| Objective1.To investigate the injury effect of palmitic acid on osteoblasts.2.To investigate the protective mechanism of liraglutide against palmitic acid-induced osteoblast apoptosis.Methods Culture of mouse osteoblast cell MC3T3-E1 in vitro.（1）The optimal time and concentration of palmitic acid stimulation were determined by CCK-8,Western Blot,flow cytometry（FCM）and reactive oxygen species（ROS）level observed by fluorescence microscopy.（2）CCK-8 assay and real-time fluorescence polymerase chain reaction（q RT-PCR）were used to determine the optimal time and concentration of liraglutide;and then the cells were divided into three groups: control group（CON）,palmitic acid group（PA）,and liraglutide + palmitic acid group（Lira+PA）.The MC3T3-E1 cells were treated with palmitic acid,liraglutide + palmitic acid respectively,the proliferation rate was detected by CCK-8 assay;the expression of GLP1 R was detected by immunofluorescence,Western Blot and q RT-PCR;the level of intracellular c AMP was detected by Elisa;FCM was used to detect the effect of the drugs on the rate of apoptosis;the protein expression levels of related key molecules GLP1 R,p-PKA,p-β-Catenin（Ser675）,OPG,RANKL,C-Capase3,Bax,Bcl2 were detect by Western Blot,and the m RNA expression levels of GLP1 R,β-Catenin,and PKA were detected by q RT-PCR.（3）To investigate the protective mechanism of liraglutide against osteoblast damage.（1）The GLP1 R inhibitor Exendin9-39 was firstly added: the effect of the inhibitor on cell proliferation was detected by CCK-8,the expression of proteins related to GLP1R/c AMP/PKA/β-Catenin signal pathway were detected by Western Blot,the level of c AMP was detected by Elisa;the m RNA expression levels of β-Catenin,PKA and osteoblast markers were detected by q RT-PCR;the effects on the apoptosis rate were measured by FCM;ROS was observed by immunofluorescence microscopy.（2）The PKA inhibitor H89 was added: the effect of the inhibitor on cell proliferation rate was detected by CCK-8 assay;the expression levels of related key protein were detected by Western Blot,and the m RNA expression levels of β-Catenin,PKA and osteoblast markers were detected by q RT-PCR;their effects on apoptosis rate were examined by flow cytometry.Results（1）Palmitic acid stimulation time was determined by CCK-8,Western Blot,flow cytometry.and ROS resulted to be 24 h at a concentration of 0.4 m M.（2）The optimal concentration of liraglutide was determined to be 100 n M based on CCK-8 and q RT-PCR results.Compared to the PA group,liraglutide improved the rate of cell proliferation and reduced the rate of apoptosis as well as the level of ROS;increased intracellular c AMP levels;up-regulated p-PKA,p-β-Catenin（Ser675）,OPG,anti-apoptotic protein Bcl2 expression levels,while the expression levels of RANKL,C-Capase3 and Bax were reduced.（3）Compared with the Lira+PA group,Exendin9-39（100 n M）decreased cell proliferation rate,down-regulated GLP1R/c AMP/PKA/β-Catenin pathway,increased apoptosis rate,increased levels of apoptosis key protein C-Capase3 and Bax protein,and decreased Bcl2.The m RNA levels of PKA,β-Catenin,OPG,and RANKL showed the same changes as protein expression.Exendin9-39 elevated intracellular ROS levels.（4）When the PKA inhibitor H89（20 μM）was added,the cell proliferation rate was downregulated compared to the Lira+PA group;H89 blocked the protective effect of liraglutide,as shown by decreased protein levels of p-PKA,p-β-Catenin（Ser675）,Bcl2,while the expression levels of apoptosis key protein C-Capase3 and Bax showed the opposite trend.Meanwhile,the m RNA levels of PKA,β-Catenin showed the same changes as well as protein expression,and the m RNA level of OPG was down-regulated while RANKLwas up-regulated.Conclution Liraglutide protected osteoblasts from apoptosis induced by excessive palmitic acid,possibly by binding to GLP-1 receptors,thus downstream c AMP/PKA/β-Catenin signaling pathway. |
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