C5aR antagonist inhibits LPS-induced inflammation in human gingival fibroblasts via NF-κB and MAPK signaling pathways

Abstract Objective Abnormal complement activation is associated with periodontitis. W54011 is a novel non-peptide C5aR antagonist (C5aRA) that exhibits favorable anti-inflammatory effects in various inflammatory models. However, whether W54011 inhibits periodontitis has not yet been fully elucidated. To address this, we have investigated the probable anti-inflammatory mechanism of W54011 in LPS-treated inflammation in human gingival fibroblasts (HGFs). Methodology HGFs were isolated from healthy gingival tissue samples using the tissue block method and were identified with immunofluorescence staining. The CCK8 assay and reverse transcription-PCR (RT-PCR) were used to select the optimal induction conditions for Lipopolysaccharide (LPS) and C5aRA (according to supplementary data S1, S2 and S3). The levels of inflammatory cytokines, C5aR, and the activation of NF-κB/MAPK signaling pathways were determined by RT-quantitative PCR (RT-qPCR) and Western blotting. Results Immunofluorescence results showed that vimentin and FSP-1 were positive in HGFs and Keratin was negative in HGFs. Immunofluorescence staining demonstrated that C5aRA inhibited LPS-stimulated nuclear translocation of p-p65. RT-qPCR and Western blotting showed that C5aRA reduced the expression of IL-1β, IL-6, TNF-α, C5aR, p-p65, p-IκBα, p-JNK, p-c-JUN, and TLR4 in LPS-induced HGFs. Conclusion These findings suggested that C5aRA attenuated the release of inflammatory cytokines in LPS-induced HGFs by blocking the activation of the NF-κB and MAPK signaling pathways.

### 摘要
#### 研究目的
补体异常激活与牙周炎密切相关。W54011是一种新型非肽类C5a受体拮抗剂（C5a receptor antagonist, C5aRA），在多种炎症模型中展现出良好的抗炎活性。然而，W54011是否能够抑制牙周炎，目前尚未完全阐明。为此，本研究针对脂多糖（Lipopolysaccharide, LPS）诱导的人牙龈成纤维细胞（human gingival fibroblasts, HGFs）炎症模型，探究W54011潜在的抗炎作用机制。

#### 研究方法
本研究采用组织块法从健康牙龈组织样本中分离HGFs，并通过免疫荧光染色对其进行鉴定。采用CCK8实验与逆转录聚合酶链反应（reverse transcription-PCR, RT-PCR）筛选LPS及C5aRA的最佳诱导条件（详见补充数据S1、S2及S3）。通过逆转录实时定量聚合酶链反应（RT-quantitative PCR, RT-qPCR）与蛋白质印迹法（Western blotting）检测炎症细胞因子、C5aR的表达水平以及NF-κB/MAPK信号通路的激活情况。

#### 研究结果
免疫荧光结果显示，HGFs的波形蛋白（vimentin）与成纤维细胞特异性蛋白1（FSP-1）呈阳性表达，角蛋白（Keratin）呈阴性表达。免疫荧光染色证实，C5aRA可抑制LPS刺激下p-p65的核转位。RT-qPCR与Western blotting结果表明，在LPS诱导的HGFs中，C5aRA可降低IL-1β、IL-6、TNF-α、C5aR、p-p65、p-IκBα、p-JNK、p-c-JUN及TLR4的表达水平。

#### 研究结论
上述研究结果表明，C5aRA可通过阻断NF-κB与MAPK信号通路的激活，减轻LPS诱导的HGFs中炎症细胞因子的释放。