Li F, Chen J L, Zeng X L, Bao H R, Liu X J
Department of Gerontal Respiratory Medicine, the Frist Hospital of Lanzhou University, Lanzhou 730000, China.
Zhonghua Yi Xue Za Zhi. 2018 Sep 11;98(34):2743-2748. doi: 10.3760/cma.j.issn.0376-2491.2018.34.013.
To investigate the effects of Toll-like receptor 4 (TLR4)-phosphatidylinositol 3-kinase (PI3K) -Ras-related C3 botulinum toxin 1 (Rac1) signaling pathway on macrophage cytoskeleton rearrangement and phagocytosis. Mouse macrophage cell line RAW264.7 was divided into blank group, negative control group and TLR4-RNA interference (RNAi) group. The lentivirus carrying TLR4 short hairpin RNA (shRNA) and nonsense control sequence were respectively transfected into TLR4-RNAi group and negative control group. The cells in blank group were not transfected. The silencing efficiency of TLR4 was detected by Western blot. Real-time fluorescence quantitative PCR was used to detect the expression of PI3K and Rac1 mRNA in each group. The expressions of PI3K, p-Rac1 and Rac1 protein were detected by Western blot. Cytoskeleton was observed by laser scanning confocal microscopy. Mean fluorescence intensity (MFI) and the percentage of cells phagocytosing flurescein inothiocyanate-labeled Eseherichina coli (FITC-.) (Phagocytosed cell %) were detected by flow cytometry. The RAW264.7 cells can be successfully transfected by TLR4-shRNA lentivirus, and the transfection efficiency ranged from 80% to 90%. The silencing efficiency of TLR4 gene was (63±4)%. After silencing the TLR4 gene, the relative expression of TLR4 mRNA and protein (0.20±0.03, 0.37±0.04), PI3K mRNA and protein (0.64±0.06, 0.75±0.06), Rac1 mRNA, protein and p-Rac1 protein (0.75±0.04, 0.76±0.01, 0.74±0.05) in TLR4-RNAi group were significantly lower than those in negative control group and blank group (all <0.01). The change of cytoskeleton: after silencing the TLR4 gene, the celluar pseudopods were short and stiff, with the impaired capacity of phagocytosing FITC-.. Cells in blank group and negative control group extended good pseudopodia, and the capacity of phagocytosing FITC-. was normal. The MFI and Phagocytosed cells % of TLR4-RNAi group[(7 453±564), (70.20±2.27)%]were significantly lower than those in the blank group and the negative control group (all <0.01). Positive correlations were existed between mRNA, protein expression of TLR4, PI3K, Rac1 and MFI, Phagocytosed cells% (all <0.05) in all groups. TLR4-PI3K-Rac1 pathway involves in the cytoskeleton rearrangement and impairs the phagocytosis of macrophages.
探讨Toll样受体4（TLR4）-磷脂酰肌醇3激酶（PI3K）-Ras相关C3肉毒杆菌毒素底物1（Rac1）信号通路对巨噬细胞骨架重排及吞噬作用的影响。将小鼠巨噬细胞系RAW264.7分为空白组、阴性对照组和TLR4-RNA干扰（RNAi）组。分别将携带TLR4短发夹RNA（shRNA）的慢病毒和无义对照序列转染至TLR4-RNAi组和阴性对照组。空白组细胞未转染。采用蛋白质免疫印迹法检测TLR4的沉默效率。采用实时荧光定量PCR检测各组PI3K和Rac1 mRNA的表达。采用蛋白质免疫印迹法检测PI3K、p-Rac1和Rac1蛋白的表达。通过激光扫描共聚焦显微镜观察细胞骨架。采用流式细胞术检测平均荧光强度（MFI）及吞噬异硫氰酸荧光素标记的大肠杆菌（FITC-E. coli）的细胞百分比（吞噬细胞%）。TLR4-shRNA慢病毒可成功转染RAW264.7细胞，转染效率为80%~90%。TLR4基因的沉默效率为（63±4）%。沉默TLR4基因后，TLR4-RNAi组TLR4 mRNA和蛋白（0.20±0.03，0.37±0.04）、PI3K mRNA和蛋白（0.64±0.06，0.75±0.06）、Rac1 mRNA、蛋白及p-Rac1蛋白（0.75±0.04，0.76±0.01，0.74±0.05）的相对表达均显著低于阴性对照组和空白组（均P<0.01）。细胞骨架变化：沉默TLR4基因后，细胞伪足短而僵硬，吞噬FITC-E. coli的能力受损。空白组和阴性对照组细胞伪足伸展良好，吞噬FITC-E. coli的能力正常。TLR4-RNAi组的MFI和吞噬细胞%[（7 453±564），（70.20±2.27）%]均显著低于空白组和阴性对照组（均P<0.01）。各组TLR4、PI3K、Rac1的mRNA及蛋白表达与MFI、吞噬细胞%均呈正相关（均P<0.05）。TLR4-PI3K-Rac1信号通路参与巨噬细胞骨架重排并损害其吞噬作用。
