Effect Of Aspirin On Osteogenic Differentiation Of Bone Marrow Stromal Cells In Ovariectomized Rats

With the increase in the average human life, more and more people entered the agingstate. Incidence of osteoporosis and that combined with fracture in women aftermenopause were higher, the incidence of male to female ratio was approximately to1:8.There were many types of drug using for treatment of osteoporosis, but there are manyproblems, such as the slow onset after taking medication, long course of treatment, sideeffects, high cost, and so on. In recent years, epidemiological survey found that long-termuse of low-dose aspirin can significantly improve bone mineral density of old people,especially of older women. This indicated that aspirin may have potentialanti-osteoporosis effect. Our previous study found that using rat osteoporosis model aftercastration, the oral administration of aspirin therapy can significantly improve the bone mineral density of rat trabecular and cortical bone. In normal human bone marrow stromalstem cells (bone marrow stem cell, BMSC)，aspirin can open the osteogenic genes andimprove mineral accumulation. The transplantation of mouse bone marrow stem cells intonude mice subcutaneously, the bone formation ability in aspirin group was significantlyhigher than that in the control group. Yet the molecular mechanism remains unclear.Aspirin was a centuries-old drug with fewer side effects. It is a very commonly used drugin the elderly people, but with no indication in the treatment of osteoporosis. Therefore, tounderstand the exact effect of aspirin on the prevention and treatment of osteoporosis,especially to uncover the molecular mechanism of this effect is of great important forproviding the feasibility of clinical treatment of postmenopausal osteoporosis using the olddrug—aspirin.Objective: To evaluate the effect of different concentrations of aspirin (Asp) onproliferation of bone marrow stromal cells (BMSCs) in ovariectomized（OVX）rats, andobserve the osteogenic differentiation by alkaline phosphatase (ALP) activities andcalcium nodules，and detection the OPG、RANKL gene and protein expression of BMSCs.Methods: Postmenopausal osteoporosis rat model was established. After12week,the bone marrow cells were extracted from the femur and tibia and single cell suspensionwas made and plated. BMSCs were cultured and passed, followed by osteogenic inductionof the BMSC. Six experimental groups were set up as the different concentrations ofaspirin group (0.25,0.5,1,2and5mmol/L) and the control group. At each time point, thecell growth was observed by the inverted microscope and the proliferation abilities weredetected by MTT. The osteogenic differentiation activities were analyzed by alkalinephosphatase (ALP) kit. The ALP staining and Alizarin red staining for calcified nodules ofthe induced BMSCs were performed at7d and21d of inducing and OPG、RANKL geneand protein expression of osteogenic cells were detected by RT-PCR and ELISA assay at14d of inducing, respectively. The experimental data were analyzed by software of SPSS13.0.Results: Aspirin cannot stimulate the proliferation ability of BMSCs of the OVXrats，yet5mmol/L of aspirin will inhibit the cell proliferation ability. In low-concentration aspirin groups (0.25，0.5,1,2mmol/L aspirin), ALP activities were significantly increasedthan the control group at5,7,9,12,14days (P<0.05). Yet with time, ALP activities weredownward trend overall. ALP staining show that positive reaction of active osteoblasts, asmall amount of brown granule in the cytoplasm and nucleus were bluish violet that inaspirin groups(1、2mmol/L) were significantly stronger（P<0.01）. The calcification areaby Alizarin red staining was significantly higher in0.5,1,2mmol/L aspirin groupscompare with the control group（P<0.05）, in which1mmol/L aspirin group was the mostobvious（P<0.01）. Compared with the control group, In low-concentration aspirin groups(0.25，0.5,1,2mmol/L aspirin) the expression of OPG gene and protein were significantlyhigher, the expression of RANKL gene and protein were significantly lower,and OPG/RANKL ratio were significantly higher (P <0.05).Conclusion: Aspirin cannot promote the proliferation ability of BMSCs directly, butlow-dose of aspirin can significantly improve the osteoblastic activity of BMSCs, promotethe formation of calcium nodules and enhance calcium deposition in ovariectomized ratsin vitro. Aspirin can upward the OPG/RANKL ratio, which may impact on the OPG/RANKL/RANK axis system to adjust the dynamic balance between bone resorption andbone formation, which may be the fundamental evidence for the novel indication ofaspirin–clinical application for prevention and treatment of postmenopausal osteoporosis.