| Objective: Periodontitis is a chronic inflammatory disease of the periodontal supporting tissue with Porphyromonas gingivalis as the main pathogenic bacteria.Lipopolysaccharides(LPS),as the main component of the outer membrane of Porphyromonas gingivalis,is an important virulence factor of Porphyromonas gingivalis.Resolvin D1(Rv D1)is a pro-resolvin lipid mediator(SPM)involved in inflammatory processes,and Rv D1 can restore some diseases with anti-inflammatory and antioxidant properties,such as periodontitis.However,the detailed mechanism of Rv D1’s anti-inflammatory effect is still unclear.Therefore,we elucidated the molecular mechanism by which Rv D1 regulates the inflammatory response of human periodontal ligament cells(h PDLCs).Methods: Three sections make up this study.Part 1: The establishment of inflammatory response model of human periodontal ligament cell.The periodontal ligament cells were extracted from young healthy permanent teeth and identified.According to the previous research results of our group,h PDLCs were stimulated with LPS(0.1 μg/m L)for 24 h to establish the inflammatory response model in vitro.At the same time,the inflammatory factors interleukin 6(IL-6)and IL-1β were measured in preliminary experiment by enzyme-linked immunosorbent assay(ELISA).Part 2: RNA-sequencing analysis of the inflammatory response of periodontal ligament cell regulated by Rv D1.Three comparison groups were analyzed by RNA sequencing,the LPS group versus the control group,the LPS+Rv D1 group versus the LPS group,and the LPS+Rv D1 group versus the control group.Through GO and KEGG analysis,differentially expressed genes(DEGs)and differential signal transduction pathways were identified,followed by quantitative real-time PCR(q RT-PCR)validated selected DEGs to test the reliability of the sequencing data.Part3: Exploration and verification of the signaling pathway mediated by Rv D1 regulating the inflammatory response of periodontal ligament cell.We performed immunofluorescence(IF)and western blotting(WB)to observe the expression of lipoxin A4 receptor/formyl peptide receptor 2(ALX/FPR2),a specific cell surface receptor belonging to Rv D1.Differential signal transduction pathways were identified based on RNA sequencing results,and the results were verified by IF,WB and q RT-PCR.Results: Part 1: The cells extracted in this experiment were identified as human periodontal ligament fibroblasts.The inflammatory response model of human periodontal ligament cells was established in vitro and the experimental grouping was carried out.According to the results of ELISA,the levels of IL-6 and IL-1β were increased in the LPS group compared with the control group.However,compared to the LPS group,the LPS+Rv D1 group had significantly decreased IL-6 and IL-1β.Part2: The RNA-seq analysis indicated that there were 502,146 and 487 DEGs in the LPS group versus the control group,the LPS+Rv D1 group versus the LPS group,and the LPS+Rv D1 group versus the control group.Five DEGs were selected,namely Toll-like receptor 1(TLR-1),tumor necrosis factor(TNF-α),chemokine ligand 8(CXCL-8),IL-1β and IL10 RB,and the experiments confirmed that the sequencing data had High reliability.The results of GO and KEGG enrichment analysis indicated that the Toll like receptor 4(TLR4)-the Myeloid differentiation factor 88(My D88)‐mediated NF-κB and MAPK signaling pathways play significant roles in the regulation of inflammatory factors by Rv D1.Part 3: It was verified that the expression of ALX/FPR2 receptor was the highest in the LPS+Rv D1 group,followed by the LPS group.This suggests that Rv D1 activates the resolution of inflammation by interacting with the ALX/FPR2 receptor.The expression of TLR4 and My D88 was increased under LPS induction,and Rv D1 could reduce the expression of TLR4 and My D88 stimulated by LPS.The results showed that Rv D1 could improve the inflammatory response of human periodontal ligament cells by regulating the expression of TLR4 and My D88.For the My D88-dependent signaling pathways downstream of TLR4 are NF-κB signaling pathway and MAPK signaling pathway,the results showed that Rv D1 significantly reduces LPS-induced nuclear translocation of NF-κB(p65),while inhibiting the phosphorylation of IKBKB.Additionally,Rv D1 inhibited LPS-induced phosphorylation of extracellular signal-regulated kinase(p-ERK1/2)in the MAPK signaling pathway.For the p38 MAPK signaling pathway,the expression of p-p38 was significantly increased after LPS stimulation,but Rv D1 failed to significantly reduce the expression of p-p38 protein induced by LPS.In addition,c-Jun NH2-terminal kinase(JNK)was not significantly changed.Conclusion: Rv D1 may regulate the inflammatory response of LPS-stimulated h PDLCs through TLR4-My D88-NF-κB signaling pathway and TLR4-My D88-MAPK signaling pathway. |
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