Wang Zimeng, Sun Yi, Wang Changyuan, Yao Jialin, Hao Ru, Wang Yang, Sun Huijun
Department of Clinical Pharmacology, College of Pharmacy, Dalian Medical University, Dalian, China.
Phytother Res. 2025 Aug 6. doi: 10.1002/ptr.70055.
Metabolic associated fatty liver disease (MAFLD) is the leading cause of chronic liver disease worldwide. Various studies have demonstrated that traditional Chinese medicine alleviates MAFLD by suppressing PANoptosis. Although Oridonin (ORI) is a traditional Chinese medicine that has been used in hepatosteatosis, its underlying mechanism for treating MAFLD, especially regarding PANoptosis, remains unclear. Therefore, this study aims to investigate the ameliorative effects of ORI on MAFLD by inhibiting PANoptosis and to elucidate the underlying mechanisms. A vivo model of MAFLD was constructed by feeding mice with a high-fat diet (HFD) for 12 weeks. ORI was simultaneously intragastric gavaged for 12 weeks. A vitro model of MAFLD was constructed by palmitic acid (PA) induced AML12 cells for 24 h; ORI was simultaneously induced for 24 h. Extraction of mice plasma for detecting lipid-related and oxidative stress-related assay kits was performed. Lipid metabolism and PANoptosis-related proteins and pathways were validated both in vivo and in vitro through western blotting and immunofluorescence. The functions of the candidate proteins were further investigated using the plasmid transfection technique. Then the Biotin labeling assays were used to analyze the proteins' direct interaction with ORI. ORI treatment significantly alleviated HFD-induced inflammation and PANoptosis. Likewise, ORI effectively attenuated PANoptosis induced by PA in the AML12 cells. Additionally, over-expression of NLRP3, which is a key component in PANoptosis, could partly antagonize the inhibiting effects of ORI on steatosis and PANoptosis. Next, our findings demonstrated the increased NLRP3 acetylation level in PA-induced cells, while ORI treatment decreased the NLRP3 acetylation level. Further studies revealed that ORI could bind to and activate SIRT2 to increase the protein expression and stability of SIRT2, which in turn deacetylated and inactivated NLRP3. Moreover, over-expression of NLRP3 could partly antagonize the inhibiting effects of ORI on lipid droplet deposition and PANoptosis, which could be rescued by over-expression of SIRT2. Therefore, these findings show that ORI can alleviate MAFLD by suppressing PANoptosis via inhibiting the SIRT2/NLRP3 pathway. These findings provide new therapeutic targets and mechanisms for the treatment of MAFLD.
代谢相关脂肪性肝病（MAFLD）是全球慢性肝病的主要病因。各种研究表明，中药通过抑制PAN凋亡来减轻MAFLD。虽然冬凌草甲素（ORI）是一种已用于治疗肝脂肪变性的中药，但其治疗MAFLD的潜在机制，尤其是与PAN凋亡相关的机制仍不清楚。因此，本研究旨在通过抑制PAN凋亡来研究ORI对MAFLD的改善作用，并阐明其潜在机制。通过给小鼠喂食高脂饮食（HFD）12周构建MAFLD的体内模型。同时对小鼠进行12周的ORI灌胃。通过棕榈酸（PA）诱导AML12细胞24小时构建MAFLD的体外模型；同时进行24小时的ORI诱导。提取小鼠血浆用于检测脂质相关和氧化应激相关的检测试剂盒。通过蛋白质印迹和免疫荧光在体内和体外验证脂质代谢和PAN凋亡相关蛋白及信号通路。使用质粒转染技术进一步研究候选蛋白的功能。然后使用生物素标记试验分析蛋白质与ORI的直接相互作用。ORI治疗显著减轻了HFD诱导的炎症和PAN凋亡。同样，ORI有效减轻了PA诱导的AML12细胞中的PAN凋亡。此外，作为PAN凋亡关键成分的NLRP3的过表达可部分拮抗ORI对脂肪变性和PAN凋亡的抑制作用。接下来，我们的研究结果表明，PA诱导的细胞中NLRP3乙酰化水平升高，而ORI治疗降低了NLRP3乙酰化水平。进一步的研究表明，ORI可以结合并激活SIRT2，以增加SIRT2的蛋白表达和稳定性，进而使NLRP3去乙酰化并使其失活。此外，NLRP3的过表达可部分拮抗ORI对脂滴沉积和PAN凋亡的抑制作用，而SIRT2的过表达可挽救这种作用。因此，这些研究结果表明，ORI可通过抑制SIRT2/NLRP3通路抑制PAN凋亡来减轻MAFLD。这些研究结果为MAFLD的治疗提供了新的治疗靶点和机制。
