Bao You-Li, Cao Yin, Wu Hong-Fei
Anhui Province Key Laboratory of Research & Development of Chinese Medicine, Department of Pharmacy, Anhui University of Chinese Medicine Hefei 230012, China.
Zhongguo Zhong Yao Za Zhi. 2023 May;48(10):2820-2828. doi: 10.19540/j.cnki.cjcmm.20230103.707.
This study aims to explore the effect of "Trichosanthis Fructus-Allii Macrostemonis" combination(GX) on the activation of NOD-, LRR-, and pyrin domain-containing protein 3(NLRP3) inflammasome, the release of inflammatory cytokines, and the level of autophagy in RAW264.7 macrophage damaged by lipopolysaccharide(LPS), and the mechanism of GX against inflammatory response in macrophages. To be specific, LPS was used to induce the injury of RAW264.7 cells. Cell Counting Kit-8(CCK-8) assay was employed to measure the survival rate of cells, and Western blot to detect the protein expression of NLRP3, apoptosis-associated speck-like protein(ASC), cysteine-aspartic acid protease(caspase)-1, interleukin(IL)-18, IL-1β, microtubule-associated protein light chain 3(LC3)-Ⅱ, and selective autophagy junction protein p62/sequestosome 1 in RAW264.7 macrophages. ELISA was used to measure the levels of IL-18 and IL-1β in RAW264.7 cells. Transmission electron microscopy was applied to observe the number of autophagosomes in RAW264.7 cells. Immunofulourescence staining was used to detect the expression of LC3-Ⅱ and p62 in RAW264.7 cells. The result showed that GX significantly reduced the protein expression of NLRP3, ASC, and caspase-1 in RAW264.7 cells, significantly increased the protein expression of LC3Ⅱ, decreased the expression of p62, significantly inhibited the secretion of IL-18 and IL-1β, significantly increased the number of autophagosomes, significantly enhanced the immunofluorescence of LC3Ⅱ, and reduced the immunofluorescence of p62. Furthermore, 3-methyladenine(3-MA) could reverse the inhibitory effect of GX on NLRP3, ASC, and caspase-1 and reduce the release of IL-18 and IL-1β. In summary, GX can increase of the autophagy activity of RAW264.7 and inhibit the activation of NLRP3 inflammasome, thereby reducing the release of inflammatory cytokines and suppressing inflammatory response in macrophages.
本研究旨在探讨“瓜蒌-薤白”药对（GX）对脂多糖（LPS）损伤的RAW264.7巨噬细胞中含核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）炎性小体激活、炎性细胞因子释放及自噬水平的影响，以及GX抗巨噬细胞炎症反应的机制。具体而言，采用LPS诱导RAW264.7细胞损伤。运用细胞计数试剂盒-8（CCK-8）法检测细胞存活率，采用蛋白质免疫印迹法检测RAW264.7巨噬细胞中NLRP3、凋亡相关斑点样蛋白（ASC）、半胱天冬酶（caspase）-1、白细胞介素（IL）-18、IL-1β、微管相关蛋白轻链3（LC3）-Ⅱ及选择性自噬连接蛋白p62/隔离小体1的蛋白表达。采用酶联免疫吸附测定法（ELISA）检测RAW264.7细胞中IL-18和IL-1β水平。运用透射电子显微镜观察RAW264.7细胞中自噬体数量。采用免疫荧光染色法检测RAW264.7细胞中LC3-Ⅱ和p62的表达。结果显示，GX可显著降低RAW264.7细胞中NLRP3、ASC和caspase-1的蛋白表达，显著增加LC3Ⅱ的蛋白表达，降低p62的表达，显著抑制IL-18和IL-1β的分泌，显著增加自噬体数量，显著增强LC3Ⅱ的免疫荧光，降低p62的免疫荧光。此外，3-甲基腺嘌呤（3-MA）可逆转GX对NLRP3、ASC和caspase-1的抑制作用，并减少IL-18和IL-1β的释放。综上所述，GX可增强RAW264.7细胞的自噬活性，抑制NLRP3炎性小体的激活，从而减少炎性细胞因子的释放，抑制巨噬细胞的炎症反应。
