探讨桃红饮体外通过VEGFC通路对巨噬细胞介导的淋巴管生成的抑制作用  

Taohongyin inhibits the VEGFC pathway in vitro and reduces macrophage-mediated lymphangiogenesis

作　　者：李斯锦 冯骁腾 王怡茹 刘萍[1] LI Sijin;FENG Xiaoteng;WANG Yiru;LIU Ping(Longhua Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)

机构地区：[1]上海中医药大学附属龙华医院,上海200032

出　　处：《北京中医药大学学报》2023年第12期1670-1683,共14页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金面上项目(No.82074200,No.81873117);国家自然科学基金青年项目(No.82204849)。

摘　　要：目的探讨桃红饮(桃仁、红花、川芎、当归尾、威灵仙)治疗动脉粥样硬化,调控巨噬细胞介导的淋巴管生成的分子机制。方法采用超高效液相色谱质谱法鉴定桃红饮有效成分。以慢病毒转染方法,构建血管内皮生长因子C(VEGFC)基因过表达的RAW264.7巨噬细胞(OE-VEGFC)与空载体对照RAW264.7巨噬细胞(OE-VEHICLE)。用人氧化低密度脂蛋白(20 mg/L)与脂多糖(100μg/L)干预RAW264.7巨噬细胞或OE-VEGFC构建动脉粥样硬化细胞模型(造模_(RAW264.7)及造模_(OE-VEGFC))。CCK-8法测定桃红饮的最佳给药质量浓度。构建RAW264.7巨噬细胞与小鼠淋巴管内皮细胞(MLECs)Transwell共培养模型。划痕实验检测细胞损伤修复情况;结晶紫染色检测细胞的迁移情况;管形成实验测定细胞的管腔形成能力;酶联免疫吸附剂测定法检测细胞上清液中VEGFC、白细胞介素6(IL-6)、诱导型一氧化氮合酶(iNOS)的含量;免疫荧光标记法检测细胞VEGFC含量;实时荧光定量PCR检测VEGFC、淋巴管内皮细胞表面受体(FLT4)、平足蛋白(PDPN)、IL-6、iNOS的mRNA表达;蛋白质印迹法检测VEGFC、FLT4、HIF-1α、淋巴管内皮透明质酸受体-1(LYVE-1)、同源异型盒基因转录因子-1(Prox-1)、IL-6、iNOS的蛋白表达。结果桃红饮中含有22种主要有效成分,其中,羟基红花黄色素A(23.3%)及D-苦杏仁苷(23.8%)含量较高。选择20 mg/L作为桃红饮的给药最佳质量浓度。OE-VEGFC及OE-VEHICLE转染效率为80%以上,OE-VEGFC巨噬细胞构建成功。与正常对照组相比,造模_(RAW264.7)组MLECs向划痕区域愈合的速度增快、细胞迁移数增多、管腔生成数增加(均P<0.05),VEGFC荧光表达量增加,细胞上清中VEGFC、IL-6、iNOS含量增加(均P<0.05),VEGFC、FLT4、PDPN、IL-6、iNOS的mRNA表达增加(均P<0.05),VEGFC、FLT4、HIF-1α、LYVE-1、Prox-1、IL-6、iNOS的蛋白表达增加(均P<0.05);桃红饮可逆转上述指标(均P<0.05)。造模_(OE-VEGFC)组与造模_(RAW264.7)组相Objective We aimed to explore whether Taohongyin(peach seed,safflower,Sichuan lovage rhizome,Chinese angelica root-tip,and Chinese clematis root)treats atherosclerosis by regulating macrophage-mediated lymphangiogenesis.Methods The effective components of Taohongyin were identified by UPLC-Q-TOF-MS._(RAW264.7)macrophages were transfected with a lentiviral vascular endothelial growth factor C(VEGFC)overexpression construct,a VEGFC knockdown construct(OE-VEGFC),and empty vector(OE-VEHICLE).The atherosclerotic cell model was constructed on_(RAW264.7)macrophages(Model_(RAW264.7))or on OE-VEGFC(Model_(OE-VEGFC))by intervention with human oxidized low density lipoprotein(20 mg/L)and lipopolysaccharide(100μg/L).The optimal concentration of Taohongyin was determined by the CCK-8 method.A Transwell co-culture model of_(RAW264.7)macrophages and mouse lymphatic endothelial cells(MLECs)was constructed.Wound healing was assessed by the scratch test.Migration of cells was analyzed by a Transwell assay followed by crystal violet staining.Tube formation was analyzed by the tube formation assay.The contents of VEGFC,interleukin-6(IL-6),and inducible nitric oxide synthase(iNOS)in the supernatant were determined by ELISA.The VEGFC content was determined by immunofluorescence labeling.The mRNA levels of VEGFC,lymphatic endothelial cell surface receptor(FLT4),podoplanin(PDPN),IL-6,and iNOS were determined by real-time fluorescence quantitative PCR.The protein expression levels of VEGFC,FLT4,HIF-1α,lymphatic vessel endothelial hyaluronic receptor 1(LYVE-1),homeobox gene transcription factor 1(Prox-1),IL-6,and iNOS were determined by Western blotting.Results UPLC-Q-TOF-MS identified 22 main active components in Taohongyin,in which hydroxysafflor yellow A(23.3%)and D-amygdalin(23.8%)were at the highest levels.A Taohongyin concentration of 20 mg/L was selected as the optimal concentration.The transfection efficiency of OE-VEGFC and OE-VEHICLE was more than 80%,indicating that the_(RAW264.7)macrophage VEGFC overexpression model was succ

关 键 词：桃红饮 动脉粥样硬化 RAW264.7巨噬细胞 小鼠淋巴管内皮细胞 淋巴管 羟基红花黄色素A 

分 类 号：R285.5[医药卫生—中药学]