黑老虎抗炎有效部位筛选及机制研究  

Screening and Mechanism Study of Anti-inflammatory Effective Fraction of Kadsura coccinea

作　　者：孙雅慧 董金金 曹春芽 刘建新 金岸 吴卫华 SUN Yahui;DONG Jinjin;CAO Chunya;LIU Jianxin;JIN An;WU Weihua(School of Pharmacy,Xuzhou Medical University,Xuzhou 221004 Jiangsu,China;Hunan Provincial Key Laboratory for Synthetic Biology of Traditional Chinese Medicine,School of Pharmaceutical Sciences,Hunan University of Medicine,Huaihua 418000 Hunan,China)

机构地区：[1]徐州医科大学药学院,江苏徐州221004 [2]湖南医药学院药学院,中药合成生物学研究湖南省重点实验室,湖南怀化418000

出　　处：《中药新药与临床药理》2022年第12期1589-1598,共10页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(81803847);湖南省自然科学基金项目(2019JJ40203);湖南省教育厅优秀青年项目(20B414);国家大学生创新创业训练计划项目(201912214019);湖南省大学生创新创业训练计划项目(湘教通[2019]100号-2811)。

摘　　要：目的 筛选黑老虎根的抗炎有效部位并探讨其抗炎机制。方法 采用热回流和超声萃取法制备黑老虎醇提乙酸乙酯萃取物(EC)和黑老虎乙酸乙酯提取物(ET);通过硅胶柱快速制备液相色谱仪分离纯化ET相关组分。采用脂多糖(LPS,100 ng·mL^(-1))诱导RAW264.7巨噬细胞作为体外炎症模型。细胞分为空白对照组、LPS模型组及不同浓度黑老虎提取物预处理组,药物于LPS刺激前2 h加入培养基。采用MTT法检测细胞活性;ELISA法检测细胞上清液中一氧化氮(NO)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)及单核细胞趋化蛋白1(MCP-1)水平;采用Western Blot及qRT-PCR法检测COX-2、iNOS、NF-κB/MAPK通路、Nrf2-HO-1/NQO-1通路相关蛋白及mRNA表达水平。结果 (1)EC、ET对LPS诱导的RAW264.7细胞NO生成的抑制率相近,二者的IC50值分别为34.07、34.69μg·mL^(-1);与空白对照组比较,100μg·mL^(-1)EC、ET组的细胞活力均显著降低(P<0.001),而在1~50μg·mL^(-1)浓度范围EC、ET组的细胞活力均无明显变化(P>0.05);与LPS模型组比较,ET 50μg·mL^(-1)组细胞的IL-6水平明显降低(P<0.05),ET 30、50μg·mL^(-1)组细胞的MCP-1水平明显降低(P<0.01,P<0.001),具有明显的抗炎作用;而EC不同浓度组细胞的IL-6、MCP-1、TNF-α水平均无明显变化(P>0.05)。(2)采用快速制备液相色谱仪分离出ET的8个组分(ET1~ET8);ET2~ET8组分均有一定的抗炎活性,ET5对NO生成的抑制作用最强,IC50值为17.02μg·mL^(-1);与LPS模型组比较,ET5 10、20、40μg·mL^(-1)组细胞的IL-6、MCP-1、TNF-α水平均明显降低(P<0.05,P<0.01,P<0.001);ET5在7.5~40μg·mL^(-1)浓度范围对有/无LPS诱导的RAW264.7细胞活性均无明显影响(P>0.05)。(3)与LPS模型组比较,20、40μg·mL^(-1)ET5组细胞的iNOS mRNA及蛋白表达显著下调(P<0.01,P<0.001),10、20μg·mL^(-1)ET5组细胞的COX-2 mRNA表达显著下调(P<0.001),对COX-2蛋白表达无影响(P>0.05);ET5各浓度组细胞的p65、IKBα、p38、ERK、JNK蛋Objective Screen the anti-inflammatory effective fraction of Kadsura coccinea and to explore its antiinflammatory mechanism. Methods Alcohol extraction ethyl acetate extract of Kadsura coccinea(EC) and ethyl acetate extract of Kadsura coccinea(ET)were prepared by hot reflux and ultrasonic extraction;ET-related fractions were separated and purified by rapid preparative liquid chromatography on silica gel columns. Lipopolysaccharide(LPS,100 ng·mL^(-1))-induced RAW264.7 macrophages were used as an in vitro inflammation model. Cells were divided into a blank control group,a LPS model group and a pretreatment group with different concentrations of Kadsura coccinea extract, and the drug was added to the culture medium 2-hour before LPS stimulation. The cell viability was detected by MTT;the levels of nitric oxide(NO),interleukin 6(IL-6),tumour necrosis factor α(TNF-α)and monocyte chemotactic protein 1(MCP-1) in cell supernatant were detected by ELISA;the related protein and m RNA expression levels of COX-2,iNOS,NF-κB/MAPK pathway,Nrf2-HO-1/NQO-1 pathway were detected by Western Blot and qRT-PCR. Results(1)EC and ET inhibited NO production in LPS-induced RAW264.7 cells at similar rates,with IC50values of 34.07 and 34.69 μg·mL^(-1),respectively;compared with the blank control group,cell viability was significantly reduced in both the 100 μg ·mL^(-1) EC and ET groups(P<0.001), while the cell viability of the EC and the ET groups was not significantly changed in the concentration range of 1-50 μg·mL^(-1)(P>0.05);compared with the LPS model group,the IL-6 level in the ET 50 μg·mL^(-1)group was significantly decreased(P<0.05),and the MCP-1 level in the ET 30 and 50 μg·mL^(-1)groups was significantly decreased(P<0.01,P<0.001),which had a significant anti-inflammatory effect;in contrast,the levels of IL-6,MCP-1 and TNF-αproduced by the cells of the different EC concentration groups were not significantly changed(P>0.05).(2)Eight fractions of ET(ET1-ET8)were separated by rapid preparative liquid chromatography;ET

关 键 词：黑老虎 抗炎作用 有效部位 乙酸乙酯提取物 Nrf2-HO-1/NQO-1通路 RAW264.7巨噬细胞 

分 类 号：R285.5[医药卫生—中药学]