Berberine ameliorates aGVHD by gut microbiota remodelling, TLR4 signalling suppression and colonic barrier repairment for NLRP3 inflammasome inhibition

Abstract

Berberine (BBR), an isoquinoline alkaloid, is used to treat gastrointestinal disorders as an herbal medicine in China. The aim of this study was to investigate the anti-inflammatory activities of BBR in a mouse model with acute graft-versus-host disease (aGVHD). Mice were intravenously injected with bone marrow cells from donors combined with splenocytes to develop aGVHD. The body weight, survival rate and clinical scores were monitored. Then the levels of inflammatory cytokines, histological changes (lung, liver and colon), colonic mucosal barrier and gut microbiota were analysed. Moreover, the toll-like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (Myd88)/nuclear factor-κB signalling pathway, NLRP3 inflammasome and its cytokines' expressions were determined. The results showed that the gavage of BBR lessened GVHD-induced weight loss, high mortality and clinical scores, inhibited inflammation and target organs damages and prevented GVHD-indued colonic barrier damage. Additionally, BBR modulated gut microbiota, suppressed the activation of the TLR4 signaling pathway and inhibited NLRP3 inflammasome and its cytokine release. This study indicated that BBR might be a potential therapy for aGVHD through NLRP3 inflammasome inhibition.

Keywords: NLRP3 inflammasome; TLR4 signaling pathway; acute graft-versus-host disease; berberine; cytokines; gut microbiota.

FIGURE 1

The mortality and morbidity of GVHD decreased in BMT by berberine (N = 10/group). (A) Changes in body weight every 4 days. (B) Percentage of survival. (C) Mean clinical scores every 4 days. (D) The flow chart of the following experimental procedures. Data are shown as mean ± SD and were analysed by Multiple t tests. *p < 0.05 and **p < 0.01 between model and berberine groups at the indicated time points. The graph (A and C) show the data combined with three independent experiments (10–15 animals in each group)

FIGURE 2

Effect of berberine on proinflammatory cytokines in serum (N = 5/group). IL‐1β (A), IL‐18 (B), IFN‐γ (C), TNF‐α (D), MCP‐1 (E) and IL‐6 (F). Data are shown as mean ± SD and were analysed by one‐way ANOVA. ^* p < 0.05; ^** p < 0.01

FIGURE 3

Attenuation of pathologic damage in GVHD target organs treated by berberine (N = 10/group). (A–C) Histology of lung in each group (100×). (D–F) Histology of liver in each group (400×). (G–I) Histology of colon in each group (400×). (J) The pathology score of the lung, liver and colon in three groups using SSS as detailed in Methods section. Data are shown as mean ± SD and were analysed by one‐way ANOVA. ^* p < 0.05 and ^** p < 0.01

FIGURE 4

Berberine repaired the colonic barrier damage in GVHD mice. (A‐C) Expression of Occludin in each group (200×) and (D‐F) expression of ZO1 in each group (200×) (N = 10/group). Protein expression of Claudin‐1 (G) and Claudin‐2 (H) in colon tissue (N = 3/group). (I) Serum LBP concentration was determined by ELISA (N = 5/group). Data are shown as mean ± SD and were analysed by one‐way ANOVA. ^* p < 0.05; ^** p < 0.01

FIGURE 5

Berberine recovered the gut microbiome changes in GVHD Mice. Differentially expressed taxa with the LDA scores >2.0 and adjusted p values <0.05 between the control and model mice (A), and between the model mice and berberine treatment mice (B). Differences are represented by the colour of over‐represented taxa. Circles show phylogenetic levels from phylum (innermost layer) to genera (outermost layer)

FIGURE 6

Suppression of gut NLRP3 inflammasome, TLR4 signalling and inflammatory cytokines in GVHD mice colonic tissues by berberine (N = 3/group). Protein expression of NLRP3 (A), ASC (B), and Caspase‐1 (C), and mRNA expression of TLR4/MyD88/NF‐κB (D) in colon tissue. The levels of inflammatory cytokines (IL‐1β, TNF‐α and IL‐6) from the colon tissue of GVHD mice were determined through ELISA (E) and Q‐PCR (F), respectively. We used the β‐actin as an internal control. Data are shown as mean ± SD. ^* p < 0.05; ^** p < 0.01; ns represents not significant (Figure A–C were analysed using one‐way ANOVA, Figure D–F were analysed by Multiple t tests)