Department of Pharmacology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an 710061, Shaanxi, PR China.
Xinjiang Rongcheng Hake Pharmaceutical Co. Ltd, Altay region 836500, Xinjiang, PR China.
Biochim Biophys Acta Mol Cell Res. 2023 Jun;1870(5):119460. doi: 10.1016/j.bbamcr.2023.119460. Epub 2023 Mar 21.
20-Hydroxyecdysone (20E) is known to have numerous pharmacological activities and can be used to treat diabetes and cardiovascular diseases. However, the protective effects of 20E against endothelial dysfunction and its targets remain unclear. In the present study, we revealed that 20E treatment could modulate the release of the endothelium-derived vasomotor factors NO, PGI and ET-1 and suppress the expression of ACE in TNF-α-induced 3D-cultured HUVECs. In addition, 20E suppressed the expression of CD40 and promoted the expression of SIRT6 in TNF-α-induced 3D-cultured HUVECs. The cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) and molecular docking results demonstrated that 20E binding increased SIRT6 stability, indicating that 20E directly bound to SIRT6 in HUVECs. Further investigation of the underlying mechanism showed that 20E could upregulate SIRT6 levels and that SIRT6 knockdown abolished the regulatory effect of 20E on CD40 in TNF-α-induced HUVECs, while SIRT6 overexpression further improved the effect of 20E. Moreover, we found that 20E could reduce the acetylation of NF-κB p65 (K310) through SIRT6, but the catalytic inactive mutant SIRT6 (H133Y) did not promote the deacetylation of NF-κB p65, suggesting that the inhibitory effect of 20E on NF-κB p65 was dependent on SIRT6 deacetylase activity. Additionally, our results indicated that 20E inhibited NF-κB via SIRT6, and the expression of CD40 was increased in HUVECs treated with SIRT6 siRNA and NF-κB inhibitor. In conclusion, the present study demonstrates that 20E exerts its effect through SIRT6-mediated deacetylation of NF-κB p65 (K310) to inhibit CD40 expression in ECs, and 20E may have therapeutic potential for the treatment of cardiovascular diseases.
20-羟基蜕皮甾酮（20E）具有多种药理学活性，可用于治疗糖尿病和心血管疾病。然而，20E 对内皮功能障碍的保护作用及其靶点尚不清楚。在本研究中，我们揭示了 20E 处理可以调节 TNF-α诱导的 3D 培养 HUVECs 中内皮衍生的血管舒缩因子 NO、PGI 和 ET-1 的释放，并抑制 ACE 的表达。此外，20E 抑制 TNF-α诱导的 3D 培养 HUVECs 中 CD40 的表达，促进 SIRT6 的表达。细胞热转移分析（CETSA）、药物亲和反应靶标稳定性（DARTS）和分子对接结果表明，20E 结合增加了 SIRT6 的稳定性，表明 20E 直接与 HUVECs 中的 SIRT6 结合。进一步研究其潜在机制表明，20E 可以上调 SIRT6 水平，SIRT6 敲低消除了 20E 对 TNF-α诱导的 HUVECs 中 CD40 的调节作用，而 SIRT6 过表达进一步增强了 20E 的作用。此外，我们发现 20E 可以通过 SIRT6 降低 NF-κB p65（K310）的乙酰化，而催化失活突变体 SIRT6（H133Y）不能促进 NF-κB p65 的去乙酰化，这表明 20E 对 NF-κB p65 的抑制作用依赖于 SIRT6 去乙酰化酶活性。此外，我们的结果表明，20E 通过 SIRT6 抑制 NF-κB，并且在 HUVECs 中用 SIRT6 siRNA 和 NF-κB 抑制剂处理后，CD40 的表达增加。总之，本研究表明，20E 通过 SIRT6 介导的 NF-κB p65（K310）去乙酰化发挥作用，抑制内皮细胞中 CD40 的表达，20E 可能具有治疗心血管疾病的潜力。
