Berberine promotes immunological outcomes and decreases neuroinflammation in the experimental model of multiple sclerosis through the expansion of Treg and Th2 cells

Abstract

Introduction: Among the most frequent demyelinating autoimmune disorders of the central nervous system (CNS) is multiple sclerosis. Experimental autoimmune encephalomyelitis (EAE) is used as an animal model of multiple sclerosis. Berberine is an alkaloid found in some medicinal plants with anti-inflammatory effects.

Methods: C57BL/6 female mice were used and divided into three groups: (1) The control group received PBS, (2) the low-dose treatment group received 10 mg/kg of berberine, and (3) The high-dose treatment group received 30 mg/kg of berberine. Myelin Oligodendrocyte Glycoprotein and complete Freund's adjuvant were subcutaneously administered to induce EAE. Mice were given intraperitoneal injections of pertussis toxin on the day of immunization and 2 days later. Histological studies showed low lymphocyte infiltration and demyelination of CNS in the treated groups.

Results: The clinical scores of the treatment group with low-dose berberine (T1: 2 ± 0.13) and high-dose berberine (T2: 1.5 ± 0.14) were significantly (p < .001) lower than the control group (CTRL: 4.5 ± 0.13). Treatment groups decreased pro-inflammatory cytokines (IFN-γ, TNF-α, interleukin [IL]-17) (p < .001) as well as increased anti-inflammatory cytokine expression (IL-4, IL-10, IL-27, IL-33, IL-35, TGF-β) (p < .01) when compared to the CTRL group. Treatment groups with berberine reduced expression of the Th1 and Th17 cytokines and transcription factors (p < .001) and increased expression of transcription factors and Th2 and Treg cytokines (p < .01) in contrast to CTRL group.

Conclusion: Berberine appears to have a protective effect on disease development and alleviating disease status in EAE, which appears to be due to the cell expansion and function of Treg and Th2 cells in addition to berberine's anti-inflammatory properties.

Keywords: Berberine; experimental autoimmune encephalomyelitis; multiple sclerosis; myelin oligodendrocyte glycoprotein.

Figure 1

Berberine inhibited the development of experimental autoimmune encephalomyelitis (EAE) in MOG‐immunized C57BL/6 mice. Female C57BL/6 mice were treated with 10 and 30 mg/kg berberine in treatment groups on Day 1 simultaneous with EAE induction until Day 25 postimmunization. Mice were monitored for signs of EAE, and the results for all mice, were presented as (A) mean clinical score and (B) body weight. Results were expressed as mean ± SEM. *p < .05; ***p < .001, compared with control group. 24 mice were divided into three groups: (1) Control group (CTRL), (2) low‐dose berberine treatment group (T1), and (3) high‐dose berberine treatment group (T2).

Figure 2

Comparative histopathology of the brain demonstrated berberine suppresses central nervous system (CNS) inflammation and demyelination. Histopathological evaluation of the brain from treated groups (low and high dose Berberine) and control was performed. Brains from each group, were collected on Day 25 postimmunization, fixed in paraformaldehyde, and embedded in paraffin. Five‐micrometer sections from different regions of the brain from each of the groups were stained with (A) hematoxylin & eosin (H&E) to enumerate infiltrating leukocytes and with (B) Luxol fast blue to assess demyelination by Light microscope (10X, scale bar). (C) CNS inflammatory foci and infiltrating inflammatory cells were quantified. Pathological scores including inflammation and demyelination were analyzed and shown with a bar graph as mean scores of pathological inflammation or demyelination ± SEM. Data are representative of three independent experiments. ***p < .001 compared with control group. 24 mice were divided into three groups: (1) Control group (CTRL), (2) low‐dose berberine treatment group (T1), and (3), high‐dose berberine treatment group (T2).

Figure 3

Berberine suppresses T‐cell proliferation. Spleen cells were harvested on day 25 postimmunization and cultured in PHA 20 μg/ml) as a positive control or with myelin oligodendrocyte glycoprotein (MOG) (20 μg/ml) for 72 h on 96‐well plates. Proliferation responses were tested using a cell proliferation ELISA, BrdU (colorimetric) kit (Roche Applied Science). Proliferation assay was conducted in triplicate wells. Data presented as mean optical density ± SEM. ***p < .001 compared with control group. 24 mice were divided into three groups: (1) Control group (CTRL), (2) low‐dose Berberine treatment group (T1), and (3) high‐dose berberine treatment group (T2).

Figure 4

Berberine suppressed pro‐inflammatory cytokines production and enhanced anti‐inflammatory cytokines production in splenocytes and lymph nodes from experimental autoimmune encephalomyelitis (EAE) mice. Splenocytes and lymph nodes from immunized mice from all groups (24 mice) were isolated on Day 25 postimmunization and restimulated with myelin oligodendrocyte glycoprotein (MOG)_35–55 (20 μg/ml) for 72 h. Culture supernatants were collected and indicated cytokine levels were measured by ELISA. Cytokine assays were conducted in duplicate wells. (A) Pro‐inflammatory cytokines as IFN‐γ TNF‐α, and IL‐17 ± SEM and (B) anti‐inflammatory cytokines as IL‐4, IL‐10, IL‐27, IL‐33, IL‐35, and TGF‐β were measured from supernatants of cultures from splenocytes and lymph nodes. Results from lymph nodes were similar to splenocytes and data was not shown. *p < .05; ***p < .001 compared with control group. 24 mice were divided into three groups: (1) Control group (CTRL), (2) low‐dose berberine treatment group (T1), and (3) high‐dose berberine treatment group (T2).

Figure 5

Gene expression of cytokines and transcription factors in central nervous system (CNS). On Day 25 postimmunization, brains, and spinal cords were collected and mRNA levels of cytokines and transcription factors were assessed by real‐time quantitative PCR. The assay was run in triplicate and fold change expression of genes was determined compared control group. (A) Th1 and Th17 related cytokines and transcription factors; IFN‐γ, STAT4, T‐bet, IL‐17, STAT3, ROR‐γt, TNF‐α (B) Th2 and Treg related cytokines and transcription factors; IL‐4, STAT6, GATA3, IL‐10, IL‐33, TGF‐β, FoxP3, and PD1. Results were expressed as fold change compared with the control group. ** p < .01; ***p < .001. 24 mice were divided into three groups: (1) Control group (CTRL), (2) low‐dose berberine treatment group (T1), and (3) high‐dose berberine treatment group (T2).