葛根素通过ERK1/2和p38 MAPK信号通路刺激成骨分化和骨形成的机制
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摘要
目的探究葛根素通过ERK1/2和p38 MAPK信号通路刺激成骨分化和骨形成的机制。方法分离培养成人成骨细胞(MC3T3-E1),运用MTT法对比加入不同浓度葛根素后细胞增殖能力和对生长曲线的影响。通过测定碱性磷酸酶活性检测葛根素对成骨细胞分化的影响。通过测定钙沉积量分析葛根素对骨形成的影响。通过ERKl/2阻断剂PD8089,p38抑制剂SB203580的加入分析ERK1/2和p38 MAPK信号通路刺激成骨分化和骨形成的机制。利用蛋白质免疫印迹(WB)检测细胞骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)的表达。结果不同浓度葛根素干预成骨细胞,可以不同程度上促进其增殖,1μmol/L葛根素组效果最明显。第1天和第3天较空白组增殖趋势不明显,第5天和第7天较空白组有明显差异。葛根素可以激活碱性磷酸酶活性,初级成骨细胞分化。葛根素可以促进钙的沉积,刺激骨形成。使用ERK1/2阻断剂PD8089后,或用抑制剂SB203580阻断p38 MAPK信号通路后,细胞增殖、碱性磷酸酶含量以及钙沉积量均较葛根素组有所下降。葛根素组(T group) BMP-2表达高于对照组(P <0. 05);葛根素+PD8089组(T+PD group)低于T组高于对照组;葛根素+SB203580组(T+SB group)钙沉积量较T组明显降低(P<0. 05),与对照组BMP-2表达量相比亦降低(P<0. 05)。结论葛根素在骨细胞周期过程中,ERK1/2和p38 MAPK信号通路对骨分化和骨形成起到了调控作用。
Objective To explore the mechanism by which puerarin stimulates osteogenesis and bone formation through the ERK1/2 and p38 MAPK signaling pathways. Methods Adult osteoblasts( MC3 T3-E1) cell culture was used in this study.The proliferative ability and the influence of growth curve of the cells after added different concentrations of puerarin were compared by MTT staining. The effect of puerarin on the differentiation of osteoblasts was assessed bymeasuring alkaline phosphatase activity and the effect of puerarin on bone formation was analyzed by measuring calcium deposition. Through the addition of ERK1/2 blocker PD8089 and p38 inhibitor SB203580,the mechanisms of involvement of the ERK1/2 and p38 MAPK signaling pathways in stimulating osteogenesis and bone formation were analyzed. Western blotting was used to detect the expression of bone morphogenetic protein-2( BMP-2). Results Different concentrations of puerarin promoted the proliferation of osteoblasts to different degrees. The effect of puerarin at 0. 1 mol/L was the most significant. The trend of proliferation was not pronounced on the first and third days compared with the level in the blank group,but significant differences emerged between the fifth and seventh days. Puerarin activated alkaline phosphatase activity and promoted the differentiation of primary osteoblasts. It also promoted calcium deposition and stimulated bone formation. After using the ERK1/2 blocker PD8089 or blocking the p38 MAPK signaling pathway with the inhibitor SB203580,cell proliferation,alkaline phosphatase content,and calcium deposition were lower than those of the puerarin group. The expression of BMP-2 in group puerarin( T) was higher than that of the control group( P<0. 05),and that of group puerarin+PD 8089( T + PD) was lower than that of group T,while the amount of calcium deposition in the group puerarin+SB 203580( T+SB) was significantly lower than that in group T( P<0. 05),and there was a decrease( P<0. 05)in the BMP-2 expression compared with that in the control group. Conclusions In the cell cycle in bone,puerarin and the ERK1/2 and p38 MAPK signaling pathways play regulatory roles in bone differentiation and bone formation.
Objective To explore the mechanism by which puerarin stimulates osteogenesis and bone formation through the ERK1/2 and p38 MAPK signaling pathways. Methods Adult osteoblasts( MC3 T3-E1) cell culture was used in this study.The proliferative ability and the influence of growth curve of the cells after added different concentrations of puerarin were compared by MTT staining. The effect of puerarin on the differentiation of osteoblasts was assessed bymeasuring alkaline phosphatase activity and the effect of puerarin on bone formation was analyzed by measuring calcium deposition. Through the addition of ERK1/2 blocker PD8089 and p38 inhibitor SB203580,the mechanisms of involvement of the ERK1/2 and p38 MAPK signaling pathways in stimulating osteogenesis and bone formation were analyzed. Western blotting was used to detect the expression of bone morphogenetic protein-2( BMP-2). Results Different concentrations of puerarin promoted the proliferation of osteoblasts to different degrees. The effect of puerarin at 0. 1 mol/L was the most significant. The trend of proliferation was not pronounced on the first and third days compared with the level in the blank group,but significant differences emerged between the fifth and seventh days. Puerarin activated alkaline phosphatase activity and promoted the differentiation of primary osteoblasts. It also promoted calcium deposition and stimulated bone formation. After using the ERK1/2 blocker PD8089 or blocking the p38 MAPK signaling pathway with the inhibitor SB203580,cell proliferation,alkaline phosphatase content,and calcium deposition were lower than those of the puerarin group. The expression of BMP-2 in group puerarin( T) was higher than that of the control group( P<0. 05),and that of group puerarin+PD 8089( T + PD) was lower than that of group T,while the amount of calcium deposition in the group puerarin+SB 203580( T+SB) was significantly lower than that in group T( P<0. 05),and there was a decrease( P<0. 05)in the BMP-2 expression compared with that in the control group. Conclusions In the cell cycle in bone,puerarin and the ERK1/2 and p38 MAPK signaling pathways play regulatory roles in bone differentiation and bone formation.
引文
[1] Yang B,Du S,Lu Y,et al.Influence of paeoniflorin and menthol on puerarin transport across MDCK and MDCK-MDR1 cells as blood-brain barrier in vitro model[J].J Pharm Pharmacol,2018,70(3):349-360.
[2] Lv H,Che T,Tang X,et al. Puerarin enhances proliferation and osteoblastic differentiation of human bone marrow stromal cells via a nitric oxide/cyclic guanosine monophosphate signaling pathway[J]. Mol Med Rep,2015,12(2):2283-2290.
[3] Yuan Y,Wu ZJ,Yao HY,et al. Activation of p38 mitogenactivated protein kinase contribute to BMP4-induced alkaline phosphatase expression in MC3T3-E1 preosteoblast[J]. Chin Med J,2006,119(4):324-327.
[4] Zhang Y,Yan M,Yu QF,et al.Puerarin prevents LPS-induced osteoclast formation and bone loss via inhibition of akt activation[J].Biol Pharm Bull,2016,39(12):2028-2035.
[5] Lee HW,Suh JH,Kim HN,et al. Berberine promotes osteoblast differentiation by Runx2 activation with p38 MAPK[J]. J Bone Miner Res,2008,23(8):1227-1237.
[6] Choi SC,Kim SJ,Choi JH,et al. Fibmblast growth factor-2and-4 promote the proliferation of bone marrow mesenchymal stem cells by the activation of the P13K-Akt and ERKl/2 signaling pathways[J].Stem Cells Dev,2008,17(4):725-736.
[7] Liao XB,Zhou XM,Li JM,et al. Taurine inhibits osteoblastic difierentiation of vascular smooth muscle cells via the ERK pathway[J].Amino Acids,2008,34(4):525-530.
[8]王文,任玲,工健楠.MAPK信号通路与细胞凋亡的关系[J].中国实用医药,2010,5(15):260-261.
[9]张曼.血管内皮细胞的ERK信号转导通路调节机制研究[J].中国实用医药,2009,4(25):250-252.
[10] Tian F,Xu LH,Wang B,et al.The neuroprotective mechanism of puerarin in the treatment of acute spinal ischemia-reperfusion injury is linked to cyclin-dependent kinase 5[J]. Neurosci Lett,2015,584(1):50-55.
[11] Tian F1,Xu LH,Zhao W,et al. The neuroprotective mechanism of puerarin treatment of acute spinal cord injury in rats[J].Neurosci Lett,2013,543(5):64-68.
[12] Xu H,Zhao M,Liang S,et al. The Effects of puerarin on rat ventricular myocytes and the potential mechanism[J]. Sci Rep,2016,6(10):35475.
[13] Deng Y,Lei T,Li H,et al.ERK5/KLF2 activation is involved in the reducing effects of puerarin on monocyte adhesion to endothelial cells and atherosclerotic lesion in apolipoprotein Edeficient mice[J]. Biochim Biophys Acta,2018,1864(8):2590-2599.
[14] Wan Q,Liu Z,Yang Y.Puerarin inhibits vascular smooth muscle cells proliferation induced by fine particulate matter via suppressing of the p38 MAPK signaling pathway[J]. BMC Complement Altern Med,2018,18(1):146.
[15] Shan Z,Cheng N,Huang R, et al. Puerarin promotes the proliferation and differentiation of MC3T3-E1 cells via microRNA-106b by targeting receptor activator of nuclear factor-κB ligand[J].Exp Ther Med,2018,15(1):55-60.
[2] Lv H,Che T,Tang X,et al. Puerarin enhances proliferation and osteoblastic differentiation of human bone marrow stromal cells via a nitric oxide/cyclic guanosine monophosphate signaling pathway[J]. Mol Med Rep,2015,12(2):2283-2290.
[3] Yuan Y,Wu ZJ,Yao HY,et al. Activation of p38 mitogenactivated protein kinase contribute to BMP4-induced alkaline phosphatase expression in MC3T3-E1 preosteoblast[J]. Chin Med J,2006,119(4):324-327.
[4] Zhang Y,Yan M,Yu QF,et al.Puerarin prevents LPS-induced osteoclast formation and bone loss via inhibition of akt activation[J].Biol Pharm Bull,2016,39(12):2028-2035.
[5] Lee HW,Suh JH,Kim HN,et al. Berberine promotes osteoblast differentiation by Runx2 activation with p38 MAPK[J]. J Bone Miner Res,2008,23(8):1227-1237.
[6] Choi SC,Kim SJ,Choi JH,et al. Fibmblast growth factor-2and-4 promote the proliferation of bone marrow mesenchymal stem cells by the activation of the P13K-Akt and ERKl/2 signaling pathways[J].Stem Cells Dev,2008,17(4):725-736.
[7] Liao XB,Zhou XM,Li JM,et al. Taurine inhibits osteoblastic difierentiation of vascular smooth muscle cells via the ERK pathway[J].Amino Acids,2008,34(4):525-530.
[8]王文,任玲,工健楠.MAPK信号通路与细胞凋亡的关系[J].中国实用医药,2010,5(15):260-261.
[9]张曼.血管内皮细胞的ERK信号转导通路调节机制研究[J].中国实用医药,2009,4(25):250-252.
[10] Tian F,Xu LH,Wang B,et al.The neuroprotective mechanism of puerarin in the treatment of acute spinal ischemia-reperfusion injury is linked to cyclin-dependent kinase 5[J]. Neurosci Lett,2015,584(1):50-55.
[11] Tian F1,Xu LH,Zhao W,et al. The neuroprotective mechanism of puerarin treatment of acute spinal cord injury in rats[J].Neurosci Lett,2013,543(5):64-68.
[12] Xu H,Zhao M,Liang S,et al. The Effects of puerarin on rat ventricular myocytes and the potential mechanism[J]. Sci Rep,2016,6(10):35475.
[13] Deng Y,Lei T,Li H,et al.ERK5/KLF2 activation is involved in the reducing effects of puerarin on monocyte adhesion to endothelial cells and atherosclerotic lesion in apolipoprotein Edeficient mice[J]. Biochim Biophys Acta,2018,1864(8):2590-2599.
[14] Wan Q,Liu Z,Yang Y.Puerarin inhibits vascular smooth muscle cells proliferation induced by fine particulate matter via suppressing of the p38 MAPK signaling pathway[J]. BMC Complement Altern Med,2018,18(1):146.
[15] Shan Z,Cheng N,Huang R, et al. Puerarin promotes the proliferation and differentiation of MC3T3-E1 cells via microRNA-106b by targeting receptor activator of nuclear factor-κB ligand[J].Exp Ther Med,2018,15(1):55-60.