Estrogen And Oxidative Stess Effect The Proliferation And Differentiation Of Osteoblasts By Regulating Peroxiredoxin Type 1 Though AKT/NF-kB Signaing Pathway

PurposeThe purpose of this study was to evaluate the effects of estrogen and oxidative stress on the biological behavior of mouse osteoblasts such as apoptosis,proliferation and differentiation in vivo and in vitro,and the expression of peroxiredoxin 1 during the process.In addition,the knockout and overexpression of peroxiredoxin 1 gene in mouse MC3T3-E1 pre-osteoblasts were performed to study the effects of this protein on the biological behavior of osteoblast.And the related signaling pathways were studied to further explore the estrogen and oxidative stress regulation of osteoblast differentiation specific mechanism of action.Methods1.In this study,ovariectomized(OVX)surgery was performed in 8-week-old female mice to prepare animal model of estrogen-deficient osteoporosis.The histopathology and immunohistochemical staining of the mouse tibial paraffin sections were used to observe the pathological changes of bone tissue and the expression of superoxide reductase after estrogen deficiency.In addition,in vitro cultured mouse MC3T3-E1 pre-osteoblast cell line was used to verify the in vivo results.2.The MC3T3-E1 pre-osteoblastic cell line was cultured in vitro using CCK8,alkaline phosphatase test,alizarin red S staining,Sirius red staining,flow cytometry,real-time PCR and Western-blot and other methods to detect the impact of oxidative stress on apoptosis,proliferation and differentiation of MC3T3-E1 cells cultured in vitro,and estrogen on the process of intervention.3.By CRISPR/Cas9 and other methods,preparation of PRX1 knockout and overexpression of mouse MC3T3-E1 pre-osteoblast cell line,by the above method to detect PRX1 on cultured MC3T3-EI in vitro effects of cell Apoptosis,Proliferation and Differentiation.4.The bioinformatics method was used to analyze the interaction between the estrogen receptor and PRX1.The KEGG analysis was used to screen the possible signaling pathways.5.Using Western-blot method to detect the expression of Cyto-c or Trx/ASK1/MAPK signaling pathway-related proteins in osteoblasts using the MC3T3-E1 cell with and without LPS stimulation.The expression level of oxidative stress through PRX1 osteoblast apoptosis mechanism.6.Inhibition of AKT signaling pathway by AKT specific inhibitor LY294002 and inhibition of NF-?B signaling pathway by PDTC,a specific inhibitor of NF-?B,and detection of AKT1 phosphorylation in osteoblasts by Western-blot and NF-kB signaling pathway AKT1 and p65 protein phosphorylation levels of estrogen on osteoblasts expression of peroxiredoxin 1-dependent signaling pathways.Results1.After 4 weeks of ovariectomy in mice,estrogen deficient animal models of osteoporosis were prepared.The staining results of the proximal tibial metaphyseal tissue sections showed that compared with the control group,the osteopenia and bone turnover rate increased and the expression of peroxiredoxin gene was up-regulated in the OVX group.PRX1 and PRX5 show different distribution patterns in osteoblast structure,while PRX1 mainly accumulates in the nucleus and PRX5 accumulates in the cytoplasm.In vitro cultured MC3T3-E1 cells showed that oxidative stress stimulated the expression of PRX1,whereas estrogen attenuated this effect.2.Oxidative stress(H2O2/LPS)increased the expression of PRX1 in MC3T3-E1 cells and caused the apoptosis of osteoblasts.However,estrogen could reduce the expression of osteogenic cells Apoptosis of cells.Compared with the control group,the ALP activity and alizarin red S staining of MC3T3-E1 osteoblasts in the hydrogen peroxide treatment group decreased.Real-time PCR results showed that mRNA levels of Runx2,type I collagen and osteocalcin decreased.Western-blot results also consistent with the PCR results.3.The results of preparing mutant cell lines showed that the mRNA and protein expression level of PRX1 in knockout/overexpressed cells was significantly lower/higher than that in wild type cells.After PRX1 knockdown,ROS levels in osteoblasts increased,leading to increased apoptosis,cell proliferation was significantly inhibited.Compared with wild-type cells,the knocked-out and overexpressed cells showed decreased ALP activity and alizarin red S staining.Real-time PCR results showed that mRNA expression of Runx2,type I collagen and osteocalcin decreased after osteopontin mRNA was up-regulated in overexpression cells.Western-blot results also showed that knockdown and overexpression of PRX1 inhibited osteoblast differentiation.4.Functional annotation of estrogen receptor and PRX1 using database information showed that TLR4/ASK 1 and MAPK signaling pathways may be involved in PRX1-regulated osteoblast apoptosis;estrogen and PRX1 may regulate osteoblast differentiation through PI3K/AKT and NF-?B signaling pathways.5.The expression level of Cyto-c in PRX1-KO cells was significantly higher than that in PRX1-KO cells without or with LPS stimulation.The expression of p-ASK1 in PRX1-OE cells was much higher than that in PRX1-KO cells and wild type However,the expression of p-ASK1,JNK and p38 was decreased in PRX1-OE cells after LPS stimulation,while the activation and expression of wild-type cells were significantly increased.6.The results of Western-blot showed that phosphorylation of AKT1 and p65 protein and expression of peroxiredoxin 1 protein in MC3T3-E1 cell line were decreased after AKT signaling pathway was inhibited by AKT specific inhibitor LY294002.However,the phosphorylation of AKT1 protein and the expression of peroxiredoxin 1 protein were not changed except that the phosphorylation level of p65 protein was significantly decreased after NF-?B specific inhibitor PDTC inhibited NF-?B signaling pathway.Conclusion1.Oxidative stress can induce osteoblast apoptosis and inhibit its differentiation by stimulating the expression of PRX1 protein in MC3T3-E1 osteoblasts,whereas estrogen can reduce osteoblast apoptosis,protection of osteoblast differentiation function normally by inhibiting its expression.2.The PRX family may be involved in the development of estrogen-deficient osteoporosis,and different members of this family may play different roles in this pathological process.Among them,PRX1 is an important regulator of osteoblast function and plays an important role in the process of cell apoptosis,proliferation and differentiation.3.After PRX1 knockdown,the level of ROS in osteoblasts is increased,and the apoptosis of osteoblasts is affected by Cyto-c pathway.PRX1 itself can affect the apoptosis of osteoblasts through the pathway of Trx/ASK1/JNK.The effect of estrogen on the expression of PRX1 in osteoblasts and the regulation of osteoblast differentiation is related to the AKT1/NF-?B signaling pathway.