Study On The Mechanism Of Doxorubicin Restrains Osteogenesis And Promotes Osteoclastogenesis In Vitro

Part I Study on the inhibition of bone marrow mesenchymal stem cell proliferation and osteogenesis by doxorubicinObjective Clinical evidence indicates that doxorubicin(DOX)as a chemotherapeutic agent can cause severe bone damage in cancer patients.In this study,BMSCs were treated with different concentrations of DOX(0～50nM)to clarify the effect of DOX on the proliferation and osteogenic differentiation,and to investigate the mechanism of doxorubicin affecting osteoblast differentiation.Methods Femoral whole bone marrow cells collected from femur of SD rats(6-8 weeks)under aseptic conditions,and BMSCs were isolated and purified by the whole adherent method.The extracted cells were further subcultured and cryopreserved.The surface markers were identified by flow cytometry.BMSCs were treated with different concentrations of DOX in osteogenic medium(OM).The effects of DOX on the proliferation of BMSCs were detected by cck-8 method and Live/Dead staining.The effects of DOX on osteogenic differentiation were detected by ALP staining,alizarin red staining and ALP activity.The expression of osteogenic specific mRNA was illuminated by qRT-PCR,and the expression of osteogenic specific protein was detected by Western blot and immunofluorescence.Results(1)DOX(10nmol/L)inhibits the ALP activity of BMSCs differentiation into osteoblasts(137.52±28.23 U/L in the DOX group and 572.55±44.69 U/L in the control group)and decreases the number of calcium nodules(absorbance:0.61±0.18 in the DOX group and 2.45 ± 0.06 in the control group).Moreover,the expression of osteogenic specific genes(ALP,col-1 and OCN)are decreased.The differences were statistically significant(P<0.05).(2)DOX can inhibit the expression of smadl/5/9,bmp-2,Osteorix,Runx2.The expressions of osteogenic differentiation gene increased following with adding bmp-2.(3)DOX can promote the proteinexpression of RANKL and inhibit the protein expression of OPG.Conclusion DOX can inhibit the proliferation and the differentiation into osteoblasts through the bmp-2/smads signaling pathway of BMSCS in vitro.Part Ⅱ Doxorubicin promoting the activation of bone marrow monocytes into osteoclastsObjective A recent study showed that bone density and bone mass decreased significantly in premenopausal breast cancer patients treated with DOX,suggesting that there is a causal relationship between the therapeutic dose of DOX and systemic bone loss.But the mechanism of differentiation of bone marrow mononuclear cells(BMMs)into osteoclasts is not clear.This study,BMMs were treated with different concentrations of DOX(0～10nM)to determine the effect on proliferation and osteoclast differentiation and the molecular mechanism of BMMs.Methods The extracted BMMs were isolated and purified by full adherence method.BMMs were directly or indirectly incubated in osteoclast induction medium containing different concentrations of DOX.The effects of DOX on the proliferation and activity of BMMs were detected by cck-8 method and Live/Dead staining.Multiple nuclei fusion of osteoclasts were labeled by tartrate-resistant acid phosphate(TRAP)staining.Osteofibroactin rings of osteoclasts were observed by immunofluorescence technique.Expressions of osteoclast-specific genes were detected by qRT-PCR,and expressions of specific osteoclast proteins were detected by WB.Results DOX can directly/indirectly promote the osteoclast formation(Direct:Number:DOX group:161.25±15.58,control group:105.26±25.79,Area ratio:DOX group:0.91±0.23,control group:0.65±0.11.Indirect:Number:DOX group:152.48±22.70,control group:105.21±21.33,Area ratio:DOX group:0.85±0.02,control group:0.55±0.11)and expression of osteoclast-related genes TRAP,NFATc1 and c-Fos.Conclusion DOX can promote BMMs to osteoclast differentiation in vitro.Part Ⅲ Shikonin protected against doxorubicin-induced bone loss in vitro and its mechanismObjective:Doxorubicin has many toxic side effects including bone destruction and bone loss.Therefore,there is a need for a synergistic therapeutic regimen that can both reduce the dose required for chemotherapy and improve doxorubicin-associated osteoporosis.In this study,BMSCs were treated with doxorubicin(10nM)and/or shikonin(250nM)to elucidate the effect of inhibiting BMSCS osteogenic differentiation and to explore the related molecular mechanisms.Methods:The experiment was divided into control group,DOX group,shikonin group and DOX+shikonin group.The effects of osteogenic differentiation were illuminated by ALP staining,alizarin red staining and ALP activity.Expressions of the specfic mRNA and proteins of osteogenic differentiation were illumianted by qRT-PCR and WB/immunofluorescence.Results:Shikonin can alleviate the inhibitory effect of DOX on osteogenic differentiation in vitro,characterized by ALP positive cells increased and calcium nodule formation(DOX group:0.65±0.21,DOX+shikonin group:1.66±0.73)and ALP activity increased(DOX group:210.451±22.08 U/L,DOX+shikonin group:385.92±88.29 U/L),and promote the expression of ALP,col-1 and the OCN.Compared with DOX group,the expression of related factors of Wnt/β-catenin signaling pathway were increased in DOX+shikonin group.Conclusion:Shionin may alleviate the inhibitory effect of DOX on differentiation of BMSCs into osteoblasts through the Wnt/β-catenin signaling pathway in vitro.