Oxymatrine prevents NF-κB nuclear translocation and ameliorates acute intestinal inflammation

Abstract

Oxymatrine is a traditional Chinese herbal product that exhibits anti-inflammatory effects in models of heart, brain and liver injury. We investigated the impact of oxymatrine in an acute model of intestinal injury and inflammation. Oxymatrine significantly decreased LPS-induced NF-κB-driven luciferase activity, correlating with diminished induction of Cxcl2, Tnfα and Il6 mRNA expression in rat IEC-6 and murine BMDC. Although oxymatrine decreased LPS-induced p65 nuclear translocation and binding to the Cxcl2 gene promoter, this effect was independent of IκBα degradation/phosphorylation. DSS-induced weight loss and histological damage were ameliorated in oxymatrine-treated C57BL/6-WT-mice. While this effect correlated with reduced colonic Il6 and Il1β mRNA accumulation, global NF-κB activity as measured in NF-κB(EGFP) mice was unaffected. Our data demonstrate that oxymatrine reduces LPS-induced NF-κB nuclear translocation and activity independently of IκBα status, prevents intestinal inflammation through blockade of inflammatory signaling and ameliorates overall intestinal inflammation in vivo.

Figure 1. Oxymatrine inhibits LPS-mediated Cxcl2, Il6 and Tnfα expression in IEC-6 Cells.

IEC-6 cells were stimulated for 1 and 4 hr with 5 μg/mL LPS ± 4 mg/mL OMT and real-time PCR performed to analyze Cxcl2, Tnfα, Il6 and Il1b mRNA accumulation standardized to Gapdh expression. Measurements expressed as fold induction over control unstimulated cells. Results combined from 2 independent experiments, and represent 3 independent experiments. *p < 0.05, **p < 0.01 compared to LPS controls.

Figure 2. Oxymatrine inhibits LPS-induced pro-inflammatory cytokine expression in BMDCs.

Naïve BMDCs were stimulated with 1 μg/mL LPS for 1 or 4 h ± 4 mg/mL OMT and real-time PCR performed to analyze Il6, Il1b and Tnfα mRNA accumulation standardized to Gapdh expression. Results are combined data from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared to LPS control.

Figure 3. Oxymatrine inhibits NF-κB activation independent of IκBα phosphorylation.

(a) IEC-18 cells were transduced using an NF-κB luciferase construct and stimulated with 5 μg/mL LPS ± 4 mg/mL OMT. Luciferase production was measured and reported as fold induction over control normalized to amount of extract protein (light units/μg). Results are combined from 3 independent experiments. (b) Western blot analysis of phospho-IκBα and total IκBα was performed using IEC-18 cells stimulated with 5 μg/mL LPS for 30, 60 or 90 minutes ± 4 mg/mL OMT. β-Actin was used as a loading control. Densitometric analysis of protein levels is shown on the right side. Representative of 3 independent experiments. (c) Western blot analysis of phospho-p38, total p38 and β-Actin was performed using IEC-6 cells stimulated with LPS for 30 or 60 minutes ± OMT. Densitometric analysis of protein levels is shown on the right side. Representative of 3 independent experiments. **p < 0.01 compared to LPS control.

Figure 4. Oxymatrine inhibits p65 nuclear translocation.

(a) IEC-6 cells were treated with 5 μg/mL LPS ± 4 mg/mL OMT for 30 minutes. Cells were fixed, permeabilized and immunofluorescent staining performed for p65 (inner panels). Hoescht nuclear staining was also performed (outer panels). Representative of 3 independent experiments. (b) p65 cytosolic and nuclear quantifications were measured as p65 nuclear accumulation in 3 random fields of view per slide. Graph represents combined data from 3 independent experiments. (c) Chromatin immunoprecipitation (ChIP) analysis of p65 binding to the Cxcl2 promoter of IEC-18 cells stimulated with LPS ± OMT for 30 and 60 min. PCR amplification products were separated using agarose gel electrophoresis and gel scan results are presented in inverse color. Representative of 2 independent experiments. **P < 0.01 compared to LPS control.

Figure 5. Oxymatrine ameliorates DSS-induced pathology.

WT C57BL/6 mice were pre-treated with saline or OMT for 2 days prior to addition of 3% DSS to drinking water. (a) Daily weights were measured (n = 5-7/group) and plotted as percentage bodyweight change from initial weight, expressed as group % mean ± SEM. (b) Representative colon swiss roll sections stained with H&E (original magnification 100X). (c) Group colon histopathological scores. Bars represent mean ± SEM. (d) Real-time PCR analysis of colonic tissue Tnf, Il6 and Il1b expression standardized to Gapdh expression. Expressed as fold induction over mock treated control mice. Representative of 2 independent experiments. (e) Immunhistochemical analysis of Ki-67 expression was performed using colon sections from control untreated, DSS-treated and DSS + 200 mg/Kg OMT-treated mice. Graph represents quantification of Ki-67⁺ cells within intact distal colonic crypts. 3 sections were stained and counted per condition. (f) Macroscopic analysis of representative colons from experimental NF-κB^EGFP mouse groups, fluorescent analysis (right panel). Representative of 2 independent experiments. *p < 0.05, **p < 0.01 compared to DSS-only control.