinds of extracellular signals to regulate cell proliferation,differentiation and apoptosis.mTORCl signals has been shown to participate in many life activities of the body.In some studies of bone metabolic diseases,mTORC1 can regulate disease progression by regulating osteoblast proliferation,apoptosis and differentiation.The purpose of this study is to explore the regulatory role of zoledronic acid on osteoblasts and its underlying mechanisms,and to explore the causes of osteonecrosis caused by zoledronic acid from molecular pathology,and provide a theoretical basis for the experimental research and clinical prevention and treatment of zoledronic acid induced osteonecrosis.Methods1、Human osteoblast MG-63 cell line was cultured and treated with different concentrations of zoledronic acid.2、The effects of zoledronic acid on the activity of osteoblasts were detected by MTT.3、Flow cytometry was used to detect the apoptosis of osteoblasts by zoledronic acid.4、The effect of zoledronic acid on the activity of alkaline phosphatase in osteoblasts was detected.5、The influence of zoledronic acid on osteoblast gene markers,ALP and OCN gene expression levels were detected by RT-PCR.6、Western blot was used to detect the protein levels of ALP,OCN and mTORC1 activity in osteoblasts treated with zoledronic acid.7、The effect of zoledronic acid on the differentiation of osteoblasts was detected after the inhibition of mTORC1 activity by rapamycin or siRNA silencing。8、Mice were treated with rapamycin and zoledronic acid in vivo,and the relationship between the activity of mTORCl and the expression of osteocalcin was detected.9、Data were analyzed using one-way analysis of variance to compare groups and all values are expressed as mean± SEM.All statistical analyses were performed using SPSS 20.0(IBM SPSS,USA)and P<0.05 was considered to indicate a statistically significant difference.Results1、High concentrations of zoledronic acid inhibited osteoblast activity.Low concentrations of zoledronic acid had no effect on the activity of osteoblasts,while long-term administration of low concentrations of zoledronic acid inhibited osteoblast activity.2、High-dose zoledronic acid promoted osteoblast apoptosis in a time-dependent manner.Low-dose zoledronic acid had no significant effect on osteoblast apoptosis.3、Low-dose zoledronic acid could enhance alkaline phosphatase activity of osteoblasts,while high-dose zoledronic acid inhibited alkaline phosphatase activity of osteoblasts.4、Low-dose zoledronic acid was used to increase the expression of osteocalcin and alkaline phosphatase in osteoblasts and promote the differentiation and maturation of osteoblasts,while high-dose zoledronic acid inhibited osteocalcin and alkaline phosphatase expression lebvels in osteoblasts.5、Low concentration of zoledronic acid inhibited mTORC1 activity of osteoblast,and high concentration of zoledronic acid promoted mTORC1 activity of osteoblasts;Rapamycin inhibited the activation of osteoblast induced by high concentration of zoledronic acid and increased the expression of osteocalcin;Osteoblast with ablation of Raptor(inhibition of mTORCl activity)treated with high concentration of zoledronic acid showed an increased expression of osteocalcin and alkaline phosphatase.6.、Compared with the mice injected with zoledronic acid alone,mTORC1 activity in the trabecular regions of long bone was reduced and the expressions of osteocalcin increased in the mice injected with both rapamycin and zoledronic acid.ConclusionsThe conclusions of this study were mainly proved by in vitro experiments:High concentrations of zoledronic acid inhibit osteoblastic activity and promote osteoblastic apoptosis,as well as preventing osteoblastic differentiation by stimulating mTORCl activity.However,MG-63 cells short-time-treated with low doses of zoledronic acid more likely to survive and differentiate and mature.The regulation of zoledronic acid on osteoblasts is dependent on the mTORCl signal to inhibit the reduction of mTORCl activation.The sensitivity of osteoblasts to zoledronic acid. |
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