Intervention of oncostatin M-driven mucosal inflammation by berberine exerts therapeutic property in chronic ulcerative colitis

Abstract

Ulcerative colitis (UC) is a chronic and etiologically refractory inflammatory gut disorder. Although berberine, an isoquinoline alkaloid, has been revealed to exert protective effects on experimental colitis, the underlying molecular mechanism in chronic intestinal inflammation remains ill-defined. This study was designed to uncover the therapeutic efficacy and immunomodulatory role of berberine in chronic UC. Therapeutic effects of oral administration of berberine were investigated in dextran sodium sulfate (DSS)-induced murine chronic UC and the underlying mechanisms were further identified by si-OSMR transfection in human intestinal stromal cells. Berberine significantly attenuated the experimental symptoms and gut inflammation of chronic UC. Berberine treatment could also maintain the intestinal barrier function and rectify tissue fibrosis. In accordance with infiltrations of antigen-presenting cells (APCs), innate lymphoid cells (ILCs), and activated NK cells in colonic lamina propria, increased expression of OSM and OSMR were observed in the inflamed tissue of chronic UC, which were decreased following berberine treatment. Moreover, berberine inhibited the overactivation of human intestinal stromal cells through OSM-mediated JAK-STAT pathway, which was obviously blocked upon siRNA targeting OSMR. The research provided an infusive mechanism of berberine and illustrated that OSM and OSMR intervention might function as the potential target in chronic UC.

Fig. 1. Berberine ameliorated the experimental symptoms and gut inflammation in DSS-induced murine chronic UC.

a Berberine (50 mg/kg) were orally administrated to DSS-induced UC mice (from day 15 to the end of experiment) and normal control mice were given normal drinking water only. Body weight change (% initial body weight) and disease activity index (DAI) were monitored at the indicated time points. b Representative images of colon morphology. c Quantitative colon length and the ratio of the colon weight to the length. d Serum cytokines determined by Luminex assay. e Serum biochemical indicators, including ALB, ALP, and TC. f Cytokine secretion levels in full-thickness colon tissue culture. g The protein levels of cytokines in colonic homogenates. h Representative images of bioluminescent imaging with L-012 and quantitative analysis of positive signals using an IVIS Spectrum CT system. i The expression of inflammasome, including NLRP3, ASC, cleaved caspase-1, assayed by western blot and GAPDH was used as a loading control. Data were presented as the mean ± SEM, and n = 15 mice per group. *p < 0.05 and **p < 0.01, compared with the vehicle group, were measured by one-way ANOVA.

Fig. 2. Berberine maintained intestinal mucosal barrier function through upregulating the expression of tight junctions.

a Representative images of H&E staining of colon sections (×50 and ×100 magnification) and histological scores. b Representative images of ultrastructural examination of intestinal epithelial layer by transmission electron microscopy (TEM). c Representative images of in vivo imaging with FITC-dextran administration and quantitative assay of imaging with FITC-dextran. d Serum fluorescent intensity of FITC-dextran. e Colonic sections were immunofluorescent staining with ZO-1, E-cadherin, and occluding, and the nuclei were visualized by DAPI staining. f Western blot assay of tight junction proteins, claudins, and MUC2 expression. g Gene expression level of tight junction proteins by RT-PCR. h Gene expression level of MUC2 by RT-PCR. Data were presented as the mean ± SEM, and n = 15 mice per group. *p < 0.05 and **p < 0.01, compared with the vehicle group, were measured by one-way ANOVA.

Fig. 3. Berberine suppressed intestinal tissue fibrosis through interfering with OSM-mediated signaling pathway.

a Representative images of picrosirius red staining of colonic tissue. b Representative images of immunohistochemistry staining with α-SMA (upper) and western blot assay of α-SMA expression (bottom). c Gene expression of OSM, OSMR, FAP, and PDPN by RT-PCR. d The expression level of OSM and OSMR, visualized and separated by agarose gel. e OSM levels in serum, full-thickness colon culture, colonic homogenates, and fecal extracts by ELISA. f Western blot assay of the expression level of OSM, OSMR, and OSM-mediated downstream signaling pathway. g Representative images of immunofluorescence staining with OSMR and p-STAT1. Data were presented as the mean ± SEM and n = 15 mice per group. *p < 0.05 and **p < 0.01, compared with the vehicle group, were measured by one-way ANOVA.

Fig. 4. Berberine interfered with the interaction between human intestinal stromal cells and immune cells, in which OSMR functioned as an indispensible factor.

a Human CCD-18Co cells were transfected with NC or si-OSMR for 72 h and then stimulated with OSM (10 ng/ml) for 1 h in the presence of berberine (50 μM). The cells were collected for western blot assay (a) and immunofluorescent staining with p-STAT1 (b). c Calcein AM-labeled U937, THP-1, and Jurkat T cells were incubated on OSM-stimulated transfected CCD-18Co cells for 30 min. Adhered immune cells were visualized under Olympus IX73 microscope. d Representative images of Calcein AM-labeled U937, THP-1, and Jurkat T cells migrated to the supernatants from OSM-stimulated transfected CCD-18Co cells for 2 h. e The counting numbers of U937, THP-1, and Jurkat T cells chemotactic to the lower chamber containing OSM-stimulated transfected CCD-18Co cells supernatants. f The mRNA expression of certain chemokines in OSM-stimulated transfected CCD-18Co cells. g Protein level of CXCL10 in OSM-stimulated transfected CCD-18Co cells. h The mRNA expression of ICAM-1 in OSM-stimulated transfected CCD-18Co cells. i Western blot assay of ICAM-1 expression in OSM-stimulated transfected CCD-18Co cells. Data were presented as mean ± SEM of three independent experiments. *p < 0.05 and **p < 0.01, compared with OSM-stimulated CCD-18Co cells, were measured by one-way ANOVA.

Fig. 5. Berberine inhibited the infiltration of inflammatory cells to the inflamed colon tissue in DSS-induced chronic colitis.

a Flow cytometry and quantitative analysis of gut lamina propria mononuclear cells (LPMCs), including monocytes (CD11b⁺), neutrophils (CD11b⁺Gr-1⁺), macrophages (CD11b⁺F4/80⁺), and dendritic cells (CD11b⁺CD11c⁺). b Representative images of immunofluorescence staining with CD11b and Ly6G. c Flow cytometry and quantitative analysis of the proportion of ILCs (CD45⁺Lineage⁻CD127⁺), including ILC1 (T-bet⁺), ILC2 (GATA3⁺), and ILC3 (RORγT⁺), in LPMCs. d The mRNA expression level of T-bet, GATA3, and RORγT by RT-PCR. e Flow cytometry and quantitative analysis of the proportion of activation of NK cells (CD335, CD27, and CD11b, gated on CD3^‒NK1.1⁺). f The mRNA expression level of NKp46, KLRG-1, and Eomes by RT-PCR. Data were presented as the mean ± SEM, and n = 15 mice per group. *p < 0.05, and **p < 0.01, compared with the vehicle group, were measured by one-way ANOVA.

Fig. 6. Berberine downregulated the expression levels of various adhesion molecules, chemokines, and their receptors.

a The mRNA expression level of ICAM-1, MadCAM-1, and CD62E in colonic tissue. b Western blot assay of the protein expression level of ICAM-1, MadCAM-1, and CD62E in colonic tissue. c Representative images of immunofluorescence staining with ICAM-1. d Gene expression level of various chemokines and their receptors in colon tissues by RT-PCR. e Representative images of immunofluorescence staining with CCR5. f Flow cytometry analysis of CCR5, CXCR3, and CCR6, gated on CD3⁺ cells. g Flow cytometry analysis of CCR5, CCR2, and CX3CR1, gated on CD11b⁺ cells. Data were presented as the mean ± SEM, and n = 15 mice per group. *p < 0.05 and **p < 0.01, compared with the vehicle group, were measured by one-way ANOVA.

Fig. 7. Diagram of OSM-driven intestinal mucosal inflammation interfered by berberine in treating chronic UC.

In the development of chronic UC, dysfunction of intestinal barrier and rouse of gut resident immune cells contributed to initiating pathogenic colitis. Further activation of resident and newly infiltrated immune cells, including neutrophils, dendritic cells, macrophages, Th cell, led to accumulation of OSM, which subsequently induced diverse inflammatory responses in OSMR-expressing stromal cells. Berberine could suppress multiple sources of OSM and activation of stromal cells, accounting for decreased expression of chemokines, adhesive molecules, and persistent damage to epithelium homeostasis. In depth, berberine impeded the phosphorylation of JAK-STAT, ERK, and AKT signaling through interfering with the binding mode of OSM and OSMR.