Wuxi School of Medicine, Jiangnan University, Wuxi, Jiangsu, PR China.
Department of Natural Medicine, School of Pharmacy, Fudan University, Shanghai, PR China.
Phytomedicine. 2024 Jun;128:155403. doi: 10.1016/j.phymed.2024.155403. Epub 2024 Feb 1.
Cardiovascular disease is one of the main causes of global mortality, and there is an urgent need for effective treatment strategies. Gut microbiota-dependent metabolite trimethylamine-N-oxide (TMAO) promotes the development of cardiovascular diseases, and shizukaol C, a natural sesquiterpene isolated from Chloranthus multistachys with various biological activities, might exhibit beneficial role in preventing TMAO-induced vascular inflammation.
The purpose of this study was to investigate the anti-inflammatory effects and the underlying mechanisms of shizukaol C on TMAO-induced vascular inflammation.
The effect and underlying mechanism of shizukaol C on TMAO-induced adhesion molecules expression, bone marrow-derived macrophages (BMDM) adhesion to VSMC were evaluated by western blot, cell adhesion assay, co-immunoprecipitation, immunofluorescence assay, and quantitative Real-Time PCR, respectively. To verify the role of shizukaol C in vivo, TMAO-induced vascular inflammation model were established using guidewire-induced injury on mice carotid artery. Changes in the intima area and the expression of GSTpi, VCAM-1, CD68 were examined using haematoxylin-eosin staining, and immunofluorescence assay.
Our data demonstrated that shizukaol C significantly suppressed TMAO-induced adhesion molecule expression and the bone marrow-derived macrophages (BMDM) adhesion in vascular smooth muscle cells (VSMC). Mechanically, shizukaol C inhibited TMAO-induced c-Jun N-terminal kinase (JNK)-nuclear factor-kappa B (NF-κB)/p65 activation, and the JNK inhibition was dependent on the shizukaol C-mediated glutathione-S-transferase pi (GSTpi) expression. By further molecular docking and protein-binding analysis, we demonstrated that shizukaol C directly binds to Keap1 to induce Nrf2 nuclear translocation and upregulated GSTpi expression. Consistently, our in vivo experiment showed that shizukaol C elevated the expression level of GSTpi in carotid arteries and alleviates TMAO-induced vascular inflammation.
Shizukaol C exerts anti-inflammatory effects in TMAO-treated VSMC by targeting Keap1 and activating Nrf2-GSTpi signaling and resultantly inhibits the downstream JNK-NF-κB/p65 activation and VSMC adhesion, and alleviates TMAO-induced vascular inflammation in vivo, suggesting that shizukaol C may be a potential drug for treating TMAO-induced vascular diseases.
心血管疾病是全球死亡的主要原因之一，因此迫切需要有效的治疗策略。肠道微生物群依赖性代谢物三甲胺 N-氧化物（TMAO）促进心血管疾病的发展，而从金粟兰科植物中分离出的具有多种生物活性的天然倍半萜 shizukaol C 可能在预防 TMAO 诱导的血管炎症方面发挥有益作用。
本研究旨在探讨 shizukaol C 对 TMAO 诱导的血管炎症的抗炎作用及其机制。
通过 Western blot、细胞黏附实验、共免疫沉淀、免疫荧光实验和定量实时 PCR 分别评估 shizukaol C 对 TMAO 诱导的黏附分子表达、骨髓来源的巨噬细胞（BMDM）与血管平滑肌细胞（VSMC）黏附的影响。使用导丝诱导小鼠颈总动脉损伤建立 TMAO 诱导的血管炎症模型，以验证 shizukaol C 在体内的作用。采用苏木精-伊红染色和免疫荧光实验检测血管内膜面积和 GSTpi、VCAM-1、CD68 的表达变化。
数据表明，shizukaol C 可显著抑制 TMAO 诱导的黏附分子表达和骨髓来源的巨噬细胞（BMDM）与血管平滑肌细胞（VSMC）的黏附。在机制上，shizukaol C 抑制了 TMAO 诱导的 c-Jun N 末端激酶（JNK）-核因子-κB（NF-κB）/p65 激活，而 JNK 抑制依赖于 shizukaol C 介导的谷胱甘肽 S-转移酶 pi（GSTpi）表达。通过进一步的分子对接和蛋白结合分析，我们证明 shizukaol C 直接与 Keap1 结合，诱导 Nrf2 核易位并上调 GSTpi 的表达。一致地，我们的体内实验表明，shizukaol C 可提高颈动脉中 GSTpi 的表达水平，并减轻 TMAO 诱导的血管炎症。
shizukaol C 通过靶向 Keap1 并激活 Nrf2-GSTpi 信号通路，抑制 TMAO 处理的 VSMC 中的炎症反应，从而抑制下游的 JNK-NF-κB/p65 激活和 VSMC 黏附，并减轻体内 TMAO 诱导的血管炎症，提示 shizukaol C 可能是治疗 TMAO 诱导的血管疾病的潜在药物。
