Suppressing By Isopsoralen Of Bone Marrow Adiposity And Attenuating Of The Adipogenic Commitment Of Bone Mesenchymal Stem Cells

Background:Osteoporosis which leads to the increasing risk of bone fracture and any other complications has become a major clinical problem, affecting over200million people worldwide.The current strategy for treating osteoporosis aim at bone resorption regulated by osteoclasts and bone formation regulated by osteoblasts, such as teriparatide, strontium, bisphosphonates. Since these synthetic medicines show several side effects that reduce their efficacy, for instance, osteonecrosis of the jaw bone induced by large doses of bisphosphonates, more healthier and safer agents active in preventingor/and treating osteopenic disorders are highly anticipated.Studies have indicated that the decreasing bone volume in osteoporosis bone is associated with the increasing adipose tissue in bone marrow. Osteoblasts and marrow adipocytes are both differentiated from a common precursor: bone marrow mesenchymal stem cells (BMSCs). It has been revealed that, comparing with the osteogenic capability of the mesenchymal stem cells (MSCs) derived from healthy women that of postmenopausal women with osteoporosis is much lower.Evidences have indicated a large degree of plasticity of osteoblasts and adipocytes that fully differentiated osteoblats from hMSCs were capable of dedifferentiation and transdifferentiation into adipocytes and vice versa. There is a reciprocal relationship and balance between the differentiation of adipocytes and osteoblasts.Most previous studies suggest bone marrow adipose purely play the role of filling the marrow cavity, lacking of hematopoietic function. However Elbaz et al have shown a lipotoxic effect from marrow adipocytes on osteoblast differetion and function, and the free fatty acids released by the adipocytes can inhibit osteoblasts proliferation and induced osteoblasts apoptosis.The existing results demonstrate excess bone marrow adipocytes are considered to be a significant negative risk factor for skeleton health. Accordingly, Inhibition of adipocytes differentiation in bone marrow meanwhile accelerating osteogenesis may be a therapeutic approach for age-related osteoporosis.The fruit of Psoralea corylifolia L.is a widely used Chinese herbal medicine, called Buguzhi meaning "medicine for strengthening bone" in China, has a special effect in treating fractures, bone and joint diseases. It is also used in many other kinds of disorders such as skin diseases, cardiovascular diseases, tumor, asthma and so on.Isopsoralen is the main active ingredient extracted from the seeds of P. corylifolia L as Psoralen which is its isomer. There have been lots of studies proposing the positive role of psoralen in promoting osteoblasts differention and stimulatory effect on bone formation.However, only a few researches have indicated the beneficial effect of Isopsoralen for the development of bone, without any mention of the underlying mechanisms of this action.In addition, to the best of our knowledge, there are no reports involving the roles and mechanisms of Isopsoralen on bone marrow adipogenesis. In the present study, by employing osteoporotic mice models induced by ovariectomy (OVX) with Isopsoralen treatment, we aimed to observe the effect of Isopsoralen on bone marrow adipogenesis in vitro. Furthermore, we evaluate the effect of Isopsoralen on the differentiation of osteoblasts and adipocytes deprived from BMSCs of C57/BL6mice in vitro under the osteogenic and adipogenic conditions respectively, and clarify the proboble underlying cellular and molecular mechanism during this period, which may give rise to a novel approach for curing osteopenic disorders.Objective:To study the effect of Isopsoralen on differentiation to adipocyte and osteoblast of bone marrow stromal cell from mice, and the expressing of runt-related transcription factor2(RUNX2), peroxisome proliferator-activated receptor γ(PPAR-γ),4E/BP1(Thr37/46) and P-S6(S235/236) on the influence of Isopsoralen; moreover, to explorethe effect of Isopsoralen on bone metabolism and the mechanism of anti-osteoporosis; finally, using ananimal model to proveour point of view.Methods:1. Materials and reagentsIsopsoralen (ISO) was obtained from the company of Sigma (molecular weight,186.1635) and the purity of this compound is more than99%. Stock solutions of ISO were prepared in dimethyl sulfoxide (DMSO) and stored at-20℃.Two-month-old female C57/BL6mice (n=18) were provided by the Experimental Animal Center of Southern Medical University. The mice were randomly divided into sham, OVX and OVX+ISO (n=6per group) groups. The mice in OVX+ISO group were administered intragastrically with ISO at dose of20mg/kg per day for5days before OVX and were maintained for2months after OVX. The mice in sham groups had removal of some fat tissue around the ovaries.2. Cell cultureThe BMSCs were isolated from C57/BL6mice (4weeks) as previously reported with modification. Mice were killed by CO2, then femora and tibias were cut aseptically, and the whole bone marrow was flushed out with a-Minimum Essential Medium (a-MEM), supplemented with10%fetal bovine serum (FBS) and antibiotics (100U/ml of penicillin-streptomycin). Cell culture medium was replaced every3days. The cells were seeded in96-well plates and6-well plates at a density of1×104and1×106cells/well, respectively, and cultured in a humidified atmosphere of5%CO2and95%air at37℃.For osteogenic differentiation, BMSCs were grown to90-100%confluence in6-well-plates and the culture medium was then replaced with osteogenic medium (a-MEM supplemented with15%FCS plus1%Penicillin/Streptomycin,100nmol/L dexamethasone,50μg/ml ascorbate-2-phosphate, and10mmol/L beta-glycerol phosphate). The medium was changed every3days.For adipocyte-induced culture, putative BMSC lines were harvested and seeded in6-well cell culture plates. On attaining90%confluency, the cells were treated with adipogenic differentiation medium: BMSCs maintenance medium supplemented with1μmol/L dexamethasone,100μmol/L indomethacin, and500μmol/L1-methyl-3-isobutylxanthine (IBMX),5μg/mL bovine insulin (USV). For the initial induction,500μmol/L IBMX was added together with the adipogenic differentiation medium. The medium was changed every4th day.3. Cell proliferation assayPrimary BMSCs were seeded in96-well plates at a density of1×104cells/well. After2-day culture, cells were treated with ISO at concentrations of0μmol/L,0.1μ/L,1μmol/L,10μmol/L,100μmol/L and1000μmol/L for48h. Cell proliferation assay by using a Caspase-8Colorimetric Assay Kit (KeyGEN Biotech, China), according to the manufacturer’s protocol. Absorbance in wells was measured at450nm.4. ALP staining assay The BMSCs were cultured in the presence of osteogenic inducers and ISO (0μmol/L,5μmol/L,10μmol/L, and20μmol/L) for14days. ALP activity was also evaluated in cells stained using an ALP staining kit (Beyotime, China), according to the manufacturer’s protocol. As for ALP staining, the cells were washed in PBS, then fixed in4%paraformaldehyde at indoor temperature for20minutes, and rinsed in distilled water. ALP staining mixture was added for30minutes at RT and under the conditions of protection from light. The cells were rinsed in distilled water and PBS to reduce nonspecific staining.5. Oil Red O StainingThe BMSCs were cultured in the presence of adipogenic inducers and ISO (0μmol/L,5μmol/L,10μmol/L, and20μmol/L) for7and14days. Fat droplets formed in differentiated adipocytes from BMSCs were observed by Oil Red O staining. Cells were fixed in4%formaldehyde for15min, washed in PBS, and stained with a0.6%(w/v) oil red O solution (60%isopropanol,40%water) for1hour at37℃. Cells were then washed with PBS to remove unbound dye and then1mL of isopropyl alcohol was added to the culture plates.6. Western blot analysisWestern blotting was performed with a SDS-PAGE Electrophoresis System.20ug protein samples were resuspended in a reduced sample buffer, and then electrophoresed on a7.5～10%Tris gel with Tris running buffer, blotted to PVDF membrane, and sequentially probed with primary antibodies against RUNX2,4E/BP1(Thr37/46), P-S6(S235/S236), PPAR-y and β-actin. Horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies were then added and secondary antibodies were detected using enhanced chemiluminescence.7. Microcomputed Tomography (μCT) AnalysesMicro-CT (Scanco Medical; μCT80) was performed on distal portion of femur, mice were sacrificed by CO2. The distal femur was selected for scanning and was corrected for the CT value, a70kV scanning voltage,30W power,429μA current, and5μm scan thickness. Our analysis included various bone parameters:trabecular thickness (Tb.Th); trabecular separation (Tb.Sp), bone volume (BV)/total volume (TV) and trabecular number (Tb.N).8. Immunofluorescence and microscopyBMSCs were grown on collagen-coated coverslips, washed with PBS, fixed in4%paraformaldehyde at room temperature for15min, washed, permeabilized with0.1%TritonX100(5min, RT) and blocked with blocking buffer (5%FBS in PBS) for30min. The cells were sequentially probed with primary antibodies against RUNX2, PPAR-y then washed with PBS for3times. After this, FITC-conjugated sheep anti-rabbit IgG and TRITC-conjugated sheep anti-mouse IgG were then added and incubated for1h at RT, then flushed for3times with PBS. After that the cells were incubated with DAPI (1μg/mL) for15min, rinsed with PBS for3times per5min. Immunofluorescence was performed on femur histology sections and using a standard protocol. Cells and femur histology were imaged with a laser-scanning confocal microscopy (Olympus; FV1000).9. Bone marrow adiposity analysisFor bone marrow adiposity analysis, distal portion of femurs were fixed, decalcified, and sectioned to2μm and subjected to hematoxylin and eosin staining following standard histology protocols. To quantify proximal metaphyseal adipocyte parameters: adipocyte number (AD#, per mm2), percent adipocyte volume per tissue volume (AV/TV). A uniform number of fields were screened in all sections by three individuals, starting three fields from the left end and three fields from the top endocortical surface, excluding the adipocytes with disruption in the fields. To avoid any bias in the final analysis, all sections were read in a blinded way without knowledge of the groups (Sham, CON or CON+ISO). Pictures were all obtained at20×magnifications using a stereomicroscope.10. Statistical analysisWe used One-way analysis of variance. A homogeneity of variance test was first used to check the data homogeneity of variance by IBM SPSS Statistics21.0software. Once the variance was confirmed as equal, then the Least-significant difference test was used for data analysis. If the variance was confirmed as unequal, then the Dunnett’sT3test was used for data analysis. The results are presented as mean±SD and P<0.05were considered statistically significant.Results:1. Cell proliferationWe measured the effect of ISO on cell proliferation of primary mouse BMSCs by CCK8assay. As shown in papper, ISO did not have significant effects on cell growth at the concentrations of1～100μmol/L after48-h treatment in primary BMSCs.2. ALP staining in vitroTo determine if ISO has effects on BMSCs differentiated into osteoblasts, the ALP staining was performed. Since an increase of ALP activity is an important indicator of osteoblast differentiation. We found that ISO enhanced the ALP activity dose-dependently in primary BMSCs and the most significant effects was at the concentration of20μmol/L at2week of osteogenic induction. These results suggest that ISO significantly stimulates BMSCs differentiated into osteoblasts.3. Oil red O in vitroISO inhibited the adipocytic differentiation of BMSCs induced by adipogenic inducers in a concentration-dependent manner. As shown in papper, there were less lipid droplets that appeared in the cytoplasm of adipocytes and stained with oil red O in the cells treated with ISO adipocytic differentiation at7days. After the2weeks adipocytic differentiation, adipogenesis rate was on the minimum level in the presence of20μmol/L ISO compared to control. Rough speaking, these appearances indicated that ISO inhibited BMSCs differentiated into adipocytes.4. Micro-CTTwo months after the surgical operation, BV was observed significant decrease. ISO treatment could be significantly rescued this bone loss. The values for Tb.Th and Tb.Sp in the OVX group were dramatically decreased or increased compared with those in the sham group. After ISO treatment for2months, these two indicators were significantly improved.5. Bone histomorphometriesRepresentative HE images of bone in the OVX group and the OVX+ISO group indicated that OVX caused a loss in the number of bone trabeculae, whereas ISO reduced this damage. Even more important, the OVX+ISO group contained less adipocytes compared with OVX group. Thus, we assumed that ISO significantly inhibited this bone loss.6. Immunofluorescence of runt-related transcription factor2(Runx2) in vivo Runx2is very important for bone remodeling, functions by expressing the differentiation of osteoblasts. We found that Runx2appeared within osteoblasts along the surface of bone lacuna, and the intensity of positive staining for Runx2was reduced in the OVX group, and it was significantly elevated in the OVX+ISO group, of course that statistical difference was existed.7. Immunofluorescence of peroxisome proliferator-activated receptor gamma (PPAR-y) in vivoA significant increasing of PPAR-y was found in the OVX+ISO groups compared with the OVX group. The majority of cells that surrounded the bone surface demonstrated positivity for PPAR-y in the OVX and OVX+ISO group and traditionally PPAR-γ were indispensable for adipocytes differentiation.8. Western Blot of RUNX2, PPAR-y, P-S6(S235/236),4E/BP1(Thr37/46) in vitro To determine if ISO has effects on BMSCs differentiation, the Western Blot assay performed. ISO promoted BMSCs differentiation in a dose-dependent manner, demonstrated by the up-regulation of osteoblast-specific marker RUNX2. The RUNX2activity was found to be increased in the cells treated with ISO at14days. ISO had the most significant effects at the concentration of20μmol/L.One week after adipogenic induction, the PPAR-y activity was found to be decreased in the cells treated with ISO in a dose-dependent manner. We also found that the expressions of PPAR-y were dramatically decreased in the presence of ISO at day14in a dose-dependent manner and the most minimum expression was also at the concentration of20μmol/L of ISO.To further determine if ISO-induced BMSCs differentiated into osteoblasts and inhibits BMSCs differentiated into adipocytes was dependent on its inhibition of mTORs signaling, we measured the expression of P-S6(S235/236),4E/BP1(Thr37/46) in vitro. We found that P-S6(S235/236) was decreased and4E/BP1(Thr37/46) went up at14days of osteogenic induction. More importantly, P-S6(S235/236) falling and4E/BP1(Thr37/46) up-regulation was observed after7days and2weeks of adipocytic differentiation. The above results were reflected in a dose-dependent manner.Conclusion:the present study showed that Isopsoralen could attenuate bone marrow adipogenesis which is dependent on the increasing the level of RUNX2and the reducing level of PPAR-y at tissue and cellular levels, leading to shifting the lineage differentiation of BMSCs towards osteoblasts instead of adipocytes, moreover, we suggest that mTORCl signaling may partly be the underlying specific signaling pathway mechanism in this process.