Anti-aging Effect Of Active Peptides And Ferulic Acid On Osteoblasts

Objective:To verify the antioxidant protective effect of active peptides and ferulic acid on osteoblasts,explore the mechanism of active peptides and ferulic acid to resist osteogenesis decline caused by oxidative stress,compare and analyze their molecular mechanisms regulating bone metabolism.It is intended to provide more experimental basis for clarifying the internal relationship between antioxidant and osteoblast aging and bone metabolism,provide theoretical basis for screening osteoporosis(OP)candidate drugs from natural antioxidant molecules,and provide new ideas and strategies for clinical prevention and treatment of osteoporosis.Methods:?Primary cell culture:osteoblasts were isolated from rat skull by enzymatic digestion and cultured in vitro,and cells were identified by alkaline phosphatase staining and alizarin red staining.?Establishment of oxidative damage model for osteoblasts:cells were exposed to different concentrations of H2O2 for 1 h,then cell survival was tested by cck8 to determine the H2O2 concentration required for damaged cells.?Experimental cell grouping:P3 cells were divided into normal group(Ctrl),model group(H2O2),active peptide group ?(PP-?),active peptide group ?(PP-?),active peptide group ?(PP-?))And ferulic acid group(FA).Cells in the normal group were untreated.Cells in the model group were model cells of oxidative damage.Oxidative damaged cells in the active peptide group and ferulic acid group were treated with peptides and ferulic acid at different concentrations respectively,then cell viability were measured by the CCK8 method,and the secretion level of ALP was measured by enzyme(alkaline phosphatase,ALP)assay kit to select the appropriate concentration.?Cell cycle detection:Cell cycle is detected by flow cytometry after active peptide and ferulic acid are applied to cells for 72 hours.?Detection of intracellular reactive oxygen species(ROS):After reactive peptides and ferulic acid were applied to cells for 72 hours,the content of ROS in the cells was measured by flow cytometry.?Apoptosis detection:Flow cytometry was used to detect the proportion of apoptotic cells and Hoechst dye was used to stain apoptotic cells.?Cell senescence detection:Cells were stained with ?-galactosidase staining kit to detect cell senescence.?Detection of osteoblast mineralization:Alizarin Red S(ARS)was used to stain the mineralizing matrix of each group and take photos.?Transcriptome results analysis:Differential analysis,GO enrichment analysis,KEGG pathway enrichment analysis,and trend analysis combined with KEGG pathway analysis were performed on the results of each group.?Quantitative real-time PCR was used to detect the expression of genes related to osteogenesis and genes related to apoptosis regulation.11Western-Blot was used to detect the expression of osteogenesis-related proteins ALP,osterix(OSX),collagen-I(Coll-I),osteopontin(OPN),matrix metalloproteinase-1(MMP-1),and matrix metalloproteinase-2(MMP-2);Apoptosis-related proteins B-cell lymphoma-2(Bcl-2),Bcl2-Associated X(Bax),cysteinyl aspartate specific proteinase-3(Caspase-3),protein kinase B(Akt),and phosphorylated protein kinase B(pAkt).Results:?The morphology of the cells was observed under a microscope,ALP staining and mineralized matrix showed that the isolated primary cells had normal morphology and good mineralization function.Therefore,the cells extracted from rat skull were osteoblasts.?The damage concentration of hydrogen peroxide was 100?M??The administration concentration of PP-? was 50?M,PP-? and PP-? was 20?M,FA was 640?M.The results of CCK8 detection showed that the cell activity was significantly weakened after H2O2 injury(p<0.05),and significantly recovered after administration of active peptides and FA(p<0.05).ALP detection showed that the secretion of ALP by osteoblasts were decreased significantly after H2O2 injury(p<0.05),and increased significantly after administration in each group(p<0.05).?Cell cycle test showed that oxidative damage can significantly increase the proportion of cells in G1 phase(P<0.05),PP-?and PP-? can significantly reduce the proportion of cells in S phase(P<0.05),PP-? and FA can significantly increase the proportion of cells in G2 phase(P<0.05).?Flow cytometry detection of intracellular ROS showed that oxidative damage can keep cells at a high level of ROS for a long time,while both active peptides and ferulic acid can significantly reduce it(P<0.05).?Flow cytometry detection of apoptosis and apoptosis fluorescent staining results showed that oxidative damage can induce apoptosis(P<0.05),while active peptide and ferulic acid can significantly reduce the occurrence of apoptosis(P<0.05).?P-galactosidase staining results showed that the Ctrl group had less green stained areas and less stained cells,while the model group had larger green stained areas and more stained cells.Both active peptide and ferulic acid could reduce the stained areas and stained cells,among them,the effects of PP-?,PP-?,and FA are better,the effect of PP-? is average,which indicates that a large number of cells enter the senescence stage after oxidative damage,so the secretion of ?-galactosidase is significantly increased(P<0.05).Active peptide and ferulic acid can delay the aging process of cells and significantly reduce the secretion of?-galactosidase(P<0.05).?Mineralized matrix alizarin red staining results showed that the osteoblasts after oxidative injury had weakened mineralization ability and could not form ideal mineralized nodules.The PP-I group was failed to form an ideal mineralized matrix,The mineralized matrix of PP-? and PP-? group is larger and darker than the model group.Generally speaking,the active peptide cannot restore the cell's mineralization ability within 21 days.PP-? has the worst effect.However,ferulic acid can restore partial mineralization within 21 days,so that osteoblasts form a smaller mineralized matrix.?Q-PCR results showed that Bax and Caspase-3 transcription increased significantly after oxidative damage(P<0.05),while Runx-2,ALP,OPG,Collagen-?,and OPN decreased significantly(P<0.05).After administration,both active peptide and FA significantly reduced the transcription of Bax and Caspase-3(P<0.05),while significantly increased the transcription of ALP(P<0.05).In addition,PP-? significantly increased Bcl-2,OSX and OPG transcription(P<0.05),PP-? significantly increased the transcription of OSX and OPG(P<0.05).FA significantly increased the transcription of OSX,OPG,Collagen-I and OPN(P<0.05),while significantly reduced the transcription of Bcl-2(P<0.05).?WB results showed that oxidative damage reduced the expression of Bcl-2,Collagen-I,OPN,Akt,and pAkt in cells,while increased the expression of Bax,Caspase-3,MMP-1,and MMP-2.The above conditions were reversed after ferulic acid treatment.PP-? and PP-? can improve the expression of osteogenesis related proteins OSX,ALP and Collagen-?,and improve the expression of apoptosis-related proteins Akt and pAkt.11Transcriptome results show that compared with normal cells,the expression of a large number of genes in oxidatively damaged cells has changed.when compared with oxidatively damaged cells,the expression of many genes after the action of active peptides and ferulic acid on cells has also occurred significant changes.According to GO enrichment analysis and KEGG pathway enrichment analysis,it can be seen that H2O2,active peptide,and FA have changed the function of cells and certain cell signaling pathways.After analysis we found that the cell pathways that PP-? acts on which closely related to osteogenesis and apoptosis are ECM-receptor interaction,Focal adhesion,P53 signaling pathway,cellular-senescence and PI3K-Akt signal pathway.The cell pathways closely related to osteogenesis and apoptosis affected by PP-?include ECM-receptor interaction,Apoptosis-multiple species,P53 signaling pathway,Focal adhesion and PI3K-Akt signal pathway.The cell pathways that ferulic acid acts on which closely related to osteogenesis and apoptosis are ECM-receptor interaction,Focal adhesion and PI3K-Akt signal pathway.Conclusions:Both active peptides and ferulic acid show the effects of resisting cell oxidative damage,reducing apoptosis,delaying cell senescence,and promoting the transcription of osteogenic factors.Active peptides can better improve the cell proliferation,anti-apoptotic and anti-aging capabilities of oxidatively damaged osteoblasts.Ferulic acid can simultaneously improve the anti-apoptosis,anti-senescence,cell differentiation and mineralization ability of oxidatively damaged osteoblasts.The mechanism of PP-?may be related to ECM-receptor interaction,Focal adhesion,cellular-senescence and PI3K-Akt signal pathway.The mechanism of PP-? may be related to ECM-receptor interaction,Apoptosis-multiple species,Focal adhesion,and PI3K-Akt signal pathway.The mechanism of ferulic acid may be related to the pathways of ECM-receptor interaction,Focal adhesion and PI3K-Akt signal pathway.