| ObjectiveAs the aging population becomes more and more serious,osteoporosis and its fractures have become a serious disease affecting the health of the elderly.The researchers found that anthocyanins have anti-osteoporosis effects,but it has been still unclear whether it can regulate osteoblasts that play an important role in the development of osteoporosis The mitogen-activated protein kinase(MAPK)pathway regulates the biological processes of osteoblast proliferation and differentiation.Cyanidin-3-O-Glucoside(C3G),a member of anthocyanins,regulates cells via the MAPK pathway in various cells such as RAW264.7 and smooth muscle cells.In this study,primary human osteoblasts and mouse osteoblast MC3T3-E1 cells were used to evaluate the role of C3G in the proliferation and differentiation of osteoblasts and the role of MAPK in C3G regulation of osteoblasts.MethodsIn this study,primary osteoblasts were extracted from the femoral head cancellous bone tissue from patients with osteoporosis undergoing artificial femoral head replacement after hip fracture by collagenase-hyaluronidase digestion.The obtained primary osteoblasts were subcultured and used until the third passage.The extracted cells were identified by alizarin red staining after induction of mineralization.Mouse osteoblast MC3T3-E1 was obtained from the Chinese Academy of Sciences cell bank.After treatment of two kinds of osteoblasts with different concentrations of C3G(0-400?mol/L)for 24h,48h and 72h,the cell proliferation rate was evaluated by MTT colorimetry.MC3T3-E1 cells were treated with different concentrations of C3G(0-200?mol/L)for 48h,and the cell growth was evaluated by real-time cell analysis(RTCA).Two osteoblasts were pretreated with ERK1/2 inhibitor PD98059,JNK inhibitor SP600125 and P38 inhibitor SB202192 for 4 h.Then,the cell proliferation rate was calculated after treatment of the two osteoblasts with C3G at a concentration of 0?mol/L or 100 ?mol/L for 24 h.Human primary osteoblasts were treated with different concentrations of C3G(0-200 ?mol/L)for 7 days,and the density of mineralized nodules was evaluated after alizarin red staining.MC3T3-E1 cells were treated with different concentrations of C3G(0-200 ?mol/L)for 24 h,and OC and CTX-I levels were determined by ELISA,and ALP activity was determined by colorimetry.MC3T3-El cells were treated with C3G at a concentration of 0?mol/L or 100?mol/L for 24h.The expression levels of ALP,Runx2 and BMP2 were determined by real-time PCR.After treatment with the inhibitor pretreatment scheme and the C3G treatment protocol as described above,the expression levels of OC,RSK2,ATF4,ATF2,OSX,FOXO1,and DLX5 were determined by real-time PCR.The abundance and phosphorylation levels of ERK1/2,JNK and P38 were determined by Western Blot.Results1.Isolation,culture and identification of human osteoblastsThe cancellous bone of the femoral head is digested with collagenase-hyaluronidase to obtain osteoblasts.The fusiform primary osteoblasts adhered to the wall 24 hours later and showed a tendency to aggregate growth in some areas.It took about 7-14 days for the cells to climb the bottom of the bottle.It took 7 days for these long fusiform or polygonal cells to spread the bottom of the flask.These cells were rich in cytoplasm and have only one nucleus.At low magnification,the cells exhibited"turbo"growth.After induction of mineralization,alizarin red staining was positive.2.Effect of C3G treatment on proliferation of MC3T3-E1 cells and human osteoblastsThe MTT results showed that the proliferation rate of both cells increased after C3G treatment,and the C3G concentration group was significantly different from the control group(P<0.05),As time went on,the gap between the C3G concentration groups and the control group gradually decreased.At 72h,the difference between 25?mol/L or 50?mol/L C3G concentration group and control group disappeared(P>0.05),but 200?mol/L and 400?/L still had a certain enhancement effect on osteoblast proliferation compared with control group(P<0.05),RTCA results showed that C3G increased the cell index of MC3T3-E1 cells,and the effect of C3G treatment was obvious at 24h.3.Effect of C3G on the proliferation of osteoblasts after pretreatment with MAPK signaling pathway inhibitorThe cell proliferation rate was significantly increased in the DMSO-C3G group compared with the DMSO-control group(P<0.001).However,after pretreatment with PD98059,SB202192 or SP600125,the cell proliferation rate of each inhibitor-C3G group was still significantly higher than that of the inhibitor-control group(P<0.001).4.Effect of C3G treatment on mineralization ability of human osteoblastsAfter treatment of human primary osteoblasts by C3G for 7 days,it produced more calcified nodules than the control group.The results showed that cells of the 100 ?mol/L or 200?/L group were observed to have more structure stained with alizarin red dye under the microscope than the control group.5.Effect of C3G treatment on ALP,OC and CTX-1 in MC3T3-E1 cellsAfter 24 hours of C3G treatment,the intracellular ALP activity or CTX-1 expression level decreased slightly with the increase of C3G concentration,but the difference was not statistically significant(P>0.05),Compared with the control group,the expression level of OC in each group treated with C3G was significantly increased(P<0.05).6.Effect of C3G treatment on ALP,BMP-2 and Runx2 mRNA expression in MC3T3-E1 cellsThe intracellular ALP mRNA levels were significantly decreased in each group treated with C3G compared with the control group,and the fold change(FC)was 0.55(P<0.05).There was no significant change in BMP-2 mRNA levels,FC=1.05(P=0.86),then Runx2 mRNA levels were significantly decreased,FC=0.35(P<0.05).7.Effect of C3G on the expression of OC,RSK2,ATF4,ATF2,OSX,FOXO1 and DLX5 mRNA in MC3T3-E1 cells after inhibitor pretreatmentThe differences in gene expression between the DMOS pretreatment groups(DMSO-control group and DMSO-C3G group)were:ATF4(FC=1.15,P<0.05),OC(FC=1.73,P<0.05),DLX5.(FC=1.26,P<0.01)and FOX 01(FC=1.30,P<0.05).There was no significant difference in mRNA expression levels between OC(FC=0.99,P=0.93)and ATF4(FC=1.05,P?0.70)in the C3G group compared with the inhibitor-control group after PD98059 pretreatment.The inhibitor-control group showed a significant increase in RSK2 mRNA levels compared with the DMSO-control group(FC= 1.90,P<0.01),and this change was not observed in the C3G group compared with DMSO-control group.After pretreatment with SB202192,the expression levels of OSX(FC=2.11,P<0.01)and DLX5(FC= 1.32,P<0.01)were increased in the C3G group compared with the inhibitor-control group.After pretreatment with SP6001 25,there was no statistically significant difference in the mRNA levels of FOX01(FC=1.31,P=0.13)and ATF2(FC= 1.30,P=0.52)in the C3G group compared with the inhibitor-control group.8.Regulation of MAPK pathway protein expression and phosphorylation level in MC3T3-E1 cells by C3GThe phosphorylation level of ERK1/2 increased after C3G treatment(P<0.05),and the phosphorylation levels of JNK(P=0.27)and P38(P=0.46)did not change significantly.There was no significant change in the total ERK1/2(P=0.08),JKN(P=0.37),and P38(P=0.64)levels.Conclusions1.C3G promotes proliferation of human primary osteoblasts and MC3T3-E1 cells in vitro.2.C3G reduces the expression of Runx2 and ALP,a marker protein in the early stage of osteoblast differentiation.Moreover,C3G enhances the expression of OC,a marker protein in the late stage of osteoblast differentiation and enhances the mineralization ability of osteoblasts.3.C3G may regulate osteoblast differentiation through the ERK1/2 pathway... |
PDF Full Text Request