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. 2016 Jun 20;6:28370.
doi: 10.1038/srep28370.

Alpinetin Attenuates Inflammatory Responses by Suppressing TLR4 and NLRP3 Signaling Pathways in DSS-induced Acute Colitis

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Free PMC article

Alpinetin Attenuates Inflammatory Responses by Suppressing TLR4 and NLRP3 Signaling Pathways in DSS-induced Acute Colitis

Xuexiu He et al. Sci Rep. .
Free PMC article

Abstract

Alpinetin, a composition of Alpinia katsumadai Hayata, has been reported to have a number of biological properties, such as antibacterial, antitumor and other important therapeutic activities. However, the effect of alpinetin on inflammatory bowel disease (IBD) has not yet been reported. The purpose of this study was to investigate the anti-inflammatory effect and mechanism of alpinetin on dextran sulfate sodium (DSS)-induced colitis in mice. In vivo, DSS-induced mice colitis model was established by giving mice drinking water containing 5% (w/v) DSS for 7 days. Alpinetin (25, 50 and 100 mg/kg) were administered once a day by intraperitoneal injection 3 days before DSS treatment. In vitro, phorbol myristate acetate (PMA)-differentiated monocytic THP-1 macrophages were treated with alpinetin and stimulated by lipopolysaccharide (LPS). The results showed that alpinetin significantly attenuated diarrhea, colonic shortening, histological injury, myeloperoxidase (MPO) activity and the expressions of tumor necrosis factor (TNF-α) and interleukin (IL-1β) production in mice. In vitro, alpinetin markedly inhibited LPS-induced TNF-α and IL-1β production, as well as Toll-like receptor 4 (TLR4) mediated nuclear transcription factor-kappaB (NF-κB) and NOD-like receptor protein 3 (NLRP3) inflammasome activation. In conclusion, this study demonstrated that alpinetin had protective effects on DSS-induced colitis and may be a promising therapeutic reagent for colitis treatment.

Figures

Figure 1
Figure 1. The chemical structure of alpinetin.
Figure 2
Figure 2. Alpinetin ameliorated the progression of DSS-induced colitis in mice.
(A) Mice were administered 5% DSS in drinking water (ad libitum) for 7 days with/without alpinetin (25, 50 and 100 mg/kg/day p.o.). Changes in DAI were evaluated daily. (B) Colons were obtained after 7 days of DSS administration and their lengths were measured. The values presented are the mean ± S.E.M (n = 10 in each group). Number sign (#) indicates P < 0.01 vs. control group. Single asterisk (*) indicates P < 0.05, anddouble asterisks (**) indicate P < 0.01 vs. DSS group.
Figure 3
Figure 3. Effect of alpinetin on histopathologic changes in colon tissues in DSS-induced mice colitis.
Colon tissue of control group (A), the DSS group (B), the DSS + alpinetin 25 mg/kg group (C), the DSS + alpinetin 50 mg/kg group (D), the DSS + alpinetin 100 mg/kg group (E) and the histological scores (F).
Figure 4
Figure 4. Effects of alpinetin on MPO activition in colon tissues of DSS-induced colitis.
The values presented are the mean ± S.E.M (n = 10 in each group). Number sign (#) indicates P < 0.01 vs. control group. Single asterisk (*) indicates P < 0.05, anddouble asterisks (**) indicate P < 0.01 vs. DSS group.
Figure 5
Figure 5. Effects of alpinetin on the levels of TNF-αand IL-1β in the homogenate of DSS-induced mice colon tissues.
The values presented are the mean ± S.E.M (n = 10 in each group). Number sign (#) indicates P < 0.01 vs. control group. Single asterisk (*) indicates P < 0.05, anddouble asterisks (**) indicate P < 0.01 vs. DSS group.
Figure 6
Figure 6. Effects of alpinetin on THP-1 cells viability.
THP-1 cells were exposed with or without alpinetin (50, 100 and 200 μg/ml) for 24 h. The cells were not affected by the alpinetin treatment.
Figure 7
Figure 7. Effects of alpinetin on the levels of TNF-α and IL-1β in the cell supernatants.
The productions of TNF-α and IL-1β were detected by ELISA (A). The mRNA levels of TNF-α and IL-1βwere detected by RT-PCR (B).The values presented are the mean ± S.E.M (n = 10 in each group). Number sign (#) indicates P < 0.01 vs. control group. Single asterisk (*) indicates P < 0.05, anddouble asterisks (**) indicate P < 0.01 vs. LPS group.
Figure 8
Figure 8. Alpinetin inhibited NF-κB pathway.
Colon tissue was collected to determine the NF-κB positive signal by immunohistochemistry (A). Protein levels of NF-kB p65 and IκBα in THP-1 cells were determined by Western blotting (B). The values presented are the mean ± S.E.M (n = 10 in each group). Number sign (#) indicates P < 0.01 vs. control group. Single asterisk (*) indicates P < 0.05, anddouble asterisks (**) indicate P < 0.01 vs. LPS group.
Figure 9
Figure 9. Alpinetin inhibited LPS-induced activation of TLR4 expression with Western blotting.
The values presented are the mean ± S.E.M (n = 10 in each group). Number sign (#) indicates P < 0.01 vs. control group. Single asterisk (*) indicates P < 0.05, anddouble asterisks (**) indicate P < 0.01 vs. LPS group.
Figure 10
Figure 10. Alpinetin inhibited NLRP3 inflammasome activation.
Colon tissue was collected to determine the NLRP3 positive signal by immunohistochemistry (A). Protein levels of NLRP3, ASC, caspase-1 in THP-1 cells were determined by Western blotting (B). The mRNA level of NLRP3 was determined by RT-PCR (C). The values presented are the mean ± S.E.M (n = 10 in each group).Number sign (#) indicates P < 0.01 vs. control group. Single asterisk (*) indicates P < 0.05, anddouble asterisks (**) indicate P < 0.01 vs. LPS group.

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