目的 基于单磷酸腺苷活化蛋白激酶（AMPK）/NOD样受体蛋白3（NLRP3）通路探讨黄芪甲苷（AS-Ⅳ）对牙龈卟啉单胞菌（Pg）-脂多糖（LPS）诱导的人牙龈成纤维细胞（HGFs）损伤的保护作用机制。方法 AS-Ⅳ（0、5、10、20、40、80 μmol·L^-1）处理HGFs（加或不加Pg-LPS造模） 24 h，CCK-8法检测细胞存活率，筛选AS-Ⅳ作用浓度。将HGFs随机分为对照组、模型组、AS-Ⅳ（10、20、40 μmol·L^-1）组、AS-Ⅳ（40 μmol·L^-1） +CC（AMPK抑制剂，Compound C，10 μmol·L^-1，AMPK抑制剂）组。对照组正常培养，模型组和给药组细胞用10 μg·mL^-1 Pg-LPS处理12 h后，进一步加相应药物处理24 h。CCK-8和克隆形成实验检测细胞的增殖；划痕实验检测细胞的迁移；流式细胞仪检测细胞的凋亡率； ELISA法检测细胞上清液中白细胞介素（IL）-8、IL-18、IL-6的表达； Western blotting检测细胞中AMPK、NLRP3、凋亡相关斑点样蛋白（ASC）、Caspase-1蛋白的表达。结果 与对照组相比，模型组细胞存活率、克隆数、划痕愈合率、p-AMPK/AMPK表达显著降低（P＜0.05），凋亡率、IL-8、IL-18、IL-6、NLRP3、ASC、Caspase-1表达显著升高（P＜0.05）；与模型组相比，AS-Ⅳ组细胞存活率、克隆数、划痕愈合率、p-AMPK/AMPK表达显著升高（P＜0.05），凋亡率、IL-8、IL-18、IL-6、NLRP3、ASC、Caspase-1表达显著降低（P＜0.05）；与AS-Ⅳ 40 μmol·L^-1组相比，AS-Ⅳ+CC组细胞存活率、克隆数、划痕愈合率、p-AMPK/AMPK表达显著降低（P＜0.05），凋亡率、IL-8、IL-18、IL-6、NLRP3、ASC、Caspase-1表达显著升高（P＜0.05）。结论 AS-Ⅳ可能通过调控AMPK/NLRP3通路，促进Pg-LPS诱导的HGFs增殖、迁移，抑制细胞凋亡和炎症反应。

Objective To investigate the protective effect and mechanism of astragaloside IV (AS-Ⅳ) on Pg-LPS induced human gingival fibroblasts (HGFs) injury based on the adenosine monophosphate-activated protein kinase (AMPK)/NOD-like receptor protein 3 (NLRP3) pathway. Methods HGFs were treated with AS-IV (0, 5, 10, 20, 40, 80 μmol·L^-1) for 24 h, with or without Pg-LPS stimulation, and cell viability was assessed by CCK-8 assay to determine the optimal concentration of AS-IV. HGFs were randomly divided into control group, model group, AS-IV groups (10, 20, 40 μmol·L^-1), and AS-IV (40 μmol·L^-1) + CC (Compound C, 10 μmol·L^-1, an AMPK inhibitor) group. The control group was cultured under normal conditions; cells in the model and treatment groups were exposed to 10 μg·mL^-1 Pg-LPS for 12 h, followed by corresponding drug treatments for 24 h. Cell proliferation was evaluated using CCK-8 and colony formation assays; cell migration was assessed by scratch wound healing assay; apoptosis rate was measured by flow cytometry; levels of interleukin (IL)-8, IL-18, and IL-6 in cell supernatants were detected by ELISA; and expression of AMPK, NLRP3, ASC, and Caspase-1 proteins was analyzed by Western blotting. Results Compared with the control group, the model group showed significantly reduced cell viability, colony number, and scratch healing rate, as well as decreased p-AMPK/AMPK expression (P < 0.05), while apoptosis rate and expression of IL-8, IL-18, IL-6, NLRP3, ASC, and Caspase-1 were significantly increased (P < 0.05). Compared with the model group, AS-IV-treated groups exhibited significantly improved cell viability, colony number, and scratch healing rate, along with increased p-AMPK/AMPK expression (P < 0.05), while apoptosis rate and expression of IL-8, IL-18, IL-6, NLRP3, ASC, and Caspase-1 were significantly reduced (P < 0.05). Compared with the AS-IV 40 μmol·L^-1 group, the AS-IV+CC group showed significantly decreased cell viability, colony number, and scratch healing rate, along with reduced p-AMPK/AMPK expression (P < 0.05), whereas apoptosis rate and expression of IL-8, IL-18, IL-6, NLRP3, ASC, and Caspase-1 were significantly elevated (P < 0.05). Conclusion AS-Ⅳ may promote Pg-LPS induced proliferation and migration of HGFs cells, inhibit cell apoptosis and inflammatory response by regulating the AMPK/NLRP3 pathway.

R965

山西省高等学校科技创新计划项目（编号2023L477）