达格列净在ox-LDL诱导形成的THP-1源性泡沫细胞焦亡中的作用  

The role of dapagliflozin in ox-LDL-triggered pyroptosis of THP-1-derived foam cells

作　　者：龚才伟 赵广建 刘大男 欧航君 赵权威 李辉 Gong Caiwei;Zhao Guangjian;Liu Danan;Ou Hangjun;Zhao Quanwei;Li Hui(Dept of Cardiology,Affiliated Hospital of Guizhou Medical University,Guiyang 550004;Institute of Medical Sciences,Guizhou Medical University,Guiyang 550004)

机构地区：[1]贵州医科大学附属医院心内科,贵阳550004 [2]贵州医科大学医学科学研究所,贵阳550004

出　　处：《安徽医科大学学报》2023年第8期1366-1373,共8页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81660083);贵州省科技计划项目(编号:黔科合平台人才[2020]5014);贵州省高层次创新型人才培养计划项目(编号:黔科合人才[2015]4026号)。

摘　　要：目的探讨达格列净(DAPA)在氧化低密度脂蛋白(ox-LDL)诱导形成的人髓系白血病单核细胞(THP-1)源性泡沫细胞焦亡中的作用。方法通过ox-LDL诱导THP-1源性巨噬细胞构建THP-1源性泡沫细胞焦亡模型。设置实验分组为:空白对照(NC)组、ox-LDL组和药物干预(ox-LDL+DAPA)组;以油红O法检测巨噬细胞泡沫化水平;细胞增殖与毒性检测试剂盒(CCK-8)检测DAPA对泡沫细胞活力的影响;Hoechst 33342/PI双染检测THP-1源性泡沫细胞焦亡;细胞免疫荧光双染检测DAPA对泡沫细胞焦亡中焦亡关键因子Caspase-1表达的影响;采用微量酶标法检测细胞培养基中乳酸脱氢酶(LDH)活性;采用qRT-PCR和Western blot分别检测核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、半胱氨酸天冬氨酸蛋白水解酶-1(Caspase-1)、凋亡相关斑点样蛋白(ASC)、消皮素D(GSDMD)、白细胞介素(IL)-18、IL-1βmRNA和蛋白表达水平。结果CCK-8法检测结果提示DAPA的最佳干预浓度为10μmol/L;油红O染色结果示THP-1巨噬细胞源性泡沫细胞焦亡模型构建成功;与NC组比较,ox-LDL组中NLRP3、Caspase-1、ASC、GSDMD、IL-18、IL-1β mRNA和蛋白表达水平均升高(P<0.05),碘化丙啶(PI)阳性细胞数及LDH活性增加(P<0.05),Caspase-1的荧光强度增强,细胞胞质内的红色脂滴增多;予DAPA干预后,ox-LDL+DAPA组中前述焦亡相关因子的mRNA和蛋白表达水平均降低(均P<0.05),PI阳性细胞数及LDH活性降低(P<0.05),Caspase-1的荧光强度减弱,细胞胞质内的红色脂滴减少(P<0.05)。结论DAPA对ox-LDL诱导形成的THP-1源性泡沫细胞焦亡具有抑制作用。Objective To investigate the role of dapagliflozin(DAPA)in ox-LDL-induced pyroptosis of human myeloid leukemia monocytes(THP-1)derived foam cells.Methods THP-1-derived foam cell pyroptosis model was constructed by ox-LDL-induced THP-1derived macrophages.The experimental groups were set as follows:the blank control group(NC),the ox-LDL group(ox-LDL),and the drug intervention group(ox-LDL+DAPA).Oil Red O staining was used to detect the foam cell levels of macrophages.The cell proliferation and toxicity assay kit was used to detect the effect of DAPA on foam cell viability.Hoechst 33342/propidium iodide(PI)double staining was used to detect THP-1 derived foam cell pyroptosis.Cell immunofluorescence double staining was used to detect the effect of DAPA on the expression of pyroptosis key factor Caspase-1 in foam cells.The activity of lactate dehydrogenase(LDH)in the cell culture medium was detected using a microplate enzyme-linked immunosorbent assay.qRT-PCR and Western blot were used to detect the mRNA and protein expression levels of Nod-like receptor pyrin domain containing 3(NLRP3),cystein-containing aspartate-specific protease-1(Caspase-1),apoptosis-associatedspeck-like protein containing CARD(ASC),gasdermin-D(GSDMD),interleukin(IL)-18 and IL-1β,respectively.Results The CCK-8 assay indicated that the optimal intervention concentration of DAPA was 10μmol/L.Oil Red O staining confirmed the successful construction of the THP-1-derived foam cell pyroptosis model.Compared with the blank control group,the expression levels of NLRP3,Caspase-1,ASC,GSDMD,IL-18,IL-1βmRNA and protein significantly increased in ox-LDL group(P<0.05),as well as the number of PI-positive cells and LDH activity(P<0.05),the fluorescence intensity of Caspase-1 and the number of redlipid droplets in the cytoplasm of the cells.However,these effects were significantly reversed after DAPA intervention in the ox-LDL+DAPA group(P<0.05).Conclusion DAPA inhibits ox-LDL-induced pyroptosis in THP-1-derived foam cells.

关 键 词：达格列净 人髓系白血病单核细胞 泡沫细胞 细胞焦亡 氧化低密度脂蛋白 

分 类 号：R541.4[医药卫生—心血管疾病]