| PARTⅠSalvia miltiorrhiza promotes osteogenic differentiation and proliferation of MC3T3-E1 preosteoblast by the ERK pathwayObjective:To study whether or not Salvia miltiorrhiza(SM)can promote osteogenic differentiation and proliferation of MC3T3-E1 preosteoblast,and examine the relationship between the mechanism and extracellular signal-regulated kinase(ERK)signal pathway.Method :Different groups with different Salvia miltiorrhiza concentration(75mg/L、150mg/L、300mg/L),the control group without Salvia miltiorrhiza,the proliferation of MC3T3-E1 cell was analyzed by cell counting kit-8(CCK8)assy,After 21 days culture,the mineralization of MC3T3-E1 was examined by alizarin red staining,and the calcium nodes were counted.The mRNA expressions of bone gla protein(BGP)and runt related transcription factor 2(Runx2)were examined by realtime fluorescence quantitative PCR(qPCR).MC3T3-E1 cells were treated with SM in high concentration(300mg/L),the protein expression of phosphorylated extracellular signal-regulated kinase(p-ERK1/2)and p-p38 were detected by Western bloting.After added inhibitor of ERK1/2 and p38 pathway,the mRNA expressions of BGP and Runx2 were examined by qPCR again.Results: Compared to control group,MC3T3-E1 preosteoblasts treated with SM had higher level of proliferation,and SM in 300mg/L showed extremely significant difference(p=0.006).MC3T3-E1 cells treated with SM in different concentration had more calcium nodes than the control group(p=0.000),the quantity was(11.40±2.30),(21.00±2.24),(35.60±1.52),(6.40±1.14)(control group)respectively.In general,the groups treated with SM had higher level of the mRNA expressions of osteoblasts-related gene BGP and Runx2.For BGPm RNA,the raletive expression were(1.02±0.04),(1.11±0.07),(1.26±0.05)respectively,the two groups(treated with SM in150mg/L and 300mg/L)haded significant statistical differences(p=0.019,p=0.000).ForRunx2 mRNA,the raletive expression were(1.38±0.25),(2.28±0.13,(2.48±0.06)respectively,the group treated with SM in 75mg/L showed significant differences(p=0.012),while the two groups treated with SM in 150mg/L and 300mg/L respectively,showed very significant statistical differences(p=0.000).The expression level of protein p-ERK1/2 was also remarkably elevated in SM group compared to the control group(p=0.014),but The expression level of protein p-p38 of the two groups was not significant difference.The two groups were compared(300mg/LSM vs 300mg/LSM+PD98059),the mRNA expressions of BGP and Runx2 obviously decreased(P=0.000).but between the two groups(300mg/L SM vs 300mg/L SM+ SB203580),there was no significant difference about the m RNA expressions of BGP and Runx2.Conclusion: Salvia miltiorrhiza can promote osteogenic differentiation,proliferation and mineralization of MC3T3-E1 preosteoblast by the ERK1/2 signal pathway and not by the p38 signal pathwayPARTⅡ Salvia miltiorrhiza prevents iron overload-induced apoptosis and decline of osteogenic capacity of MC3T3-E1 by blocking p38 MAPK and JAK2/STAT3 signalObjective:To study Salvia miltiorrhiza(SM)whether or not can prevent iron accumulation-induced decline of osteogenic differentiation,proliferation and mineralization of MC3T3-E1 preosteoblast,and examine the mechanism.Method: Different groups were set,group F1: 50μmol/L ammonium ferric citrate(FAC),group F2: 200μmol/L FAC,group D1+F2 : 75mg/L SM+200μmol/L FAC,group D2+F2:150mg/L SM+200μmol/L FAC,group D3+F2:300mg/L SM+200μmol/L FAC,CON as the control without any treatment.The proliferation of MC3T3-E1 cell was analyzed by cell counting kit-8(CCK8)assy,the mineralization of MC3T3-E1 was examined by alizarin red staining,the apoptosis rate of the cells was examined by flow cytometry.The mRNA expressions of BGP,Runx2 and Col1 a were examined by realtime fluorescence quantitative PCR(qPCR).The protein expression of p-p38、p-JAK2、p-STAT3 were detected by Western blotting in group F2,D2+F2 and CON.Result: Compared to group F2,the proliferation of group D1+F2 and group D2+F2had obviously rise[(87.38±1.03)% vs(82.35±1.79)%,(86.91±1.00)% vs(82.35±1.79)%,p<0.01].The generation rate of calcium nodes of group D2+F2 was higher than group F2 observably.Compered to the groups treated with iron of high concentration only(group F1,F2),the groups treated with iron and SM together(group D1+F2,D2+F2,D3+F2)had lower apoptosis rate obviously(p<0.01).The relative expression of osteogenesis-related genes(BGP,Runx2,Col1a)of different groups(groupD1+F2,D2+F2,D3+F2),that were added SM of different concentration and FAC,had higher level than group F2(p <0.05)[(for BGP,(1.95±0.04,2.08±0.17,2.78±0.10)vs 0.78±0.08;for RunX2,(0.64±0.02,0.75±0.02,0.83±0.03)vs 0.58±0.02;for Col1 a,(0.39±0.02,0.74±0.02,0.94±0.02)vs 0.35±0.03].Western blotting measure result : Compared to the control group,the protein expression level of p-p38,p-JAK2 and p-STAT3 in group F2 was significantly rised(p < 0.01),however,when added 150mg/L SM to culture medium(group D2+F2),expression level of p-p38、p-JAK2 declined obviously(p<0.01).Conclusion: Salvia miltiorrhiza can prevent iron overload-induced apoptosis of MC3T3-E1 preosteoblast,meanwhile reduce the decline of proliferation,differentiation and mineralization which induced by excess iron.the mechanism is related to suppressing the p38 MAPK and JAK2/STAT3 signal pathway.PART Ⅲ Salvia miltiorrhiza suppress RAW264.7 proliferation osteoclastic differentiation and function by reducing the production of ROS in the cells and suppressing NFATc1 signalObjective:To explore the mixture of water-soluble effective component of Salvia miltiorrhiza,whether or not it can affect proliferation,differentiation and osteoclastic function of osteoclast,and probe the mechanism.Method: using RANKL(receptor activator of NF-κB ligand)induce RAW264.7 to osteoclast as the model of osteoclast in vitro.Used Salvia miltiorrhiza in differentconcentration(75mg/L、150mg/L、300mg/L respectively)treat cells of different groups,and compare to control group.The proliferation of RAW264.7 cell was analyzed by cell counting kit-8(CCK8)assy,Osteoclast differentiation and number counting was assessed by tartrate resistant acid phosphatase(TRAP)staining and electron microscope,used fluorescence probe DCFH-DA to detect Reactive oxygen specie(ROS)in cells,and used fluorescence microscope and multiscan spectrum to measure the ROS level.The mRNA expression of cathepsin k(CTK),matrix metalloprotease-9(MMP9)and TRAP was detected by fluorescent quantitative polymerase chain reaction(qPCR).The expression of CTK、TRAP and NFATc1 protein was detected by western blot.Result: Salvia miltiorrhiza can suppress the proliferation of RAW264.7 cells concentration-dependently.The result of TRAP staining indicated that Salvia miltiorrhiza in different concentration could suppress the generation and maturity of osteoclast in dose-dependent manner,the average number of osteoclast in one well,which was the treated with different concentration of Salvia miltiorrhiza,was few than the control group obviously.According to the result of quantitative polymerase chain reaction(qPCR),Salvia miltiorrhiza could suppress the mRNA transcription of cathepsin K(CTK),matrix metallopeptidase-9(MMP9)and tartrate resistant acid phosphatase(TRAP),the three important gene of osteoclast.According the result of observation by fluorescence microscope and assessement by multiscan spectrum,Salvia miltiorrhiza significantly reduced intracellular ROS level,especially in high concentration(300mg/L).Conclusion:Salvia miltiorrhiza can suppress RAW264.7 cell to differentiate to osteoclast,and suppress the proliferation and differentiation of RAW264.7 cell,and restrain the osteoclastic function.one of the mechanism was relating to reduce the production of ROS in the cells and suppress NFATc1 signal. |
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