| Diabetes mellitus(DM)is a chronic disease that progresses worldwide at alarming rates.DM affects bone metabolism by increasing bone resorption,reducing bone formation,and finally leading to osteoporosis.Osteoporosis,a chronic progressive bone metabolic disease common in the elderly,is characterized with bone loss,and damaged bone microstructure,resulting in increased bone fragility and a significantly increased fracturerisk.Many glucagon-like peptide-1(GLP-1)receptor agonists(GLP-1RA)are currently used to treat type 2 diabetes and obesity.Moreover,both in vitro and in vivo studies have suggested that GLP-1 RA can improve bone metabolism.Liraglutide is a long-acting GLP-1RA that continuously activates GLP-1R.Bone formation is a series of events,including differentiation of mesenchymal precursor cells into osteoblast precursors,maturation of osteoblasts,and formation and mineralization of the matrix.Many signaling pathways including Wnt / β-catenin、Notch、bone morphogenetic proteins(BMP)/tumor growth factor(TGF)–β、PI3K/Akt/m TOR、mitogen-activated protein kinase(MAPK)、 Hedgehog(Hh)and other signaling pathways are involved in the osteogenic differentiation.Our previous studies found that liraglutide can promote the proliferation,differentiation and inhibit the apoptosis of mouse pre-osteoblast MC3T3-E1 cells.The present study aims to explore whether Notch,Wnt / β-catenin and Hh signaling pathways are involved in the regulation of liraglutide onMC3T3-E1 cells.Part Ⅰ Liraglutide regulates the proliferation and differentiation of preosteoblasts MC3T3-E1 through Notch signaling pathwayObjective: To explore the role of Notch pathway in the regulation of liraglutide on pre-osteogenic differentiation.Methods:1.MC3T3-E1 cells were treated with different concentrations of liraglutide(0,10,100,500,1000 nM)and cell viability were evaluated after treatment for 24,48,or 72 h using MTT.2.MC3T3-E1 cells were induced to osteoblast by 50 mg/L-ascorbic acid and 10 mM β-glyceryl phosphate in the presence of different concentrations of liraglutide(0,10,100,500,1000 nM)for 28 days.The activity of alkaline phosphatase(ALP),an indicator of early osteogenic differentiation,was measured at 0,7,and 14 days after induction.Alizarin red staining was used to detect the extracellular matrix mineralized nodules at 21 day after induction.The mRNA levels of osteogenic differentiation markers collagen 1(COL1),osteocalcin(OC),Runt-related transcription factor 2(RUNX2)and osteoprotegerin(OPG)were detected by quantitative real time-PCR(qRT-PCR),and the mRNA and protein levels of GLP-1R were determined by Western blot(WB)and qRT-PCR at 0,7,14,21 and 28 days after induction.3.MC3T3-E1 cells were treated with 100 nM liraglutide.The expression of Notch-related proteins(Notch1,JAG1,and HES1)were determined using WB and intracellular cAMP activity was measured at 0,20,40,and 60 minutes,respectively.4.MC3T3-E1 cells were treated with Notch inhibitor DAPT(50 μM)in the presence or absence of 100 nM liraglutide.Cell viability were evaluated using MTT and mRNA levels of COL1,OC,RUNX2 and OPG geneswere detected using qRT-PCR after treatment for 48 h.5.MC3T3-E1 cells were transfected with NC siRNA or siRNA of GLP-1R for 48 h.The expression of Notch-related proteins(Notch1,JAG1,and HES1)were determined using WB and intracellular cAMP activity was measured.Results:1.Liraglutide significantly promoted the proliferation of MC3T3-E1 cells.Cells treated with 100 nM liraglutidefor 48 h showed the highest proliferation.2.The changes of mRNA levels of osteogenic differentiation factors Col-1,OC,RUNX2,and OPG genes and ALP activity confirmed the induced MC3T3-E1 cells were undergoing osteogenic and mature.Liraglutide treatment can significantly increase the mRNA levels of these biomarkers and the mineralized area of the matrix as well as the mineralized nodules.During osteogenic differentiation,the mRNA and protein levels of GLP-1R gradually increased,and liraglutide treatment could significantly increase the mRNA and protein levels of GLP-1R at the 7th and 14 th days.3.Liraglutide significantly increased the protein expression levels of Notch-related molecules Notch1,JAG1,and HES1,peaked at 20 minutes after treatment,and then gradually decreased.But the expression levels of proteins at 60 minutes were still significantly higher than those in the control group.It also significantly increased intracellular cAMP activity,peaking at 20 minutes and almost returning to normal levels at 60 minutes.4.Liraglutide significantly increased the proliferation of MC3T3-E1 cells and the mRNA levels of COL1,OC,RUNX2 and OPG genes,but simultaneous DAPT treatment reduced MC3T3-E1 cell proliferation and mRNA levels of COL1,OC,RUNX2 and OPG genes,indicating DAPT reduces the increase of liraglutide on the proliferation and osteogenic differentiation of MC3T3-E1 cells.5.Liraglutide significantly up-regulated the protein expressions of Notch1,JAG1,and HES1,while GLP-1R knockdown significantly reduced the protein expressions of Notch1,JAG1,and HES1,as well as the up-regulation of cAMP activity by liraglutide.Part Ⅱ Liraglutide promotes the proliferation and differentiation of MC3T3-E1 cells through cross-linking with Notch signaling pathway by β-cateninObjective: To investigate whether the Notch pathway and β-catenin interact in the regulation of liraglutide onthe proliferation and differentiation of MC3T3-E1 cells.Methods:1.MC3T3-E1 cells were treated with Notch inhibitor DAPT(50μM)in the presence or absence of 100 nM liraglutide for 48 h.The mRNA level of TCF7L2 was detected by qRT-PCR,the protein expression of β-catenin were detected by WB and the localization of β-catenin were detected by immunofluorescence assay.2.MC3T3-E1 cells were transfected with NC siRNA or siRNA ofβ-catenin in the presence of 100 nM liraglutide for 48 h.mRNA and protein levels of Notch signaling pathway proteins(Notch1,JAG1 and HES1)were detected using qRT-PCR and WB.Results:1.Liraglutide significantly increased the mRNA level of TCF7L2 and the protein expression of β-catenin.Itpromotedthe trans-localization of β-catenin from the cytoplasm to the nucleus.DAPT and liraglutide treatment significantly reduced the mRNA level of TCF7L2 and the protein expression of β-catenin which was still localized in the cytoplasm after co-treatment.2.Liraglutide significantly up-regulated the expression of Notch1,JAG1 and HES1,while β-catenin knockdown significantly reduced the protein expression of Notch1,JAG1 and HES1.Part Ⅲ Notch signaling pathway and Hedgehog signaling pathway through Gli1 cross-linking to affect the proliferation and differentiation of liraglutide onMC3T3-E1 cellsObjective: To investigate whether Hh signaling pathway is involved in the regulation of osteoblasts by liraglutide,and further explore the interaction between Hh signaling and Notch signaling during this process.Methods:1.MC3T3-E1 cells were treated with 100 nM liraglutide for 48 h.The mRNA and protein expression levels of Hh signaling pathway proteins Hh and Gli1 were detected by qRT-PCR and WB.2.MC3T3-E1 cells were transfected with NC siRNA or siRNA of Gli1 in the presence of 100 nM liraglutide for 48 h.mRNA levels of osteogenic differentiation-related genes(RUNX2 and OCN)were detected using qRT-PCR.3.MC3T3-E1 cells were treated with Hh pathway inhibitor cyclopamine(10μM)in the presence or absence of 100 nM liraglutide for 48 h.The mRNA and protein expression levels of Hh signaling pathway protein Gli1 and Notch pathway proteins Notch1,JAG1 and HES1 were detected by qRT-PCR and WB.4.MC3T3-E1 cells were treated with Notch inhibitor DAPT(50μM)in the presence or absence of 100 nM liraglutide for 48 h.The mRNA and protein expression levels of Hh signaling pathway proteins Hh and Gli1 were detected by qRT-PCR and WB.5.MC3T3-E1 cells were transfected with NC siRNA or siRNA of Gli1 in the presence of 100 nM liraglutide.mRNA and protein expression levels of Notch1,JAG1 and HES1 were detected by qRT-PCR and WB.Results:1.Liraglutide significantly increased the mRNA and protein expression levels of Hh signaling pathway proteins Hh and Gli1.2.Gli1 knockdown significantly reduced the mRNA expression levels of osteogenic differentiation proteins RUNX2 and OCN,inhibiting the promotion of liraglutide on the osteoblast differentiation.3.Liraglutide significantly activate Hh and Notch signaling pathways,while simultaneous cyclopamine treatment can significantly reduce the mRNA and protein expression levels of Gli1,Notch1,JAG1,and HES1,and inhibited the activation of Hh and Notch pathways by liraglutide.4.Liraglutide significantly increased the mRNA and protein expression levels of Hh and Gli1 and activate the Hh pathway,but simultaneous DAPT treatment can significantly reduce the mRNA and protein expression levels of Hh and Gli1,and inhibited the activation of Hh pathway by liraglutide.5.Interfering with Gli1 can significantly down-regulate the mRNA and protein expression levels of Notch1,JAG1,and HES1,indicating that liraglutide can also activate the Notch signaling pathway by activating the Hh pathway.Part Ⅳ The role of Notch signaling pathway in the inhibition of liraglutide onMC3T3-E1 apoptosisObjective: To investigate the role of Notch signaling pathway and cross-linking molecules GLP-1R,β-catenin and Gli1 in the inhibition of liraglutide onMC3T3-E1 apoptosis.Methods:1.MC3T3-E1 cells were induced apoptosis using serum deprivation for24 h,and then treated with notch inhibitor DAPT in the presence or absence of liraglutide for 48 h.Cell apoptosis was evaluated using flow cytomerty.The expression of Notch-related proteins(Notch1,JAG1,and HES1)and apoptosis-related proteins(Bax,Bcl-2,caspase-3)were detected using WB.2.MC3T3-E1 cells were induced apoptosis using serum deprivation for24 h,and then transfected with siRNAs of GLP-1R,β-catenin and Gli1 individually,in the presence or absence of liraglutide for 48 h.Cell apoptosis was evaluated using flow cytomerty.Results:1.Liraglutide significantly reduce the serum-starvation induced apoptosis of MC3T3-E1 cells,increased the ratio of Bcl-2/Bax protein and reduced the ratio of caspase-3/pro-caspase-3.Simultaneous treatment of DAPT and liraglutide increased the apoptosis,reduced Bcl-2 / Bax protein ratio and increased the ratio of caspase-3 / pro-caspase-3,inhibiting the inhibitory effect of liraglutide on the serum starvation induced apoptosis of MC3T3-E1 cells.2.Liraglutide significantly reduced the serum starvation-induced apoptosis of MC3T3-E1 cells,and knockdown of GLP-1R,β-catenin or Gli1 significantly increased the apoptosis.Conclusions:1.Liraglutide promotes the proliferation and differentiation of and inhibits serum starvation-induced apoptosis of MC3T3-E1 cells by activating the Notch signaling pathway,which is regulated by GLP-1R.2.Liraglutide activates the Wnt signaling pathway through the Notch signaling pathway with the cross-link of β-catenin,which promotes the proliferation,differentiation and inhibits serum starvation-induced apoptosis of MC3T3-E1 cells.3.Liraglutide activates the Hh signaling pathway through Notch signaling pathway with the cross-link of Gli1,which promotes the proliferation,differentiation and inhibits serum starvation-induced apoptosis of MC3T3-E1 cells. |
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