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Curr Issues Mol Biol
2024 Nov 24;4612:13405-13417. doi: 10.3390/cimb46120799.
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Anti-Inflammatory Effects of Apostichopus japonicus Extract in Porphyromonas gingivalis-Stimulated RAW 264.7 Cells.
Kim MJ
,
Kim HJ
.
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Apostichopus japonicus has been used both as a food and in traditional medicine. However, its anti-inflammatory effects in periodontal diseases have not been studied. We examined the anti-inflammatory properties of Apostichopus japonicus extract in RAW 264.7 cells stimulated by Porphyromonas gingivalis. The cytotoxicity of Apostichopus japonicus extract was evaluated using the MTS assay. Its effect on NO production was then measured using the NO assay. The mRNA expression of inducible nitric oxide synthase (iNOS) and the pro-inflammatory cytokines IL-1β and IL-6 were assessed using quantitative real-time PCR (qRT-PCR). Western blotting was performed to investigate the expression of regulatory proteins involved in the NF-κB and MAPK signaling pathways. Apostichopus japonicus extract significantly inhibited NO production without cytotoxicity in RAW 264.7 cells. Following Porphyromonas gingivalis stimulation, treatment with the extract decreased iNOS mRNA expression and protein levels, which are responsible for NO production. The extract also suppressed the mRNA expression of pro-inflammatory cytokines. Additionally, Apostichopus japonicus extract inhibited NF-κB activation by regulating signaling molecules such as IKK and IκBα, while also preventing the phosphorylation of MAPK, including ERK, p38, and JNK, showing anti-inflammatory potential. Therefore, it may be a promising natural candidate for the development of new preventive and therapeutic strategies for periodontitis.
Figure 1. Typical preliminary HPLC chromatogram of a crude extract of A. japonicus. A 10 μL sample was injected into a reverse-phase column at a flow rate of 1 mL/min and monitored at 205 nm.
Figure 2. Effect of Apostichopus japonicus extract on the viability of RAW 264.7 cells. RAW 264.7 cells were treated with various concentrations of A. japonicus extract (0.0125–0.2 mg/mL) for 24 h, following which cell viability was determined by an MTS assay. Data are presented as the mean ± SD (n = 3). *** p < 0.001, compared with untreated cells.
Figure 3. Inhibitory effect of A. japonicus extract on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in Porphyromonas gingivalis-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with various concentrations of A. japonicus extract (0.0125–0.1 mg/mL) for 2 h and then stimulated with P. gingivalis (1 × 107 CFU/mL) for 24 h. (A) NO concentrations were measured using a Griess assay. The (B) mRNA and (C) protein levels of iNOS were determined by quantitative real-time PCR (qRT-PCR) and a Western blot, respectively. iNOS levels indicate changes in protein expression normalized to β-actin levels. Data are presented as the mean ± SD, in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to the P. gingivalis-stimulated cells.
Figure 4. Effects of A. japonicus extract on interleukin (IL)-1β and IL-6 production in P. gingivalis-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with various concentrations of A. japonicus extract (0.0125–0.1 mg/mL) for 2 h and then stimulated with P. gingivalis (1 × 107 CFU/mL) for 24 h. The mRNA levels of (A) IL-1β and (B) IL-6 were measured by qRT-PCR. IL-1β and IL-6 mRNA levels were normalized to β-actin mRNA levels. Data are presented as the mean ± SD, in triplicate. ** p < 0.01 and *** p < 0.001, compared with P. gingivalis-stimulated cells.
Figure 5. Effect of A. japonicus extract on nuclear factor kappa B (NF-κB) expression in P. gingivalis-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with various concentrations of A. japonicus extract (0.0125–0.1 mg/mL) for 2 h and then stimulated with P. gingivalis (1 × 107 CFU/mL) for 20 min. Subsequently, nuclear and cytoplasmic fractions were separated and subjected to Western blot analyses. NF-κB p65 levels were assessed in the nuclear and cytoplasmic fractions. Proliferating cell nuclear antigen (PCNA) and β-actin were used as loading controls for the nuclear and cytoplasmic extracts, respectively. The relative quantification of NF-κB p65 levels in the nuclear fraction was normalized to PCNA levels. Data are presented as the mean ± SD of triplicate experiments. *** p < 0.001, compared with P. gingivalis-stimulated cells.
Figure 6. Effects of A. japonicus extract on NF-κB inhibitor α (IκBα) and IκB kinase (IKK)α/β phosphorylation in P. gingivalis-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with various concentrations of A. japonicus extract (0.0125–0.1 mg/mL) for 2 h, followed by stimulation with P. gingivalis (1 × 107 CFU/mL) for 10–15 min. Subsequently, cells were harvested and cell lysates were prepared for a Western blot analysis of the indicated proteins. (A) phosphorylated (p)-IκBα and IκBα levels in the cytoplasmic extracts. Relative quantification of IκBα phosphorylation normalized to β-actin levels. (B) p-IKKα/β levels in the cytoplasmic extracts. Relative quantification of IKKα/β phosphorylation normalized to β-actin levels. Data are expressed as the mean ± SD of triplicate experiments. ** p < 0.01 and *** p < 0.001, compared with P. gingivalis-stimulated cells.
Figure 7. Effects of A. japonicus on the mitogen-activated protein kinase (MAPK) signaling pathway in P. gingivalis-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with various concentrations of A. japonicus extract (0.0125–0.1 mg/mL) for 2 h and then stimulated with P. gingivalis (1 × 107 CFU/mL) for 15 min. Cell lysates were analyzed using a Western blot analysis to assess the effect on the expression levels of (A) extracellular signal-regulated kinase (ERK), phosphorylated (p)-ERK, (B) p38, p-p38, and (C) c-Jun N-terminal kinase (JNK), p-JNK. All signals were normalized to β-actin levels, which served as an internal control. Data are expressed as the mean ± SD of triplicate experiments. ** p < 0.01 and *** p < 0.001, compared with the P. gingivalis-stimulated cells.
Figure 8. Schematic overview of the mechanisms underlying the anti-inflammatory effects of Apostichopus japonicus extract in Porphyromonas gingivalis-stimulated RAW 264.7 cells. IKK, IκB kinase; IκBα, NF-κB inhibitor α; NF-κB, nuclear factor kappa B; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IL, interleukin; iNOS, inducible nitric oxide synthase; NO, nitric oxide.
