Institute of Rheumatology and Immunology, The Affiliated Hospital of North Sichuan Medical College and Institute of Basic Medicine and Forensic Medicine, North Sichuan Medical College, Nanchong, 637001, Sichuan, China.
Medical Imaging Key Laboratory of Sichuan Province, North Sichuan Medical College, Nanchong, 637001, Sichuan, China.
J Transl Med. 2024 Aug 22;22(1):785. doi: 10.1186/s12967-024-05303-7.
Activation of the NLRP3 inflammasome is critical in the inflammatory response to gout. Potassium ion (K) efflux mediated by the TWIK2 channel is an important upstream mechanism for NLRP3 inflammasome activation. Therefore, the TWIK2 channel may be a promising therapeutic target for MSU crystal-induced inflammation. In the present study, we investigated the effect of ML335, a known K2P channel modulator, on MSU crystal-induced inflammatory responses and its underlying molecular mechanisms.
By molecular docking, we calculated the binding energies and inhibition constants of five K2P channel modulators (Hydroxychloroquine, Fluoxetine, DCPIB, ML365 and ML335) with TWIK2. Intracellular potassium ion concentration and mitochondrial function were assessed by flow cytometry. The interaction between MARCH5 and SIRT3 was demonstrated by immunoprecipitation and Western blotting assay. MSU suspensions were injected into mouse paw and peritoneal cavity to induce acute gout model.
ML335 has the highest binding energy and the lowest inhibition constant with TWIK2 in the five calculated K2P channel modulators. In comparison, among these five compounds, ML335 efficiently inhibited the release of IL-1β from MSU crystal-treated BMDMs. ML335 decreased MSU crystal-induced K efflux mainly dependent on TWIK2 channel. More importantly, ML335 can effectively inhibit the expression of the mitochondrial E3 ubiquitin ligase MARCH5 induced by MSU crystals, and MARCH5 can interact with the SIRT3 protein. ML335 blocked MSU crystal-induced ubiquitination of SIRT3 protein by MARCH5. In addition, ML335 improved mitochondrial dynamics homeostasis and mitochondrial function by inhibiting MARCH5 protein expression. ML335 attenuated the inflammatory response induced by MSU crystals in vivo and in vitro.
Inhibition of TWIK2-mediated K efflux by ML335 alleviated mitochondrial injury via suppressing March5 expression, suggesting that ML335 may be an effective candidate for the future treatment of gout.
NLRP3 炎性小体的激活在痛风的炎症反应中至关重要。TWIK2 通道介导的钾离子（K）外流是 NLRP3 炎性小体激活的重要上游机制。因此，TWIK2 通道可能是 MSU 晶体诱导炎症的有前途的治疗靶点。在本研究中，我们研究了已知的 K2P 通道调节剂 ML335 对 MSU 晶体诱导的炎症反应及其潜在的分子机制的影响。
通过分子对接，我们计算了五种 K2P 通道调节剂（羟氯喹、氟西汀、DCPIB、ML365 和 ML335）与 TWIK2 的结合能和抑制常数。通过流式细胞术评估细胞内钾离子浓度和线粒体功能。通过免疫沉淀和 Western blot 测定证实了 MARCH5 和 SIRT3 之间的相互作用。将 MSU 混悬液注入小鼠爪和腹腔中诱导急性痛风模型。
在计算的五种 K2P 通道调节剂中，ML335 与 TWIK2 的结合能最高，抑制常数最低。相比之下，在这五种化合物中，ML335 可有效抑制 MSU 晶体处理的 BMDM 中 IL-1β 的释放。ML335 降低 MSU 晶体诱导的 K 外流主要依赖于 TWIK2 通道。更重要的是，ML335 可以有效抑制 MSU 晶体诱导的线粒体 E3 泛素连接酶 MARCH5 的表达，并且 MARCH5 可以与 SIRT3 蛋白相互作用。ML335 阻止了 MSU 晶体诱导的 MARCH5 对 SIRT3 蛋白的泛素化。此外，ML335 通过抑制 MARCH5 蛋白表达来改善线粒体动力学平衡和线粒体功能。ML335 减轻了体内和体外 MSU 晶体诱导的炎症反应。
ML335 通过抑制 March5 表达减轻 TWIK2 介导的 K 外流，从而减轻线粒体损伤，这表明 ML335 可能是痛风未来治疗的有效候选药物。
