Effects of N-acetyl-L-cysteine polysulfides on periodontitis in a mouse model

Abstract

Background: Polysulfides are reported to be involved in various important biological processes. N-acetyl-l-cysteine polysulfide with 2 sulfane sulfur atoms (NAC-S2) regulates diverse toll-like receptor (TLR) signaling pathways. Here, we aimed to determine the role of NAC-S2 in periodontitis and explore the potential mechanism.

Methods: A periodontitis mouse model was established by ligating the subgingival between the first and second molars in wild-type, TLR4^-/- , and Myd88^-/- mice.

Results: NAC-S2 did not affect the proportion of macrophages (CD11b⁺ F4/80⁺ ) or neutrophils (CD11b⁺ GR-1⁺ ) in the bone marrow. Mechanically, lipopolysaccharides (LPS), Zymosan A, or poly I: C induced tumor necrosis factor (TNF), interleukin (IL)-6, and IL-1β expression in bone marrow-derived macrophages (BMDMs) could be inhibited by NAC-S2. On the other hand, NAC-S2 suppressed the phosphorylation levels of IκB-α, p65, and IκB kinase (IKK)-β induced by LPS in BMDMs, while LPS induced phosphorylation of ERK1/2, p38, and transforming growth factor β-activated kinase 1 (TAK1) could not be affected by NAC-S2. In wild-type periodontitis mice, NAC-S2 administration decreased the cemento-enamel-junction-alveolar bone crest (CEJ-ABC) distance and the relative mRNA expression of TNF, IL-6, and IL-1β, while such phenomena could not be observed in TLR4 deficiency or Myd88 deficiency mice.

Conclusions: All of these results indicate that NAC-S2 ameliorates TLR4/NF-κB pathway mediated inflammation in mouse periodontitis model.

Keywords: NAC-S2; NF-κB; TLR4; inflammation; periodontitis.

Figure 1

NAC Polysulfides do not affect the development of myeloid cells in the bone marrow. (A) Reaction scheme of the synthesis of NAC polysulfides (NAC‐S2). (B) Flow cytometry analysis of macrophages (CD11b⁺F4/80⁺), and neutrophil (CD11b⁺GR‐1⁺) (n = 4). These subpopulations were firstly based on gating the living CD11b⁺ groups. (C) conventional DC (cDC, CD11c⁺CD11b^hiB220^–) and plasmacytoid DCs (pDCs, CD11c⁺CD11b^–B220⁺) in bone marrow from 6 to 8‐weeks mice intraperitoneally injected with PBS (0.1 mL, solvent control) or NAC polysulfides (120 mmol/kg body weight) for 1 week (n = 4). These subpopulations were based on gating all living cells first. Data from all panels were presented as representative FACS plots (left) and mean ± SD values based on multiple samples (right). Similar results were obtained in three independent experiments. Two‐tailed Student's t tests were performed was applied for statistical comparison between groups. ns, no significance.

Figure 2

NAC‐S2 inhibited the expression of various pro‐inflammatory cytokines. Primary BMDMs were pretreated with 0.1 or 0.5 mM NAC‐S2 for 24 h, then treated with LPS and/or oxNAC. (A) The expression of indicated cytokines in primary NAC‐S2‐pretreated BMDMs treated with indicated reagents was measured by qRT‐PCR (n = 5). (B) ELISA of the indicated cytokines in the supernatants of NAC‐S2‐pretreated BMDMs stimulated with LPS for 24 h (n = 5). All data are presented as fold relative to the glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) mRNA level. Data are presented as mean ± SD values and representative of at least three independent experiments. One‐way ANOVA followed by a Tukey's test was performed for statistical comparison between groups. *p < .05; **p < .01; ***p < .005.

Figure 3

NAC‐S2 also impaired the expression of pro‐inflammatory cytokines induced by various stimulators. Primary BMDMs were pretreated with 0.1 or 0.5 mM NAC‐S2 for 24 h, then treated with zymosan, poly I:C, and/or oxNAC. (A and B) The expression of indicated cytokines in primary NAC‐S2‐pretreated BMDMs treated with indicated reagents was measured by qRT‐PCR (n = 3). All data are presented as fold relative to the GAPDH mRNA level. Data are presented as mean ± SD values and representative of at least three independent experiments. One‐way ANOVA followed by a Tukey's test was applied for statistical comparison between groups. *p < .05; **p < .01; ***p < .005. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.

Figure 4

NAC‐S2 suppressed the phosphorylation of IKKs and NF‐kB signal. Primary BMDMs were pretreated with 0.5 mM NAC‐S2 for 24 h, then insulted with LPS. (A and B) Western blot analysis of the indicated phosphorylated (P‐) and total proteins in whole‐cell lysates in NF‐κB signal (A) and MAPKs signal (B) of NAC‐S2‐pretreated BMDMs treated with indicated reagents for the indicated periods. (C) IB analysis of the activated levels of IKKs and TAK1 in total‐cell extracts of indicated BMDMs. BMDMs, bone marrow‐derived macrophages.

Figure 5

NAC‐S2 ameliorated inflammatory response in the periodontitis model. (A) Periodontitis mice were treated as indicated. 6 to 8‐week mice were intraperitoneally injected with PBS (0.1 mL, solvent control), and NAC polysulfides (120 mmol/kg body weight) for 1 week before inducing the periodontitis model. The cemento‐enamel‐junction–alveolar bone crest was analyzed by micro‐CT (n = 6 for each group). Averages and SEM are shown. (B) mRNA levels in gingival tissues from mice with periodontitis were examined by qRT‐PCR. All data are presented as fold relative to the GAPDH mRNA level (n = 3). Data are presented as mean ± SD values and representative of at least three independent experiments. One‐way ANOVA followed by a Tukey's test was performed for statistical comparison between groups. *p < .05. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.