The Effect Of Aspirin On Differentiation Or Maturation Of Rat Osteoclasts And On The Gene Expression Of Rank MRNA

BackgroundOsteoclasts and osteoblasts regulate bone resorption and formation to allow boneremodelling and homeostasis. The balance between bone resorption and formation isdisturbed by abnormal recruitment of osteoclasts. Osteoclast differentiation is dependenton the receptor activator of nuclear factor NF-kappa B (RANK; also called TNFRSF11A)ligand (RANKL) as well as the macrophage colony-stimulating factor (M-CSF). TheOPG/RANKL/RANK system and RANK signalling induce osteoclast formation mediatedby various cytokines. The RANK/RANKL pathway has been primarily implicated inmetabolic, degenerative and neoplastic bone disorders or osteolysis. The central role ofOPG/RANK/RANKL interaction in osteoclastogenesis makes RANK an attractive targetfor potential therapies in treatment of osteoporosis.The earlier research of our group shows that low concentration of aspirin treatment can effectively increase the ovariectomized rat bone density, improve the mechanicalstrength of bone, improve three-dimensional structure of trabecular bone and promote thetrabecular bone remodeling. Low and medium concentration of aspirin stimulate theosteogenic differentiation of BMSCs, indicating that aspirin had the ability to enhance thebone metabolism activity. However, the prevention and treatment effect and its mechanismof aspirin on osteoporosis remain unclear.ObjectiveThe purpose of this study was to assess the effect of aspirin inhibition of RANKexpression in rat osteoclasts in vitro on differentiation of osteoclasts and activity of boneresorption, to explore the possible molecular mechanism of aspirin for the prevention andtreatment of postmenopausal osteoporosis, and to aspirin innovation applied to theprevention and treatment of postmenopausal osteoporosis to provide a new theoreticalbasis.Methods(1) Rat myelomonocytes were cultured on glass coverslips and bone slices withsoluble receptor activator of NF-κB ligand (sRANKL) and macrophage colony stimulatingfactor (M-CSF).(2) Estrogen (10-6mmol/L) and aspirin (0.25mmol/L,0.5mmol/L,1.0mmol/L, and1.5mmol/L) were added to the cultures.(3) The tartrate-resistant acidphosphatase (TRAP) staining was performed to observe cell morphology and staining, andcount the number of osteoclasts.(4) The absorption lacuna in bone slices were observedwith optical microscope and scanning electron microscope (SEM), and count the area ofbone resorption lacunae.(5) The expression of RANK mRNA was determined by reversetranscription-polymerase chain reaction (RT-PCR).(6) Protein level of RANK wasmeasured using enzyme-linked immunosorbent assay (ELISA).Results(1) Compared with the normal control group, the number of osteoclasts and pit areasin estrogen group and aspirin groups are lower than that of the normal control group, thedifference is statistically significant (P <0.05). With the increase of aspirin concentration,the number of TRAP-positive multinucleated osteoclasts and lacunae resorption pits’ area are gradually reduced, and the difference is statistically significant within these groups (P<0.05). Compared to the estrogen group, low concentration of aspirin group (0.25mmol/L) has no significant difference, while the number of TRAP-positive multinucleatedosteoclasts and the areas of lacunae resorption pits are gradually decreasing in the otherconcentrations of aspirin groups (0.5mmol/L,1mmol/L,1.5mmol/L), the difference isstatistically significant (P <0.05).(2) The result of RT-PCR shows that compared with the normal control group, theRANK mRNA expression in osteoclasts of estrogen group and each aspirin group(0.5mmol/L,1mmol/L, and1.5mmol/L) are both reduced, and with increasing aspirinconcentration, it is gradually reduced in a dose-dependent manner (P <0.05).(3) The result of ELISA shows that compared with the normal control group, proteinlevel of RANK in osteoclasts of estrogen group and each aspirin group (0.5mmol/L,1mmol/L, and1.5mmol/L) are both reduced, and with increasing aspirin concentration, itis gradually reduced in a dose-dependent manner (P <0.05).ConclusionsAspirin can suppress the RANK mRNA expression of rat osteoclast in vivo cellculture system. It can also inhibit the differentiation and maturation of osteoclast, and as aconsequence decrease the bone resorption activity in a dose-dependent manner. This maybe the key molecular mechanism for the anti-osteoporosis capacity of aspirin throughinhibiting the osteoclast activity, which may provide the feasibility of clinical treatment ofpostmenopausal osteoporosis using the old drug—aspirin—for a totally novel clinicalindication.