Protective role of berberine on ulcerative colitis through modulating enteric glial cells-intestinal epithelial cells-immune cells interactions

Abstract

Ulcerative colitis (UC) manifests as an etiologically complicated and relapsing gastrointestinal disease. The enteric nervous system (ENS) plays a pivotal role in rectifying and orchestrating the inflammatory responses in gut tract. Berberine, an isoquinoline alkaloid, is known as its anti-inflammatory and therapeutic effects in experimental colitis. However, little research focused on its regulatory function on ENS. Therefore, we set out to explore the pathological role of neurogenic inflammation in UC and the modulating effects of berberine on neuro-immune interactions. Functional defects of enteric glial cells (EGCs), with decreased glial fibrillary acidic protein (GFAP) and increased substance P expression, were observed in DSS-induced murine UC. Administration of berberine can obviously ameliorate the disease severity and restore the mucosal barrier homeostasis of UC, closely accompanying by maintaining the residence of EGCs and attenuating inflammatory infiltrations and immune cells overactivation. In vitro, berberine showed direct protective effects on monoculture of EGCs, bone marrow-derived dendritic cells (BMDCs), T cells, and intestinal epithelial cells (IECs) in the simulated inflammatory conditions. Furthermore, berberine could modulate gut EGCs-IECs-immune cell interactions in the co-culture systems. In summary, our study indicated the EGCs-IECs-immune cell interactions might function as a crucial paradigm in mucosal inflammation and provided an infusive mechanism of berberine in regulating enteric neurogenic inflammation.

Keywords: APCs, antigen-presenting cells; BDNF, brain-derived neurotrophic factor; BMDCs, bone marrow-derived dendritic cells; Berberine; CGRP, calcitonin gene-related peptide; DSS, dextran sulfate sodium; EGCs, enteric glial cells; ENS, enteric nervous system; Enteric glial cells; Enteric nervous system; GDNF, glial cell derived neurotrophic factor; GFAP, glial fibrillary acidic protein; IBD, inflammatory bowel diseases; IECs, intestinal epithelial cells; LMPC, lamina propria mononuclear cells; MAPK, mitogen-activated protein kinases; MLNs, mesenteric lymph nodes; MPO, myeloperoxidase; Mucosal inflammation; UC, ulcerative colitis; Ulcerative colitis; VIP, vasoactive intestinal polypeptide.

Graphical abstract

Figure 1

Berberine exerted protective effects and enhanced residence of EGCs in DSS-induced ulcerative colitis. (A) Mice were treated with 3% DSS (from Day 0–7) and sterile water for additional 4 days. Berberine (100 mg/kg) was administrated daily and body weight (shown as the percentage of initial body weight) and DAI were monitored. (B) Typical pictures of colon. (C) Colon length. (D) Spleen index expressed as spleen weight (mg)/body weight (g). (E) Typical histological sections stained with H&E (50 × and 400 × magnification). (F) Statistics of histological score. (G) Immunohistochemistry staining of GFAP, substance P, GDNF, and BDNF of colonic tissue (scale = 75 μm). (H) Western blot assay of the protein level of GDNF, substance P, and BDNF. (I) The mRNA expression of Gfap, Gdnf, substance P, and their related genes in colon. (J) GDNF, substance P, and BDNF in colonic homogenates, determined by ELISA. All data were presented as mean ± SEM (n = 8 mice per group). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the vehicle group. Normal, mice with no treatment; Vehicle, mice treated only with DSS; Berberine, mice treated with DSS and berberine.

Figure 2

Berberine modulated the function of EGCs and decreased the expression of substance P in vitro. (A)–(C), Rat EGC cell line, CRL-2690, were treated with indicated concentrations of berberine. (A) Western blot assay of GFAP and GDNF of EGCs at various timepoints. (B) The mRNA expression level of Gfap and Gdnf of EGCs after 4 h incubation was determined, *P < 0.05, **P < 0.01, and ***P < 0.001 compared with medium control. (C) Immunofluorescence staining with GFAP and GDNF in EGC (Scale = 25 μm). (D) and (E) Rat splenocytes were activated with ConA stimulation for 24 h, and subsequently the supernatants were added into EGC cells culture. (D) Apoptosis assay of EGCs following annexin V and PI staining after 24 h incubation. (E) The mRNA expression of substance P and Gdnf of EGCs. (F) CRL-2690 cells were culture with recombinant BDNF (100 μg/mL) in the presence or absence of indicated concentrations and real-time PCR was performed to determine the expression of substance P. (E) and (F) *P < 0.05, **P < 0.01, and ***P < 0.001 compared with splenocytes supernatants or BDNF-primed cells. All data were presented as mean ± SEM of three independent experiments.

Figure 3

Berberine maintained the intestinal barrier homeostasis in DSS-colitis mice and TNF-α and/or neuropeptides-cultured epithelial cells. (A) Left, typical in vivo imaging pictures with bioluminescent imaging by injecting L-012 sodium (25 mg/kg). Right, quantification of bioluminescent images by ROI determination. (B) Intestinal permeability measurement by oral administration of FITC-dextran (600 mg/kg). Fluorescent images (left) and serum fluorescence intensity (right) of FITC-dextran were determined. (C) Left, colonic homogenates were analyzed by Western blotting to detect the expression of tight junction protein (ZO-1, E-cadherin, and occludin). Right, quantitative analysis of tight junction protein expression. (D) Immunofluorescence staining with ZO-1 and E-cadherin of colonic sections (Scale = 100 μm). (E) and (F) Barrier function in vitro was assayed in TNF-α and IFN-γ-primed Caco-2 cells. FITC-dextran permeability (E) and trans-epithelial electrical resistance (TEER, F) were determined. (G) HT-29 cells were cultured with berberine, substance P, or GDNF in the absence or presence of TNF-α induction. E-cadherin expression by immunofluorescence staining was performed (Scale = 75 μm). (A)–(D), all data were presented as mean ± SEM; n = 8 mice per group. (E)–(G), all data were presented as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001: (A)–(C), compared with the vehicle group; (E) and (F), compared with TNF-α/IFN-γ-stimulated cells.

Figure 4

Berberine suppressed the colonic leukocytes infiltration by regulating chemotaxis and adhesion. The percentage of macrophage (CD11b⁺F4/80⁺), neutrophils (CD11b⁺Gr-1⁺), and dendritic cells (CD11b⁺CD11c⁺) were assayed in MLNs (A) and colonic lamina propria (B). (C) Colonic sections were immunofluorescence stained with Ly6G, CD11b, and F4/80 and the nuclei were stained with DAPI (Scale = 100 μm). (D) The percentage of CD25-positive cells, naïve T cells (CD44^‒CD62L⁺) and effector T cells (CD44⁺CD62L^‒) gated on CD3⁺CD4⁺ subpopulation, Th1 (CD4⁺IFN-γ⁺) and Th17 cells (CD4⁺IL-17⁺) in MLNs. (E) The percentage of CD3⁺CD4⁺ cells, CD3⁺γδTCR⁺ cells, and CD3⁺IL-17⁺ cells in colonic lamina propria. (F) Heatmap of the mRNA expression of chemokines, chemokine receptors, and adhesion molecules. (G) Immunofluorescence staining with CCR6 and DAPI of colonic tissue (Scale = 100 μm). (H) Western blot assay of certain chemokines, chemokine receptors, and adhesion molecules expression. All data were presented as mean ± SEM; n = 8 mice per group.

Figure 5

Berberine inhibited chemotaxis and adhesion in TNF-α and/or neuropeptides-cultured human HT-29 cells. (A) Calcein AM-labeled THP-1 cells were incubated onto HT-29 monolayer cells for 30 min. THP-1 cells adhered to the TNF-α-activated HT-29 cells were observed after washing (Scale = 100 μm). (B) Immunofluorescence staining with ICAM-1 of TNF-α-activated HT-29 cells (Scale = 25 μm). (C) Western blot assay of CD62E and ICAM-1 expression in TNF-α-activated HT-29 cells. (D) Representative images of THP-1 cells migrated to the HT-29 cell supernatants as described in Methods (Scale = 100 μm). (E) The counting number of THP-1 cells chemotactic to the lower chamber containing HT-29 cell supernatants. (F) Secretion of IP-10 and IL-8 in the supernatants of TNF-α-activated HT-29 cells. (G) and (H) HT-29 cells were cultured with berberine, substance P, or GDNF, in the absence or presence of TNF-α induction. Western blot assay of CD62E and ICAM-1 (G), and the mRNA expression of IP-10 (H) were performed. All data were presented as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, compared with TNF-α-stimulated cells.

Figure 6

Berberine inhibited the overexpression of inflammatory mediators and related signaling pathway in DSS-induced colitis. (A) Cytokines secretion in the full-thickness colon tissue culture by ELISA. (B) Cytokines protein levels in the colonic homogenates. (C) mRNA was isolated from the colon for real-time PCR analysis of indicated gene expression. The mRNA expression level in normal group was set as 100%. (D) The mRNA expression of Inos, Cox-2, and inflammasome-related genes. Western blot assay of neuropeptides-related signaling pathways, including PI3K-AKT-mTOR (E), MAPK, and P65 NF-κB signaling (F) were performed. All data were presented as mean ± SEM; n = 8 per group. *P < 0.05, **P < 0.01, and ***P < 0.001, compared with the vehicle group.

Figure 7

Diagram of gut EGCs–IECs–immune cells interaction affected by berberine to ameliorate UC. Upon DSS exposure, epithelial barrier disturbance and functional defects of EGCs and inflammatory cell infiltrations were observed. Berberine ameliorated the disease severity and intestinal inflammation, accompanying by directly restoring the mucosal barrier homeostasis, maintaining the residence of EGCs and attenuating inflammatory infiltrations and immune cells overactivation, respectively. In depth, berberine could modulate gut EGCs–IECs–immune cell interactions, providing an infusive mechanism of berberine in regulating enteric neurogenic inflammation. Briefly, berberine could prevent EGCs apoptosis, inhibiting substance P expression and increasing GDNF expression in the presence of activated immune cells. By contrast, berberine showed anti-inflammatory effects involving in GDNF and substance P. Moreover, berberine, along with GDNF and substance P could interfere with the communication between immune cell and epithelial cells, including the barrier function, adhesion and chemotaxis. Abbreviations: EGCs, enteric glial cells; IEC, intestinal epithelial cells.