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Rosmarinic acid suppresses colonic inflammation in dextran sulphate sodium (DSS)-induced mice via dual inhibition of NF- κ B and STAT3 activation

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ABSTRACT

Ulcerative colitis (UC), a type of inflammatory bowel disease (IBD), is a chronic inflammatory disorder of the colon. Although UC is generally treated with anti-inflammatory drugs or immunosuppressants, most of these treatments often prove to be inadequate. Rosmarinic acid (RA) is a phenolic ester included in various medicinal herbs such as Salvia miltiorrhiz and Perilla frutescens. Although RA has many biological and pharmacological activities, the anti-inflammatory effect of RA in colonic tissue remains unclear. In this study, we investigated the anti-inflammatory effects and underlying molecular mechanism of RA in mice with dextran sulphate sodium (DSS)-induced colitis. In the DSS-induced colitis model, RA significantly reduced the severity of colitis, as assessed by disease activity index (DAI) scores, colonic damage, and colon length. In addition, RA resulted in the reduction of the inflammatory-related cytokines, such as IL-6, IL-1β, and IL-22, and protein levels of COX-2 and iNOS in mice with DSS-induced colitis. Furthermore, RA effectively and pleiotropically inhibited nuclear factor-kappa B and signal transducer and activator of transcription 3 activation, and subsequently reduced the activity of pro-survival genes that depend on these transcription factors. These results demonstrate that RA has an ameliorative effect on colonic inflammation and thus a potential therapeutic role in colitis.

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Effect of RA on NF-κB activation and expression of its relative gene products in DSS-induced colitis mice.(A) The manifestation and translocation to the nucleus of p65 in colon tissues were observed by immunohistochemistry estimation. (B) Immunoscore of p65 in colon of DSS-induced mice was estimated. (C) Nuclear (N) and cytosol (C) extracts were prepared from colon tissues on 7 days of DSS administration and translocation of p65 to the nucleus and phosphorylation of IκB were estimated by western blot analysis using specific antibodies. C23 and α-tubulin were used as internal controls. (D) NF-κB-related proteins were determined by western blot analysis using specific antibodies. β-actin was used as internal controls. Relative ratio level was determined by densitometric analysis (Bio-rad Quantity One® Software) normalized to internal controls. Values are the mean ± SD (n = 10); ##P < 0.01, ###P < 0.001 vs control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs the DSS-induced group; significances between treated groups were determined using ANOVA and Dunnett’s post hoc test.
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f5: Effect of RA on NF-κB activation and expression of its relative gene products in DSS-induced colitis mice.(A) The manifestation and translocation to the nucleus of p65 in colon tissues were observed by immunohistochemistry estimation. (B) Immunoscore of p65 in colon of DSS-induced mice was estimated. (C) Nuclear (N) and cytosol (C) extracts were prepared from colon tissues on 7 days of DSS administration and translocation of p65 to the nucleus and phosphorylation of IκB were estimated by western blot analysis using specific antibodies. C23 and α-tubulin were used as internal controls. (D) NF-κB-related proteins were determined by western blot analysis using specific antibodies. β-actin was used as internal controls. Relative ratio level was determined by densitometric analysis (Bio-rad Quantity One® Software) normalized to internal controls. Values are the mean ± SD (n = 10); ##P < 0.01, ###P < 0.001 vs control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs the DSS-induced group; significances between treated groups were determined using ANOVA and Dunnett’s post hoc test.

Mentions: NF-κB signalling is a key process during inflammation and thus constitutes an attractive target for anti-inflammatory interventions8. To clarify the molecular mechanisms of RA, we evaluated its effect on NF-κB activation in DSS-induced colitis mice. As shown in Fig. 5A, IHC analysis demonstrated that RA markedly suppressed the protein expression of NF-κB p65 in the inflamed mucosa and inhibited translocation of NF-κB p65 to the nucleus. In addition, immunoreactivity score of p65 was assessed in the representative pictures of each group according to Table 3. The mice in the DSS-induced group had a markedly increased score compared to that of the control group; the score of the RA-treated mice was significantly decreased in comparison (Fig. 5B). In western blot analysis, the colons of DSS-administered mice had a sharp increase in the translocation of p65 from cytosol to the nucleus. In contrast, RA treatment markedly inhibited the DSS-induced nuclear translocation of p65 in colonic tissues (Fig. 5C). Moreover, we determined that RA inhibits the phosphorylation and degradation of IκB in the colonic tissue of DSS-induced mice. We also found that DSS increased the expression levels of NF-κB-related proteins, such as survivin, Bcl-2 family proteins, and XIAP, whereas RA treatment reduced these increases in the colonic tissue of DSS-induced mice (Fig. 5D).


Rosmarinic acid suppresses colonic inflammation in dextran sulphate sodium (DSS)-induced mice via dual inhibition of NF- κ B and STAT3 activation
Effect of RA on NF-κB activation and expression of its relative gene products in DSS-induced colitis mice.(A) The manifestation and translocation to the nucleus of p65 in colon tissues were observed by immunohistochemistry estimation. (B) Immunoscore of p65 in colon of DSS-induced mice was estimated. (C) Nuclear (N) and cytosol (C) extracts were prepared from colon tissues on 7 days of DSS administration and translocation of p65 to the nucleus and phosphorylation of IκB were estimated by western blot analysis using specific antibodies. C23 and α-tubulin were used as internal controls. (D) NF-κB-related proteins were determined by western blot analysis using specific antibodies. β-actin was used as internal controls. Relative ratio level was determined by densitometric analysis (Bio-rad Quantity One® Software) normalized to internal controls. Values are the mean ± SD (n = 10); ##P < 0.01, ###P < 0.001 vs control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs the DSS-induced group; significances between treated groups were determined using ANOVA and Dunnett’s post hoc test.
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f5: Effect of RA on NF-κB activation and expression of its relative gene products in DSS-induced colitis mice.(A) The manifestation and translocation to the nucleus of p65 in colon tissues were observed by immunohistochemistry estimation. (B) Immunoscore of p65 in colon of DSS-induced mice was estimated. (C) Nuclear (N) and cytosol (C) extracts were prepared from colon tissues on 7 days of DSS administration and translocation of p65 to the nucleus and phosphorylation of IκB were estimated by western blot analysis using specific antibodies. C23 and α-tubulin were used as internal controls. (D) NF-κB-related proteins were determined by western blot analysis using specific antibodies. β-actin was used as internal controls. Relative ratio level was determined by densitometric analysis (Bio-rad Quantity One® Software) normalized to internal controls. Values are the mean ± SD (n = 10); ##P < 0.01, ###P < 0.001 vs control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs the DSS-induced group; significances between treated groups were determined using ANOVA and Dunnett’s post hoc test.
Mentions: NF-κB signalling is a key process during inflammation and thus constitutes an attractive target for anti-inflammatory interventions8. To clarify the molecular mechanisms of RA, we evaluated its effect on NF-κB activation in DSS-induced colitis mice. As shown in Fig. 5A, IHC analysis demonstrated that RA markedly suppressed the protein expression of NF-κB p65 in the inflamed mucosa and inhibited translocation of NF-κB p65 to the nucleus. In addition, immunoreactivity score of p65 was assessed in the representative pictures of each group according to Table 3. The mice in the DSS-induced group had a markedly increased score compared to that of the control group; the score of the RA-treated mice was significantly decreased in comparison (Fig. 5B). In western blot analysis, the colons of DSS-administered mice had a sharp increase in the translocation of p65 from cytosol to the nucleus. In contrast, RA treatment markedly inhibited the DSS-induced nuclear translocation of p65 in colonic tissues (Fig. 5C). Moreover, we determined that RA inhibits the phosphorylation and degradation of IκB in the colonic tissue of DSS-induced mice. We also found that DSS increased the expression levels of NF-κB-related proteins, such as survivin, Bcl-2 family proteins, and XIAP, whereas RA treatment reduced these increases in the colonic tissue of DSS-induced mice (Fig. 5D).

View Article: PubMed Central - PubMed

ABSTRACT

Ulcerative colitis (UC), a type of inflammatory bowel disease (IBD), is a chronic inflammatory disorder of the colon. Although UC is generally treated with anti-inflammatory drugs or immunosuppressants, most of these treatments often prove to be inadequate. Rosmarinic acid (RA) is a phenolic ester included in various medicinal herbs such as Salvia miltiorrhiz and Perilla frutescens. Although RA has many biological and pharmacological activities, the anti-inflammatory effect of RA in colonic tissue remains unclear. In this study, we investigated the anti-inflammatory effects and underlying molecular mechanism of RA in mice with dextran sulphate sodium (DSS)-induced colitis. In the DSS-induced colitis model, RA significantly reduced the severity of colitis, as assessed by disease activity index (DAI) scores, colonic damage, and colon length. In addition, RA resulted in the reduction of the inflammatory-related cytokines, such as IL-6, IL-1&beta;, and IL-22, and protein levels of COX-2 and iNOS in mice with DSS-induced colitis. Furthermore, RA effectively and pleiotropically inhibited nuclear factor-kappa B and signal transducer and activator of transcription 3 activation, and subsequently reduced the activity of pro-survival genes that depend on these transcription factors. These results demonstrate that RA has an ameliorative effect on colonic inflammation and thus a potential therapeutic role in colitis.

No MeSH data available.


Related in: MedlinePlus