Journal: Immunity, Inflammation and Disease

Article Title: Effects of N‐acetyl‐L‐cysteine polysulfides on periodontitis in a mouse model

doi: 10.1002/iid3.959

Figure Lengend Snippet: 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 hi B220 – ) 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.

Article Snippet: NAC‐S2 (Wako) was prepared by reacting NAC with sulfide in the presence of NaNO 2 , following the published protocol.

Techniques: Flow Cytometry, Injection, Two Tailed Test

Journal: Immunity, Inflammation and Disease

Article Title: Effects of N‐acetyl‐L‐cysteine polysulfides on periodontitis in a mouse model

doi: 10.1002/iid3.959

Figure Lengend Snippet: 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.

Article Snippet: NAC‐S2 (Wako) was prepared by reacting NAC with sulfide in the presence of NaNO 2 , following the published protocol.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Immunity, Inflammation and Disease

Article Title: Effects of N‐acetyl‐L‐cysteine polysulfides on periodontitis in a mouse model

doi: 10.1002/iid3.959

Figure Lengend Snippet: 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.

Article Snippet: NAC‐S2 (Wako) was prepared by reacting NAC with sulfide in the presence of NaNO 2 , following the published protocol.

Techniques: Expressing, Quantitative RT-PCR

Journal: Immunity, Inflammation and Disease

Article Title: Effects of N‐acetyl‐L‐cysteine polysulfides on periodontitis in a mouse model

doi: 10.1002/iid3.959

Figure Lengend Snippet: 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.

Article Snippet: NAC‐S2 (Wako) was prepared by reacting NAC with sulfide in the presence of NaNO 2 , following the published protocol.

Techniques: Western Blot, Derivative Assay

Journal: Immunity, Inflammation and Disease

Article Title: Effects of N‐acetyl‐L‐cysteine polysulfides on periodontitis in a mouse model

doi: 10.1002/iid3.959

Figure Lengend Snippet: 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.

Article Snippet: NAC‐S2 (Wako) was prepared by reacting NAC with sulfide in the presence of NaNO 2 , following the published protocol.

Techniques: Injection, Micro-CT, Quantitative RT-PCR

Journal: Redox Biology

Article Title: Formation of a reducing microenvironment and regulation of protein supersulfidation by gut microbial supersulfides

doi: 10.1016/j.redox.2026.104123

Figure Lengend Snippet: Supersulfides mediate interbacterial crosstalk (A) The levels of CysSSH in the culture supernatant were quantified by LC-ESI-MS/MS. (B) Each bacterial strain was anaerobically incubated with fecal suspension with or without cystine, and intracellular reactive sulfur levels were quantified as NEM-S-NEM by LC-ESI-MS/MS. (C) L. reuteri was anaerobically incubated with NAC-S2 and intracellular sulfane sulfur levels were quantified by LC-ESI-MS/MS. Data are expressed as the means ± SEM. Statistical significance was assessed using one-way ANOVA with the Tukey's multiple comparisons test in (A) and (C). ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: The medium was replaced with fresh MRS broth containing 250 μM NAC-S2 (Peptide Institute, Inc., Osaka, Japan), and the cells were anaerobically incubated for 30 min at 37 °C, followed by washing with D-PBS(−).

Techniques: Tandem Mass Spectroscopy, Incubation, Suspension

Journal: Redox Biology

Article Title: Formation of a reducing microenvironment and regulation of protein supersulfidation by gut microbial supersulfides

doi: 10.1016/j.redox.2026.104123

Figure Lengend Snippet: Profiling and environmental modulation of gut bacterial supersulfidome (A) Gel image showing supersulfidated proteins from various bacterial strains isolated using a pull-down method. Black bands indicate supersulfidated proteins. (B) Violin plot of quantified protein abundances (log-scale) for the identified proteins ( L. reuteri ; n = 917, E. bolteae ; n = 2064). The dashed line represents the median, and the dotted lines represent the first and third quartiles, respectively. (C) L. reuteri was anaerobically incubated with NAC-S2, and total supersulfidated cysteine residues were quantified by LC-ESI-MS/MS. (D) Rank plots of log2-transformed protein abundance ratio values represent changes in protein supersulfidation levels in L. reuteri following NAC-S2 stimulation. Proteins are sorted by their fold change values. Red indicates upregulated proteins (>2-fold), and blue indicates downregulated proteins (<0.5-fold). Detailed information regarding individual proteins is listed in . (E) Top 10 supersulfidated proteins enriched in the cystine (+) condition. All identified proteins were ranked based on their -fold change compared to the cystine (−) group. Detailed information concerning individual proteins is listed in . Data are expressed as means ± SEM. Statistical significance was assessed using Mann–Whitney U test in (B) or unpaired Student's t -test in (C). ∗∗∗ p < 0.001.

Article Snippet: The medium was replaced with fresh MRS broth containing 250 μM NAC-S2 (Peptide Institute, Inc., Osaka, Japan), and the cells were anaerobically incubated for 30 min at 37 °C, followed by washing with D-PBS(−).

Techniques: Isolation, Incubation, Tandem Mass Spectroscopy, Transformation Assay, Quantitative Proteomics, MANN-WHITNEY