Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China; Department of Rheumatism and Immunology, Peking University Shenzhen Hospital, Shenzhen, China.
J Ethnopharmacol. 2020 Oct 5;260:113039. doi: 10.1016/j.jep.2020.113039. Epub 2020 Jun 1.
Zicao is the dried root of Lithospermum erythrorhizon Sieb, et Zucc, Arnebia euchroma (Royle) Johnst, or Arnebia guttata Bunge and commonly used to treat viral infection, inflammation, arthritis and cancer in China.Shikonin (SKN) is a major active chemical component isolated from zicao. Previous research showed that SKN has anti-inflammatory, immunomodulatory and analgesic effects, and inhibits the development of arthritis and the condition of collagen arthritis (CIA) mice; nevertheless, its role in the angiogenesis of rheumatoid arthritis (RA) has not been elucidated.
The purpose of this study was to investigate the antiangiogenic activity of SKN in CIA rats and various angiogenesis models.
The anti-arthritic effect of SKN on CIA rats was tested by arthritis score, arthritis incidence, radiological observation and histopathology evaluation of inflamed joints. Vessel density evaluated with CD31 immunohistochemistry/immunofluorescence in joint synovial membrane tissues of CIA rats, chick chorioallantoic membrane assay, rat aortic ring assay, and the migration, invasion, adhesion and tube formation of human umbilical vein endothelial (HUVEC) cells induced by tumor necrosis factor (TNF)-α were used to measured the antiangiogenenic activity of SKN. Moreover, the effect of SKN on the expression of angiogenic mediators, such as vascular endothelial growth factor (VEGF), VEGFR2, TNF-α, interleukin (IL)-1β, platelet derived growth factor (PDGF) and transforming growth factor (TGF)-β in sera and joint synovia of rats, and in TNF-α-induced MH7A/HUVEC cells were measured by immunohistochemistry, enzyme linked immunosorbent assay, Western blot and/or real-time polymerase chain reaction (PCR). Through the analysis of protein and mRNA levels of phosphoinositide 3-kinase (PI3K), Akt and PTEN, and the autophosphorylation of ERK1/2, JNK and p38 in joint synovia of rats and in TNF-α-induced HUVEC cells, the molecular mechanism of its inhibition was elucidated by using Western blot and/or real-time PCR.
SKN significantly reduced the arthritis score and arthritis incidence, and inhibited inflammation, pannus formation, cartilage and bone destruction of inflamed joints in CIA rats. Partially, SKN remarkably decreased the immature blood vessels in synovial membrane tissues of inflamed joints from CIA rats. It also suppressed in vivo angiogenesis in chick embryo and VEGF-induced microvessel sprout formation ex vivo. Meanwhile, SKN inhibited TNF-α-induced migration, invasion, adhesion and tube formation of HUVEC cells. Moreover, SKN significantly decreased the expression of angiogenic activators including VEGF, VEGFR2, TNF-α, IL-1β, PDGF and TGF-β in synovia of CIA rats and/or in MH7A/HUVEC cells. More interestingly, SKN downregulated PI3K and Akt, and simultaneously upregulated PTEN both at protein and mRNA levels in synovia tissues and/or in TNF-α-induced HUVEC cells. It also suppressed the phosphorylation and gene level of TNF-α-induced signaling molecules, as ERK1/2, JNK, and p38 in synovium and/or in TNF-α-induced HUVEC cells.
These findings indicate for the first time that SKN has the anti-angiogenic effect in RA in vivo, ex vivo and in vitro by interrupting the PI3K/AKT and MAPKs signaling pathways.
ETHNOPHARMACOLOGICAL 相关性：紫草是紫草科、紫草属植物紫草、新疆紫草或内蒙紫草的干燥根，在中国常用于治疗病毒感染、炎症、关节炎和癌症。紫草素（SKN）是从紫草中分离出的一种主要活性化学成分。先前的研究表明，SKN 具有抗炎、免疫调节和镇痛作用，并抑制关节炎和胶原性关节炎（CIA）小鼠的发展；然而，其在类风湿性关节炎（RA）血管生成中的作用尚未阐明。
本研究旨在探讨 SKN 在 CIA 大鼠和各种血管生成模型中的抗血管生成活性。
通过关节炎评分、关节炎发生率、放射性观察和炎症关节的组织病理学评估来测试 SKN 对 CIA 大鼠的抗关节炎作用。用 CD31 免疫组化/免疫荧光法评估 CIA 大鼠关节滑膜组织中的血管密度，用鸡胚绒毛尿囊膜试验、大鼠主动脉环试验和肿瘤坏死因子（TNF）-α诱导的人脐静脉内皮（HUVEC）细胞的迁移、侵袭、粘附和管形成来测定 SKN 的抗血管生成活性。此外，通过免疫组化、酶联免疫吸附试验、Western blot 和/或实时聚合酶链反应（PCR）测定 SKN 对大鼠血清和关节滑膜中血管生成介质（如血管内皮生长因子（VEGF）、VEGFR2、TNF-α、白细胞介素（IL）-1β、血小板衍生生长因子（PDGF）和转化生长因子（TGF）-β）的表达，以及 TNF-α诱导的 MH7A/HUVEC 细胞的表达。通过 Western blot 和/或实时 PCR 分析 SKN 对大鼠关节滑膜和 TNF-α诱导的 HUVEC 细胞中磷酸肌醇 3-激酶（PI3K）、Akt 和 PTEN 的蛋白和 mRNA 水平以及 ERK1/2、JNK 和 p38 的自身磷酸化的影响，阐明其抑制作用的分子机制。
SKN 显著降低了 CIA 大鼠的关节炎评分和关节炎发生率，并抑制了炎症、滑膜增生、软骨和骨破坏。部分地，SKN 显著减少了 CIA 大鼠滑膜组织中不成熟的血管。它还抑制了鸡胚中的体内血管生成和 VEGF 诱导的微血管芽形成。同时，SKN 抑制了 TNF-α诱导的 HUVEC 细胞的迁移、侵袭、粘附和管形成。此外，SKN 显著降低了 CIA 大鼠滑膜组织和/或 MH7A/HUVEC 细胞中血管生成激活剂的表达，包括 VEGF、VEGFR2、TNF-α、IL-1β、PDGF 和 TGF-β。更有趣的是，SKN 下调了 PI3K 和 Akt 的蛋白和 mRNA 水平，同时上调了 PTEN 的蛋白和 mRNA 水平，这在滑膜组织和/或 TNF-α诱导的 HUVEC 细胞中均有体现。它还抑制了 TNF-α诱导的信号分子 ERK1/2、JNK 和 p38 的磷酸化和基因水平，包括在滑膜和/或 TNF-α诱导的 HUVEC 细胞中。
这些发现首次表明，SKN 通过中断 PI3K/AKT 和 MAPKs 信号通路，在体内、体外和体外具有抗 RA 的血管生成作用。
