【摘要】 目的 基于Unc-51样自噬激活激酶1(ULK1)/自噬相关蛋白13(ATG13)/微管相关蛋白1轻链3B(LC3B)通路探讨槐定碱对结核分枝杆菌感染巨噬细胞自噬的作用机制。方法 小鼠巨噬细胞系RAW264.7分为对照组、感染组、低、中、高浓度(25、50、100μg/mL)槐定碱组、高浓度槐定碱(100μg/mL)+ULK-101(ULK1抑制剂,1μmol/L)组。对照组不需结核分枝杆菌H37Ra菌株感染,其他组RAW264.7细胞均需H37Ra感染;H37Ra感染6 h后,感染组正常培养,其他组进行对应药物干预处理24 h。检测结核分枝杆菌菌落数;酶联免疫吸附试验检测细胞上清液中白细胞介素(IL)-6、肿瘤坏死因子-α(TNF-α)、IL-1β水平;透射电镜观察自噬小体形成;单丹磺酰戊二胺染色观察RAW264.7细胞自噬小体荧光表达;免疫荧光染色观察RAW264.7细胞LC3B表达;Western blot实验检测RAW264.7细胞Beclin-1、p62、p-ULK1、ATG13、LC3B-Ⅱ/LC3B-Ⅰ、磷酸化的腺苷酸活化蛋白激酶(p-AMPK)、磷酸化的哺乳动物雷帕霉素靶蛋白(p-mTOR)表达。结果 与对照组相比,感染组细胞上清液中IL-6、TNF-α、IL-1β水平及细胞p62、p-mTOR蛋白表达水平升高,细胞自噬小体数量与平均荧光强度、LC3B表达平均荧光强度、Beclin-1、p-ULK1、ATG13、LC3B-Ⅱ/LC3B-Ⅰ、p-AMPK蛋白表达水平降低(P<0.05);与感染组相比,低、中、高浓度槐定碱组细胞中结核分枝杆菌菌落数、上清液中IL-6、TNF-α、IL-1β水平、p62、p-mTOR蛋白表达水平降低,细胞自噬小体数量与平均荧光强度、LC3B表达平均荧光强度、Beclin-1、p-ULK1、ATG13、LC3B-Ⅱ/LC3B-Ⅰ、p-AMPK蛋白表达水平升高,且高浓度槐定碱组变化趋势最为明显(P<0.05)。ULK-101可逆转高浓度槐定碱对H37Ra感染RAW264.7细胞自噬的促进作用及对过度炎症反应的抑制作用。结论 槐定碱可能通过激活ULK1/ATG13/LC3B自噬通路,增强巨噬细胞对结核分枝杆菌的清除能力,同时抑制过度的炎症反应。更多还原

【Abstract】 Objective To investigate the mechanism of Sophoridine on autophagy in macrophages infected with Mycobacterium tuberculosis(M. tuberculosis) based on the UNC-51-like autophagy activating kinase 1(ULK1)/autophagy-related protein 13(ATG13)/microtubule-associated protein 1 light chain 3B(LC3B) pathway. Methods Mouse macrophage RAW264.7 cells were divided into a control group, an infection group, low-, medium-, and high-concentration Sophoridine groups(25, 50 and 100 μg/mL), and a high-concentration Sophoridine(100 μg/mL) + ULK-101(ULK1 inhibitor, 1 μmol/L) group. The control group was not infected with M. tuberculosis H37Ra strain, while RAW264.7 cells in other groups were infected with H37Ra. At 6 h after H37Ra infection, the infection group was cultured normally, while the other groups received corresponding drug interventions for 24 h. The number of M. tuberculosis colonies was measured. The levels of interleukin(IL)-6, tumor necrosis factor-α(TNF-α), and IL-1β in the cell supernatant were detected using enzyme-linked immunosorbent assay(ELISA). Autophagosome formation was observed using transmission electron microscopy. Monodansylcadaverine(MDC) staining was used to observe the fluorescence expression of autophagosomes in RAW264.7 cells, and immunofluorescence staining was performed to observe LC3B expression in RAW264.7 cells. Western blot was used to detect the expression of Beclin-1, p62, p-ULK1, ATG13, LC3B-Ⅱ/LC3B-Ⅰ, phosphorylated adenosine monophosphate-activated protein kinase(p-AMPK), and phosphorylated mammalian target of rapamycin(p-mTOR) in RAW264.7 cells. Results Compared with the control group, the levels of IL-6, TNF-α, and IL-1β in the supernatant of RAW264.7 cells and the expression levels of p62 and p-mTOR proteins in RAW264.7 cells were significantly increased in the infection group, while the number of autophagosomes, the mean fluorescence intensity of autophagosomes, the mean fluorescence intensity of LC3B expression, and the expression of Beclin-1, p-ULK1, ATG13, LC3B-Ⅱ/LC3B-Ⅰ, and p-AMPK proteins were significantly decreased(P<0.05). Compared with the infection group, the number of M. tuberculosis colonies in RAW264.7 cells, the levels of IL-6, TNF-α, and IL-1β in the supernatant, and the expression of p62 and p-mTOR proteins in RAW264.7 cells were significantly decreased in the low-, medium-, and highconcentration Sophoridine groups, while the number of autophagosomes, the mean fluorescence intensity of autophagosomes, the mean fluorescence intensity of LC3B expression, and the expression of Beclin-1, p-ULK1, ATG13, LC3B-Ⅱ/LC3B-Ⅰ, and p-AMPK proteins were significantly increased, with the most pronounced changes observed in the high-concentration Sophoridine group(P<0.05). ULK-101 reversed the promoting effect of high-concentration Sophoridine on autophagy in H37Ra-infected RAW264.7 cells and its inhibitory effect on excessive inflammatory responses. Conclusion Sophoridine may enhance the ability of macrophages to clear M. tuberculosis and inhibit excessive inflammatory responses by activating the ULK1/ATG13/LC3B autophagy pathway.更多还原