（河北中石油中心医院口腔科，廊坊 065000）
摘　要：为探究藤黄酸对脂多糖诱导人牙周膜干细胞炎症反应和凋亡的影响及Wnt/β-catenin信号通路的调控作用，体外培养hPDLSCs细胞，分为预实验和正式实验两步进行。预实验分为对照组（不做干预）、脂多糖组（10 μg/mL脂多糖）、低/中/高浓度藤黄酸组（10 μg/mL 脂多糖+0.5、1.0、2.0 μmol/L藤黄酸）。正式实验分为对照组、脂多糖组、藤黄酸组（10 μg/mL 脂多糖+2.0 μmol/L藤黄酸）、抑制剂组（10 μg/mL脂多糖+2 μmol/L Wnt/β-catenin信号通路抑制剂XAV939）、藤黄酸+抑制剂组（10 μg/mL脂多糖+2.0 μmol/L藤黄酸+2 μmol/L XAV939）和藤黄酸+激活剂组（10 μg/mL脂多糖+2.0 μmol/L藤黄酸+20 μmol/L Wnt/β-catenin信号通路激活剂SKL2001），干预24 h。用活细胞计数试剂盒（CCK-8）法检测细胞活力；用酶联免疫吸附试验（ELISA）检测炎症因子肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）和白细胞介素-6（IL-6）的表达水平；用Hoechst 33258染色法检测细胞凋亡率；用蛋白免疫印迹（Wb）法检测B淋巴细胞瘤-2（Bcl-2）和半胱氨酸天冬氨酸蛋白酶-3（Caspase-3）及Wnt/β-catenin信号通路相关蛋白的表达水平。预实验中，脂多糖组的细胞活力较对照组降低（P<0.05），炎症因子TNF-α、IL-1β和IL-6的表达水平增加（P<0.05）；高浓度藤黄酸组细胞活力较脂多糖组增加（P<0.05），炎症因子TNF-α、IL-1β和IL-6的表达水平降低（P<0.05），所以选择高浓度藤黄酸组作为最佳作用浓度进行正式实验。正式实验中，脂多糖组细胞活力和抗凋亡Bcl-2蛋白的表达水平较对照组降低（P<0.05），炎症因子TNF-α、IL-1β和IL-6的表达水平、细胞凋亡率以及Caspase-3、Wnt和β-catenin蛋白的表达量增加（P<0.05）；藤黄酸组和抑制剂组的细胞活力和抗凋亡Bcl-2蛋白的表达水平较脂多糖组增加（P<0.05），炎症因子TNF-α、IL-1β、IL-6的表达水平、细胞凋亡率以及Caspase-3、Wnt和β-catenin蛋白的表达水平降低（P<0.05）；相较于藤黄酸组，藤黄酸+抑制剂组上述指标的变化趋势更加显著（P<0.05），藤黄酸+激活剂组则显著扭转了这些指标的变化趋势（P<0.05）。藤黄酸或可通过抑制Wnt/β-catenin信号通路信号转导促进脂多糖诱导的人hPDLSCs的细胞活性，抑制其凋亡及炎症反应。本研究可为牙周炎的治疗提供新的理论参考。
关键词：牙周炎；藤黄酸；脂多糖；牙周膜干细胞；Wnt/β-catenin信号通路
中图分类号：R781.4                          文献标志码：ADOI：10.3969/j.issn.1007-7146.2024.06.011

（Department of Stomatology, Hebei Petro China Central Hospital, Langfang 065000, China）
Abstract: To investigate the effects of luteic acid on inflammation and apoptosis of human periodontal stem cells induced by lipopolysaccharide and the regulation of Wnt/β-catenin signaling pathway, hPDLSCs were cultured in vitro, which was divided into two steps: pre-experiment and formal experiment. The pre-experiment was divided into control group (no intervention), lipopolysaccharide group (10 μg/mL lipopolysaccharide), low/medium/high concentration luboflavic acid group (10 μg/mL lipopolysaccharide +0.5, 1.0, 2.0 μmol/L luboflavic acid). The formal experiment was divided into control group, lipopolypaccharide group, luteic acid group (10 μg/mL lipopolypaccharide +2.0 μmol/L luteic acid), inhibitor group (10 μg/mL lipopolypaccharide +2 μmol/L Wnt/β-catenin signaling pathway inhibitor XAV939), luteic acid + inhibitor group (10 μg/mL lipopolysaccharide +2.0 μmol/L luteic acid +2 μmol/L XAV939) and luteic acid + activator group (10 μg/mL lipopolysaccharide +2.0 μmol/L luteic acid +20 μmol/L Wnt/β-catenin signaling pathway activator SKL2001), they were treated for 24 h. Cell viability was measured by CCK-8. The expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were detected by enzyme-linked immunosorbent assay (ELISA). The apoptosis rate was detected by Hoechst 33258 staining. The expression levels of B lymphoblastoma-2 (Bcl-2), cysteine aspartate protease-3 (Caspase-3) and Wnt/β-catenin signaling pathway were detected by Western blot (Wb). In the preliminary experiment, the cell viability of LPS group decreased compared with control group (P<0.05), and the expression levels of inflammatory factors TNF-α, IL-1β and IL-6 increased (P<0.05). Compared with LPS group, the cell viability of high-concentration luteic acid group increased (P<0.05), and the levels of inflammatory factors TNF-α, IL-1β and IL-6 decreased (P<0.05). Therefore, the high-concentration luteic acid group was selected as the optimal concentration for formal experiment. In the formal experiment, the cell viability and anti-apoptotic Bcl-2 protein expression levels in LPS group decreased compared with those in control group (P<0.05), while the expression levels of inflammatory factors TNF-α, IL-1β and IL-6, apoptosis rate and the expression levels of Caspase-3, Wnt and β-catenin proteins increased (P<0.05). Compared with LPS group, the cell viability and anti-apoptotic Bcl-2 protein expression levels in luteic acid group and inhibitor group increased (P<0.05), while the expression levels of inflammatory factors TNF-α, IL-1β, IL-6, cell apoptosis rate and the expression levels of Caspase-3, Wnt and β-catenin proteins decreased (P<0.05). Compared with the luteic acid group, the change trend of the above indexes in the luteic acid + inhibitor group was more significant (P<0.05), and the change trend of these indexes in the luteic acid + activator group was significantly reversed (P<0.05). Luteic acid may promote the cellular activity of human hPDLSCs induced by lipopolysaccharide and inhibit their apoptosis and inflammation by inhibiting the signal transduction of Wnt/β-catenin signaling pathway. This study can provide a new theoretical reference for the treatment of periodontitis.
Key words: periodontitis; gambogic acid; lipopolysaccharide; periodontal membrane stem cells; Wnt/β-catenin signaling pathway
（Acta Laser Biology Sinica, 2024, 33(6): 567-576）