Anti-inflammatory properties of uvaol on DSS-induced colitis and LPS-stimulated macrophages

Abstract

Background: Apocynum venetum leaves are used as a kind of phytomedicine and the main ingredient in some traditional Chinese medicine products for the relief of colitis. To understand the bioactive constituents of A. venetum L., we did a phytochemistry study and investigated anti-Inflammatory effects of compounds and explored the underlying mechanisms.

Methods: We isolated compounds from ethanol extract of A. venetum L. leaf and detected the most effective compound by NO inhibition assay. We investigated anti-Inflammatory effects on dextran sulfate sodium (DSS)-induced colitis mice and lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The disease activity index was determined by scores of body weight loss, diarrhea and rectal bleeding; histological damage was analyzed by H&E staining; macrophages change in the colon were analyzed by immunohistochemistry (IHC); myeloperoxidase activity was measured by myeloperoxidase assay kits; levels of proinflammatory cytokines were determined by qPCR and ELISA; protein production such as COX-2, iNOS, STAT3 and ERK1/2 were determined by western blotting.

Results: We isolated uvaol from ethanol extract of A. venetum L. leaf and found uvaol has excellent potential of inhibiting NO production. We further found uvaol could attenuate disease activity index (DAI), colon shortening, colon injury, and colonic myeloperoxidase activity in DSS-induced colitis mice. Moreover, uvaol significantly reduces mRNA expression and production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, and MCP-1) and infiltration of macrophages in colonic tissues of colitis mice. Studies on LPS challenged murine macrophage RAW246.7 cells also revealed that uvaol reduces mRNA expression and production of pro-inflammatory cytokines and mediators. Mechanically, uvaol inhibits the pro-inflammatory ERK/STAT3 axis in both inflamed colonic tissues and macrophages.

Conclusions: A. venetum leaf contains uvaol and uvaol has potent anti-inflammatory effects on DSS-induced experimental colitis and LPS-stimulated RAW264.7 macrophage cells. These results suggest uvaol is a prospective anti-inflammatory agent for colonic inflammation.

Keywords: Anti-inflammatory; Apocynum venetum L.; Colonic inflammation; Macrophage; Uvaol.

Fig. 1

Isolation of uvaol from A.venetum L. a Isolation flow chart of uvaol. AVL-PE-layer: petroleum ether extract from ethanol extract of A. venetum L. leaf, AVL-TCM-layer: chloroform extract from ethanol extract of A. venetum L. leaf, AVL-EA-layer: ethyl acetate extract from ethanol extract of A. venetum L. leaf, AVL-MeOH-layer: methanol extract from ethanol extract of A. venetum L. leaf, b chromatograms of a standard uvaol and Fra.6 that contains target compound (210 nm)

Fig. 2

Effects of uvaol on body weight gain (a), disease activity index (DAI) (b), colon length (c), myeloperoxidase (MPO) activity (d) and histological damage (magnification of 10 × for representative H&E staining images) (e) of DSS-treated mice. Uvaol was administered to mice from day 8 to day 14. Weight and DAI scores are measured every 2 days. DAI was determined by combining scores of (i) body weight loss, (ii) stool consistency, and (iii) stool bleeding. On day 14, the mice were sacrificed, the colon lengths and histological score of colons were measured. Data are expressed as mean ± SEM, n = 7.^###P < 0.001 and^##P < 0.01, compared with control group; ***P < 0.001, **P < 0.01 and *P < 0.05, compared with DSS group

Fig. 3

Effects of uvaol on pro-inflammatory macrophage infiltration (a), mRNA expression and production of pro-inflammatory mediators (b–c) in colon tissues of DSS-treated mice. Colitis was induced in all groups except the control group. Uvaol was administered to mice from day 8 to day 14. On day 14, the mice were sacrificed, and mRNA expression and production of pro-inflammatory mediators such as MCP-1, TNF-α, IL-1β and IL-6 in colon tissues were determined by RT-PCR and ELISA. Pro-inflammatory macrophages in colon tissues were stained by F4/80 (magnification of 20 × for representative F4/80 staining images). Integrated optical density(IOD)were analyzed by Image pro-Plus v.6.0. Data are expressed as mean ± SEM, n = 7.^###P < 0.001,^##P < 0.01 and ^#P < 0.05, compared with control group; ***P < 0.001, **P < 0.01 and *P < 0.05, compared with DSS group

Fig. 4

Uvaol inhibits mRNA expression and production of inflammatory mediators of LPS-stimulated RAW264.7 cells. a Cytotoxicity of uvaol on RAW264.7 cells. Uvaol was incubated with RAW264.7 cells for 24 h, and cell viability was detected by CCK8 kit. Data are expressed as mean ± SEM, n = 6. b NO release in LPS- stimulated RAW264.7 cells. Data are expressed as mean ± SEM, n = 4. c The microscopic morphological changes of LPS-induced RAW264.7 cells (magnification 20 ×). d mRNA expression of TNF-α, MCP-1, IL-6 and IL-1β of LPS-stimulated RAW264.7 cells. e TNF-α, IL-6 and IL-1β protein concentration in supernatants of LPS-stimulated RAW264.7 cells. f COX-2 and iNOS expressions of LPS-stimulated RAW264.7 cells. Data are expressed as mean ± SEM, n = 3. The images shown are representatives of three independent experiments.^###P < 0.001 and^##P < 0.01, compared with control group; ***P < 0.001, **P < 0.01 and *P < 0.05, compared with LPS- stimulated group

Fig. 5

Uvaol inhibits STAT3 and ERK activation in colon tissues of DSS-treated mice and LPS-stimulated RAW264.7 cells. a STAT3, p-STAT3, ERK and p-ERK expressions in colon tissues of DSS-treated mice. Colitis was induced in all groups except the control group. Uvaol and CsA were administered to mice from day 8 to day 14. On day 14, the mice were sacrificed, and protein expression of STAT3, p-STAT3, ERK and p-ERK in colon homogenates were determined by western blotting. Data are expressed as mean ± SEM, n = 6. b STAT3, p-STAT3, ERK and p-ERK expression in LPS-stimulated RAW264.7 cells. RAW264.7 cells were treated with 1 μg/mL of LPS in the absence or presence of designated concentrations of uvaol for 3 h, Data are expressed as mean ± SEM, n = 3. The images shown are representatives of three independent experiments.^###P < 0.001 and^##P < 0.01, compared with control group; ***P < 0.001, **P < 0.01 and *P < 0.05, compared with DSS or LPS- stimulated group

Fig. 6

Possible mechanism of uvaol in alleviating DSS-induced experimental colitis via regulation of macrophage pro-inflammatory responses. DSS treatment induced macrophage infiltration and pro-inflammatory responses in the colon. Upon the stimulation by inflammatory stimulus such as TNF-α, LPS in the inflamed colonic circumstance, ERK/STAT3 signal was activated to promote transcription of pro-inflammatory genes TNF-α, IL-1β, IL-6, etc. and then the release of pro-inflammatory cytokines TNF-α, IL-6, IL-1β, COX-2 and iNOS. Uvaol could inhibit ERK and STAT3 activation to suppress the transcription of pro-inflammatory genes and the release of pro-inflammatory cytokines and further improve clinical symptoms and suppress colon inflammation of DSS-treated mice