Wu Chun-Lin, Hu Ya-Nan, Liu Yi-Qiang, Li Hui, Wen Quan
School of Basic Medical Sciences, Guangzhou University of Chinese Medicine Guangzhou 511400, China.
Ziwei Community Health Service Center of Longgang District People's Hospital Shenzhen 518100, China.
Zhongguo Zhong Yao Za Zhi. 2024 Dec;49(23):6439-6449. doi: 10.19540/j.cnki.cjcmm.20240912.702.
The aim of this study was to investigate the underlying mechanism of chrysophanol(Chr) in reducing inflammation and foam cell formation induced by oxidized low-density lipoprotein(ox-LDL) and to investigate the targets and pathways related to effects of Chr on coronary atherosclerosis, providing a theoretical basis for the development of new clinical drugs. RAW264.7 macrophages were cultured in vitro, and after determining the appropriate concentrations of Chr and ox-LDL for treating RAW264.7 macrophages using a cell counting kit-8(CCK-8), the macrophages were treated with different concentrations of Chr(10, 15 μmol·L(-1)) and ox-LDL(with or without 80 mg·mL(-1)) for 24 h. RAW264.7 macrophages were divided into four groups: control group, model group(80 mg·mL(-1) ox-LDL), treatment group(80 mg·mL(-1) ox-LDL+10 μmol·L(-1) Chr), and treatment group(80 mg·mL(-1) ox-LDL+15 μmol·L(-1) Chr). Lipid accumulation in each group was detected by oil red O staining. CD36 expression was analyzed by flow cytometry. Western blot was used to detect the expression of scavenger receptor class A1(SR-A1), scavenger receptor class B type Ⅰ(SR-B1), autophagy-related protein 5(Atg5), Beclin-1, autophagy adaptor protein p62(P62), the ratio of microtubule-associated protein light chain 3(LC3)Ⅱ to LC3Ⅰ(LC3Ⅱ/LC3Ⅰ), nuclear factor kappa B P65(NF-κB P65), inhibitor of κB kinase β(IKKβ), nuclear factor of κB inhibitor(IκB), high mobility group box protein 1(HMGB1), phosphatidylinositol 3-kinase(PI3K), protein kinase B(Akt), and phosphorylated mammalian target of rapamycin(mTOR). Real-time quantitative polymerase chain reaction(RT-qPCR) was used to detect the mRNA expression levels of ATP-binding cassette transporter A1(ABCA1), ATP-binding cassette transporter G1(ABCG1), interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), HMGB1, inducible nitric oxide synthase(iNOS), arginase 1(Arg1), macrophage galactose-type lectin-1(Mgl-1), and NF-κB P65. Immunofluorescence analysis was performed to determine the localization of HMGB1 in RAW264.7 cells in each group. The autophagy inhibitor 3-methyladenine(3-MA) was added as a control for reverse validation, and the RAW264.7 macrophages were divided into four groups again: control group, model group(80 mg·mL(-1) ox-LDL), treatment group(80 mg·mL(-1) ox-LDL + 15 μmol·L(-1) Chr), and inhibitor group(80 mg·mL(-1) ox-LDL+15 μmol·L(-1) Chr+3-MA). The results showed that Chr effectively reduced foam cell formation by regulating the expression levels of SR-A1, ABCA1, ABCG1, the LC3Ⅱ/LC3Ⅰ ratio, Atg5, Beclin-1, and p62, and inhibited the NF-κB/HMGB1-PI3K/Akt/mTOR signaling pathway. Moreover, the inhibitory effects of Chr on autophagy and the NF-κB/HMGB1-PI3K/Akt/mTOR pathway were reversed by the autophagy inhibitor 3-MA. In conclusion, Chr exhibits therapeutic potential for the treatment of atherosclerosis by inducing autophagy and modulating the NF-κB/HMGB1 and PI3K/Akt/mTOR pathways to inhibit the formation of macrophage inflammatory foam cells.
本研究旨在探讨大黄酚（Chr）减轻氧化型低密度脂蛋白（ox-LDL）诱导的炎症和泡沫细胞形成的潜在机制，并研究Chr对冠状动脉粥样硬化作用的相关靶点和途径，为开发新的临床药物提供理论依据。体外培养RAW264.7巨噬细胞，使用细胞计数试剂盒-8（CCK-8）确定用于处理RAW264.7巨噬细胞的Chr和ox-LDL的合适浓度后，用不同浓度的Chr（10、15 μmol·L⁻¹）和ox-LDL（含或不含80 mg·mL⁻¹）处理巨噬细胞24小时。将RAW264.7巨噬细胞分为四组：对照组、模型组（80 mg·mL⁻¹ ox-LDL）、治疗组（80 mg·mL⁻¹ ox-LDL + 10 μmol·L⁻¹ Chr）和治疗组（80 mg·mL⁻¹ ox-LDL + 15 μmol·L⁻¹ Chr）。通过油红O染色检测每组中的脂质积累。采用流式细胞术分析CD36表达。用蛋白质免疫印迹法检测A类清道夫受体1（SR-A1）、Ⅰ型B类清道夫受体（SR-B1）、自噬相关蛋白5（Atg5）、Beclin-1、自噬衔接蛋白p62（P62）、微管相关蛋白轻链3（LC3）Ⅱ与LC3Ⅰ的比值（LC3Ⅱ/LC3Ⅰ）、核因子κB P65（NF-κB P65）、κB激酶β抑制剂（IKKβ）、κB核因子抑制剂（IκB）、高迁移率族蛋白1（HMGB1）、磷脂酰肌醇3-激酶（PI3K）、蛋白激酶B（Akt）和磷酸化雷帕霉素靶蛋白（mTOR）的表达。采用实时定量聚合酶链反应（RT-qPCR）检测ATP结合盒转运体A1（ABCA1）、ATP结合盒转运体G1（ABCG1）、白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）、HMGB1、诱导型一氧化氮合酶（iNOS）、精氨酸酶1（Arg1）、巨噬细胞半乳糖型凝集素-1（Mgl-1）和NF-κB P65的mRNA表达水平。进行免疫荧光分析以确定每组RAW264.7细胞中HMGB1的定位。加入自噬抑制剂3-甲基腺嘌呤（3-MA）作为反向验证对照，将RAW264.7巨噬细胞再次分为四组：对照组、模型组（80 mg·mL⁻¹ ox-LDL）、治疗组（80 mg·mL⁻¹ ox-LDL + 15 μmol·L⁻¹ Chr）和抑制剂组（80 mg·mL⁻¹ ox-LDL + 15 μmol·L⁻¹ Chr + 3-MA）。结果表明，Chr通过调节SR-A1、ABCA1、ABCG1、LC3Ⅱ/LC3Ⅰ比值、Atg5、Beclin-1和p62的表达水平有效减少泡沫细胞形成，并抑制NF-κB/HMGB1-PI3K/Akt/mTOR信号通路。此外，自噬抑制剂3-MA逆转了Chr对自噬和NF-κB/HMGB1-PI3K/Akt/mTOR通路的抑制作用。总之，Chr通过诱导自噬和调节NF-κB/HMGB1以及PI3K/Akt/mTOR通路来抑制巨噬细胞炎性泡沫细胞的形成，从而展现出治疗动脉粥样硬化的潜力。
