Natural Sulfurs Inhibit LPS-Induced Inflammatory Responses through NF-κB Signaling in CCD-986Sk Skin Fibroblasts

Abstract

Lipopolysaccharide (LPS)-induced inflammatory response leads to serious damage, up to and including tumorigenesis. Natural mineral sulfur, non-toxic sulfur (NTS), and methylsulfonylmethane (MSM) have anti-inflammatory activity that may inhibit LPS-induced inflammation. We hypothesized that sulfur compounds could inhibit LPS-induced inflammatory responses in CCD-986Sk skin fibroblasts. We used Western blotting and real-time PCR to analyze molecular signaling in treated and untreated cultures. We also used flow cytometry for cell surface receptor analysis, comet assays to evaluate DNA damage, and ELISA-based cytokine detection. LPS induced TLR4 activation and NF-κB signaling via canonical and protein kinase C (PKC)-dependent pathways, while NTS and MSM downregulated that response. NTS and MSM also inhibited LPS-induced nuclear accumulation and binding of NF-κB to proinflammatory cytokines COX-2, IL-1β, and IL-6. Finally, the sulfur compounds suppressed LPS-induced ROS accumulation and DNA damage in CCD-986Sk cells. These results suggest that natural sulfur compounds could be used to treat inflammation and may be useful in the development of cosmetics.

Keywords: CCD-986Sk; LPS; MSM; NF-κB; NTS; ROS; TLR4; anti-inflammatory effect.

Figure 1

Sulfur compounds inhibited LPS-induced expression of TLR4. (A) Western blot analysis in CCD-986Sk cells treated with 10, 100, and 1000 ng/mL LPS for 48 h showing TLR4 and NF-κB expression. (B) MTT assay showing cell viability with 10 ng/mL LPS and increasing concentrations of NTS or MSM. Data are representative of three independent experiments. *** p < 0.001 (ANOVA test). # The mean difference is significant at the 0.001 level. (C) Flow cytometry showing inhibition of LPS-induced expression of TLR4 by 3 µg/mL NTS and 200 mM MSM for 48 h. (D) Real-time PCR data of mRNA after treatment with 10 ng/mL LPS and 3 µg/mL NTS or 200 mMMSM for 48 h showing the relative expression of TLR4 normalized to GAPDH. *** p < 0.001 (ANOVA test). # The mean difference is significant at the 0.001 level. (E) Western blotting analysis of TLR4 expression in CCD-986Sk cells treated with 10 ng/mL LPS and NTS (1.5 and 3 ng/mL) or MSM (100 and 200 mM) for 48 h. Expression levels were determined by densitometry and normalized to β-actin. Data are representative of three independent experiments. *** p < 0.001 (ANOVA test). # The mean difference is significant at the 0.001 level.

Figure 2

Sulfur compounds inhibited LPS-induced ROS. (A) Flow cytometry analysis of cellular ROS in CCD-986Sk cells after treatment with 10 ng/mL LPS and 3 µg/mL NTS or 200 mM MSM for 48 h. (B) Real-time PCR of iNOS mRNA after treatment with 10 ng/mL LPS and 3 µg/mL NTS or 200 mM MSM after 48 h, data normalized to GAPDH. *** p < 0.001 (ANOVA test). # The mean difference was significant at the 0.001 level. (C) Western blotting analysis of iNOS expression in CCD-986Sk cells treated with 10 ng/mL LPS and NTS (1.5 and 3 ng/mL) or MSM (100 and 200 mM) for 48 h. The representative expression levels of proteins following NTS or MSM treatment were determined by densitometry and normalized to β-actin. Data are representative of three independent experiments. *** p < 0.001 (ANOVA test). # The mean difference was significant at the 0.001 level.

Figure 3

Sulfur compounds inhibited LPS-induced DNA damage. (A) Comet assay imaged by electron microscopy for 10× and 40× magnification showing fragmented DNA migrated out of the nucleoid body and formed a comet tail after treatment with 10 ng/mL LPS, 3 µg/mL NTS, or 200 mM MSM for 48 h. Representative comet length and comet-positive cells were analyzed as the fold change versus the control. Statistical analysis was performed using the ANOVA test (*** p < 0.001). # Mean difference was significant at the 0.001 level. (B) Western blot analysis in CCD-986Sk cells showing that 3 µg/mL of NTS or 200 mM of MSM inhibited LPS-induced expressions of p-ATM, p-Chk2, p-BRCA1, and p-p53. Expression was determined by densitometry and normalized to β-actin. The values are presented as means ± SEM of three independent experiments performed in duplicate (n = 3). *** p < 0.001 (ANOVA test). # Mean difference was significant at the 0.001 level.

Figure 4

Sulfur compounds inhibited the LPS-induced canonical and PKC-dependent NF-κB pathways. (A) Western blotting analysis in CCD-986Sk cells treated with 1.5 and 3 µg/mL of NTS or 100 and 200 mM of MSM for 48 h showing the inhibition of 10 ng/mL LPS-induced p-IKKα/β, IκBα, and p-IκBα expression. Protein expression was determined by densitometry and normalized to β-actin. Values are presented as means ± SEM of three independent experiments performed in duplicate (n = 3). *** p < 0.001 (ANOVA test). Relative expressions of p-IκBα protein were determined via densitometry and normalized to total IκBα protein. *** p < 0.001. # Mean difference was significant at the 0.001 level. (B) Western blotting analysis in CCD-986Sk cells treated with 1.5 and 3 µg/mL of NTS or 100 and 200 mM of MSM for 48 h showing inhibition of 10 ng/mL LPS-induced p-PKC-α, p-ERK, and p-p38 expression along with unchanged expressions of PKC-α, ERK, and p38 proteins. Protein expression was determined by densitometry and normalized to β-actin. The values are presented as means ± SEM of three independent experiments performed in duplicate (n = 3). *** p < 0.001 (ANOVA test). Relative expressions of p-PKC-α, p-ERK, and p-p38 were determined via densitometry and normalized to their total PKC-α, ERK, and p38 proteins. *** p < 0.001. # Mean difference was significant at the 0.001 level.

Figure 5

Sulfur compounds inhibited the LPS-induced NF-κB, COX-2, and proinflammatory cytokines. (A) Real-time PCR data of mRNA after treatment with 10 ng/mL LPS and 3 µg/mL NTS and 200 mM MSM for 48 h showing the relative expression levels of NF-κB, COX-1, and COX-2 normalized to GAPDH mRNA. *** p < 0.001 (ANOVA test). # The mean difference was significant at the 0.001 level. (B) Western blotting analysis in CCD-986Sk cells treated with 10 ng/mL LPS and NTS (1.5 and 3 ng/mL) or MSM (100 and 200 mM) for 48 h showing the expression of NF-κB, COX-1, and COX-2. The representative expression levels of proteins following NTS or MSM treatment were determined by densitometry and normalized to β-actin. Data are representative of three independent experiments. *** p < 0.001 (ANOVA test). # The mean difference was significant at the 0.001 level. (C) Real-time PCR data of mRNA after treatment with 10 ng/mL LPS and 3 µg/mL of NTS and 200 mM of MSM for 48 h showing the relative expression levels of IL-1β and IL-6 normalized to GAPDH mRNA. *** p < 0.001 (ANOVA test). # The mean difference was significant at the 0.001 level. (D) Western blotting analysis in CCD-986Sk cells treated with 10 ng/mL LPS and NTS (1.5 and 3 ng/mL) or MSM (100 and 200 mM) for 48 h showing the expression of IL-1β and IL-6. The representative expression levels of proteins following NTS or MSM treatment were determined by densitometry and normalized to β-actin. Data are representative of three independent experiments. *** p < 0.001 (ANOVA test). # The mean difference was significant at the 0.001 level. (E) ELISA analysis showing the inhibition of the LPS-induced expression of IL-1β and IL-6 by 3 µg/mL of NTS and 200 mM of MSM. Data are representative of three independent experiments. *** p < 0.001 (ANOVA test). # The mean difference was significant at the 0.001 level.

Figure 6

Sulfur compounds inhibited the LPS-induced nuclear accumulation of NF-κB. (A) Western blotting analysis of nuclear extracts harvested from CCD-986Sk cells treated with 3 µg/mL NTS or 200 mM MSM or 40 µM TLR4 inhibitor (TLR4-C34) for 48 h showing the inhibition of 10 ng/mL LPS-induced nuclear translocation of NF-κB. (B) Protein expression after NTS, MSM, and TLR4-C34 treatment were determined by densitometry and normalized to TBP. Data are representative of three independent experiments. *** p < 0.001 (ANOVA test). # The mean difference was significant at the 0.001 level.

Figure 7

Molecular mechanism of LPS-induced regulation of the inflammatory response by TLR4/NF-κB through canonical and PKC-mediated pathways. The anti-inflammatory activities of NTS and MSM were achieved by inhibiting these pathways and binding of NF-κB to proinflammatory cytokines.