Protective Effect Of Puerarin On Hypoxia-reoxygenation Injury Of Osteoblasts

Objective: To investigate the protective effect of puerarin on hypoxia-reoxygenation osteoblasts.Methods : Primary cells were obtained by tissue block adherence method and enzymatic digestion method.After purification of osteoblasts by differential adherence method,hematoxylin and eosin staining was used to identify the purity of osteoblasts,ALP staining(alkaline phosphatase staining)and Alizarin Red staining identifies the ability of cells to secrete alkaline phosphatase and mineralization.After the osteoblasts in the exponential growth state were set in the normal group,the others were treated with puerarin(10-6-10-10 M),and the OD value of the cells was measured by CCK-8 at 3 hours,12 hours,and 24 hours.The hypoxia-reoxygenation model was established,and the effective concentration of puerarin on hypoxia-reoxygenated osteoblasts was further studied by selecting the effective concentration in 3 hours.The normal group,hypoxia-reoxygenation group,hypoxia-reoxygenated puerarin group and puerarin were established.After the hypoxia,the effect was added for 3 hours,and then placed in a normal oxygen incubator.MTT was used to detect the changes of cell viability,and flow cytometry was used to detect apoptosis,ROS and cell cycle changes in each group.RT-PCR and immunoprotein detection were used to detect the expression of osteogenic specific gene bone morphogenetic protein 2(BMP-2)and core binding factor α1(RUNX-2)m RNA.Western blotting was used to detect the expression of type I collagen(Collagen type I),bone morphogenetic protein 2(BMP-2),core binding factor α1(RUNX-2),and transforming growth factor β1 (TGF-β1).Another cell-forming cells were resuspended by trypsin digestion,re-inoculated into 24-well plates,stained with ALP(alkaline phosphatase staining),and photographed under a microscope.The cells were then modeled and digested and planted in 24-well plates.After 17 days,alizarin red stained and photographed.Image J analyzes the changes in ALP secretion and mineralization.After cell modeling,immunoblotting was used to detect the expression changes of Cleaved-caspases-3,Bcl-2 and Bax.The exponential growth phase cells were selected and divided into four groups,normal group,hypoxia-reoxygenation group,hypoxia-reoxygenation + puerarin group,and PD98059 pretreatment for hypoxia-reoxygenated puerarin group.Immunoglobulin imprinting detects the role of the ERK signaling pathway and detects changes in Bcl-2 after PD98069 treatment.The cells were further divided into four groups,normal group,hypoxia-reoxygenation group,hypoxia-reoxygenation puerarin group,and LY294002 pretreatment for hypoxia-reoxygenation and puerarin group.Immunoglobulin imprinted to detect the role of the ERK signaling pathway and to detect changes in Bcl-2 after PD98059 treatment.Immunofluorescence was used to observe changes in Bcl-2.Results:The The extracted cells were more consistent in morphology,suggesting that the cells were of higher purity.ALP staining(alkaline phosphatase staining)and Alizarin Red staining(alkaline red staining)demonstrated that the obtained osteoblasts had mineralization function and ALP secretion function.,to prove that the obtained cells have the function of mature osteoblasts.CCK-8 detection of puerarin has an effect on the proliferation of osteoblasts.At 12 hours and 24 hours,puerarin at 10-6-10-10 M can effectively promote the proliferation of osteoblasts,and the effect is 3 hours,only 10-8-10-9M puerarin can effectively promote the proliferation of osteoblasts.Hypoxia-reoxygenation can effectively reduce the biological activity of osteoblasts.Puerarin can effectively inhibit the negative effects of hypoxia-reoxygenation.The activity of cells in hypoxia-reoxygenation group is significantly lower than that of normal group.Puerarin can also inhibit the proliferation of osteoblasts caused by hypoxia-reoxygenation,and increase the cells that penetrate cells into S and G2.Puerarin can also inhibit osteoblast apoptosis caused by hypoxia-reoxygenation,resulting in hypoxia-reoxygenation.A large number of osteoblasts were apoptotic,and in the puerarin group,apoptosis was decreased;The ability of puerarin to inhibit apoptosis was detected by immunoblotting.The result was hypoxia-reoxygenation,which resulted in low expression of Bcl-2 in cells,increased expression of Bax,and increased expression of Cleaved-caspases3,an activated form of caspases.Effectively inhibits apoptosis induced by hypoxia-reoxygenation,puerarin enhances Bcl-2 expression,decreases Bax expression,and decreases expression of Cleaved-caspases3;puerarin also inhibits osteoblast-associated protein type I caused by hypoxia-reoxygenation The expression of collagen(Collagen type I),bone morphogenetic protein 2(BMP-2),core binding factor α1(RUNX-2),and transforming growth factor β1(TGF-β1)decreased.The expression levels of bone morphogenetic protein 2(BMP-2)and core binding factor α1(RUNX-2)were detected by RT-PCR.The mineralization level of osteoblasts and osteogenesis-related genes BMP-2,RUNX-The trend of 2 is the same,Hypoxia-reoxygenation can reduce the expression of p-AKT and p-ERK in osteoblasts,while puerarin can increase the expression of p-AKT and p-ERK in osteoblasts.LY294002 and PD98059 can partially inhibit puerarin.The role of p-AKT and p-ERK decreased.Western blot showed that hypoxia-reoxygenation decreased the expression of Bcl-2,while puerarin could increase the expression of Bcl-2,while LY294002 and PD98059 could inhibit the expression of Bcl-2.The expression of Bcl-2 was confirmed by immunofluorescence.The immunoblot is consistent.Conclusions:Osteoblasts can be obtained by tissue block adherence method and enzymatic digestion.Puerarin can effectively promote the proliferation of osteoblasts.Puerarin can inhibit the apoptosis of osteoblasts induced by hypoxia-reoxygenation and reduce the level of intracellular ROS.Promote cell proliferation and promote mineralization of osteoblasts.AKT and ERK signaling pathways can affect puerarin apoptosis in hypoxic reoxygenation osteoblasts.