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

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C5aR antagonist inhibits LPS-induced inflammation in human gingival fibroblasts via NF-κB and MAPK signaling pathways
Chen, Yan; Liu, Yang; Li, Hao; Huna, Risu; Tan, Xiaohan; Li, Ning; Zhang, Yiying; Jiao, Xiaohui; Liu, Mingyue, 2023, "C5aR antagonist inhibits LPS-induced inflammation in human gingival fibroblasts via NF-κB and MAPK signaling pathways", https://doi.org/10.48331/scielodata.XR5TEV, SciELO Data, V1
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Description
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). Materials and methods: 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 S 1 and S 2). 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: In 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. (2022-12-24)
Subject Medicine, Health and Life Sciences
Keyword Receptor, Anaphylatoxin C5a/antagonists & inhibitors, Fibroblasts, Gingiva/cytology, Periodontitis, NF-kappa B, Mitogen-Activated Protein Kinases
Related Publication Chen Y, Liu Y, Li H, Huna R, Tan X, Li N, Zhang Y, Jiao X, Liu M. C5aR antagonist inhibits LPS-induced inflammation in human gingival fibroblasts via NF-κB and MAPK signaling pathways. J Appl Oral Sci. 2023;31:e20220404. doi: 10.1590/1678-7757-2022-0404 doi: 10.1590/1678-7757-2022-0404
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Figure 1. Primers sequences used for RT-PCR and RT-qPCR.
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Figure 2. Isolation and culture of human gingival fibroblasts (HGFs).(A) The appearance of primary HGFs cultured for 2 days.(B) The appearance of primary HGFs cultured for 4 days.(C) The appearance of primary HGFs cultured for 7 days.(D) The appearance of primary HGFs cultured for10 days.(E) After 14 days of culture, the primary HGFs reached 80-90% confluency.(F) The appearance of HGFs after passaging. Scale bar = 100μm.
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Figure 3. Identification of the 2th passage cells from HGFs with immunofluorescent staining.(A-C) The green positive expression of FSP-1 in the cytoplasmof HGFs.(D-F) Keratin is unstained in the cytoplasm.(G-I) The positive staining of vimentin in the cytoplasm of HGFs was red fluorescenc.The positive staining of nucleus of HGFs was blue fluorescenc. Scale bar =100μm. Fluorescence microscope × 200.
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Figure 4. The content of C5a in the cell supernatant of the different group was measured by ELISA assay. Data represent the mean ± SEM. of three independent experiments. +P < 0.05 vs. the control group. *P < 0.05 vs. the LPS+C5aRA group.
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Figure 5. Effects of C5aR antagonists on the expression of C5aR and inflammatory factors of LPS-induced HGFs by RT-qPCR and Western blotting.(A) C5aRA inhibited expression of C5aR, TNF-α, IL-1β, and IL-6 mRNA in LPS-stimulated HGFs. (B) C5aR antagonists inhibited expression of C5aR, TNF-α, IL-1β, and IL-6 protein in LPS-stimulated HGFs. Data represent the mean ± S.E.M. of three independent experiments. +P < 0.05 vs. the control group. *P < 0.05 vs. the LPS+C5aRA group.
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Figure 6. Effects of C5aR antagonists on NF-κB signaling pathway activation and TLR4 expression in LPS-stimulated HGFs.(A) Effects of C5aR antagonists on mRNA levels of p65, p-p65, IκBα, p-IκBα and TLR4 in LPS-stimulated HGFs.(B) Effects of C5aR antagonists on protein levels of p65, p-p65, IκBα, p-IκBα and TLR4 in LPS-stimulated HGFs. Data represent the mean ± S.E.M. of three independent experiments. +P < 0.05 vs. the control group. *P < 0.05 vs. the LPS+C5aRA group.
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Figure 7. The effects of C5aR antagonists on p-p65 translocation into the nucleus were used to detect by immunoflfluorescence staining.(A-C) LPS+C5aR antagonists group.(D-F) The control group.(G-I) LPS group. The Blue staining represented the nucleus; The green staining was p-p65. Scale bar = 50μm. Fluorescence microscope × 400.
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Figure 8. Effects of C5aR antagonists on MAPK signal pathway activation in LPS-stimulated HGFs.(A) Effects of C5aR antagonists on mRNA levels of JNK, p-JNK, c-JUN and p-c-JUN in LPS-stimulated HGFs. (B) Effects of C5aR antagonists on protein levels of JNK, p-JNK, c-JUN, and p-c-JUN in LPS-stimulated HGFs. Data represent the mean ± S.E.M. of three independent experiments. +P < 0.05 vs. the control group. *P < 0.05 vs. the LPS+C5aRA group.
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Figure S1. Effects of LPS and C5aRA on the activity of HGFs using the CCK8 assay. (A) HGFs were treated with LPS at concentrations of 0.1, 1, and 5 μg/mL for 24, 48, and 72 h, respectively. (B) HGFs were treated with C5aRA at concentrations of 0.1, 1, and 5 μg/mL for 24, 48, and 72 h, respectively. Data represent the mean ± SEM of three independent experiments. * P < 0.05 vs. the control group, ** P < 0.01 vs. the control group.
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Figure S2. Effects of different concentrations of LPS on the expression of C5aR, IL-1β, IL-6, and TNF-α mRNA, determined using RT-PCR analysis. (A) C5aR; (B) IL-6; (C) IL-1β; and (D) TNF-α mRNA expression. Data represent the mean ± SEM of three independent experiments. * P < 0.05 vs. the control group. ∆ P < 0.05 vs. the 1 μg/mL (48 h) LPS group.
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