The Role Of Puerarin On Osteoblast And Osteoclast And Mechanism Research

1ObjectiveIn this study, we examined the effect of Puerarin on osteoblast profliferation and differentiation and osteoclast formation in vitro; examined the protection of Puerarin on osteoblast inhibited by TNF-α. and further explore its effect on bone metabolism; observed expressions of differentiation markers of osteoblast, to find the possible mechanism of Puerarin’s function, to provide the evidence for a deeper understanding of Puerarin’s role in bone metabolism.2Methods2.1Primary osteoblastic cells were obtained from new bone mice and cultured.Establish the osteoblastic cells model to observe the morphology of the osteoblasts.2.2To study the effect of different concentration of Puerarin on profliferation and differentiation of osteoblastic cells. Cultured the osteoblastic cells together with10-7~10-5mol/L Puerarin for1~7days. To assay cells proliferation by MTT on1,3,5and7days;To assay cells ALP activity by PNPPon1,3,6days.2.3To study the effect of different concentration of TNF-α on profliteration and differentiation of osteoblastic cells.Cutured the osteoblastic cells with Ong/mL,5ng/mL,10ng/mL,30ng/mL and50ng/mL cytokines TNF-a. To assay cells proliferation by MTT on48h.To assay cells ALP activity by PNPP on72h.2.4To study the effect of Puerarin on expression of differentiation markers of osteoblast.Cutured the osteoblastic MC3T3-E1cells with10-6mol/L Puerarin.Extracted the total UNA from the cells on5,7.10and12days. To evaluate the ALP. RANKL. OPG. OPN. BSP, BGPmRNA expressions by reverse-transcription polymerase chain reaction (RT-PCR).2.5Induced osteoclasl formation. To induced osteoclasts formation by mice bone marrow cells and murine macrophage cell lines RAW264.7respetively. adding the cytokines M-CSF and RANKL. then detected osteoclasts by staining for lartrale-resistani acid phosphalase (TRAP).2.6To observe the effect of Puerarin of osteoclasl formation. Induce osteoclasts in the presence of RANKL.M-CSF and10-7~10-5mol/L Pue-rarin. Observe the effect of Puerarin of osteoclast formation after6days.3Results3.1Different concentration of Puerarin(10-7~10-5mol/L)promoted both prolifer-ation and ALP activity within a dose dependent manner.3.2TNF-αinhibited the proliferation and ALP activity signincantly,appeared a dose dependent relationsip within5~50ng/mL3.3Puerarin inhibited the reducetion of the number of cells induced by5ng/ml TNF-α The effect was significantly in1~5days.Puerarin alleviate the inbibition of TNF-αon ALP activity.but the effect showed no signincant difference.3.4Puerarin down-regulatd the RANL mRNA cxpression and up-regulated the OPG m RNA expression.Puerarin increased the ALP.OPN.BSP.BGP mRNA expression. For the OPN mRNA、Pucrarin raiscd its peak period and shorten its higll expression time.3.5Puerarin inhibited osteoclast formation and reduceed the number of osteoclasts.4Conclusions4.1Puerarin can stimulate osteoblastic cell profliferation,enhanced ALP activity.and the cffcets also showed dose-depcndent relationship.4.2Puerarin can reduced the numbdrs of osteoblasts decrased subiectcd to TNF-α trcatment.suggesting Puerarin may protnote the funetion of osteoblastic ccll and ngainst inhibilon by TNF-α4.3Puerarin can promote ALP activity in presence of TNF-α.but the offect showed no signifteant difference while compared to the TNF-αonly group.The result indicate that Puerarin may mainly against the reducetion of cells number.and shows no obviously effcet on ALP activity in presence of TNF-α.4.4Puerarin down-regulates PANKL mRNA cxprcssion while not signifieatly increases OPG mPNA expression of MC3T3-E1mous steoblast-like cells.The result hows Puerarin can improve the OPG/RNKL expression ration,suggesting that it mav have effeet on osteoclast formation and affect bonc metabolism.4.5Puerarin can up-regulate ALP.BSP.and BGP mRNA expression in MC3T3-E1mouse steoblast-like cells.suggesting that puerarin may promote lsteolast differentiation and mineralization to enhancc osteoblas function,and improving bone metabolism.According to the prcvious results.regulation of OPN mRNA is complicated:it can shorten the time of high expression of OPN mRNA,increasing the amount of the peak of expression,suggesting that puerarin may have a dual regulatory role of OPN mRNA expression.4.6Different concentration of Puerarin can decreased the number of osteoclasts. suggesting that Puerarin can inhibit bone resorption by reducing osteoclast formation.