目的 探讨梓醇对脂多糖（lipopolysaccharide，LPS）诱导的软骨细胞炎症损伤、细胞外基质（extracellular matrix，ECM）代谢失衡及细胞焦亡的影响，并探讨其与腺苷酸活化蛋白激酶（AMP-activated protein kinase，AMPK）/核因子-κB（nuclear factor-κB，NF-κB）/NOD样受体热蛋白结构域3（NOD like receptor family pyrin domain containing 3，NLRP3）信号轴的关系。方法 通过网络药理学筛选梓醇、膝骨关节炎与细胞焦亡的交集靶点并进行富集分析。采用LPS建立炎症损伤并诱导焦亡模型，给予梓醇（20、50 μmol/L）预处理1 h，Western blotting检测ECM相关蛋白II型胶原α1链（collagen type II alpha 1 chain，COL2A1）、聚集蛋白聚糖（aggrecan，ACAN）、基质金属蛋白酶3（matrix metalloproteinase 3，MMP3）和MMP13及AMPK/NF-κB/NLRP3信号轴关键蛋白表达；乳酸脱氢酶（lactate dehydrogenase，LDH）释放实验评估细胞膜完整性相关损伤；ELISA法检测细胞上清液中白细胞介素-1β（interleukin-1β，IL-1β）和IL-18水平；qRT-PCR检测NLRP3、IL-1β、IL-6和肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）的mRNA表达。以半胱氨酸天冬氨酸蛋白酶-1（cystein-asparate protease-1，Caspase-1）抑制剂VX-765为阳性对照，并采用AMPK抑制剂Compound C进一步验证机制。结果 与对照组比较，LPS刺激可诱导ECM合成下降、分解增强，并显著激活NLRP3相关焦亡通路（P＜0.001）。与模型组比较，梓醇可显著上调COL2A1、ACAN表达并下调MMP3、MMP13表达（P＜0.05、0.01、0.001），同时显著降低LDH、IL-1β与IL-18的释放（P＜0.01、0.001），并抑制NLRP3、cleaved Caspase-1及消皮素D的N端片段（N-terminal fragment of gasdermin D，GSDMD-N）等焦亡相关指标（P＜0.05、0.001）；此外，梓醇可显著上调p-AMPK/AMPK及磷酸化乙酰辅酶A羧化酶（phosphorylated acetyl-CoA carboxylase，p-ACC）/ACC表达（P＜0.05、0.001），并下调p-p65/p65表达（P＜0.001），同时下调NLRP3、IL-1β、IL-6、TNF-α炎症相关基因表达（P＜0.05、0.001）。AMPK抑制剂Compound C可部分逆转梓醇的上述作用（P＜0.05、0.01、0.001）。结论 梓醇可能通过激活AMPK并促进下游ACC磷酸化，抑制NF-κB/NLRP3相关通路，从而保护软骨细胞基质稳态。

Objective To investigate the effect of catalpol on lipopolysaccharide (LPS)-induced inflammatory injury, extracellular matrix (ECM) metabolic dysregulation and pyroptosis in chondrocytes, and to explore whether these effects involve the AMP-activated protein kinase (AMPK)/nuclear factor-κB (NF-κB)/NOD like receptor family pyrin domain containing 3 (NLRP3) signaling axis. Methods Network pharmacology was used to screening the intersection targets of catalpol, knee osteoarthritis and pyroptosis and enrichment analysis were performed. LPS was used to establish an inflammatory injury and induce pyroptosis model, followed by pretreatment with catalpol (20, 50 μmol/L) for 1 h. Protein expressions of collagen type II alpha 1 chain (COL2A1), aggrecan (ACAN), matrix metalloproteinase 3 (MMP3), MMP13 and key molecules in AMPK/NF-κB/NLRP3 signaling axis were detected by Western blotting. Lactate dehydrogenase (LDH) release assay was performed to assess cell membrane integrity-related injury. The levels of interleukin-1β (IL-1β) and IL-18 in supernatants were detected by ELISA. The mRNA levels of NLRP3, IL-1β, IL-6 and tumor necrosis factor-α (TNF-α) were detected by qRT-PCR. The cystein-asparate protease-1 (Caspase-1) inhibitor VX-765 was used as a positive control, the mechanism was further validated using AMPK inhibitor Compound C. Results Compared with control group, LPS stimulation could induce a decrease in ECM synthesis and an increase in ECM decomposition, significantly activate the NLRP3 related pyroptosis pathway (P < 0.001). Compared with model group, catalpol could significantly upregulate the expressions of COL2A1, ACAN and downregulate the expressions of MMP3, MMP13 (P < 0.05, 0.01, 0.001), while significantly reduce the release of LDH, IL-1β and IL-18 (P < 0.01, 0.001), and inhibit NLRP3, cleaved Caspase-1, N-terminal fragment of gasdermin D (GSDMD-N) and other pyroptosis related indicators (P < 0.05, 0.001). In addition, catalpol could significantly upregulate the expressions of p-AMPK/AMPK and phosphorylated acetyl CoA carboxylase (p-ACC)/ACC (P < 0.05, 0.001), and downregulate the expression of p-p65/p65 (P < 0.001), while downregulate the expressions of NLRP3, IL-1β, IL-6, TNF-α inflammation related genes (P < 0.05, 0.001). AMPK inhibitor Compound C could partially reverse the above-mentioned effects of catalpol (P < 0.05, 0.01, 0.001). Conclusion Catalpol may protect the matrix homeostasis of chondrocytes by activating AMPK and promoting downstream ACC phosphorylation, inhibiting NF-κB/NLRP3 related pathways.

R285.5

国家自然科学基金面上项目（82575094）；广东省自然科学基金面上项目（2024A1515012062）；中医证候全国重点实验室项目（SLKY2025A0002）；广州中医药大学2025年校级“揭榜挂帅”研究生创新能力提升项目（A3-0317-25-429-008）