小檗碱通过SPHK2/S1P/ERK5通路对ox-LDL诱导的巨噬细胞胞葬功能障碍的影响OA
Effects of berberine on low-density lipoprotein(ox-LDL)-induced efferocytosis dysfunction in macrophages via the SPHK2/S1P/ERK5 pathway
目的 探究小檗碱(BBR)对氧化低密度脂蛋白(ox-LDL)诱导的巨噬细胞胞葬功能障碍及炎症反应的作用及机制.方法 以ox-LDL诱导人THP-1巨噬细胞胞葬功能障碍为模型.将细胞分为对照组、ox-LDL组、ox-LDL+BBR(5、10、20 μmol/L)组、ox-LDL+BBR+ABC294640 组、ox-LDL+BBR+ABC294640+S1P组、ox-LDL+BBR+BIX02189 组,ox-LDL组用 80 μmol/L ox-LDL 处理24 h,其他组用 80 μmol/L ox-LDL处理 2 h后,根据实验需要加入BBR、SPHK2 抑制剂ABC294640(25 μmol/L)、外源性S1P(80 nmol/L)或MEK5 抑制剂BIX02189(100 nmol/L)共同孵育 24 h.通过荧光实验和流式细胞术检测巨噬细胞胞葬效率;用ELISA检测S1P含量;通过Western blot检测MerTK、AXL、TYRO3、SPHK1/2、MEK5、ERK5 和p-ERK5 蛋白表达水平.结果 与对照组相比,ox-LDL组胞葬效率降低(P<0.01,P<0.001),MerTK、AXL、TYRO3、MEK5、p-ERK5/ERK5、SPHK2 蛋白表达水平降低(P<0.01,P<0.001),S1P含量降低(P<0.01).与ox-LDL组相比,20 μmol/L BBR处理组胞葬效率显著增加(P<0.01),MerTK、AXL、TYRO3、MEK5、p-ERK5/ERK5、SPHK2 蛋白表达水平升高(P<0.05,P<0.01).与ox-LDL+20 μmol/L BBR组相比,加入ABC294640 后胞葬效率降低(P<0.01),MerTK、AXL、TYRO3、MEK5、p-ERK5/ERK5、SPHK2 蛋白表达水平下降(P<0.05,P<0.01).与 ox-LDL+20 μmol/L BBR+ABC294640 组相比,加入S1P后胞葬效率增加(P<0.01,P<0.05),MerTK、AXL、TYRO3、MEK5、p-ERK5/ERK5、SPHK2 蛋白表达水平升高(P<0.05,P<0.01).与ox-LDL+20 μmol/L BBR组相比,加入BIX02189 后胞葬效率降低(P<0.01),MerTK、AXL、TYRO3、MEK5、p-ERK5/ERK5 蛋白表达水平下降(P<0.05,P<0.01).结论 BBR可能通过SPHK2/S1P/ERK5 途径发挥作用,减轻ox-LDL诱导的巨噬细胞胞葬功能障碍.
Objective To investigate the effects and mechanisms of berberine(BBR)on oxidized low-density lipoprotein(ox-LDL)-induced macrophage efferocytosis dysfunction and inflammatory response.Methods Using an ox-LDL-induced efferocytosis dysfunction model in human THP-1-derived macrophages,the cells were divided into the following groups:control,ox-LDL,ox-LDL+BBR(5,10,20 μmol/L),ox-LDL+BBR+ABC294640,ox-LDL+BBR+ABC294640+S1P,and ox-LDL+BBR+BIX02189 groups.The ox-LDL group was treated with 80 μmol/L ox-LDL for 24 h.In other groups,cells were pretreated with 80 μmol/L ox-LDL for 2 h,followed by co-incubation for 24 h with BBR,the SPHK2 inhibitor ABC294640(25 μmol/L),exogenous S1P(80 nmol/L),or the MEK5 inhibitor BIX02189(100 nmol/L),as required.Macrophage efferocytosis efficiency was assessed using fluorescence microscopy and flow cytometry.S1P levels were measured by ELISA.Protein expression levels of MerTK,AXL,TYRO3,SPHK1/2,MEK5,ERK5,and p-ERK5 were determined by Western blot.Results Compared with the control group,the ox-LDL group exhibited decreased efferocytosis efficiency(P<0.001,P<0.01),reduced protein expression levels of MerTK,AXL,TYRO3,MEK5,p-ERK5/ERK5,and SPHK2(P<0.01,P<0.001),and decreased S1P levels(P<0.01).Compared with the ox-LDL group,treatment with 20 μmol/L BBR significantly increased efferocytosis efficiency(P<0.01)and elevated protein expression levels of MerTK,AXL,TYRO3,MEK5,p-ERK5/ERK5,and SPHK2(P<0.05,P<0.01).Compared with the ox-LDL+20 μmol/L BBR group,the addition of ABC294640 decreased efferocytosis efficiency(P<0.01)and reduced protein expression levels of MerTK,AXL,TYRO3,MEK5,p-ERK5/ERK5,and SPHK2(P<0.05,P<0.01).Compared with the ox-LDL+20 μmol/L BBR+ABC294640 group,the addition of S1P increased efferocytosis efficiency(P<0.01,P<0.05)and elevated protein expression levels of MerTK,AXL,TYRO3,MEK5,p-ERK5/ERK5,and SPHK2(P<0.05,P<0.01).Compared with the ox-LDL+20 μmol/L BBR group,the addition of BIX02189 decreased efferocytosis efficiency(P<0.01)and reduced protein expression levels of MerTK,AXL,TYRO3,MEK5,and p-ERK5/ERK5(P<0.05,P<0.01).Conclusion BBR may exert its effects through the SPHK2/S1P/ERK5 pathway to alleviate ox-LDL-induced macrophage efferocytosis dysfunction.
付傲妮;刘婉婷;李婧;杨皓天;李朝荃;谢玉鑫;雷偲;易光辉
南华大学衡阳医学院心血管疾病研究所,湖南省动脉硬化重点实验室,湖南动脉硬化疾病国际科技合作基地,湖南 衡阳 421001南华大学衡阳医学院心血管疾病研究所,湖南省动脉硬化重点实验室,湖南动脉硬化疾病国际科技合作基地,湖南 衡阳 421001||南华大学湖南省分子靶向新药研究合作创新中心药物与药理研究所,湖南 衡阳 421001南华大学衡阳医学院基础医学院,湖南 衡阳 421001南华大学衡阳医学院心血管疾病研究所,湖南省动脉硬化重点实验室,湖南动脉硬化疾病国际科技合作基地,湖南 衡阳 421001南华大学衡阳医学院心血管疾病研究所,湖南省动脉硬化重点实验室,湖南动脉硬化疾病国际科技合作基地,湖南 衡阳 421001南华大学衡阳医学院心血管疾病研究所,湖南省动脉硬化重点实验室,湖南动脉硬化疾病国际科技合作基地,湖南 衡阳 421001||南华大学湖南省分子靶向新药研究合作创新中心药物与药理研究所,湖南 衡阳 421001南华大学衡阳医学院心血管疾病研究所,湖南省动脉硬化重点实验室,湖南动脉硬化疾病国际科技合作基地,湖南 衡阳 421001南华大学衡阳医学院心血管疾病研究所,湖南省动脉硬化重点实验室,湖南动脉硬化疾病国际科技合作基地,湖南 衡阳 421001||南华大学湖南省分子靶向新药研究合作创新中心药物与药理研究所,湖南 衡阳 421001||南华大学衡阳医学院基础医学院,湖南 衡阳 421001
医药卫生
小檗碱胞葬作用巨噬细胞鞘氨醇激酶 2细胞外信号调节激酶 51-磷酸鞘氨醇
berberineefferocytosismacrophagesphingosine kinase 2extracellular signal-regulated kinase 5sphingosine-1-phosphate
《湖南中医药大学学报》 2026 (5)
889-897,9
国家自然科学基金项目(81770490)湖南省科技计划项目(2020JJ4535).
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