Berberine improved experimental chronic colitis by regulating interferon-γ- and IL-17A-producing lamina propria CD4+ T cells through AMPK activation

Abstract

The herbal medicine berberine (BBR) has been recently shown to be an AMP-activated protein kinase (AMPK) productive activator with various properties that induce anti-inflammatory responses. We investigated the effects of BBR on the mechanisms of mucosal CD4⁺T cell activation in vitro and on the inflammatory responses in T cell transfer mouse models of inflammatory bowel disease (IBD). We examined the favorable effects of BBR in vitro, using lamina propria (LP) CD4⁺ T cells in T cell transfer IBD models in which SCID mice had been injected with CD4⁺CD45RB^high T cells. BBR suppressed the frequency of IFN-γ- and Il-17A-producing LP CD4⁺ T cells. This effect was found to be regulated by AMPK activation possibly induced by oxidative phosphorylation inhibition. We then examined the effects of BBR on the same IBD models in vivo. BBR-fed mice showed AMPK activation in the LPCD4⁺ T cells and an improvement of colitis. Our study newly showed that the BBR-induced AMPK activation of mucosal CD4⁺ T cells resulted in an improvement of IBD and underscored the importance of AMPK activity in colonic inflammation.

Figure 1

BBR suppressed inflammatory cytokines of LP CD4⁺ T cells from colitis SCID mice in vitro. (A) The cytotoxicity analysis of BBR. Colitis LP CD4⁺ T cells were stimulated with PMA plus ionomycin mixed with BBR (BBR) or not (Control). The bar graphs show number of LP CD4⁺ T cells. (B) Colitis LP CD4⁺ T cells were stimulated with PMA plus ionomycin mixed with BBR (BBR) or not (Control) for 8 h. After that, cells were collected, and intracellular staining was performed to analyze the CD3⁺CD4⁺IFN-γ⁺- or IL-17A⁺-producing cells by flow cytometry. Representative flow cytometry images are shown. Flow cytometry showed the percentage of CD3⁺CD4⁺IFN-γ⁺-or IL-17A⁺-producing cells in colitis LP CD4⁺ T cells. (C) The bar graphs show the percentage of IFN-γ- and IL-17A-producing cells in LP CD4⁺ T cells. All data are reported as the mean ± SEM. N is 6 in each group. N.S. not significant. *P < 0.05, **P < 0.01.

Figure 2

BBR suppressed the Th1/Th17-related JAK/STAT pathway of LP CD4⁺ T cells collected from colitis SCID mice. (A–D) Colitis LP CD4⁺ T cells stimulated with PMA plus ionomycin were collected and analyzed for the JAK/STAT pathway by Western blotting.The left figures show the representative Western blotting images of each protein. The cropped blots are used in the figure, and full-length blots are presented in Supplementary Fig. S2. All gels were run in the same experimental conditions (see material and methods for details). The bar graphs show the percentage of each protein expression. All data are reported as the mean ± SEM. N is 5 in each group. *P < 0.05.

Figure 3

BBR increased AMPK activity and regulated the IFN-γ and IL-17A secretion from colitis LP CD4⁺ T cells. (A) The phosphorylation of AMPK at Thr172 levels in colitis LP CD4⁺ T cells stimulated with PMA plus ionomycin mixed with BBR (BBR) or not (Control) was detected by Western blotting. The left figure is a representative blotting image. The right figure is the percentage of pAMPK/AMPK expression. (B-E) Colitis LP CD4⁺ T cells were treated with DMSO, AICAR (250 μM), AICAR (250 µm) mixed with C.C. (400 nM) or C.C. alone (400 nΜ) for 30 minutes and stimulated with PMA plus ionomycin for 4 h. After stimulation, cells were collected, and intracellular staining was performed to analyze the CD3⁺CD4⁺IFN-γ⁺- or CD3⁺CD4⁺IL17A⁺-producing cells by flow cytometry. The cells were then analyzed for AMPK at Thr172 levels by Western blotting. (B) The figure shows the representative flow cytometry. (C) The bar graphs show the percentage of cytokine-producing cells. (D) The figure shows the representative Western blotting images of AMPK at Thr172 levels. (E) The bar graphs show the percentage of pAMPK/AMPK expression. The cropped blots are used in the figure, and full-length blots are presented in Supplementary Fig. S3. All gels were run in the same experimental conditions (see material and methods for details). All data are reported as the mean ± SEM. N is 5 in each group. *P < 0.05, **P < 0.01.

Figure 4

BBR affected the oxidative phosphorylation and decreased the total adenosine triphosphate production. Colitis LP CD4⁺ T cells were stimulated with PMA plus ionomycin mixed with BBR (BBR) or not (Control) for 8 h. (A) After culture, the cells were collected, and the intracellular ATP was analyzed. (B) Bioenergetic profile of colitis LP CD4⁺ T cells. The OCR profile following the addition of mitochondrial inhibitors (oligomycin, FCCP, rotenone/antimycin A) was determined by a Flux analyzer xXF 96 S. (C–E) The effects of BBR on the OCR, mitochondrial ATP and ECAR profiles were separately analyzed. All data are reported as the mean ± SEM. N is 5 in each group. *P < 0.05, **P < 0.01.

Figure 5

BBR ameliorated experimental colitis in vivo with changes in gut microbiota and decreased the production of IFN-γ and IL-17A. A total of 3 × 10⁵ CD⁴⁺CD45RB^high T cells of Balb/c mice were transferred into new SCID mice to establish a colitis model (n = 6 per group). After that, a diet including BBR (BBR) or not (Control) was given to each group. The mice were monitored for up to seven weeks and then sacrificed and analyzed. (A) The gross appearance of the colon, spleen and mesenteric lymph nodes. (B) Clinical scores. (C) Histopathology of the distal colon. Original magnification: ×40. (D) Histological scores. Pictures show representative samples from each group. (E) Number of LP CD4⁺CD3⁺ T cells. (F) Intracellular staining of IFN-γ- and IL-17A-producing cells in LP CD4⁺ T cells. Flow cytometry and a bar graph showing the percentage of CD3⁺CD4⁺IFN-γ⁺- or IL-17A⁺-producing cells in colitis LP CD4⁺ T cells. (G) The AMPK expression of LP CD4⁺ T cells in both groups. The bar graph shows the percentage of pAMPK/AMPK expression. (H) The expression of CD25⁺Foxp3⁺ Treg on CD3⁺CD4⁺ T cells in the LP. (I) The expression of Bcl2⁺ on CD3⁺CD4⁺ T cells in the LP. Cropped blots are used in the figure; full-length blots are presented in Supplementary Fig. S5. All gels were run in the same experimental conditions (see Materials and methods for details). The numerical values represent the mean values of 6 samples per group. Pictures and dot plots of flow cytometry show representative samples from each group. All data are shown as the mean ± SEM for 6 mice per group. *P < 0.05, **P < 0.01.

Figure 6

(A,B) A comparison of the fecal microbial community at the phylum and genus level between colitis and BBR. (C) Alpha diversity_Observed OTU, PD whole tree, Chao1 and Shannon index. (D,E) Beta diversity_UniFrac_unweighted and UniFrac unweighted distance. Each point represents the gut microbiota community of each mouse, with black points and gray points indicating the colitis and BBR groups, respectively. All data are shown as the mean ± SEM for 6 mice per group. *P < 0.05, **P < 0.01.