摘要
目的:探讨黄芪建中汤对巨噬细胞极化及对癌症恶病质(CC)小鼠脂肪消耗的影响。方法:利用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱(UPLC-Q-Orbitrap HRMS)控制黄芪建中汤质量。(1)体外实验:制备黄芪建中汤含药血清,并通过细胞毒性实验筛选最佳浓度;培养小鼠单核巨噬细胞(RAW264.7)并随机分为6个组:空白组、经典活化巨噬细胞(M1)组、选择活化巨噬细胞(M2)组、黄芪建中汤干预空白(HQJZ+空白)组、黄芪建中汤干预M1(HQJZ+M1)组、黄芪建中汤干预M2(HQJZ+M2)组。通过实时定量聚合酶链式反应(Real-time PCR)检测各组巨噬细胞标志物:分化抗原86(CD86)、诱导型一氧化氮合成酶(iNOS)、甘露糖受体(CD206)和精氨酸酶1(Arg1)mRNA的相对表达情况。(2)体内实验:32只BALB/c小鼠随机分为正常组、模型组、醋酸甲羟孕酮(MPA)组、黄芪建中汤(HQJZ)组,除正常组外其余小鼠通过注射小鼠结肠癌细胞(CT-26)建立CC模型,随后MPA组和HQJZ组分别给予MPA(0.13 g·kg^(-1)·d^(-1))和HQJZ(13.13 g·kg^(-1)·d^(-1))灌胃,正常组和模型组给予等体积生理盐水灌胃,连续干预10 d。Real-time PCR检测小鼠附睾脂肪中巨噬细胞标志物iNOS、Arg1、CD86、CD206的表达水平及脂肪褐变相关解偶联蛋白1(UCP1)和过氧化物酶体增殖物激活受体γ(PPARγ)的mRNA相对表达情况;蛋白免疫印迹法(Western blot)检测UCP1、PPARγ蛋白的相对表达水平;微型电子计算机断层扫描(micro CT)检测小鼠剩余脂肪体积;苏木素-伊红(HE)染色评估小鼠脂肪褐变情况并计算病理评分。结果:在体外实验中,黄芪建中汤含药血清优势剂量的浓度为12.5%;Real-time PCR结果显示,与空白组比较,HQJZ+空白组Arg1 mRNA表达水平降低(P<0.05),iNOS和CD86 mRNA表达水平均升高(P<0.05);与M1组比较,HQJZ+M1组Arg1和CD206 mRNA表达水平降低(P<0.05);与M2组比较,HQJZ+M2组CD206 mRNA表达水平降低(P<0.05),CD86 mRNA表达水平升高(P<0.01)。在体内实验中,Real-time PCR结果表明,与正常组比较,模型组CD86和CD206 mRNA表达显著上升(P<0.01);与模型组比较,MPA组CD206 mRNA表达水平降低(P<0.01),CD206表达水平显著降低(P<0.01)。Western blot结果表明,与模型组比较,HQJZ组UCP1和PPARγ蛋白表达水平明显降低(P<0.05,P<0.01);micro CT结果表明,HQJZ组的白色脂肪总体积比模型组大(P<0.05);HE染色结果表明,与模型组比较,HQJZ组病理评分更低(P<0.05)。结论:黄芪建中汤可能通过促进巨噬细胞M1极化并抑制M2极化来抑制白色脂肪褐变,延缓CC小鼠脂肪消耗。
Objective:To investigate the effects of Huangqi Jianzhongtang(HQJZ)on macrophage polarization and fat consumption in cancer cachexia(CC)mice.Methods:Ultra-performance liquid chromatography-quadrupole/electrostatic field Orbitrap high-resolution mass spectrometry(UPLC-Q-Orbitrap HRMS)was used to control the quality of HQJZ.(1)In vitroexperiment:HQJZ-containing serum was prepared,and the optimal concentration was determined by cytotoxicity assay.Mouse monocyte-derived macrophages(RAW264.7)were cultured and randomly divided into six groups,including a blank group,a classically activated macrophages(M1)group,an alternatively activated macrophages(M2)group,a HQJZ+blank group,a HQJZ+M1 group,and a HQJZ+M2 group.The relative expression of macrophage marker genes CD86,inducible nitric oxide synthase(iNOS),CD206,and arginase-1(Arg1)was detected by real-time quantitative polymerase chain reaction(Real-time PCR).(2)In vivo experiment:Thirty-two BALB/c mice were randomly divided into a control group,a model group,a medroxyprogesterone acetate(MPA)group,and a HQJZ group.Except for the control group,the other mice were injected with CT-26 colon cancer cells to establish a CC model.Mice in the MPA and HQJZ groups were given MPA(0.13 g·kg^(-1)·d^(-1))or HQJZ(13.13 g·kg^(-1)·d^(-1))by gavage,respectively,while mice in the control and model groups were given an equal volume of saline by gavage,with interventions continued for 10 d.Real-time PCR was used to detect the expression of macrophage markers(iNOS,Arg1,CD86,CD206)and fat browning-related genes uncoupling protein 1(UCP1)and peroxisome proliferator-activated receptorγ(PPARγ)in epididymal adipose tissue.Western blot(WB)was used to detect protein expression levels of UCP1 and PPARγ.Micro-computed tomography(micro-CT)was used to measure residual fat volume,and hematoxylin-eosin(HE)staining was used to assess fat browning and calculate pathological scores.Results:In vitro,the dominant effective concentration of HQJZ-containing serum was 12.5%.Real-time PCR results showed that,compared with the blank group,Arg1 expression decreased in the HQJZ+blank group(P<0.05),CD206 showed a downward trend without statistical significance,while iNOS and CD86 expression were significantly increased(P<0.05).Compared with the M1 group,Arg1 and CD206 expression decreased in the HQJZ+M1group(P<0.05).Compared with the M2 group,CD206 expression decreased in the HQJZ+M2 group(P<0.05),CD86 expression increased significantly(P<0.01).In vivo,Real-time PCR results showed that,compared with the control group,CD86 and CD206expression levels were significantly increased in the model group(P<0.01).Compared with the model group,CD206 expression in the MPA group was significantly decreased(P<0.01).In the HQJZ group,CD206 was significantly decreased(P<0.01).WB results showed that,compared with the model group,protein expression of UCP1 and PPARγwas significantly reduced in the HQJZ group(P<0.05,P<0.01).micro-CTresults showed that the total white fat volume in the HQJZ group was greater than that in the model group(P<0.05).HE staining results showed that pathological scores in the HQJZ group were lower than those in the model group(P<0.05).Conclusion:HQJZ may inhibit white adipose tissue browning by promoting macrophage M1 polarization and suppressing M2 polarization,thereby delaying fat consumption in CC mice.
作者
房智彦
朱海燕
淮文英
黄聪
杨若聪
于海艳
张天娥
FANG Zhiyan;ZHU Haiyan;HUAI Wenying;HUANG Cong;YANG Ruocong;YU Haiyan;ZHANG Tiane(College of Basic Medical Sciences,Chengdu University of Traditional Chinese Medicine(TCM),Chengdu 611137,China;School of Pharmacy,Chengdu University of TCM,Chengdu 611137,China;Academic Inheritance Center of TCM,Chengdu University of TCM,Chengdu 611137,China)
出处
《中国实验方剂学杂志》
北大核心
2026年第2期61-69,共9页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(82205072,82104732)
四川省科技厅重点研发计划项目(2022NSFSC1549,2023YFS0333)。
关键词
黄芪建中汤
癌症恶病质
巨噬细胞极化
白色脂肪
脂肪褐变
Huangqi Jianzhongtang
cancer cachexia
macrophage polarization
white adipose tissue
fat browning
