| Research background and obiectivePostmenopausal osteoporosis(PMO)is a chronic bone metabolism disease characterized by decreased bone mass that leads to bone pain,microarchitectural abnormalities,and increased vulnerability.PMO is mainly caused by a large decrease in hormones in the body after menopause,resulting in excessive activation of osteoclasts,which in turn leads to osteoporosis in patients.Nowadays,the improvement of medical level,the increase of per capita orders,and the dramatic increase in the number of the elderly make osteoporosis a difficult problem affecting social development and people’s quality of life.Osteoporosis is a common metabolic bone disease characterized by excessive loss of bone protein and mineral content leading to decreasing bone density and decreasing bone support.Nowdays,despite the prevalence of this disease,drugs to treat the disease are not ideal.Although estrogens,bisphosphonates,and dinosemide have been reported to be effective in osteoporosis,the associated adverse effects,such as thromboembolism,esophageal irritation,and osteonecrosis of the jaw,have limited the use of these drugs in the treatment of osteoporosis.We should spare no effort to explore advanced drugs which has less side effects and good efficacy.It is understood that in the imbalance of bone remodeling,osteoclasts are the main bone resorbing cells,while excessive enhancement of osteoclast activity is the primary cause of osteoporosis and is the culprit leading to destructive bone diseases such as osteoporosis.Osteoclasts originate from bone marrow hematopoietic stem cells and are the only multinucleated cells found in current studies with bone resorption function.In adulthood,on the one hand,osteoblasts continue to generate bone,and on the other hand,osteoclasts continue to resorb bone,and the two maintain balance,a process that is bone remodeling.Through bone remodeling,on the one hand,the relative stability of the skeletal system can be maintained in order to exert its support,protection,and motor functions,and on the other hand,the balance of the body can be maintained.In order to seek a safe and effective anti-osteoporosis drug,we screened 100FDA-approved drugs against osteoclast differentiation,from which we found that orlistat had the effect of increasing bone mass in patients to prevent osteoporosis,and found through preliminary experiments that the effect of orlistat in inhibiting osteoclast differentiation was not caused by the toxicity of the drug to the cells.However,studies on whether orlistat can actually be used to treat osteoclast-related diseases similar to osteoporosis are still blank,and the molecular mechanisms remain unclear.We hope to explore whether orlistat can inhibit RANKL-induced OC differentiation and production,and to explore the molecular biological mechanism behind this series of effects.The reserch methodFirst,the effect of orlistat on osteoclast function was studied at the in vitro level(1)the effect of orlistat on osteoclast proliferation ability in vitro was observed,and it was clarified that its inhibition of osteoclast differentiation was not caused by its potential toxicity;(2)at the same time point,different concentrations of orlistat were applied to treat osteoclasts in vitro to clarify the correlation between orlistat concentration and the degree of inhibition;(3)the same concentration of orlistat was applied on days 1,3,and 5 of osteoclast differentiation to clarify the time-dependent manner in which it inhibited osteoclast differentiation;(4)the effect of orlistat on osteoclast actin cyclization ability was observed to clarify its effect on osteoclast cytoskeletal structure;(5)bone resorption formation experimental methods were applied to observe the effect of orlistat on bone resorption activity in differentiated and mature OC.Second,the effect of orlistat on bone mass in OVX mice was studied at the in vivo level.(1)sham,OVX,and OVX + orlistat groups were set up,respectively,and micro CT was applied to observe the effect of orlistat on bone loss after 12 weeks of treatment;whether bone mass was affected was determined by vonkossa staining;(2)sham,OVX,and OVX + orlistat groups were set up,respectively,and TRAP staining of bone tissue was applied to observe the effect of orlistat on osteoclast bone resorption activity.(3)sham,OVX,and OVX + orlistat groups were set up to observe the effect of orlistat on osteoblast bone activity using Goldner staining of bone tissue.Third,to elucidate the molecular mechanism by which orlistat inhibits osteoclast differentiationResults1.The effect of orlistat on osteoclast function at the in vitro level2.(1)MTT colorimetric results showed that for both primary cells,the proliferation of cultured cells was not significantly inhibited at orlistat concentrations less than 50 u M,when orlistat did not show cytotoxicity against BMMs and RAW264.7.(2),the less TRAP-positive MNC monocytes were observed with orlistat concentration,indicating that orlistat inhibited osteoclastogenesis in a dose-dependent manner.(3),orlistat was added to osteoclast differentiation medium at the indicated time points.The results showed that the addition of orlistat at the initial stage of differentiation could maximize osteoclast formation,while the inhibitory effect of orlistat at the later stage was weak.(4)After orlistat intervention,a significant reduction in the area and number of actin bands in RANKL-induced BBMs cells was observed,which indicated that orlistat could inhibit the formation of actin rings in a concentration-dependent manner.(5),pit formation experiments showed that the SHAM group produced many large and deep resorption pits upon stimulation with RANKL however,the depth and area of bone pits on dentin slices were significantly reduced after orlistat intervention.3.The effect of orlistat on bone mass in OVX mice was studied at the in vivo level.(1)After 12 weeks of orlistat treatment,micro CT observation revealed that the levels of relevant parameters such as osteoclast surface/bone surface area(Oc S/BS)and eroded surface/bone surface(ES/BS)were reversed to some extent.(2)The results of bone immunohistochemical TRAP staining and ELISA detection of serum demonstrated that orlistat could inhibit osteoclast bone resorption activity to some extent.(3)No significant effect of orlistat on osteoblast bone activity was observed by Goldner staining of bone tissue.4.To elucidate the molecular mechanism by which orlistat inhibits osteoclast differentiation(1)Orlistat can inhibit NF-κB activation stimulated by RANKL though inhibiting the degradation of nuclear factor inhibitor protein.At the same time,we found that orlistat is abled to inhibit RANKL stimulating activation of connecting protein which join in this program named kinase,and this inhibition is achieved by inhibiting nuclear factor inhibitor protein degradation and phosphorylation of ERK,JNK and p38.This in turn suggests that orlistat acts on precursor cells to downregulate the expression of osteoclast-specific transcription factors induced by receptor activator of nuclear factor κB ligand,thereby reducing subsequent osteoclast differentiation.ConclusionResults of a prospective study showed that orlistat was able to inhibit osteoclastogenesis in vitro and in vivo by inhibiting NF-κB and MAPK signaling pathways,indicating that orlistat is a promising candidate for the treatment of osteoclastogenic diseases similar to postmenopausal osteoporosis.The results of this study may enrich the clinical spectrum of orlistat. |
PDF Full Text Request