基于磷脂酰肌醇3激酶与苏氨酸蛋白激酶1信号通路小檗碱调节巨噬细胞极化机制研究  

Berberine regulates polarization of macrophages:a mechanistic study based on PI3K/Akt1 signaling pathway

作　　者：陈玉善[1] 王婷婷 孟中华 尚莎莎 郑峻萌 宗永华 司春婴[1] 梁亚州 关怀敏[1] Chen Yushan;Wang Tingting;Meng Zhonghua;Shang Shasha;Zheng Junmeng;Zong Yonghua;Si Chunying;Liang Yazhou;Guan Huaimin(Heart Center,the First Affiliated Hospital of Henan University of Chinese Medicine,,Zhengzhou 450000,Henan Province,China)

机构地区：[1]河南中医药大学第一附属医院心脏中心,郑州450000

出　　处：《中华老年心脑血管病杂志》2024年第6期694-698,共5页Chinese Journal of Geriatric Heart,Brain and Vessel Diseases

基　　金：国家重点研发计划(2020YFC2004706);国家自然科学基金(82004311);河南省中医药科学研究专项课题(2019JDZX2044);河南省中医药科学研究专项课题(2022JDZX026);河南省中医药科学研究专项课题(2024ZY2026);河南省卫生健康委国家中医药传承创新专项课题(2023ZXZX1001)。

摘　　要：目的探究小檗碱对氧化型低密度脂蛋白(ox-LDL)诱导的人髓系白血病单核细胞(THP-1)巨噬细胞极化的影响和可能作用机制。方法采用佛波酯(PMA)将THP-1细胞诱导成巨噬细胞,加入不同浓度的小檗碱,分别为对照组、小檗碱5μmol/L组、小檗碱10μmol/L组、小檗碱20μmol/L组、小檗碱40μmol/L组、小檗碱50μmol/L组,干预24 h或48 h。采用细胞计数8试剂盒检测细胞活力,筛选小檗碱最佳浓度和时间。将PMA诱导的THP-1巨噬细胞分为空白组、模型组、小檗碱组、抑制剂组、小檗碱+抑制剂组。酶联免疫吸附测定检测诱导型一氧化氮合酶(iNOS)和转化生长因子β_(1)(TGF-β_(1))的含量;定量聚合酶链反应检测肿瘤坏死因子α(TNF-α)、精氨酸酶1(Arg1)、磷脂酰肌醇3激酶(PI3K)、苏氨酸蛋白激酶1(Akt1)mRNA表达情况;Western blot检测Akt1和磷酸化Akt1(p-Akt1)抗体蛋白含量。结果干预24 h时,与对照组比较,小檗碱40μmol/L组和50μmol/L组巨噬细胞活性降低(P<0.05);干预48 h时,小檗碱5μmol/L组、小檗碱10μmol/L组、小檗碱20μmol/L组、小檗碱40μmol/L组、小檗碱50μmol/L组巨噬细胞活性均低于对照组[(0.89±0.02)%、(0.82±0.03)%、(0.71±0.02)%、(0.62±0.03)%、(0.53±0.02)%vs(1.01±0.01)%,P<0.05]。小檗碱20μmol/L组处理24 h时对细胞活力影响不大,作为最佳浓度和时间。与空白组比较,模型组iNOS含量和TNF-αmRNA水平升高,TGF-β_(1)含量、Arg1 mRNA、PI3K mRNA、Akt1 mRNA及p-Akt1/Akt1蛋白表达水平降低(P<0.05);与模型组比较,小檗碱组iNOS含量和TNF-αmRNA水平降低,TGF-β_(1)含量、Arg1 mRNA、PI3K mRNA、Akt1 mRNA及p-Akt1/Akt1蛋白表达水平升高(P<0.05);与小檗碱组比较,小檗碱+抑制剂组iNOS含量和TNF-αmRNA水平升高,Arg1 mRNA、PI3K mRNA、Akt1 mRNA及p-Akt1/Akt1蛋白表达水平降低(P<0.05)。结论小檗碱可通过激活PI3K/Akt1通路,抑制ox-LDL诱导的THP-1巨噬细胞炎性反应,并抑制巨噬细胞向M1型极化,促进Objective To explore the effect and possible mechanism of berberine on the macrophage polarization of human myeloid leukemia monocytic cell line THP-1 induced by oxidized low-density lipoprotein(ox-LDL).Methods THP-1 cells were induced into macrophages by PMA,and then according to different concentrations of berberine,the cells were divided into control group,and 5,10,20,40 and 50μmol/L berberine groups.After intervention for 24 or 48 h,CCK8 assay was used to detect cell viability for optimal concentration and time of berberine treatment.PMA-induced THP-1 macrophages were assigned into blank group,model group(ox-LDL),berberine group,inhibitor group(phosphatidyl inositol 3-kinase(PI3K)inhibitor LY294002)and berberine+inhibitor group(berberine+LY294002).The contents of inducible nitric oxide synthase(iNOS)and TGF-β_(1) were detected by ELISA.qPCR was employed to measure the mRNA expression of TNF-α,arginase 1(Arg1),PI3K and protein kinase B Akt1,and Western blotting was applied to detect the protein levels of Akt1 and phosphorylated protein kinase B antibody(p-Akt1).Results In 24 h after intervention,the macrophage activity was significantly lower in the 40 and 50μmol/L berberine groups than the control group(P<0.05),and after 48 h,the activity in all the 5 doses of berberine groups was obviously lower than that in the control group[(0.89±0.02)%,(0.82±0.03)%,(0.71±0.02)%,(0.62±0.03)%and(0.53±0.02)%vs(1.01±0.01)%,P<0.05].Berberine treatment of 20μmol/L for 24 h had little effect on cell viability,and the dose and the time were regarded as the best concentration and time.Compared with the blank group,iNOS content and TNF-αmRNA level were increased in the model group,while TGF-β_(1) content,mRNA levels of Arg1,PI3K and Akt1,and p-Akt1/Akt1 protein levels were decreased(P<0.05).iNOS content and TNF-αmRNA level were decreased,while TGF-β_(1) content,mRNA levels of Arg1,PI3K and Akt1 and protein levels of p-Akt1/Akt1s were increased in the berberine group than the model group(P<0.05).Compared with the berb

关 键 词：小檗碱 脂蛋白类 LDL 巨噬细胞 磷酸肌醇3-激酶类 PI3K/Akt1信号通路 

分 类 号：R285[医药卫生—中药学]