Effects Of Cyanidin-3-O-Glucoside On Osteoblasts Proliferation And Differentiation By Pigk/Ly6a

ObjectiveTo explore the effect and mechanism of Cyanidin-3-O-Glucoside(C3G)on the proliferation and differentiation of MC3T3-E1 cells,and provide a theoretical basis for the research of anthocyanins as the main component of anti-osteoporosis health care products and drugs.MethodsMC3T3-E1 cells were purchased from Shanghai Cell Bank,Chinese Academy of Sciences.The proliferation of MC3T3-E1 cells treated with 0 50 100 and 200μmol/L C3 G for 24 h was evaluated by CCK8 assay.The mineralization of cells treated with 050 100 and 200μmol/L C3 G for 14 d was evaluated by alizarin red staining.RNA-seq was used to detect the m RNA differential expression of 0 and 100μmol/L C3 G groups,GO enrichment analysis and KEGG pathway enrichment analysis were performed for the differential genes.The m RNA expression and protein expression of Pigk and Ly6 a in osteoblasts which treated with 0 and 100μmol/L C3 G was detected by q PCR and Western Blot.Pigk-si RNA was transfected into MC3T3-E1 cells,and the m RNA expression level of Pigk was detected by q PCR to evaluate the knockout effect and screen the si RNA sequence.MC3T3-E1 cells were divided into Control-si RNA group,Pigk-si RNA group,Control-si RNA +C3G group and Pigk-si RNA+C3G group.The m RNA expressions of Alp,Runx2,Atf4,Ly6 a and Pigk were detected by q PCR,and the protein expressions of Alp,Runx2,Atf4,Ly6 a and Pigk were detected by Western Blot.Results1.Effect of C3 G on proliferation and differentiation of MC3T3-E1cellsCCK8 results showed that compared with the control group,the proliferation rate of 100 and 200μmol/L C3 G groups was significantly increased,P<0.01;The results of alizarin red staining showed that there were more mineralized nodules in 100 and200μmol/L C3 G groups compared with the control group,but there was no significant effect at 50μmol/L C3 G.2.Sequencing analysis of the effect of C3 G on MC3T3-E1 cell transcriptome51 DEGs were detected by RNA-seq(P≤0.05,mean FPKM>0.5,fold change>1.2)There were 34 up-regulated genes and 17 down-regulated genes.PIGK and Ly6 a were up-regulated.GO analysis of DEGs revealed that several significantly enriched up-regulated GO terms were related to GPI glycolipid synthesis,which were all related to Pigc and Pigk,including: GPi-anchors the biosynthesis process;PH value decreased;GPI anchors metabolic processes;Glucolipid biosynthesis,etc.By analyzing KEGG pathway of DEGS,it was found that 5 significantly enriched signal pathway upregulation pathways contained glucosylphosphatidylinositol anchored biosynthesis.3.Effect of C3 G on gene and protein expression of Pigk and Ly6 a in MC3T3-E1 cellsThe results showed that compared with the control group,the m RNA expression of Pigk(P<0.01)and Ly6a(P<0.01)in the C3 G group was up-regulated,and the difference was statistically significant.Compared with the control group,the protein expression of Pigk(P<0.05)and Ly6a(P<0.05)was up-regulated,and the difference was statistically significant.4.Effect of C3 G on proliferation of MC3T3-E1 cells silenced by Pigk-si RNAThe results of CCK8 showed that compared with the Control-si RNA group,there was not any statistically significant difference in cell proliferation rate between the Pigk-si RNA group and the Control-si RNA group,P>0.05.Compared with the Control-si RNA+C3G group,the cell proliferation rate in the Pigk-si RNA+C3G group decreased,and the difference was statistically significant,P<0.05.Compared with pigk-si RNA group,there was no significant differences in cell proliferation rate between Pigk-si RNA+C3G group,P>0.05.5.C3 G effects on m RNA and protein expression in Pigk-si RNA silencing MC3T3-E1 cellsCompared with the Control-si RNA group,the m RNA expression of Alp decreased and the m RNA expression of Atf4 increased in the Pigk-si RNA group,the difference was statistically significant,P<0.05.Compared with the Control-si RNA+C3G group,the m RNA expression of Alp and Atf4 in the Pigk-si RNA +C3G group decreased,and the difference was statistically significant,P<0.05.Compared with the Pigk-si RNA group,the m RNA expression of Alp was increased,and the difference was statistically significant,P<0.05.,but there was no significant differences in Atf4 m RNA expression,P>0.05.Compared with the Control-si RNA group,the protein expression of Alp and Atf4 in the Pigk-si RNA group was increased,and the difference was statistically significant,P<0.05.Compared with the Control-si RNA+C3G group,the protein expression of Alp and Atf4 in Pigk-si RNA +C3G group decreased,P<0.05.Compared with the Pigk-si RNA group,the increase of Atf4 protein expression in the Pigk-si RNA+C3G group was statistically significant,P>0.05,but there was no statistical significance in the protein expression of Alp,P>0.05.There was no significant differences in Runx2 gene expression among the four groups,P>0.05.Compared with the Control-si RNA group,the m RNA expression and protein expression of Ly6 a and Pigk decreased in Pigk-si RNA group,P<0.05.Compared with the Pigk-si RNA group,the protein expression of Ly6 a in the Pigk-si RNA+C3G group decreased,the difference was statistically significant,P<0.05,but there was no statistical significance in the expression of m RNA and protein of Pigk,P>0.05.Compared with the Control-si RNA+C3G group,the m RNA and protein expression of Pigk and the protein expression of Ly6 a in the Pigk-si RNA+C3G group were decreased,and the differences were statistically significant,P<0.05.Conclusion1.Pigk affects the differentiation of MC3T3-E1 cells by organising the gene and protein expressions of Alp,Atf4 and Ly6 a.2.C3 G promotes gene and protein expression of Pigk and Ly6 a in MC3T3-E1.3.C3 G governs the proliferation and differentiation of MC3T3-E1 cells by affecting Ly6 a through Pigk.