异甘草素通过激活自噬抑制巨噬细胞中结核杆菌生存的实验研究  

Isoliquiritigenin Inhibits Survival of Mycobacterium tuberculosis in Macrophages by Activating Autophagy

作　　者：李彬[1] LI Bin(Department of Pulmonology,Henan Provincial Hospital of Traditional Chinese Medicine,Zhengzhou 450002,Henan,China)

机构地区：[1]河南省中医院肺病科,河南郑州450002

出　　处：《辽宁中医杂志》2024年第4期200-204,I0004,共6页Liaoning Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(81904165)。

摘　　要：目的探究异甘草素对H37Ra感染的巨噬细胞中结核杆菌的抑制作用及其涉及的机制。方法以5、10、25、50、75、100μmol/L异甘草素处理RAW264.7细胞24、48、72 h,MTT检测细胞活力。细胞被H37Ra感染6 h后:(1)利用10、25或50μM异甘草素处理细胞24 h;(2)利用50μg/mL雷帕霉素处理细胞2 h;(3)利用50μmol/L异甘草素和30 pg/mL EGF[epidermal growth factor,一种PI3K(phosphatidylinositol-3-hydroxykinase)/AKT激活剂]或5mM 3MA(一种自噬抑制剂)共处理细胞24 h。利用计数法测量细菌菌落总数;利用Western blotting检测PI3K/AKT/mTOR通路蛋白和自噬标志蛋白表达;利用免疫荧光检测LC3(Microtubule-associated protein 1 light chain 3)蛋白表达。结果不同浓度异甘草素处理不影响细胞活力。异甘草素或雷帕霉素处理感染后细胞显著减少细菌菌落总数,激活PI3K/AKT/mTOR通路,促进LC3-Ⅱ/LC3-Ⅰ蛋白相对表达量,增强LC3荧光强度,并抑制P62蛋白相对表达量。EGF处理逆转异甘草素诱导的自噬激活。3MA处理逆转异甘草素诱导的细菌菌落数下降。结论异甘草素通过抑制PI3K/AKT/mTOR通路,激活自噬,抑制感染H37Ra的巨噬细胞中结核杆菌生长。Objective To investigate the inhibitory effect of isoliquiritigenin(ISL)on Mycobacterium tuberculosis in H37Ra-infected macrophages and the mechanism involved.Methods RAW264.7 cells were treated with 5,10,25,50,75 or 100μmol/L ISL for 24,48 or 72 h,and MTT was used to detect cell viability.After cells were infected with H37Ra for 6 h:(1)cells were treated with 10,25 or 50μmol/L ISL for 24 h;(2)cells were treated with 50μg/mL rapamycin for 2 h;(3)cells were co-treated with 50μmol/L ISL and 30 pg/mL epidermal growth factor[EGF,a PI3K(phosphatidylinositol-3-hydroxykinase)/AKT pathway activator]or 5 mmol/L 3MA[an autophagy inhibitor]for 24 h.The total number of bacterial colonies was measured by counting method.The expressions of PI3K/AKT/mTOR pathway proteins and autophagy marker proteins were detected by Western blotting.Immunofluorescence was used to measure microtubule-associated protein 1 light chain 3(LC3)protein level.Results The treatment with different concentrations of ISL had no significant effect on cell viability.ISL or rapamycin treatment significantly reduced the total number of bacterial colonies,activated the PI3K/AKT/mTOR pathway,promoted the expressions of LC3-Ⅱ/LC3-Ⅰprotein expression,enhanced LC3 fluorescence intensity and reduced P62 protein expression.EGF treatment reversed ISL-induced autophagy activation.The treatment with 3MA reversed the decrease of bacterial colony number induced by ISL.Conclusion ISL induced autophagy by inhibiting PI3K/AKT/mTOR pathway activation,thereby suppressing the growth of Mycobacterium tuberculosis in H37Ra-infected macrophages.

关 键 词：异甘草素 PI3K/AKT/mTOR通路 自噬 H37RA 巨噬细胞 

分 类 号：R285[医药卫生—中药学]