The Mechanism Of AP-1/TLR4 Positive Feedback Signal Involved In The Stagnation Of Pre-osteoblast Differentiation Induced By High Glucose

Objective The purpose of this study was to explore the effect of diabetic hyperglycemia simulated by 25.5mmol/L glucose concentration on the differentiation of preosteoblasts into mature osteoblasts and the related mechanism.Methods Mouse precranial osteoblasts(MC3T3-E1)underwent osteogenic differentiation in osteogenic induction differentiation medium Induction.Osteogenic induction differentiation medium containing 25.5mmol/LD-glucose was used to simulate the hyperglycemic conditions of diabetes.Lipopolysaccharide(LPS)and resatovi(CLI095)were used as the specific activator and inhibitor of TLR4.Phorbol Ester(PMA)and curcumin(CUR)were used as specific activators and inhibitors of AP-1.MC3T3 cells were differentiated by osteogenic induction medium containing appropriate concentrations of the above reagents under high glucose conditions.normal control group(NC),high glucose group(HG),Normal control group(NC),high glucose group(HG),high glucose and TLR4 activation group(HG+LPS),high glucose and TLR4 inhibition group(high glucose and TLR4 inhibition group,HG+CLI095),high glucose and AP-1 activation group(HG+PMA),high glucose and AP-1 inhibition group(High glucose and AP-1 inhibition group,HG+CUR).Under the above conditions,the following experiments were carried out according to the different days of cultivation required by the experiment.1.MC3T3-E1 cell lines were cultured in the proliferation medium(MIMα+10%FBS+1%P/S),and the osteogenic induction differentiation culture was performed on the osteogenic induction differentiation medium(MIM α+10%FBS+1%P/S+10nmol/ lβ-sodium glycerol phosphate +50mg/L ascorbic acid)according to the conditions of each groups.2.Cell Counting Kit-8(CCK8)and colony forming unit assays(CFU)were used to evaluate the proliferation of each group.The cloned cell communities and the cells cultured for seven days were stained with alkaline phosphatase(ALP)in alkaline phosphatase to evaluate the ALP activity in all groups.3.The cells differentiated for 14 days were stained with Sirius red to evaluate type I collagen production.Alizarin red staining was performed on the cells after 21 days of differentiation to determine the deposition of calcium salts on the cell surface4.Quantitative Real-time PCR(qPCR)detection of RUNX family related transcription factor 2,RUNX2),osteoblast specific transcription factor(Osterix,Osx),osteocalcin(OCN)and osteoprotegerin(OPN)mRNA expression.5.Total protein was extracted and nuclear protein and cytoplasmic protein were isolated.Western blotting is employed to detect the subunit activating protein-1(AP-1): phosphorylated Jun proto-oncogene(p-jun),phosphorylated Jun proto-oncogene(p-Jun),Fos proto-oncogene(Fos proto-oncogene,c-jun),phosphorylated Jun proto-oncogene(p-Jun),phosphorylated Jun proto-oncogene,c-fos),phosphorylated Fos proto-oncogene(p-fos),Toll like receptor 4(TLR4).And the distribution of p-jun and p-fos in the nucleus/plasma of each groups.6.In conjunction with the National Center of Biotechnology Information(NCBI),The University of California Santa Cruz Genome Browser(UCSC)and JASPAR database analyzed the regulatory effects of AP-1 on the upstream of the TLR4 promoter.7,Use of Electrophoretic mobility shift assay(EMSA),comparison between groups of AP-1 and the combination of TLR4 promoter probe efficiency.Results1.Under the differentiation condition,compared with the NC group,the HG group did not show obvious changes in proliferation after 1,3 and 5 days,but showed a higher proliferation activity on the 7th day(P<0.05).The CFU results of the cells cultured for 7 days showed that there was almost no difference in proliferation activity between the HG group and the NC group.However,ALP staining for CFU-forming cells showed that the NC group had a higher ALP activity than the HG group(P<0.05).Compared with the HG group,the difference between the HG+CLI095 vs.The cell proliferation activity in the HG+CUR group was restored to the same level as that in the NC group,accompanied by the increase in ALP activity of the cell community.However,the HG+LPS and HG+PMA groups showed opposite results.2.ALP staining,Sirius red staining and alizarin red staining were performed on the cells of each group after 7 days,14 days and 21 days of differentiation culture.It was found that compared with NC group,HG group showed significantly lower ALP activity,collagen Ⅰ level and worse calcium deposition.Compared with the HG group,the HG+CLI095 and HG+CUR groups showed higher ALP activity and collagen Ⅰ level and more significant calcium salt deposition,while the HG+LPS and HG+PMA groups showed the opposite.3,qPCR was performed on differentiated cultured cells.Compared with NC group,HG group had lower expression levels of RUNX2(P<0.01),OSX(P<0.05),OCN(P<0.05),OPN(P<0.001)on day 7.On day 14,the HG+CLI095and HG+CUR groups showed higher expression levels of RUNX2(P<0.001,P<0.01),OSX(P<0.01,P<0.001),OCN(P<0.05,P<0.001),and OPN(P<0.05,P<0.05).The opposite was observed in the HG+LPS and HG+PMA groups.4.Western blotting results showed that there was no significant difference in total c-jun and c-fos expression among the groups(P>0.05).Compared with NC group,the expression of p-Jun(P< 0.05),p-fos(P<0.05),TLR4(P<0.0001)in total protein and the nuclear translocation of p-Jun(P<0.05)and p-fos(P<0.01)in nuclear protein were significantly increased in HG group.Compared with HG group,the expressions of p-Jun(P<0.05,P<0.001),p-fos(P<0.01,P<0.001),TLR4(P<0.01,P<0.05)in total protein and p-Jun(P<0.05,P<0.001)in nuclear protein in HG+CLI095 and HG+Cur groups were significantly increased(P<0.05,P<0.001,P<0.01)decreased and returned to the level of NC group.Although p-fos nuclear translocation decreased,it was not statistically significant.However,the opposite was observed in the HG+LPS and HG+PMA groups.5.Bioinformatics analysis showed that the TLR4 promoter region has AP-1 binding sites at positions 520-526 and 1757-1763,and some AP-1 family members showed high score(confidence score >90%)binding activity.6.Electrophoretic mobility shift assay showed that AP-1 was bound to the promoter probe of TLR4 gene in vitro,and HG group had higher binding activity than NC group.Activation of TLR4 and AP-1 under high glucose conditions increased the binding activity of AP-1 to the promoter probe of TLR4gene(HG+LPS group,HG+PMA group).In addition,inhibition of TLR4 and AP-1 under high glucose conditions decreased the binding activity of AP-1 to the promoter probe of TLR4 gene(HG+CLI095 group,HG+Cur group).Conclusions High glucose in vitro inhibits osteogenic differentiation by affecting the stage of osteogenic differentiation from preosteoblasts to mature osteoblasts.AP-1/TLR4 forms a positive feedback loop to participate in this process.