Luo Kaiteng, Xie Maodi, Yang Wei, Li Tao, Jiang Chunling
Department of Anesthesiology, West China Hospital, Sichuan University, Chengdu 610041, China.
Laboratory of Mitochondria and Metabolism, National-Local Joint Engineering Research Centre of Translational Medicine of Anesthesiology, West China Hospital, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2023 Sep;54(5):941-946. doi: 10.12182/20230960206.
To investigate the effect of silencing protein phosphatase 2cm ( 2) gene on the expression of inflammatory factors in macrophages infected with ( . ) and the mechanisms involved.
The effects of 2 knockdown on inflammatory factors, proliferation, apoptosis, and Toll-like receptor (TLR) signaling were analyzed in RAW 264.7 cells, a murine macrophage cell line, transfected with adenovirus (Ad). The cells were divided into four groups, including Ad-Ctrl group, Ad- 2 group, Ad-Ctrl+ . group and Ad- 2+ . group. Cell transfection was achieved by separately introducing control adenovirus (Ad-Ctrl) or adenovirus targeting the 2 gene (Ad- 2) and inflammation or the absence of inflammation was induced by applying or not applying . . The expression of tumor necrosis factor-alpha ( ), interleukin-1β ( -1 ), 2, 4, Toll-like receptor adaptor protein ( ) and myeloid differentiation factor 88 ( 88) was determined by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). PP2Cm protein expression was determined by Western blot. Cell proliferation was determined by cell counting kit-8 (CCK-8) assay and cell apoptosis was measured by flow cytometry.
The expression of 2gene and PP2Cm protein was downregulated in the Ad- 2 group when compared to the Ad-Ctrl group, with the diference showing statistical significance ( <0.05). When compared to those of the Ad-Ctrl+ . group, macrophages in the Ad- 2+ . group showed significantly increase in the - and -1 gene levels ( <0.01). Furthermore, the Ad- 2 group demonstrated elevated gene expression levels of 2, 4, and 88 in macrophages when compared to the Ad-Ctrl group, with the difference showing statistical significance ( <0.05). There were no statistically significant differences in cell apoptosis and proliferation between the Ad-Ctrl and Ad- 2 groups.
Silencing 2 gene promotes the inflammatory response of macrophages to . infection. Moreover, the TLR pathway plays an important role in the inflammatory activation of macrophages.
探讨沉默蛋白磷酸酶2Cm（PP2Cm）基因对感染结核分枝杆菌（M. tuberculosis）的巨噬细胞中炎症因子表达的影响及其相关机制。
在转染腺病毒（Ad）的小鼠巨噬细胞系RAW 264.7细胞中，分析PP2C沉默对炎症因子、增殖、凋亡和Toll样受体（TLR）信号传导的影响。细胞分为四组，包括Ad-Ctrl组、Ad-PP2C组、Ad-Ctrl+M. tuberculosis组和Ad-PP2C+M. tuberculosis组。通过分别导入对照腺病毒（Ad-Ctrl）或靶向PP2C基因的腺病毒（Ad-PP2C）实现细胞转染，通过施加或不施加M. tuberculosis诱导炎症或不诱导炎症。通过实时荧光定量聚合酶链反应（RT-qPCR）测定肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、IL-2、IL-4、Toll样受体衔接蛋白（MyD88）和髓样分化因子88（Myd88）的表达。通过蛋白质印迹法测定PP2Cm蛋白表达。通过细胞计数试剂盒-8（CCK-8）测定法测定细胞增殖，并通过流式细胞术测量细胞凋亡。
与Ad-Ctrl组相比，Ad-PP2C组中PP2C基因和PP2Cm蛋白的表达下调，差异具有统计学意义（P<0.05）。与Ad-Ctrl+M. tuberculosis组相比，Ad-PP2C+M. tuberculosis组中的巨噬细胞TNF-α和IL-1β基因水平显著升高（P<0.01）。此外，与Ad-Ctrl组相比，Ad-PP2C组中巨噬细胞的IL-2、IL-4、MyD88和Myd88基因表达水平升高，差异具有统计学意义（P<0.05）。Ad-Ctrl组和Ad-PP2C组之间的细胞凋亡和增殖没有统计学显著差异。
沉默PP2C基因可促进巨噬细胞对M. tuberculosis感染的炎症反应。此外，TLR途径在巨噬细胞的炎症激活中起重要作用。
