基于NF-κB、Nrf2/HO-1信号通路及Bcl-2/Caspase-3凋亡蛋白表达探讨雷公藤-川芎组分配伍对类风湿关节炎成纤维样滑膜细胞的影响  

Combination of Components from Tripterygii Radix et Rhizoma-Chuanxiong Rhizoma Affects RA-FLSs by Regulating NF-κB,Nrf2/HO-1 Signaling Pathways and Bcl-2/Caspase-3 Expression

作　　者：管咏梅 万志艳 王舒慧 朱卫丰 刘志勇 蒋成 臧振中 GUAN Yongmei;WAN Zhiyan;WANG Shuhui;ZHU Weifeng;LIU Zhiyong;JIANG Cheng;ZANG Zhenzhong(Jiangxi University of Chinese Medicine,Key Laboratory of Modern Preparation of Traditional Chinese Medicine,Ministry of Education,Nanchang 330004,China;National Key Laboratory of Modern Chinese Medicine Creation of Classic Prescriptions,Nanchang 330004,China;Laboratory Animal Science and Technology Center,Jiangxi University of Chinese Medicine,Nanchang 330004,China)

机构地区：[1]江西中医药大学,现代中药制剂教育部重点实验室,南昌330004 [2]经典名方现代中药创制全国重点实验室,南昌330004 [3]江西中医药大学实验动物科技中心,南昌330004

出　　处：《中国实验方剂学杂志》2025年第2期17-26,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82060722);博士科研启动基金项目(2023WBZR007);2023年度中药学重点学科项目(2023jzzdxk016)。

摘　　要：目的:探讨雷公藤-川芎组分配伍对类风湿关节炎成纤维样滑膜细胞(RA-FLS)的影响及其可能机制。方法:将RA-FLS细胞分为空白组、阳性药(甲氨蝶呤)组、雷公藤组、川芎组和雷公藤-川芎配伍组,以细胞增殖与活性检测(CCK-8)实验最低有效抑制浓度确定阳性药(甲氨蝶呤)组、雷公藤组、川芎组和雷公藤-川芎配伍组给药浓度后考察各给药组对细胞增殖,侵袭,凋亡状况;细胞肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)炎症因子水平、活性氧(ROS)和丙二醛(MDA)含量;蛋白免疫印迹法(Western blot)检测核因子E2相关因子2(Nrf2)、血红素氧合酶-1(HO-1)、核转录因子-κB p65(NF-κB p65)、磷酸化核转录因子-κB抑制蛋白(p-IκBα)、胱天蛋白酶-3(Caspase-3)、B细胞淋巴瘤-2(Bcl-2)蛋白表达的水平和实时荧光定量聚合酶链式反应(Real-time PCR)检测Nrf2、HO-1、NF-κB p65 mRNA表达的水平。结果:甲氨蝶呤组、雷公藤组、川芎组、雷公藤-川芎配伍组的给药浓度分别为2.50 mg·L^(-1)甲氨蝶呤,0.20 mg·L^(-1)雷公藤甲素+0.20 mg·L^(-1)雷公藤红素,5.00 mg·L^(-1)阿魏酸+20.00 mg·L^(-1)川芎嗪,0.20 mg·L^(-1)雷公藤甲素+0.20 mg·L^(-1)雷公藤红素+5.00 mg·L^(-1)阿魏酸+20.00 mg·L^(-1)川芎嗪。与空白组比较,各给药组细胞增殖、侵袭能力显著下降,凋亡能力显著上升(P<0.01);TNF-α、IL-6水平、ROS和MDA含量显著下降(P<0.01);Caspase-3、Nrf2、HO-1蛋白及mRNA表达水平均显著上升(P<0.01)、Bcl-2、NF-κB p65、p-IκBα蛋白及mRNA基因表达水平均显著下降(P<0.01)。与雷公藤组比较,雷公藤-川芎组对RA-FLS细胞增殖、侵袭能力明显降低(P<0.05),凋亡率进一步上升;Nrf2、HO-1 mRNA,Nrf2、Caspase-3蛋白表达明显升高(P<0.05),NF-κB p65、p-IκBα蛋白表达均明显下降(P<0.05)。结论:川芎雷公藤组分配伍可抑制RA-FLS细胞的细胞增殖、侵袭,促进凋亡,缓解氧化应激和炎症其机制可能与抑制NF-κB�Objective:To investigate the effects of the combination of components from Tripterygii Radix et Rhizoma and Chuanxiong Rhizoma on rheumatoid arthritis fibroblast-like synoviocytes(RA-FLSs)and the underlying mechanism.Methods:RA-FLSs were grouped as follows:blank control,positive control(methotrexate),Tripterygii Radix et Rhizoma components,Chuanxiong Rhizoma components,and components from Tripterygii Radix et Rhizoma+Chuanxiong Rhizoma.The cell-counting kit-8(CCK-8)assay was employed to the cell proliferation,invasion,and apoptosis.The levels of tumor necrosis factor(TNF)-α,interleukin(IL)-6,reactive oxygen species(ROS),and malondiadehyde(MDA)in cells were measured.Western blot was employed to determine the protein levels of nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase-1(HO-1),nuclear factor-kappa B(NF-κB)p65,phosphorylated inhibitory subunit of NF-κBα(p-IκBα),cysteinyl aspartate-specific protease-3(Caspase-3),and B-cell lymphoma 2(Bcl-2).Real-time PCR was employed to determine the mRNA levels of Nrf2,HO-1,and NF-κB p65.Results:The cells in the groups of positive control,Tripterygii Radix et Rhizoma components,Chuanxiong Rhizoma components,and components from Tripterygii Radix et Rhizoma+Chuanxiong Rhizoma were treated with 2.50 mg·L^(-1)methotrexate,0.20 mg·L^(-1)triptolide+0.20 mg·L^(-1)celastrol,5.00 mg·L^(-1)ferulic acid+20.00 mg·L^(-1)ligustrazine,0.20 mg·L^(-1)triptolide+0.20 mg·L^(-1)celastrol+5.00 mg·L^(-1)ferulic acid+20.00 mg·L^(-1)ligustrazine,respectively.Compared with the blank control group,drug administration reduced the proliferation and invasion and increased the apoptosis of cells(P<0.01),lowered the levels of TNF-α,IL-6,ROS,and MDA(P<0.01),up-regulated the mRNA and protein levels of Caspase-3,Nrf2,and HO-1(P<0.01),and down-regulated the mRNA and protein levels of Bcl-2,NF-κB p65,and p-IκBα(P<0.01).Compared with the Tripterygii Radix et Rhizoma components group,the combination of components from Tripterygii Radix et Rhizoma+Chuanxiong Rhizoma inhibited the

关 键 词：雷公藤甲素 雷公藤红素 川芎嗪 阿魏酸 类风湿关节炎成纤维样滑膜细胞(RA-FLS)细胞 

分 类 号：R285[医药卫生—中药学] R243[医药卫生—中医学] R277