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- 北京中医药大学学报 2023年46卷第12期 页码：1670-1683
- 作者机构：
上海中医药大学附属龙华医院 上海 200032
- 作者简介：
李斯锦，女，在读博士生
#刘萍，女，教授，主任医师，博士生导师，主要研究方向：中西医结合防治动脉粥样硬化相关心脑血管疾病，E-mail：liuping5156@sina.com
- 基金信息：国家自然科学基金面上项目(82074200;81873117);国家自然科学基金青年项目(82204849)
DOI：10.3969/j.issn.1006-2157.2023.12.008
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李斯锦, 冯骁腾, 王怡茹, 等. 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[J]. 北京中医药大学学报, 2023,46(12):1670-1683.
LI Sijin, FENG Xiaoteng, WANG Yiru, et al.
Taohongyin inhibits the VEGFC pathway in vitro and reduces macrophage-mediated lymphangiogenesis[J]. Journal of Beijing University of Traditional Chinese Medicine, 2023,46(12):1670-1683.李斯锦, 冯骁腾, 王怡茹, 等. 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[J]. 北京中医药大学学报, 2023,46(12):1670-1683. DOI： 10.3969/j.issn.1006-2157.2023.12.008.
LI Sijin, FENG Xiaoteng, WANG Yiru, et al.
Taohongyin inhibits the VEGFC pathway in vitro and reduces macrophage-mediated lymphangiogenesis[J]. Journal of Beijing University of Traditional Chinese Medicine, 2023,46(12):1670-1683. DOI： 10.3969/j.issn.1006-2157.2023.12.008.
摘要
目的,2,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,2,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(100 μg/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,2,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,组相比，上述指标增加(均,P,<,0.05)；与桃红饮＋造模,RAW264.7,组相比，桃红饮＋造模,OE-VEGFC,组上述指标增加(均,P,<,0.05)；与造模,OE-VEGFC,组比较，桃红饮＋造模,OE-VEGFC,组上述指标减少(均,P,<,0.05)。,结论,2,桃红饮可能通过抑制VEGFC通路减少巨噬细胞介导的淋巴管形成。
Abstract
Objective,2,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,2,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 (100 μg/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,2,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 successfully constructed. Compared with the normal control group, the wound healing capacity of MLECs was significantly increased, cell migration and tube formation were enhanced (all ,P,<,0.05), VEGFC fluorescence was increased (all ,P,<,0.05), the contents of VEGFC, IL-6, and iNOS in the supernatant were increased (all ,P,<,0.05), the mRNA expressions of VEGFC, FLT4, PDPN, IL-6, and iNOS were increased (all ,P,<,0.05), and the protein expressions of VEGFC, FLT4, HIF-1α, LYVE-1, Prox-1, IL-6, and iNOS were increased ( all ,P,<,0.05) in the Model,RAW264.7, group. After intervention with ,Taohongyin, of 20 mg/L, all the indicators were decreased (all ,P,<,0.05).Compared with the Model,RAW264.7, group, all the indicators in the Model,OE-VEGFC, group were increased(all ,P,<,0.05). Compared with the ,Taohongyin,+ Model,RAW264.7, group, all the indicators in the ,Taohongyin,+ Model,OE-VEGFC, group were increased(all ,P,<,0.05). Compared with the Model,OE-VEGFC, group, all the indicators in the ,Taohongyin,+ Model,OE-VEGFC, group were decreased(all ,P,<,0.05).,Conclusion,2,Taohongyin, may reduce macrophage-mediated lymphangiogenesis by inhibiting the VEGFC pathway.
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
Keywords
TaohongyinatherosclerosisRAW264.7 macrophage cellmouse lymphatic endothelial cellslymphatic vesselshydroxysafflower yellow A
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