达格列净对人单核细胞系TH...质水平的影响及相关机制研究_罗恩斯.pdf

转化医学杂志 2022年12月 第11卷 第6期Translational Medicine Journal,Vo1.11 NO.6,Dec 2022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罗恩斯，董志会，陈冬萍，杨群峰，万杰君 摘要 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(0g/L、37.5g/L、75.0g/L、150.0g/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(liver X receptor,LXR)、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(P0.05)，光密度(optical density,OD)值、存活率、单克隆形成数目、TC、FC、CE水平、LXR与ABCA1转录及表达水平降低(P0.05)，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(P0.05)，OD值、存活率、单克隆形成数目、TC、FC、CE水平、LXR、ABCA1转录及蛋白表达水平逐渐降低(P0.05)。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LXR、ABCA1转录及蛋白表达从而间接抑制LXR/ABCA1通路的激活有关。关键词 达格列净；单核细胞；细胞活性；作用机制 中图分类号 R733.7 文献标志码 A 文章编号 2095-3097（2022）06-0341-06doi:10.3969/j.issn.2095-3097.2022.06.003Effect and underlying mechanism of dapagliflozin in human monocytic THP-1 cellproliferation,apoptosis and lipid synthesisLUO Ensi1,DONG Zhihui2,CHEN Dongping2,YANG Qunfeng1,WAN Jiejun1(1.Department of Endocrinology,Dongguan Binhai Bay Central Hospital,Dongguan Guangdong 523900,China;2 Department of Central Laboratory,Dongguan Binhai Bay Central Hospital,Dongguan Guangdong 523900,China)Abstract 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 differentiatedinto 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(0g/L,37.5g/L,70.0g/L,150.0g/L).After 72h of cell culture,cell proliferation was measured by cell counting kit-8(CCK-8).The number ofmonoclonal cells was measured by crystal violet staining.Cell apoptosis rate was measured by flow cytometry.And thelevels 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 (LXR)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,theapoptosis rate and the miR-613 expression level were increased(P0.05),and the optical density(OD)value,survivalrate,number of monoclonal cells,levels of TC,FC and CE,and the mRNA and protein expression levels of LXR andABCA1 were decreased in dapagliflozin treatment groups(P0.05).With the increase of dapagliflozin concentration,the apoptosis rate and the miR-613 mRNA expression level were gradually increased(P0.05),while the OD value,sur基金项目 广东省医学科学技术研究基金资助项目(B2021400)；东莞市滨海湾中心医院院内科研资助项目(2020012)作者单位 523900 广东 东莞,东莞市滨海湾中心医院内分泌科(罗恩斯，杨群峰，万杰君)；中心实验室(董志会，陈冬萍)341转化医学杂志 2022年12月 第11卷 第6期Translational Medicine Journal,Vo1.11 NO.6,Dec 2022vival rate,number of monoclonal cells,serum levels of TC,FC and CE,and the mRNA and protein expression levels ofLXR and ABCA1 were gradually decreased(P0.05).ConclusionDapagliflozin 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 LXR and ABCA1 expression and then indirectly inhibiting the activation of LXR/ABCA1 pathway.Key words Dapagliflozin;Monocytes;Cell activity;Mechanism单核细胞在动脉粥样硬化的发生发展中起关键作用。单核细胞中胆固醇流出的失衡最终导致转化为载脂泡沫细胞，这些泡沫细胞积聚在动脉壁成为动脉粥样硬化病变早期的标志1-2。过氧化物酶体增殖物激活受体c(peroxisome proliferator activated receptor c,PPARc)是一种核受体，在胆固醇稳态和动脉粥样硬化形成的关键生物学过程中起关键作用3。一旦被激活，PPARc会诱导一系列参与单核细胞中胆固醇流出的基因。肝 X受体(liverX receptor,LXR)被确定为 PPARc 的一种靶基因4。LXR最典型的作用之一是促进胆固醇逆向转运(reverse cholesterol transport,RCT)，即胆固醇从外周输送到肝脏进行排泄5。RCT的第一步是单核细胞的胆固醇流出，单核细胞的胆固醇通过三磷酸腺 苷 结 合 盒 转 运 蛋 白 A1(adenosine triphosphatebinding cassette transporter A1,ABCA1)和其他转运蛋白转移到载脂蛋白A-I(apolipoprotein A-I,apoA-I)和高密度脂蛋白(high-density lipoprotein,HDL)6。ABCA1是最早发现的LXR靶基因之一，可促进单核细胞胆固醇外流并维持细胞甾醇稳态。ABCA1的突变消除了胆固醇流出并增加了发生动脉粥样硬化的风险7。miRNA是小的(约1922 nt)非编码RNA，主要通过与靶 mRNA 的 3 端非翻译的区域(3-Untranslated Region,3-UTR)结合来调节基因表达，从而导致mRNA降解或翻译抑制。miRNA在多种正常和疾病相关生物过程的细胞过程中发挥重要作用，包括代谢稳态、肿瘤发生和心脏发生。最近，越来越多的证据表明miRNA参与胆固醇代谢。使用生物信息学分析，预测几种miRNA可能靶向人LXR和ABCA1mRNA的3-UTR，并且miR-613具有较高的调节LXR和ABCA1的概率8。先前的报道表明，miR-613与甲状腺乳头状癌、胃癌、乳腺癌和脂肪生成有关。然而，尚不清楚miR-613是否通过靶向人髓系白血病单核细胞(human myeloidleukemia mononuclear cells,THP-1)中的 LXR 和ABCA1参与调节胆固醇流出。达格列净是一种选择性钠依赖性葡萄糖转运蛋白 2 抑制剂(sodiumdependent glucose transporter 2 inhibitor,SGLT2i)，作用于脏近端肾小管，抑制其