高糖环境下成骨细胞PAD4表达和转录调控对成骨功能的影响

Effect of PAD4 expression and transcriptional regulation on osteogenic function in osteoblasts exposed to high glucose

目的:探究高糖环境下成骨细胞中肽基精氨酸脱亚胺酶4(PAD4)表达和转录调控对成骨细胞的作用及其相关机制。方法:分别使用正常(2 g/L)和高糖(4 g/L)培养基培养hFOB1.19成骨细胞,分为正常组和高糖组;并在高糖环境下通过慢病毒将sh-PAD4和sh-NC转染至hFOB1.19成骨细胞,分为sh-PAD4组和sh-NC组;实时荧光定量PCR(RT-qPCR)和蛋白质免疫印迹法(western blotting)检测各组细胞PAD4、H3cit、NFAT2、OCN和ALP表达,染色质免疫共沉淀PCR技术(ChIP-qPCR)检测PAD4、H3cit、H3K27ac在NFAT2、OCN和ALP转录起始位点(TSS)的富集,细胞计数试剂盒(CCK-8)法检测细胞增殖水平,茜素红S染色法测定细胞矿化能力。结果:与正常组比较,高糖组PAD4、H3cit、NFAT2、OCN和ALP表达升高,PAD4、H3cit、H3K27ac在NFAT2、OCN和ALP TSS富集水平增加,细胞增殖水平和矿化能力升高(P<0.05)。与sh-NC组比较,sh-PAD4组PAD4、H3cit、NFAT2、OCN和ALP表达降低,PAD4、H3cit、H3K27ac在NFAT2、OCN和ALP TSS富集水平降低,细胞增殖水平和矿化能力降低(P<0.05)。结论:高糖环境下PAD4及其介导的H3cit表达增加,后者在NFAT2、OCN和ALP TSS富集,促进成骨相关基因表达、成骨细胞增殖和分化。

Objective:To explore the effect of peptidyl arginine deiminase 4(PAD4)expression and transcriptional regulation on osteoblasts in high glucose environment and its related mechanism.Methods:hFOB1.19 osteoblasts were cultured in normal(2g/L)and high glucose(4g/L)medium and divided into normal group and high glucose group;sh-PAD4 and sh-NC were transfected into hFOB1.19 osteoblasts by lentivirus in high glucose environment and divided into sh-PAD4 group and sh-NC group.The expression of PAD4,H3cit,NFAT2,OCN and ALP were detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and western blotting.The enrichment of PAD4,H3cit and H3K27ac at the transcription start site(TSS)of NFAT2,OCN and ALP was detected by chromatin immunoprecipitation PCR(ChIP-qPCR).Cell proliferation was determined by cell counting kit-8(CCK-8)assay,and cell mineralization capacity was determined by alizarin red S staining.Results:Compared with the control group,the expression of PAD4,H3cit,NFAT2,OCN and ALP in the high glucose group increased,the enrichment levels of PAD4,H3cit and H3K27ac in NFAT2,OCN and ALP TSS in the high glucose group increased,and the cell proliferation level and mineralization ability increased(P<0.05).Compared with the sh-NC group,the expression of PAD4,H3cit,NFAT2,OCN and ALP in the sh-PAD4 group decreased,the enrichment levels of PAD4,H3cit and H3K27ac in NFAT2,OCN and ALP TSS decreased,the cell proliferation level and mineralization ability decreased(P<0.05).Conclusion:The expression of PAD4 and its mediated H3cit increase in the high glucose environment, and the latter is enriched in NFAT2, OCN and ALP TSS, which promotes the expression ofosteogenic-related genes, proliferation and differentiation of osteoblasts.