摘要
目的探讨在动脉粥样硬化(AS)相关的缺氧和氧化低密度脂蛋白(ox-LDL)环境中,缺氧诱导因子-1α(HIF-1α)对巨噬细胞表面髓系DAP-12相关凝集素-1(MDL-1)表达的调控作用,并分析其对巨噬细胞生物学特性的影响。方法本研究于2022年3月在新疆医科大学临床医学研究院心血管病研究所进行。实验将RAW 264.7小鼠单核巨噬细胞分为四组:空白对照组、缺氧+ox-LDL组、缺氧+ox-LDL+DMOG组和缺氧+ox-LDL+2ME2组。通过WesternBlot技术分析各组巨噬细胞相关凝集素-1(MDL-1)、缺氧诱导因子-1α(HIF-1α)、细胞周期素D1(CCND1)、胱天蛋白酶3(Caspase3)、胱天蛋白酶1(Caspase1)、Nod样受体蛋白3(NLRP3)的表达差异。利用CCK-8和台盼蓝染色法评估细胞增殖和活力。LDH、IL-1β、IL-6、TNF-α等炎性因子的含量则通过ELISA试剂盒测定。结果本研究评估了RAW 264.7小鼠单核巨噬细胞在不同处理条件下的蛋白表达和细胞功能变化。在缺氧+ox-LDL组中,与空白对照组相比,MDL-1、HIF-1α、Caspase3、Caspase1、NLRP3的表达显著增加,同时CCND1表达显著降低(P<0.05)。此外,该处理组的LDH、IL-1β、IL-6和TNF-α水平也显著增加[(340.267±41.338)U/L比(154.667±20.044)U/L,(89.251±8.488)pg/ml比(49.623±5.070)pg/ml,(36.642±4.730)ng/ml比(20.619±0.884)ng/ml,1104.515±46.411 pg/m比(785.291±40.339)pg/ml,P<0.01],细胞增殖能力和活细胞率显著下降[OD值(0.296±0.065)比(0.570±0.032),活细胞率(69.108±7.816)%比(98.147±0.781)%,P<0.01]。DMOG处理组相较于缺氧+ox-LDL组表现出蛋白表达和炎症标志物的显著下降,细胞增殖和活细胞率得到改善(P<0.01)。相反,2ME2处理加剧了炎症反应和细胞损伤,蛋白表达和炎症指标均有所增加,细胞活性进一步降低(P<0.01)。结论HIF-1α在缺氧和ox-LDL环境下调节MDL-1表达,进而影响巨噬细胞的增殖、存活和炎性因子的分泌功能,该生物学特性可能在AS的病理过程中发挥重要作用。
Objective To investigate the regulatory effect of hypoxia-inducible factor-1α(HIF-1α)on the expression of myeloid DAP-12-associated lectin-1(MDL-1)on macrophage surfaces in the environment associated with atherosclerosis(AS),characterized by hypoxia and oxidized low-density lipoprotein(ox-LDL)and the impact of this regulation on the biological characteristics of macrophages.Methods This study was conducted in March 2022 at the Institute of Cardiovascular Diseases,Clinical Medical Research Academy,Xinjiang Medical University.The RAW 264.7 murine monocyte-macrophages were divided into four groups:the control group,hypoxia+ox-LDL group,hypoxia+ox-LDL+DMOG group and hypoxia+ox-LDL+2ME2 group.The expression levels of MDL-1,hypoxia-inducible factor-1α(HIF-1α),cyclin D1(CCND1),cysteine protease3(Caspase3),cysteine protease-1(Caspase1)and NOD-like receptor protein 3(NLRP3)were analyzed using Western Blot technique.The cell proliferation and viability were assessed using the CCK-8 assay and trypan blue staining method.The levels of inflammatory markers such as LDH,IL-1β,IL-6 and TNF-αwere determined using ELISA kits.Results The changes in protein expression and cellular functions of RAW 264.7 murine monocyte macrophages under various treatment conditions were evaluated.In the hypoxia+ox-LDL group,compared to the control group,there was a significant increase in the expression of MDL-1,HIF-1α,Caspase3,Caspase1 and NLRP3,while the expression of CCND1 was significantly reduced(P<0.05);additionally,the levels of LDH,IL-1β,IL-6 and TNF-αsignificantly increased[(340.267±41.338)U/L vs.(154.667±20.044)U/L,(89.251±8.488)pg/ml vs.(49.623±5.070)pg/ml,(36.642±4.730)ng/ml vs.(20.619±0.884)ng/ml,1104.515±46.411 pg/m vs.(785.291±40.339)pg/ml,P<0.01],but there was a significant decrease in cell proliferation capacity and viability[OD value(0.296±0.065)vs.(0.570±0.032),viability(69.108±7.816)%vs.(98.147±0.781)%,P<0.01].The hypoxia+ox-LDL+DMOG group,compared to the hypoxia+ox-LDL group,showed significant decrease in protein expression and inflammatory markers,and improvement in cell proliferation and viability(P<0.01).In contrast,there were exacerbated inflammatory responses and cellular damage in the hypoxia+ox-LDL+2ME2 group,with increased expression of proteins and inflammatory markers and further reduced cellular activity(P<0.01).Conclusion HIF-1αregulates the expression of MDL-1 in the environment of hypoxia and ox-LDL,subsequently affecting the proliferation,survival and secretion of inflammatory factors in macrophages.These biological characteristics may play a significant role in the pathophysiological process of AS.
作者
卡德艳木·阿布力米提
施林
严飞
Kadeyanmu·ABULIMITI;SHI Lin;YAN Fei(Department of Cardiology,The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China)
出处
《中国心血管病研究》
CAS
2024年第5期475-480,共6页
Chinese Journal of Cardiovascular Research
基金
新疆维吾尔自治区自然科学基金(2021D0C315)。
