Cl-amidine attenuates lipopolysaccharide-induced inflammation in human gingival fibroblasts via the JNK/MAPK, NF-κB, and Nrf2 signalling pathways.

Cl-amidine has been reported to have anti-inflammatory properties in a variety of diseases. However, the role of Cl-amidine in periodontal disease remains unclear. Here, the purpose of this study was to investigate the effect of Cl-amidine on lipopolysaccharide (LPS)-induced inflammation in human gingival fibroblasts (HGFs). The cytotoxic effect of Cl-amidine was measured with the Cell Counting Kit-8 (CCK-8) assay and Annexin V-FITC/PI staining. The protein levels of IL-6 and IL-8 in culture supernatants were measured with enzyme-linked immunosorbent assay (ELISA). The mRNA levels of inflammatory cytokines, TLR4 and MyD88 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The expression patterns of IL-6, TNF-ɑ, and IL-1β in HGFs were tested with western blot. The levels of NF-κB, MAPK, and Nrf2 pathway-related proteins were detected by western blot. Immunofluorescence (IF) staining was used to examine the nuclear translocation of NF-κB p65. Moreover, a rat gingivitis model was established to further clarify the role of Cl-amidine. Our results showed that Cl-amidine suppressed LPS-induced gingival inflammation both in vitro and in vivo. Mechanistically, Cl-amidine inhibited LPS-induced MyD88 expression, NF-κB activation, and JNK phosphorylation. Additionally, Cl-amidine upregulated Nrf2 and Ho-1 expression both with and without LPS stimulation but did not alter ROS levels or Keap1 expression. Overall, our data suggest that Cl-amidine acts as an inhibitor of LPS-induced gingival inflammation via the JNK/MAPK, NF-κB, and Nrf2 signalling pathways.