摘要
目的研究益气解毒方对电离辐射诱导的巨噬细胞极化的调控作用及该作用与Toll样受体4(TLR4)/髓样分化初级反应蛋白质88(MyD88)/核因子-κB(NF-κB)信号通路的关系。方法SPF级雄性SD大鼠55只按体质量分为空白组(30只)、安多霖组(10只)、益气解毒方组(15只),分别灌胃去离子水、安多霖混悬液(0.3456 g/kg)、益气解毒方煎液(20.88 g/kg)0.01 mL/g,1次/d,连续7 d。末次灌胃2 h后腹主动脉取血制得空白大鼠血清、安多霖大鼠血清、益气解毒方高剂量大鼠血清。取适量益气解毒方高剂量大鼠血清和空白大鼠血清分别以1∶1、1∶3比例混合得到益气解毒方中、低剂量大鼠血清。将RAW264.7细胞分为空白组(10%空白大鼠血清),模型组(10%空白大鼠血清),安多霖组(10%安多霖大鼠血清),益气解毒方低、中、高剂量组(10%益气解毒方低、中、高剂量大鼠血清)。各细胞分别用对应大鼠血清培养24 h,除空白组外,均予12 Gy^(60) Coγ射线一次性照射,建立巨噬细胞辐射损伤模型。照射后24 h,CCK-8法检测巨噬细胞存活率,划痕实验检测细胞迁移率,鬼笔环肽染色观察细胞形态,酶联免疫吸附测定检测细胞上清液中的肿瘤坏死因子-α(TNF-α)、白细胞介素-10(IL-10)含量,流式细胞术检测经典活化的巨噬细胞(M1型)占比,蛋白质印迹法检测TLR4、MyD88、NF-κB蛋白表达。结果照射后24 h,和空白组相比,模型组巨噬细胞的存活率下降、迁移率下降(P<0.01),细胞体积变大、伪足增多,细胞上清TNF-α含量升高,M1型巨噬细胞比例增多,TLR4、MyD88及NF-κB蛋白表达量升高(P<0.05,P<0.01);和模型组相比,各给药组巨噬细胞的存活率和迁移率上升(P<0.05,P<0.01),细胞体积变小、形状规则,细胞上清TNF-α含量下降,M1型巨噬细胞占比下降,TLR4、MyD88、NF-κB蛋白表达量降低(P<0.05,P<0.01),IL-10表达有上升趋势。结论益气解毒方可能通过调控TLR4/MyD88/NF-κB信号通路,一定程度上抑制电离辐射诱导的巨噬细胞M1极化,降低促炎因子分泌并促进抗炎因子分泌,缓解电离辐射导致的炎症损伤。
Objective To investigate the regulatory effect and mechanism of Yiqi Jiedu Decoction(YQJD)on ionizing radiation-induced macrophage polarization and its correlation with the Toll-like receptor 4(TLR4)/myeloid differentiation primary response protein 88(MyD88)/nuclear factor kappa-B(NF-κB)signaling pathway.Methods Fifty-five specific-pathogen-free male Sprague-Dawley rats were randomly divided into blank(n=30),anduolin(n=10),and YQJD groups(n=15).They were respectively gavaged with deionized water,anduolin suspension(0.3456 g/kg),and YQJD high-dose(20.88 g/kg)at a dose of 0.01 mL/g body weight once a day for seven consecutive days.2 hours after the last gavage,blood was collected from the abdominal aorta to prepare the control rat,andolin rat,and YQJD high-dose sera.Appropriate amounts of YQJD high-dose and control sera were mixed in a ratio of 1∶1 and 1∶3,respectively,to obtain YQJD medium-and low-dose rat serum.RAW264.7 cells were divided into blank(10%blank rat serum),model(10%blank rat serum),anduolin(10%anduolin rat serum),and YQJD-L,YQJD-M,YQJD-H groups(10%YQJD low-,medium-,and high-dose rat serum).Except for the blank group,the cells in other groups were irradiated with 12 Gy^(60) Coγ-rays once to establish the macrophage radiation injury model.At 24 h after irradiation,cell viability was detected using the CCK-8 method,and the cell migration rate was measured using the scratch test.Cell morphology was observed using phalloidin staining,tumor necrosis factor-alpha(TNF-α)and interleukin-10(IL-10)levels in the cell supernatant were quantified using enzyme-linked immunosorbent assay,and the proportion of M1 macrophages was detected using flow cytometry.TLR4,MyD88,and NF-κB protein expression were detected using Western blotting.Results Twenty-four hours after irradiation,compared with the blank group,the model group exhibited significantly reduced cell viability and migration rate(P<0.01),increased cell volume and pseudopodia formation,elevated TNF-αand IL-10 levels,an increased proportion of M1 macrophages,and upregulated TLR4,MyD88,and NF-κB protein expression(P<0.05,P<0.01).Compared with the model group,each drug-treated group showed improved cell viability and migration rate(P<0.05,P<0.01),decreased cell volume,more regular cell shape,reduced TNF-αlevels,lower M1-type macrophage proportion,and downregulated TLR4,MyD88,and NF-κB protein expression(P<0.05,P<0.01).IL-10 level showed an upward trend.Conclusion YQJD can partially inhibit M1 macrophage polarization and suppress inflammatory responses,which may be related to the TLR4/MyD88/NF-κB signaling pathway.
作者
胡瑞瑶
赵章弟
王安
李文元
雷家俊
曾嘉欢
安梓睿
胡素敏
HU Ruiyao;ZHAO Zhangdi;WANG An;LI Wenyuan;LEI Jiajun;ZENG Jiahuan;AN Zirui;HU Sumin(Beijing University of Chinese Medicine,Beijing 102488,China)
出处
《北京中医药大学学报》
北大核心
2025年第7期933-942,共10页
Journal of Beijing University of Traditional Chinese Medicine
基金
国家自然科学基金面上项目(No.12075035)
2023年北京中医药大学本博贯通学生科学研究项目(No.XBB23035)。
