Baicalein from Scutellaria baicalensis mitigates oxidative stress through the IIS pathway in a C. elegans model of ulcerative colitis

Abstract

Introduction: Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease with limited therapeutic options. Baicalein, a phenolic flavonoid extracted from Scutellaria baicalensis, has been traditionally used in Chinese medicine for its potent anti-inflammatory, anti-tumor, and antiviral properties. This plant, known as Huang-Qin, is indigenous to East Asia and has been widely used to treat various conditions such as fever, respiratory diseases, and inflammation.

Aim of the study: This study aimed to establish a C. elegans model of UC induced by dextran sodium sulfate (DSS) and to investigate the protective effects of baicalein on intestinal injury.

Materials and methods: DSS was used to induce acute intestinal injury in C. elegans. N2 and mutant strains (daf-2 and daf-16) were exposed to DSS at concentrations of 5% (w/v), which identified as optimal for inducing intestinal inflammation. The effects of 25 μM, 50 μM, and 100 μM of baicalein on intestinal barrier function, oxidative stress markers, and relevant gene expression were evaluated, including genes related to epithelial barrier integrity (clc-2, mtm-6, etc.), oxidative stress, and the IIS and p38 MAPK pathways.

Results: Baicalein significantly improved physiological condition and intestinal permeability in worm treated with 5% DSS. It restored the expression of epithelial barrier genes and reduced oxidative stress, as indicated by decreased ROS, enhancing SOD activity, daf-16 nuclear translocation etc. Baicalein's protective effects were associated with the activation of the p38 MAPK and IIS pathways. In daf-2 and daf-16 mutant strains, baicalein demonstrated partial dependence on the IIS pathway for its protective effects.

Conclusion: This study established a DSS-induced UC model in C. elegans and demonstrated that baicalein exerts protective effects on intestinal barrier integrity and oxidative stress, through the IIS and MAPK pathways. These findings support the use of C. elegans as a model for UC research and provide valuable insights into baicalein's therapeutic potential for inflammatory bowel diseases.

Keywords: C. elegans; IIS pathway; baicalein; dextran sodium sulfate; ulcerative colitis.

FIGURE 1

Effects of different DSS concentrations on C. elegans. Fourth-stage (L4) N2 C. elegans were exposed to 1%, 2%, 5%, 10%, and 30% DSS solutions for 24 h. Intestinal permeability was assessed following a rinse with M9 buffer (total magnification: ×100 and scale bar: 200 μm) (a). (b): Erioglaucine dye coverage on the nematodes was quantified to normalize the data. (c): LD50 values for each DSS concentration on C. elegans were determined. Statistical significance is denoted by '*' for comparison to the control group, where *p < 0.05 and ***p < 0.001 indicate levels of significance.

FIGURE 2

Baicalein intervention promotes growth and development in nematodes following DSS treatment. (a) Molecular structure of baicalein. L4 stage N2 C. elegans were treated with 5% DSS followed by 0.1% DMSO or varying concentrations of baicalein (25, 50, and 100 μM). Longevity (b), body length (c), and body width (d) were assessed, with data normalized accordingly. Statistical significance is denoted by '*' for comparisons with the control group and '#' for comparisons with the 5% DSS treatment group, with *p < 0.05, ***p < 0.001, #p < 0.05, ##p < 0.01, and ###p < 0.001 indicating significance levels.

FIGURE 3

Baicalein ameliorates movement impairments in nematodes following DSS treatment. L4 N2 C. elegans exposed to 5% DSS were treated with 0.1% DMSO or baicalein (25, 50, and 100 μM) respectively. Locomotive abilities were evaluated by measuring head thrashes per 30 s (a) and body bends per minute (b), with normalized data for comparison. Statistical significance is denoted by '*' for comparisons with the control group and '#' for comparisons with the 5% DSS treatment group, with *p < 0.05, ***p < 0.001, #p < 0.05, ##p < 0.01, and ###p < 0.001 indicating levels of significance.

FIGURE 4

Baicalein mitigates intestinal permeability in DSS-treated C. elegans. The effects of baicalein treatment on C. elegans exposed to 5% DSS were assessed by observing L4 stage nematodes treated with either 0.1% DMSO or varying concentrations of baicalein (25, 50, and 100 μM). (a) Intestinal permeability was observed with total magnification ×100 (scale bar: 200 μm) and ×400 (scale bar: 50 μm) magnifications. Quantification of the observed effects is shown in (b). (c) qRT-PCR analysis was used to assess expression levels of genes involved in intestinal barrier integrity, with data normalized for comparison. Statistical significance is indicated by '*' for comparisons with the control group and '#' for comparisons with the 5% DSS treatment group, with notations denoting significance levels: *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001.

FIGURE 5

Baicalein reduces oxidative stress in DSS-exposed C. elegans. The L4 stage of N2 C. elegans exposed to 5% DSS were treated with either 0.1% DMSO or baicalein (25, 50, and 100 μm) to assess oxidative stress. Lipofuscin and ROS levels were observed under fluorescence microscopy with total magnification ×100 (scale bar: 150 μm) (a). Fluorescence intensities were quantified for ROS (b) and lipofuscin (c) with data normalized for comparison. Statistical significance is denoted by '*' for comparisons with the control group and '#' for comparisons with the 5% DSS treatment group, with significance indicated as ***p < 0.001, #p < 0.05, ###p < 0.001.

FIGURE 6

Baicalein alleviates oxidative stress in DSS-treated C. elegans by modulating oxidative and antioxidant markers. The L4 stage of N2 C. elegans exposed to 5% DSS were treated with 0.1% DMSO or baicalein (25, 50, and 100 μM). Levels of MDA (a) and SOD (b) were measured. Statistical significance is denoted by '*' for comparisons with the control group and '#' for 5% DSS treatment group comparisons, with notations indicating significance: *p < 0.05, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001.

FIGURE 7

Baicalein modulates gene expression within the p38 MAPK pathway in DSS-exposed C. elegans. The L4 stage of N2 C. elegans subjected to 5% DSS were treated with 0.1% DMSO or baicalein (50 μM). mRNA levels of p38 MAPK pathway genes were measured in nematode homogenates. Statistical significance is indicated by '*' for comparisons with the control group and '#' for DSS model group comparisons, with significance denoted as *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01, ###p < 0.001.

FIGURE 8

Baicalein alleviates oxidative stress in DSS-treated C. elegans by modulating oxidative stress-related mRNA. The L4 stage of N2 C. elegans exposed to 5% DSS were treated with 0.1% DMSO or baicalein 50 μM). Levels of oxidative stress-related mRNA was measured. Statistical significance is denoted by '*' for comparisons with the control group and '#' for 5% DSS treatment group comparisons, with notations indicating significance: *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001.

FIGURE 9

Baicalein influences subcellular localization of DAF-16::GFP. The L4 stage of the DAF-16GFP strains exposed to 5% DSS were treated with 0.1% DMSO or baicalein (25, 50, and 100 μM). (a) Fluorescence microscopy images depicting DAF-16GFP nuclear translocation events in TJ356 strain nematodes with total magnification ×100 (scale bar: 200 μm) (b) Quantitative analysis of nuclear translocation frequency for DAF-16GFP fusion protein. Statistical significance is indicated by '*' for comparisons with the control group and '#' for comparisons with the 5% DSS treatment group, with notations denoting significance levels: *p < 0.05, #p < 0.05, ##p < 0.01, ###p < 0.001.

FIGURE 10

Baicalein Enhances Intestinal Barrier in DSS-Treated daf-2 Mutant. The L4 stage of the daf-2 mutant strains exposed to 5% DSS were treated with 0.1% DMSO or baicalein (50 μM). (a) Intestinal permeability was observed with total magnification ×100 (scale bar: 200 μm) and ×400 (scale bar: 50 μm) magnifications. Quantification of the observed effects is shown in (b). (c) qRT-PCR analysis was used to assess expression levels of genes involved in intestinal barrier integrity, with data normalized for comparison. Statistical significance is indicated by '*' for comparisons with the control group and '#' for comparisons with the 5% DSS treatment group, with notations denoting significance levels: *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001.

FIGURE 11

Baicalein Enhances Intestinal Barrier in DSS-Treated daf-16 Mutant Nematodes. The L4 stage of the daf-16 mutant strains exposed to 5% DSS were treated with 0.1% DMSO or baicalein (50 μM). (a) Intestinal permeability was observed with total magnification ×100 (scale bar: 200 μm) and ×400 (scale bar: 50 μm) magnifications. Quantification of the observed effects is shown in (b). (c) qRT-PCR analysis was used to assess expression levels of genes involved in intestinal barrier integrity, with data normalized for comparison. Statistical significance is indicated by '*' for comparisons with the control group and '#' for comparisons with the 5% DSS treatment group, with notations denoting significance levels: *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001.

FIGURE 12

The proposed mechanisms by which baicalein mitigates the effects of DSS-induced damage in C. elegans. Upon DSS treatment, nematodes experience increased ROS and MDA levels, leading to oxidative stress and compromised intestinal permeability. Baicalein intervention appears to counteract these effects by enhancing several physiological parameters, including lifespan, body length, body width, head swings, and body bends, while decreasing lipofuscin accumulation, a marker of aging. In terms of oxidative damage, baicalein treatment enhances SOD activity, leading to reduced ROS levels, which subsequently lowers MDA formation, an indicator of lipid peroxidation. Additionally, baicalein improves intestinal permeability by modulating the expression of gene associated with barrier function. Mechanistically, the insulin/IGF-1 signaling pathway (IIS) and the MAPK pathway are involved in the regulation of baicalin oxidative stress and antioxidant capacity. This multi-pathway approach underscores baicalein’s potential as a therapeutic compound in enhancing health span and reducing oxidative stress under DSS-induced conditions.