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脂多糖通过触发髓系细胞表达的受体-1调节巨噬细胞极化

[ lipopolysaccharide regulates macrophage polarization via triggering receptors expressed on myeloid cells-1].

作者信息

Liu L, Yang Y, Zhou J Y, Wu Y F, Zhao L

机构信息

Department of Periodontology, West China Hospital of Stomatology, Sichuan University & State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases, Chengdu 610041, China Yang Yun is working on 3E Dental, Chengdu 610041, China.

出版信息

Zhonghua Kou Qiang Yi Xue Za Zhi. 2021 Feb 9;56(2):175-181. doi: 10.3760/cma.j.cn112144-20200210-00042.

Abstract

To investigate the relationship between pattern recognition receptor triggering receptors expressed on myeloid cells-1 (TREM-1) and M1/M2 polarization in macrophages stimulated by lipopolysaccharide (Pg-LPS), so as to explore the mechanism of TREM-1 in periodontitis. Human monocytic cell line THP-1 were induced to differentiate into macrophages by phorbol-12-myristate-13-acetate and stimulated by 0 (blank control group) and 1 μg/ml Pg-LPS (LPS group), respectively. LP17, the TREM-1 inhibitor (LPS+LP17 group) and its control peptide (LPS+control peptide group) with final concentration of 0.1 μg/ml were added at the same time. After 24 hours stimulation, the expression of TREM-1, M1 markers and related cytokines [CD86, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β], M2 markers and related cytokines (CD206,IL-10) mRNA were detected by real-time quantitative-PCR (RT-qPCR), the level of TREM-1, CD86 and CD206 proteins were detected by Western blotting, and TNF-α, IL-1β and IL-10 in the macrophage culture supernatant were detected by enzyme linked immunosorbent assay. After 24 h of cell culture, the relative expressions of TREM-1 mRNA (1.40±0.14) and protein (3.85±0.24) in macrophages in the LPS group increased compared with the blank control group (1.01±0.18 and 1.00±0.05, respectively) (<0.05). Meanwhile, the expression levels of M1 markers CD86 mRNA and protein [LPS group vs blank control group were (1.42±0.01 vs 1.00±0.09) and (1.55±0.07 vs 1.00±0.10), respectively] were up-regulated (<0.01), and the expressions of mRNA and protein of M1 related cytokines TNF-α and IL-1 increased (<0.05). After the addition of TREM-1 blocker LP17, the levels of mRNA and protein of TREM-1 showed no significant changes (0.05), while the levels of CD86 mRNA (0.96±0.00) and protein (1.36±0.02) decreased (<0.05), and the levels of TNF-α and IL-1 further decreased (<0.05). For M2 marker CD206 and related cytokine IL-10, CD206 mRNA (0.56±0.05) and protein (0.25±0.04) were significantly down-regulated (<0.01) compared with the blank control group (1.02±0.25 and 1.00±0.10, respectively), and IL-10 mRNA was up-regulated compared with the blank control group (<0.05), with no significant change in protein (>0.05). After the addition of LP17, the expressions of CD206 and IL-10 mRNA in the LPS+LP17 group were further down-regulated compared with the LPS group (<0.05), and there was no statistically significant difference between the two groups in protein level (>0.05). TREM-1 and its downstream signaling pathway might be involved in M1 polarization of Pg-LPS-mediated macrophages, thus playing a pro-inflammatory role in the development of periodontitis. There is no obvious evidence that TREM-1 is involved in regulating M2 polarization of Pg-LPS-mediated macrophages.

摘要

为研究髓系细胞触发受体-1(TREM-1)与牙龈卟啉单胞菌脂多糖(Pg-LPS)刺激的巨噬细胞M1/M2极化之间的关系,以探讨TREM-1在牙周炎中的作用机制。采用佛波酯诱导人单核细胞系THP-1分化为巨噬细胞,分别用0(空白对照组)和1 μg/ml Pg-LPS(LPS组)刺激。同时加入终浓度为0.1 μg/ml的TREM-1抑制剂LP17(LPS+LP17组)及其对照肽(LPS+对照肽组)。刺激24小时后,采用实时定量聚合酶链反应(RT-qPCR)检测TREM-1、M1标志物及相关细胞因子[CD86、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β]、M2标志物及相关细胞因子(CD206、IL-10)mRNA的表达,采用蛋白质免疫印迹法检测TREM-1、CD86和CD206蛋白水平,采用酶联免疫吸附测定法检测巨噬细胞培养上清液中TNF-α、IL-1β和IL-10水平。细胞培养24小时后,LPS组巨噬细胞TREM-1 mRNA相对表达量(1.40±0.14)和蛋白相对表达量(3.85±0.24)较空白对照组(分别为1.01±0.18和1.00±0.05)升高(<0.05)。同时,M1标志物CD86 mRNA和蛋白表达水平[LPS组与空白对照组分别为(1.42±0.01比1.00±0.09)和(1.55±0.07比 1.00±0.10)]上调(<0.01),M1相关细胞因子TNF-α和IL-1的mRNA和蛋白表达增加(<0.05)。加入TREM-1阻断剂LP17后,TREM-1的mRNA和蛋白水平无明显变化(>0.05),而CD86 mRNA(0.96±0.00)和蛋白(1.36±0.02)水平降低(<0.05),TNF-α和IL-1水平进一步降低(<0.05)。对于M2标志物CD206及相关细胞因子IL-10,与空白对照组(分别为1.02±0.25和1.00±0.10)相比,CD206 mRNA(0.56±0.05)和蛋白(0.25±0.04)显著下调(<0.01),IL-10 mRNA较空白对照组上调(<0.05),蛋白水平无明显变化(>0.05)。加入LP17后,LPS+LP17组CD206和IL-10 mRNA表达较LPS组进一步下调(<0.05),两组蛋白水平差异无统计学意义(>0.05)。TREM-1及其下游信号通路可能参与Pg-LPS介导的巨噬细胞M1极化,从而在牙周炎发展中发挥促炎作用。无明显证据表明TREM-1参与调节Pg-LPS介导的巨噬细胞M2极化。

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