Effect Of Prior Administration Of Vitamin K₂ Followed By Zoledronic Acid On Osteoporosis And Its Main Mechanism Research

BackgroundOsteoporosis is defined as a skeletal disorder characterized by compromised bone strength and micro-architectural deterioration of bone tissue,predisposing to an increased risk of fracture[1].Osteoporosis is divided into two major categories:primary osteoporosis and secondary osteoporosis,while postmenopausal osteoporosis is common in primary osteoporosis.Effective anti-osteoporosis drugs increase bone mineral density,improve bone quality,and reduce the incidence of osteoporotic fractures.Anti-catabolic agents or anabolic agents can improve the bone mass,however,to maintain long-term therapeutic effects is difficult especially after the mono-therapy is withdrawn.Besides that,evidence[2,3]confirmed that prolonged the course of bisphosphonates could not get added benefit,in contrast,bone mass plateau occurred inevitably.Side effects accompanied with the prolonged period of mono-therapy,since researches[5,6]indicated that long-term administrate of teriparatide increases the risk of osteosarcoma,while treatment with bisphosponates over 5 years increased the risk of atypical fracture and bisphosphonate related osteonecrosis of the jaw.Providing a long-term,effective,and reliable strategy for postmenopausal osteoporosis and how to prevent osteoporotic fractures effectively remains a hug challenge.Sequential therapy may serve a new idea for the treatment of osteoporosis,so as to obtain greater therapeutic effects finally,since Leder[7-9]indicated that prior administration of anabolic agents then followed by anti-catabolic agents,such as prior administration of teriparatide followed by alendronate or denosumab,improves the bone mineral density of lumbar significantly,in addition,the therapeutic effects surpass the mono-therapy and simultaneously protocol completely.However,this sequential protocol has been questioned by Bo Abrahamsen[10]shortly after its publised.The reason why prior administration of anabolic agents then followed by anti-catabolic agents serve favorable therapeutic effects depends on prior administration of teriparatide provided sufficient time for complete the secondary mineralization.Once provide sufficient time,prior administration of anti-catabolic agents then followed by anabolic agents serve favorable therapeutic effects as well,therefore,sequential strategy just the order of thing.Appropriate theoretical supportting seems the missing link in sequential protocol,thus the clinical research process related to the sequential protocol of anabolic agents then followed by anti-catabolic agents is blocked recently.Besidea that,the high cost of the teriparatide followed by denosumab protocol limited its widely popularize.Nevertheless,there' s still plenty of sequential protocols exist in daily clinical practice,sometimes,convert to another mechanism agents is a reasonable choice to get more benefits and avoid the side effects induced by previous agents,and the implementation of sequential protocol inevitably occurred at this time.As a result,researches refer to sequential protocol possess a favorable clinical foundation.However,literatures refer to the sequential protocol for osteoporosis only reported the favorable therapeutic effects at present,few literature involved the mechanism and long-term follow-up,therefore,a theoretical breakthrough is urgent needed which allow the sequential protocol going further.We reorganized and extended the clue of sequential protocol for osteoporosis according to previous researches and proposed some new ideas as below:sequential proptocol should not be confined to the pattern of anabolic agents sequential anti-catabolic agents,additional protocols such as,other mechanism agents combined or sequential anabolic/anti-catabolic agents;future researches should involve the mechanisms,such as,whether one agent possess the ability to reduce or eliminate the side effects induced by previous agent,besides that,is there any synergism,additive or antagonistic effect between the two drugs;subsequently,safety is essential for long-term strategy,besides that,patients compliances and the medical cost should also be considered as well.After comprehensive consideration of various anti-osteoporosis agents,we proposed our new sequential protocol,that is,prior administration of vitamin K2 followed by zoledronic acid protocol.Vitamin K2 improves the bone formation[11,12],inhibits the process of apoptosis contribute to maintain the cell viability and the number of osteoblasts[13],furthermore,vitamin K2 inhibits the proliferation and differentiation of osteoclasts via NF-KB inhibition[14,15].As a new generation of nitrogen-containing bisphosphate,zoledronic acid possess powerful capacity to anti-bone absorption,and the feasibility of dosing as 5 mg infusion once a year ensures a favorable patients compliances to zoledronic acid.However,zoledronic acid has been reported to inhibit bone turnover for 12 months or 18 months,such negativeceffect of zoledronic acid toward osteoblasts proliferation and differentiation was also detected in much lower concentrations(e.g.above 10-8mol/L)[16].Based the evidence above,vitamin K2 and zoledronic acid exert similar effect towards osteoclasts,while playing opposite role towards osteoblasts.As a basic research of new sequential protocol,we will focus on the notion whether vitamin K2 can antagonize the inhibitory effect induced by zoledronic acid on osteoblasts.Suppose prior administration of vitamin K2 can reduce or eliminate the inhibition towards osteoblasts induced by zoledronic acid,as a result,these osteoblasts will improve the new bone formation while the anti-bone absorption induced by zoledronic acid unchanged,which in turn improve the net bone gain.For this purpose,we designed in vitro(osteoblasts)and in vivo experiments so as to verify the efficacy of new sequential protocol.Firstly,cell viability and mineralization capacity will be detected in mono-therapy,simultaneous and different sequential protocols,and the interaction between the drugs will be calculated in simultaneous and sequential protocols,furthermore,main mechanisms will be clarified by detected the changes in osteoblasts-related gene expression.Secondly,we will put different protocols into osteoporosis rat models and measured the bone mineral density,bone trabecular morphology,bone biomechanics,bone calcium and hydroxyproline content,and surface-based bone turnover marks in mono-therapy,simultaneously and different sequential protocols,so as to verify the favorable effects in vitamin K2 sequential zoledronic acid protocol.The innovations and advancements of our research are listed blew:firstly,for the first time vitamin K2 sequential zoledronic acid protocol was used for treatment of postmenopausal osteoporosis?secondly,this new strategy belongs to the category of other mechanism agents sequential anti-catabolic agents,this new sequential protocol breakthrough the routine of anabolic agents sequential anti-catabolic agents,thirdly,for the first time elucidated the mechanism via in vitro experiment;fourthly,this new sequential protocol significantly reduced the medical cost and contribute to improve the patients compliances in comparison with the teriparatide sequential denosumab.Objective:This study aimed to investigate the effect of prior administration of vitamin K2 followed by zoledronic acid on osteoblasts proliferation,differentiation and mineralization,subsequently investigate the therapeutic effects in animal experiments,finally elucidate the main mechanisms of new sequential protocol.Method:(1)In vitro experiment.Firstly,intervened osteoblasts with different concentrations of vitamin K2 and zoledronic acid for 72 hours respectively,then detected the cell viability and calculated the IC50 values subsequently.Diluted vitamin K2 and zoledronic acid into 6 concentrations according to 0.125,0.25,0.5,1,2,4 times IC50 values respectively.Secondly,with the concentrations at the same times of IC50 values,vitamin K2 and zoledronic acid will be arranged to different protocols for another 144-hour experiment,and protocols including vehicle,vitamin K2,zoledronic acid,vitamin K2 combined zoledronic a2cid,vitamin K2 sequential zoledronic acid and zoledronic acid sequential vitamin K2.With the MTT method,cell viability was detected in different protocols,and the CI index was calculated subsequently.The morphology of osteoblasts and the state of mineralized nodule formation under different concentrations and different protocols were observed with Alizarin red staining method,and the calcium content was quantitatively measured with calcium assay kit.Thirdly,detected the changes of Bcl-2,Bax,RunX2 and Sost gene expression in osteoblasts with different protocols,furthermore,elucidated the main mechanisms of favorable effects induced by vitamin K2 sequential zoledronic acid protocol.(2)In vivo experiment.Seventy-six 20-week-old healthy Wistar rats experienced the ovariectomy(OVX)and sham surgery,then rats were divided into sham,OVX,vitamin K2,zoledronic acid,vitamin K2 combined zoledronic acid,vitamin K2 sequential zoledronic acid,zoledronic acid sequential vitamin K2 groups.A 12-week intervention according to different protocols was performed.Rats were injected with tetracycline and calcein before sacrificed for surface-based bone turnover assay.At the end of experiments rats were sacrificed,femurs,tibias and peripheral blood samples were collected.Double-energy X-ray bone densitometer machine was used to measure femoral bone mineral density.Siemens preclinical Inveon PET/SPECT/CT was used to measure femoral trabecular bone parameters,then generated trabecular morphology with 3D images.The compact servohydraulic fatigue testing system was used to test the bending resistance ability of femurs.Subsequently,proximal femurs were ashed and the bone calcium content was measured with calcium assay kit.Distal femurs were hydrolyzed with hydrochloric acid,femoral hydroxyproline content was measured with hydroxyproline assay kit.Double fluorescent labeling method was used to mearsure the surface-based bone turnover in trabecular bone,and the peripheral blood was collected for blood lipid analysis and prothrombin time measurement.Results:(1)While the concentrations of vitamin K2 and zoledronic acid at the same times of IC50 values,zoledronic acid inhibit the osteoblasts proliferation,differentiation and mineralization more significantly in comparison with vitamin K2.Prior administration of vitamin K2 partially antagonized the osteoblasts inhibition induced by zoledronic acid,subsequently the cell viability and mineralization ability were significantly improved in comparsion with zoledronic acid alone.However,in vitamin K2 combined zoledronic acid protocol and zoledronic acid sequential vitamin K2 protocol,synergistic effect occurred characterized by consistent inhibition of osteoblasts,especially in simultaneously protocol,as a result decreased the cell viability and mineralization.(2)Vitamin K2 exerted its anti-apoptosis effect,improved the osteoblasts proliferation,differentiation and mineralization,these favorable results mainly due to effectively increased the Bc1-2/Bax rate,up regulated the RunX2 expression while down regulated the Sost expression.Whereas the simultaneously protocol and zolerdronic acid sequential vitamin K2 showing an opposite trend,and the trend in simultaneously protocol even worse.(3)Vitamin K2 increased the bone mineral density in OVX rats,and improved the trabecular archtecture,bone calcium content and new bone mineralization.Zoledronic acid increased the bone mineral density,mild improved the trabecular architecture,and improved the bone calcium content,whereas inhibited the new bone formation and mineralization.Prior administration of vitamin K2 followed by zoledronic acid uniquely increased the bone mineral density,improved the trabecular structure,Young' s modulus,bone calcium content and new bone mineralization.Zoledronic acid sequential vitamin K2 improve the bone mineral density,trabecular structure and bone calcium content,however,these therapeutic effects showing no significant increase in comparison with zoledronic acid alone.The simultaneously protocol showing no therapeutic effects in comparison with OVX.Conclusion:Prior administration of vitamin K2 partially antagonized the osteoblasts inhibition of zoledronic acid on osteoblasts proliferation,differentiation and mineralization,more new bone formation while the anti-resorption effect of zoledronic acid would not be affected,thus leading to a net gain of bone mass.The proposed mechanisms underlying such a beneficial protocol included improved the Bcl-2/Bax rate,up regulated RunX2 expression and down regulated the Sost expression,which in turn inhibit the process of osteoblasts apoptosis,improve the osteoblasts proliferation,differentiation and mineralization.However,zoledronic acid sequential vitamin K2 protocol lack effective therapeutic effects in comparison with zoledronic acid alone,simultaneously protocol is not recommended due to lack benefits over OVX which should be avoid.These findings supported that pretreatment with vitamin K2 followed by zoledronic acid protocol might be recommended as a long-term strategy for osteoporosis management.