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. 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. 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. PGRN down-regulates LPS-induced macrophage M1 polarization in phenotype and function via NF-κB/MAPK signaling pathways.

中文翻译：

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前颗粒蛋白可通过NF-кB和MAPK途径抑制LPS诱导的巨噬细胞M1极化。

巨噬细胞M1极化在炎性疾病中起关键作用。前颗粒蛋白（PGRN）具有潜在的抗炎作用，但是，PGRN对巨噬细胞M1极化的作用尚未得到很好的研究。我们的研究旨在调查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。在有或没有重组PGRN（rPGRN）和肿瘤坏死因子α抗体（抗TNF-α）的情况下，LPS将RAW264.7细胞极化为M1巨噬细胞。使用细胞计数试剂盒8检测（CCK-8），流式细胞术，实时荧光定量PCR检测（q-PCR），蛋白质印迹检测和酶联免疫吸附测定（ELISA）来确定不同处理对细胞增殖，表面表型标志物的表达以及炎性细胞因子的表达和分泌。分别通过蛋白质印迹和免疫荧光检测NF-κB/丝裂原活化蛋白激酶（MAPK）通路的激活和NF-κBp65的核易位。THP-1和原代骨髓单核细胞（BMDMs）也用于证明PGRN对LPS诱导的炎性细胞因子的表达和分泌的影响。在RAW264.7细胞中，浓度低于80 ng / ml的rPGRN以剂量依赖性方式显着促进细胞增殖。rPGRN显着抑制LPS诱导的表型（CD86 / CD206比）和功能（肿瘤坏死因子（TNF-α）和诱导型一氧化氮合酶（iNOS）表达）的变化。rPGRN还显着下调了LPS刺激的TNF-α分泌和IKKα/β，IкBα，p65，JNK和p38的活化磷酸化以及NF-кBp65的核易位。此外，与对照组相比，重组TNF-α（rTNF-α）显着增强了TNF-α和iNOS的表达。此外，抗TNF-α显着抑制LPS诱导的TNF-α和iNOS表达。在THP-1和BMDM细胞中，进一步证实了rPGRN对LPS增强的TNF-α和iNOS表达以及TNF-α分泌的逆转作用。PGRN通过NF-κB/ MAPK信号通路下调LPS诱导的巨噬细胞M1极化的表型和功能。