颗粒蛋白前体通过 NF-кB 和 MAPK 通路抑制 LPS 诱导的巨噬细胞 M1 极化。
Progranulin inhibits LPS-induced macrophage M1 polarization via NF-кB and MAPK pathways.
机构信息
Department of Periodontology, School of Stomatology, Shandong University, 44 West Wenhua Road, Jinan, 250012, Shandong, People's Republic of China.
Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Jinan, Shandong, China.
出版信息
BMC Immunol. 2020 Jun 5;21(1):32. doi: 10.1186/s12865-020-00355-y.
BACKGROUND
Macrophage M1 polarization plays a pivotal role in inflammatory diseases. Progranulin (PGRN) has potential anti-inflammation action, however, the effect of PGRN on macrophage M1 polarization has been poorly studied. Our study aimed to investigate the effect of PGRN on lipopolysaccharide (LPS)-induced macrophage M1 polarization and clarify the underlying mechanisms.
METHODS
RAW264.7 cells were polarized to M1 macrophage by LPS with or without recombinant PGRN (rPGRN) and tumor necrosis factor alpha antibody (anti-TNF-α). A cell counting kit-8 assay (CCK-8), flow cytometry, Quantitative Real-Time PCR assay (q-PCR), Western blot assay and enzyme-linked immunosorbent assay (ELISA) were used to determine the effect of different treatments on cell proliferation, expression of surface phenotype marker and expressions and secretion of inflammatory cytokines. The activation of NF-κB/mitogen-activated protein kinase (MAPK) pathways and the nuclear translocation of NF-κB p65 were detected by Western blot and immunofluorescence respectively. THP-1 and primary bone marrow-derived monocytes (BMDMs) were also used to demonstrate effect of PGRN on expressions and secretion of inflammatory cytokines induced by LPS.
RESULTS
In RAW264.7 cells, rPGRN at concentrations below 80 ng/ml significantly promoted cell proliferation in dose dependent fashion. rPGRN significantly inhibited LPS-induced change of phenotype (CD86/CD206 ratio) and function (tumor necrosis factor (TNF-α) and inducible nitric oxide synthase (iNOS) expressions). LPS-stimulated secretion of TNF-α and activated phosphorylation of IKKα/β, IкBα, p65, JNK and p38 and the nucleus translocation of NF-кB p65 were also significantly downregulated by rPGRN. In addition, recombinant TNF-α (rTNF-α) significantly boosted TNF-α and iNOS expression vs the control group. Moreover, anti-TNF-α significantly inhibited LPS-induced TNF-α and iNOS expression. In THP-1 and BMDM cells, reversing effect of rPGRN on LPS-enhanced expressions of TNF-α and iNOS and secretion of TNF-α was further demonstrated.
CONCLUSIONS
PGRN down-regulates LPS-induced macrophage M1 polarization in phenotype and function via NF-κB/MAPK signaling pathways.
背景
巨噬细胞 M1 极化在炎症性疾病中起着关键作用。颗粒蛋白前体（PGRN）具有潜在的抗炎作用，但 PGRN 对巨噬细胞 M1 极化的影响尚未得到充分研究。我们的研究旨在探讨 PGRN 对脂多糖（LPS）诱导的巨噬细胞 M1 极化的影响，并阐明其潜在机制。
方法
用 LPS 和重组 PGRN（rPGRN）和肿瘤坏死因子α抗体（anti-TNF-α）将 RAW264.7 细胞极化为 M1 巨噬细胞。用细胞计数试剂盒-8 法（CCK-8）、流式细胞术、定量实时 PCR 法（q-PCR）、Western blot 法和酶联免疫吸附试验（ELISA）测定不同处理对细胞增殖、表面表型标志物表达以及炎症细胞因子表达和分泌的影响。用 Western blot 和免疫荧光法分别检测 NF-κB/丝裂原活化蛋白激酶（MAPK）通路的激活和 NF-κB p65 的核转位。还用 THP-1 和原代骨髓来源单核细胞（BMDMs）证明 PGRN 对 LPS 诱导的炎症细胞因子表达和分泌的影响。
结果
在 RAW264.7 细胞中，浓度低于 80ng/ml 的 rPGRN 呈剂量依赖性显著促进细胞增殖。rPGRN 显著抑制 LPS 诱导的表型变化（CD86/CD206 比值）和功能（肿瘤坏死因子（TNF-α）和诱导型一氧化氮合酶（iNOS）表达）。LPS 刺激的 TNF-α分泌和 IKKα/β、IкBα、p65、JNK 和 p38 的磷酸化激活以及 NF-кB p65 的核转位也被 rPGRN 显著下调。此外，重组 TNF-α（rTNF-α）与对照组相比显著增加了 TNF-α和 iNOS 的表达。此外，抗 TNF-α显著抑制了 LPS 诱导的 TNF-α和 iNOS 表达。在 THP-1 和 BMDM 细胞中，rPGRN 对 LPS 增强的 TNF-α和 iNOS 表达和 TNF-α分泌的逆转作用进一步得到证实。
结论
PGRN 通过 NF-κB/MAPK 信号通路下调 LPS 诱导的巨噬细胞 M1 极化表型和功能。
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