达格列净对人单核细胞系THP-1细胞增殖、凋亡,脂质水平的影响及相关机制研究

Effect and underlying mechanism of dapagliflozin in human monocytic THP-1 cell proliferation, apoptosis and lipid synthesis

目的 达格列净对人髓系白血病单核细胞(human myeloid leukemia mononuclear cells, THP-1)细胞增殖、凋亡,脂质水平的影响及相关机制研究。方法 THP-1细胞经100ng/ml佛波酯(phorbol ester, PMA)与100mM氧化低密度脂蛋白(oxidized low density lipoprotein, ox-LDL)诱导24h后,再添加不同浓度达格列净(0μg/L、37.5μg/L、75.0μg/L、150.0μg/L)继续培养。细胞培养72h后,采用细胞计数试剂盒(cell counting kit-8, CCK-8)测定细胞增殖水平,结晶紫染色测定单克隆数目,流式细胞仪检测细胞凋亡,生化仪测定细胞总胆固醇(total cholesterol, TC)、游离胆固醇(free cholesterol, FC)、胆固醇酯(cholesterol esters, CE)水平,反转录聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)法及蛋白印迹法测定微小核糖核酸-163(micro ribonucleic acid-163, miR-613)、肝X受体α(liver X receptor α, LXRα)、三磷酸腺苷结合盒转运蛋白A1(adenosine triphosphate binding cassette transporter A1,ABCA1)转录与表达水平。结果 与未加药THP-1细胞培养组相比,达格列净加药组凋亡率、miR-613转录水平升高(P<0.05),光密度(optical density, OD)值、存活率、单克隆形成数目、TC、FC、CE水平、LXRα与ABCA1转录及表达水平降低(P<0.05),且随着达格列净浓度的增加,凋亡率、miR-613转录水平逐渐升高(P<0.05),OD值、存活率、单克隆形成数目、TC、FC、CE水平、LXRα、ABCA1转录及蛋白表达水平逐渐降低(P<0.05)。结论 达格列净可抑制人单核细胞系THP-1细胞增殖,促进人单核细胞系THP-1细胞凋亡,降低其细胞脂质水平;其机制可能与达格列净上调miR-613转录降低了LXRα、ABCA1转录及蛋白表达从而间接抑制LXRα/ABCA1通路的激活有关。

Objective To explore the effect and underlying mechanism of dapagliflozin in human myeloid leukemia mononuclear cell line(THP1) proliferation, apoptosis and lipid synthesis. Methods THP-1 cells were differentiated into macrophages using phorbol-12-myristate-13-acetate(PMA, 100 ng/mL) and oxidized low-density lipoprotein(oxLDL, 100mM) for 24 hours, and then incubated with different concentrations of dapagliflozin(0μg/L, 37.5μg/L, 70.0μg/L, 150.0μg/L). After 72h of cell culture, cell proliferation was measured by cell counting kit-8(CCK-8). The number of monoclonal cells was measured by crystal violet staining. Cell apoptosis rate was measured by flow cytometry. And the levels of total cholesterol(TC), free cholesterol(FC) and cholesteryl esters(CE) were measured by biochemical analyzer. Meantime, the gene transcription and protein expression levels of micro ribonucleic acid-163(miR-613), liver X receptor α(LXRα) and adenosine triphosphate binding cassette transporter A1(ABCA1) were determined by reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting. Results Compared with non-drugged group, the apoptosis rate and the miR-613 expression level were increased(P<0.05), and the optical density(OD) value, survival rate, number of monoclonal cells, levels of TC, FC and CE, and the mRNA and protein expression levels of LXRα and ABCA1 were decreased in dapagliflozin treatment groups(P<0.05). With the increase of dapagliflozin concentration,the apoptosis rate and the miR-613 mRNA expression level were gradually increased(P<0.05), while the OD value, survival rate, number of monoclonal cells, serum levels of TC, FC and CE, and the mRNA and protein expression levels of LXRα and ABCA1 were gradually decreased(P<0.05). Conclusion Dapagliflozin can inhibit the proliferation and promote the apoptosis of human monocytic cell line THP-1 and reduce the lipid peroxidation. The mechanism may be related to the up-regulation of miR-613, and the down-regulation of LXRα and ABCA1 expression and then indirectly inhibiting the activation of LXRα/ABCA1 pathway.