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. 2021 Aug 24:12:729414.
doi: 10.3389/fphar.2021.729414. eCollection 2021.

V体育2025版 - Naringin Exerts Therapeutic Effects on Mice Colitis: A Study Based on Transcriptomics Combined With Functional Experiments

Jianyi Dong  1 , Yuanyuan Chen  1 , Fang Yang  1 , Weidong Zhang  1 , Kun Wei  1 , Yongjian Xiong  2 , Liang Wang  1 , Zijuan Zhou  1 , Changyi Li  1 , Jingyu Wang  3 , Dapeng Chen  1
Affiliations

Naringin Exerts Therapeutic Effects on Mice Colitis: A Study Based on Transcriptomics Combined With Functional Experiments

Jianyi Dong et al. Front Pharmacol. .

Abstract

Naringin has been shown to exert protective effects in an animal model of ulcerative colitis, but detailed mechanisms remain unclear. This study aimed to investigate function and signaling mechanisms underlying naringin-induced therapeutic effects on colitis. Two mouse models were established to mimic human Inflammatory bowel disease (IBD) by treating drinking water with dextran sodium sulphate or intra-colonic administration of 2, 4, 6-trinitrobenzene sulfonic acid. Transcriptomics combined with functional experiments were used to investigate underlying mechanisms. Colitis symptoms, including weight loss and high disease activity index were significantly reversed by naringin. The inflammatory response, oxidative reactions, and epithelial cell apoptosis that occur with colitis were also alleviated by naringin. After naringin treatment, transcriptomics results identified 753 differentially expressed mRNAs that were enriched in signaling pathways, including the neuroactive ligand-receptor interaction, calcium signaling, and peroxisome proliferator-activated receptor (PPAR) signaling VSports手机版. The naringin-induced alleviation of colitis was significantly inhibited by the PPAR-γ inhibitor BADGE. In IEC-6 and RAW264. 7 cells incubated with lipopolysaccharide (LPS), NF-κB-p65, a downstream protein of PPAR-γ, was significantly increased. Naringin suppressed LPS-induced high expression of NF-κB-p65, which was inhibited by small interfering RNA targeting PPAR-γ. Our study clarifies detailed mechanisms underlying naringin-induced therapeutic effects on mice colitis, and PPAR-γ was found to be the main target of naringin by functional experiments both in vivo and in vitro. Our study supplies new scientific information for the use of naringin in colitis treatment. .

Keywords: NF-κB; RNA sequencing; inflammatory bowel disease; naringin; peroxisome proliferator-activated receptor-γ. V体育安卓版.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Toxicity of naringin in normal cells and mice. (A) Chemical structure of naringin. Naringin was administered to mice by gavage once a day for seven consecutive days. Effects of naringin on (B) body weight and (C) food intake. Expression levels of the colonic cytokines (D) TNF-α, (E) IL-1β, and (F) INF-γ were examined by ELISA, and expression levels of (G) PPAR-γ were examined by western blotting. (H) Cytotoxicity of naringin was studied by CCK8 assay in RAW 264.7 and IEC-6 cells. Data are expressed as the mean ± SD. Values in the normal control (NC) group are set to 100%, and other values are given relative to the NC group. *p <0.05 compared with NC group. n = 3 samples in western blotting experiments; n = 6 samples for other experiments. These blots are cropped, and the full-length blots are presented is in the Supplementary Figure S1.
FIGURE 2
FIGURE 2
Naringin ameliorates DSS-colitis symptoms. Colitis symptoms were recorded after 7 days of naringin treatment. Effects of naringin on (A) body weight, (B) food intake, (C) DAI, (D) colon weight-to-length ratio, and (E) gross morphology of colon tissue. (F) Total histological score was calculated as the sum of epithelial damage and histological score. (G) H and E staining of mice colonic tissue. Scale bars, 200, 100, 50 μm. Green ring indicates damaged villi structure; black arrow indicates infiltration of inflammatory cells; blue ring indicates submucosal edema. Data are expressed as mean ± SD. Values in the sham group are set to 100%, and other values are given relative to the sham group. **p < 0.01 compared with sham group; ## p < 0.01 compared with DSS-colitis group; n = 6 samples.
FIGURE 3
FIGURE 3
Effects of naringin on inflammation and apoptosis in DSS-colitis. Expression levels of the colonic cytokines (A) MPO, (B) TNF-α, (C) IL-1β, and (D) IFN-γ were determined by ELISA. Western blotting analysis of inflammation-related proteins (E) iNOS, (F) p-NF-κB-p65 (calculated as p-NF-κB-p65/NF-κB-p65); apoptosis-related protein (G) cl-caspase3, calculated as cl-caspase3/caspase3. Data are expressed as mean ± SD. Values in the sham group are set to 100% and other values are given relative to those in the sham group, *p < 0.05, **p < 0.01 compared with sham group; # p < 0.05, ## p < 0.01 compared with DSS-colitis group; n = 3 samples in western blotting experiments; n = 6 samples for other experiments. These blots are cropped, and the full-length blots are presented is in the Supplementary Figures S2–S9.
FIGURE 4
FIGURE 4
High-throughput transcriptome sequencing and KEGG pathway enrichment analysis. Naringin (40 mg/kg) was administered to mice by gavage once a day for seven consecutive days and total RNA was extracted from colonic epithelial cells. (A) Volcano plot of differentially expressed mRNAs in model group vs. naringin treatment group. (B) Heatmap of some representative genes. (C) The scatter plots showing the top 20 enriched KEGG pathways in model group vs. naringin group. The Rich factor is the ratio of differentially expressed gene numbers annotated in this pathway term to all gene number annotated in this pathway term. Greater rich factor means greater intensiveness. Q-value is corrected p value ranging from 0 to 1. (D) Target-pathway network including candidate 133 targets and 11 KEGG pathways.
FIGURE 5
FIGURE 5
High-throughput transcriptome sequencing and KEGG pathway enrichment analysis. Naringin (40 mg/kg) was administered to mice by gavage once a day for seven consecutive days and total RNA was extracted from colonic epithelial cells. (A) Volcano plot of differentially expressed mRNAs in model group vs. control group. (B) The scatter plots showing the top 20 enriched KEGG pathways in model group vs. control group. The Rich factor is the ratio of differentially expressed gene numbers annotated in this pathway term to all gene number annotated in this pathway term. Greater rich factor means greater intensiveness. Q-value is corrected p value ranging from 0 to 1.
FIGURE 6
FIGURE 6
The effect of naringin on some mRNAs in PPAR signaling pathways detected by qRT-PCR. Naringin (40 mg/kg) was administered to mice by gavage once a day for seven consecutive days. The expression levels of some mRNAs in PPAR signaling pathways including (A) PPAR-α, (B) PPAR-γ, (C) PPAR-δ, (D) Scd1, and (E) Hmgcs2 were detected by qRT-PCR (means ± SD, n = 3). **p < 0.01 compared with sham group; # p < 0.05, ## p < 0.01 compared with DSS-colitis group.
FIGURE 7
FIGURE 7
Effects of naringin on PPAR-γ expression in DSS-colitis. Naringin was administered to mice by gavage once a day for seven consecutive days. (A) Western blotting analysis of PPAR-α. (B) Western blotting analysis of PPAR-γ. (C) IF staining of PPAR-γ in colonic epithelium. Scale bars, 100 μm. (D) IHC staining of PPAR-γ in colonic epithelium. Scale bars, 200, 100, and 50 μm. Data are expressed as mean ± SD. Values in the sham group are set to 100% and other values are given relative to those in the sham group. **p < 0.01 compared with sham group; # p < 0.05, ## p < 0.01 compared with DSS-colitis group; n = 3 samples in western blotting experiments; n = 6 samples for other experiments. These blots are cropped, and the full-length blots are presented is in the Supplementary Figures S10–S13.
FIGURE 8
FIGURE 8
Effects of PPAR-γ inhibitor BAGDE (30 mg/kg) on naringin induced treatment. (A) PPAR-γ expression in the presence of PPAR-γ inhibitor BAGDE. Effects of naringin (40 mg/kg) on (B) weight loss, (C) DAI, (D) colon weight-to-length ratio and (E) gross morphology of colon tissue. (F) Total histological score was calculated as the sum of epithelial damage and histological score. (G) H and E staining analysis of the aggravated symptoms with BADGE. Scale bars, 200, 100, and 50 μm. Green ring indicates damaged villi structure; black arrow indicates infiltration of inflammatory cells; blue ring indicates submucosal edema, blue arrow indicates muscle fiber separation. Data are expressed as mean ± SD. Values in the sham group are set to 100% and other values are given relative to those in the sham group. **p < 0.01 compared with sham group; ## p < 0.01 compared with DSS-colitis group; n = 3 samples in western blotting experiments; n = 6 samples for other experiments. These blots are cropped, and the full-length blots are presented is in the Supplementary Figures S14–S15.
FIGURE 9
FIGURE 9
The anti-inflammatory effect of naringin (20 μM) was eliminated by siRNA targeting PPAR-γ. Expression levels of the colonic cytokines (A) TNF-α, (B) IL-1β, (C) IFN-γ in DSS-induced RAW264.7 cells were determined by ELISA. Expression of PPAR-γ and p-NF-κB-p65 in the absence and presence of siRNA targeting PPAR-γ in (D–F) RAW 264.7 and (G–I) IEC-6 cells. Data are expressed as mean ± SD. Values in corresponding sham or normal control (NC) group are set to 100% and other values are given relative to the control values. **p < 0.01 compared with sham/NC group; ## p < 0.01 compared with DSS/LPS control group or as indicated; n = 3 samples in western blotting experiments; n = 6 samples for other experiments. These blots are cropped, and the full-length blots are presented is in the Supplementary Figures S16–S21.
FIGURE 10
FIGURE 10
Effects of naringin on TNBS-colitis. Naringin was administered to mice by gavage once a day for seven consecutive days. Effects of naringin on (A) body weight, (B) food intake, (C) DAI, (D) colon weight-to-length ratio, and (E) gross morphology of colon tissue. (F) Total histological score was calculated as the sum of epithelial damage and histological score. (G) H and E staining of mice colonic tissue. Scale bars, 200, 100, and 50 μm. Green ring indicates damaged villi structure; black arrow indicates infiltration of inflammatory cells. Expression levels of the colonic cytokines (H) MPO, (I) TNF-α, (J) IL-1β, and (K) IFN-γ were determined by ELISA. Data are expressed as mean ± SD. Values in the sham group are set to 100% and other values are given relative to those in the sham group. **p < 0.01 compared with sham group; # p < 0.05, ## p < 0.01 compared with TNBS-colitis group; n = 6 samples.
FIGURE 11
FIGURE 11
Effects of naringin on PPAR-γ expression, inflammation and apoptosis in TNBS-colitis mouse model. (A) Western blotting analysis of PPAR-γ. (B) IF staining of PPAR-γ in colonic epithelium. Scale bars, 100 μm. (C) IHC staining of PPAR-γ in colonic epithelium. Scale bars, 200, 100, and 50 μm. Western blotting analysis of inflammation-related proteins (D) p-NF-κB-p65 (calculated as p-NF-κB-p65/ NF-κB-p65), (E) iNOS; apoptosis-related protein (F) cl-caspase3, calculated as cl-caspase3/caspase3. Data are expressed as mean ± SD. Values in the sham group are set to 100% and other values are given relative to those in the sham group. **p < 0.01 compared with sham group; # p < 0.05, ## p < 0.01 compared with TNBS-colitis group; n = 3 samples in western blotting experiments; n = 6 samples for other experiments. These blots are cropped, and the full-length blots are presented is in the Supplementary Figures S22–S31.
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