1、1478 中草药 中草药 2023 年年 3 月月 第第 54 卷卷 第第 5 期期 Chinese Traditional and Herbal Drugs 2023 March Vol.54 No.5 基于 TLR4/NF-B/NLRP3 通路的羟基红花黄色素 A 抑制血管紧张素 II 诱导的血管外膜成纤维细胞迁移研究 李伟慷1，崔清卓1#，郑玉光1，张一昕3，郭炳颜4，赵京山1,2,3*，刘 琳2*1.河北中医学院药学院，河北省中药炮制创新中心，河北 石家庄 050020 2.河北中医学院基础医学院，河北 石家庄 050020 3.河北省中药资源利用与质量评价国际联合研究中心，河北 石家
2、庄 050020 4.河北医科大学第二附属医院，河北 石家庄 050020 摘 要：目的 基于通过 Toll 样受体 4（Toll-like receptor 4，TLR4）/核因子-B（nuclear factor-B，NF-B）/NOD 样受体热蛋白结构域相关蛋白 3（NOD-like receptor thermal protein domain associated protein 3，NLRP3）通路探讨羟基红花黄色素A（hydroxysafflor yellow A，HSYA）抑制血管紧张素 II（angiotensin II，ANG II）诱导的血管外膜成纤维细胞（vascula
3、r adventitial fibroblasts，VAFs）迁移作用。方法 组织贴块法培养大鼠胸主动脉 VAFs，免疫荧光实验检测细胞中 Vimentin 和-平滑肌肌动蛋白（-smooth muscle actin，-SMA）蛋白表达，鉴定 VAFs。设置对照组、ANG II 组、HSYA 组、脂多糖（lipopolysaccharide，LPS）组和 LPSHSYA 组，对照组 VAFs 给予培养基正常培养，其余各组给予 1107 mol/L ANG II 处理 24 h，然后更换为含 100 nmol/L LPS 或 40 mol/L HSYA 的培养基，继续培养 24 h，划痕实验检
4、测 VAFs 的迁移能力；CCK-8 法检测细胞活力；Western blotting 检测 TLR4、NF-B 和 p-NF-B 蛋白表达；免疫荧光实验检测 NLRP3 炎性小体相关蛋白 NLRP3、半胱氨酸天冬氨酸蛋白酶-1（cystein-asparate protease-1，Caspase-1）及凋亡斑点样蛋白（apoptotic spot-like protein，ASC）共表达情况。结果 免疫荧光结果证实 VAFs 培养成功。划痕实验结果显示，ANG II 能够显著增加细胞的迁移率（P0.01），LPS 刺激进一步提高 VAFs 的迁移率（P0.01），HSYA 能够抑制 ANG
5、 II 及 LPS 对 VAFs 迁移的促进作用（P0.01）。Western blotting 结果显示，ANG II 显著提高细胞内 TLR4 和 p-NF-B 的表达（P0.05、0.001），并促进 NF-B 入核（P0.001）；HSYA 显著抑制 ANG II 诱导的细胞内 TLR4 和 p-NF-B 的表达（P0.05、0.01、0.001），并抑制 NF-B 入核（P0.05、0.01、0.001）。免疫荧光实验结果表明，ANG II 及 LPS 促进 NLRP3 炎症小体相关蛋白 NLRP3、Caspase-1 及 ASC 共表达，HSYA 可减少 ANG II 及 LPS
6、诱导 NLRP3、Caspase-1 及 ASC 蛋白共表达，抑制 NLRP3 炎症小体的组装。结论 HSYA 通过抑制 TLR4/NF-B/NLRP3 通路减少 ANG II 诱导的 VAFs 迁移。关键词：羟基红花黄色素 A；血管外膜成纤维细胞；血管紧张素 II；脂多糖；TLR4/NF-B/NLRP3 通路 中图分类号：R285.5 文献标志码：A 文章编号：0253-2670(2023)05-1478-09 DOI:10.7501/j.issn.0253-2670.2023.05.014 Inhibitory effect of hydroxylsafflower yellow A on
7、 migration of vascular adventitial fibroblasts induced by angiotensin II based on TLR4/NF-B/NLRP3 pathway LI Wei-kang1,CUI Qing-zhuo1,ZHENG Yu-guang1,ZHANG Yi-xin3,GUO Bing-yan4,ZHAO Jing-shan1,2,3,LIU Lin2 1.School of Pharmacy,Hebei University of Chinese Medicine,Hebei TCM Processing Innovation Cen
8、ter,Shijiazhuang 050020,China 收稿日期：2022-11-13 基金项目：河北省自然科学基金资助项目（H2020423061）；河北省高等学校科学技术研究项目重点计划项目（ZD2017056）；河北省重点研发计划项目中医药创新专项（223777149D）；河北省中医药管理局科研计划项目（2017009，2022080）；河北省教育厅青年基金资助项目（QN2022092）作者简介：李伟慷，硕士研究生，研究方向为活血化瘀中药作用机制。E-mail:*通信作者：赵京山，教授，研究方向为心血管疾病分子生物学、中药药效物质及作用机制。Tel:(0311)89926035 E-
9、mail: 刘 琳，博士，副教授，研究方向为重要系统脂代谢细胞生物学。E-mail:#共同第一作者：崔清卓，硕士研究生，研究方向为活血化瘀中药作用机制。E-mail: 中草药 中草药 2023 年年 3 月月 第第 54 卷卷 第第 5 期期 Chinese Traditional and Herbal Drugs 2023 March Vol.54 No.5 1479 2.Basic Medical College,Hebei University of Chinese Medicine,Shijiazhuang 050020,China 3.International Joint Rese
10、arch Center on Resource Utilization and Quality Evaluation of Traditional Chinese Medicine of Hebei Province,Shijiazhuang 050020,China 4.The Second Affiliated Hospital of Hebei Medical University,Shijiazhuang 050020,China Abstract:Objective To investigate the effect of hydroxysafflor yellow A(HSYA)o
11、n migration of advangiolar fibroblasts(VAFs)induced by angiotensin (ANG)based on Toll-like receptor 4(TLR4)/nuclear factor-B(NF-B)/NOD-like receptor thermal protein domain associated protein 3(NLRP3)pathway.Methods VAFs were cultured from rat thoracic aorta by tissue patch method,protein expressions
12、 of Vimentin and-smooth muscle actin(-SMA)was detected by laser confocal assay for identification of VAFs.Control group,ANG II group,HSYA group,lipopolysaccharide(LPS)group and LPS+HSYA group were set up.VAFs in control group were given normal culture medium,and the other groups were given 1107 mol/
13、L ANG II for 24 h,and then changed to contain 100 nmol/L LPS or 40 mol/L HSYA medium,continue to culture for 24 h,and migration ability of VAFs was tested by scratch test;CCK-8 method was used to detect cell viability;Western blotting was used to detect TLR4,NF-B and p-NF-B protein expression;The co
14、-expression of NLRP3 inflammatory corpuscle-associated protein NLRP3,cystein-aspartate protease-1(Caspase-1)and apoptotic spot-like protein(ASC)were detected by immunofluorescence assay.Results The results of immunofluorescence confirmed that VAFs were successfully cultured.The scratch test results
15、showed that ANG II could significantly increase the cell migration rate(P 0.01),LPS stimulation can further increase the migration of VAFs(P 0.01),HSYA can inhibit the promotion of ANG II and LPS on VAFs migration(P 0.01).Western blotting results showed that ANG II significantly increased intracellu
16、lar TLR4 and p-NF-B expressions(P 0.05,0.001),and promoted NF-B entry(P 0.001);HSYA significantly inhibited the intracellular TLR4 and p-NF-B expressions induced by ANG II(P 0.05,0.01,0.001),and inhibited NF-B entered the nucleus(P 0.05,0.01,0.001).The results of immunofluorescence test showed that ANG II and LPS promoted the co-expression of NLRP3,Caspase-1 and ASC proteins associated with NLRP3 inflammatory bodies,and HSYA could reduce the co-expression of NLRP3,Caspase-1 and ASC proteins indu