Shengyu decoction ameliorates knee osteoarthritis by inhibiting endoplasmic reticulum stress via Piezo1 channels

Background: Shengyu decoction (SYD) is a classic and excellent prescription of traditional Chinese Medicine (TCM). The innovative use of SYD by Chinese medical master Prof. Qi Shi in the treatment of knee osteoarthritis (KOA) has achieved considerable clinical outcomes. However, the current weakness is the lack of study on the active ingredients and mechanisms of SYD. Purpose: To evaluate the role of SYD in reducing KOA cartilage damage as well as to explore the active ingredients and mechanisms of SYD. Methods: The KOA rat model and chondrocyte model were established. This study employed various molecular biology techniques to clarify the role of SYD in vivo and in vitro. Furthermore, the active ingredients and mechanisms of SYD were analyzed through ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), RNA sequencing, molecular docking and surface plasmon resonance (SPR) experiment. Finally, rescue experiments were conducted to verify the mechanisms. Results: The results revealed that SYD could significantly reduce cartilage tissue lesions, inhibit inflammation and regulate proliferation-apoptosis balance. Transcriptome analysis showed an increase in the expression of Piezo1, Xbp1, Atf6 in KOA and SYD downregulated them. UPLC-Q-TOF-MS analysis revealed four bioactive compounds of SYD, which were further confirmed to directly interact with Piezo1 through molecular docking and SPR assays. Furthermore, SYD downregulated the calcium ion concentration, Piezo1 and ERS intensity. Meanwhile, in the rescue experiment, Yoda1, the agonist of Piezo1, antagonized the pharmacological effects of SYD. Conclusions: The present results provides strong evidence that SYD protected articular cartilage via inhibiting the Piezo1-mediated ERS signaling pathway. Overall, our work emphasizes the pivotal role of TCM in addressing medical challenges and provides new ideas for the treatment of KOA.背景：生愈汤（Shengyu decoction, SYD）是中医药（traditional Chinese Medicine, TCM）领域经典优良的方剂。中国医学大师齐实教授创新性地将其应用于膝骨关节炎（knee osteoarthritis, KOA）的治疗，取得了可观的临床疗效。然而当前研究存在明显短板：生愈汤的活性成分与作用机制尚未得到系统阐明。 目的：评估生愈汤减轻膝骨关节炎软骨损伤的作用，并探究其活性成分与潜在作用机制。 方法：本研究构建了膝骨关节炎大鼠模型与软骨细胞模型，采用多种分子生物学技术在体内、体外层面明确生愈汤的药理作用。此外，通过超高效液相色谱-四极杆飞行时间质谱（ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, UPLC-Q-TOF-MS）、RNA测序、分子对接及表面等离子体共振（surface plasmon resonance, SPR）实验分析生愈汤的活性成分与作用机制，并最终通过挽救实验验证其作用通路。 结果：研究结果显示，生愈汤可显著减轻软骨组织病变、抑制炎症反应，并调控细胞增殖-凋亡平衡。转录组学分析表明，膝骨关节炎模型中Piezo1、Xbp1、Atf6的表达水平显著上调，而生愈汤可下调这些基因的表达。超高效液相色谱-四极杆飞行时间质谱分析筛选出生愈汤的4种生物活性成分，经分子对接与表面等离子体共振实验证实，这些成分可直接与Piezo1靶向结合。进一步研究发现，生愈汤可降低细胞内钙离子浓度、Piezo1表达水平及内质网应激（endoplasmic reticulum stress, ERS）强度。在挽救实验中，Piezo1激动剂Yoda1可拮抗生愈汤的药理作用。 结论：本研究结果提供了强有力的证据，证实生愈汤通过抑制Piezo1介导的内质网应激信号通路发挥关节软骨保护作用。综上，本研究凸显了中医药在应对医学挑战中的关键价值，同时为膝骨关节炎的治疗提供了全新研究思路。