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. 2025 Sep 5;10(36):40981–40994. doi: 10.1021/acsomega.5c03232

Naringenin Alleviates the Autophagy-Associated AMPK-Akt/mTOR Pathway to Regulate Inflammation and Barrier Function, Decreasing DSS-Induced Intestinal Fibrosis

Ke-Ying Wang †,‡, Chun-Xiang Huang †,‡, Xiao-Jia Hu §, Jun-Yang Liu †, Xiao-Hui Qin †, Chen-Xi Tong †, Zhi-Qiang Liu †, Wen He †,‡, Tie-Min Jiang ‡,*, Jia-Le Song †,‡,∥,*
PMCID: PMC12444503  PMID: 40978409

Abstract

The development of strictures due to intestinal fibrosis remains a significant clinical challenge in patients with ulcerative colitis (UC). The purpose of this experiment was to investigate the protective effects of naringenin (NAR, 40 mg/kg), a natural flavonoid predominantly present in grapes and oranges, against dextran sodium sulfate (DSS, 2.5%)-induced intestinal fibrosis in UC mice. Oral administration of NAR effectively mitigated clinical symptoms and histological damage in UC mice by reducing the colonic F4/80 and MPO levels. Additionally, NAR lowered the serum concentrations of proinflammatory cytokines and inhibited NLRP3 inflammasome activation in the colon. NAR regulates the Nrf2/Keap1 pathway to combat oxidative damage caused by DSS and enhance autophagy through the AMPK-Akt/mTOR pathway, ultimately decreasing intestinal fibrosis in UC mice by inhibiting α-SMA and Collagen-I. Taken together, our findings demonstrate that NAR can prevent the occurrence and progression of intestinal fibrosis. This effect is achieved by adjusting the AMPK-Akt/mTOR pathway and the promotion of autophagy at the molecular level.

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1. Introduction

Ulcerative colitis (UC) is caused by a conjunction of genetic, infectious, and environmental factors, leading to a severe immune response in the body. Due to clinical symptoms such as blood in the stool, diarrhea, and abdominal pain, people with UC often have a lower quality of life. There is an elevated risk of bowel cancer development in patients with UC. Studies have shown that up to 11% of patients with chronic and progressive UC can develop fibrotic disease. Fibrosis has been identified in the colons of patients with a shorter duration of ulcerative colitis. Fibrosis is a sign of chronic inflammation of mucosal tissues. Tissue damage and loss of function can result from prolonged or excessive inflammatory responses. Inhibition of the fibrotic process has largely been elusive despite significant advances in UC treatment. Early prevention remains the best way to improve colitis-associated intestinal fibrosis.

Autophagy maintains cellular homeostasis by degrading and recycling intracellular components. It is also induced to produce energy in response to starvation, growth factor deficiencies, or high energy demands, thereby supporting metabolic processes. Autophagy removes polyubiquitinated protein aggregates that form during stress and disease, degrades invading pathogens, and is implicated in the secretion of pathogen-induced proinflammatory cytokines, antigen presentation, and lymphocyte development. Autophagy is crucial for maintaining intestinal barrier integrity, antimicrobial defense, and the mucosal immune response. Defects in autophagy can exacerbate the progression of colitis through intestinal dysfunction and the induction of inflammation. Research has identified significantly reduced levels of autophagy in the intestinal epithelial cells of mice with DSS-induced UC, which exacerbates intestinal inflammation in mice. Intestinal submucosal inflammation can compromise the structural integrity of the epithelial barrier. This can lead to changes in intestinal permeability and abnormal immune responses.

Natural compounds and functional dietary ingredients for treating colitis have gained much attention from researchers. , The restriction of intestinal fibrosis could be achieved by flavonoids and other polyphenolic compounds, such as quercetin resveratrol, curcumin, berberine, and other compounds known for their antioxidant and antiinflammatory properties. Naringenin (NAR) is a flavonoid compound commonly found in grapefruit, pummelo and oranges. NAR and resveratrol exhibit strong antioxidant and antiinflammatory effects, yet resveratrol and curcumin have low bioavailability due to issues like poor solubility. The sugar group in naringin, a glycoside form of naringenin, causes steric hindrance, making it less potent than naringenin. Gut microbes in the intestine convert naringin to naringenin, which is then absorbed. Several reports suggest that NAR has a range of pharmacological properties, including antiinflammatory, antioxidant, antiobesity, and antidiabetic effects, as well as the prevention of fibrosis in organs and tissues. Studies have revealed that NAR may be effective at treating colitis symptoms by preserving intestinal barrier integrity and repairing the epithelial barrier structure of the injured intestine, thus leading to therapeutic effects on colitis. , Nonetheless, there is a scarcity of evidence on the effects and mechanisms of NAR in improving colonic fibrosis caused by colitis. The purpose of this research is to examine how NAR plays a role in improving DSS-induced intestinal fibrosis, and the findings will offer fresh perspectives and strategies for the prephylaxis and treatment of intestinal fibrosis.

2. Materials and Methods

2.1. Chemical Reagents

5-Aminosalicylic acid (5-ASA, BD23033, purity ≥99%), naringenin (NAR, BD246816, purity ≥98%), and carboxymethyl cellulose (CMC) were supplied by Bide Pharmatech Ltd. (Shanghai, China). Hematoxylin and eosin (H&E) were obtained from Servicebio Technology Co., Ltd. (Wuhan, China). Dextran sulfate sodium (DSS, molecular weight: 3600–50,000 molecular weight) was acquired from MP Biomedicals (Irvine, CA, USA). The Periodic Acid Schiff (PAS, G1281) Stain Kit and Alcian Blue (AB, G1560) Stain Kit were acquired from Solarbio Science Co. Ltd. (Beijing, China). Masson’s Trichrome Stain Kit (BP-DL022) was obtained from Nanjing SenBeijia Biological Technology Co., Ltd. (Nanjing, China). RIPA lysis buffer (P0013B) and the BCA assay kit (P0009) were bought from Beyotime Biotech. Inc. (Shanghai, China).

2.2. Animal Grouping and Experimental Design

Male C57BL/6 mice (6 weeks old, 18–22 g) were bought from Henan SCBS Biotechnology Co., Ltd. (Henan, China). All mice were individually housed in a standard specific pathogen-free (SPF) environment (12 h light/dark cycle, 25 ± 1 °C) with ad libitum access to food and water. Thirty-five mice were randomly assigned to the normal, DSS, DSS + 5-ASA (50 mg/kg), and DSS + NAR (40 mg/kg) groups after 1 week of acclimatization. In order to induce a mouse model of UC, a 2.5% DSS solution (w/v) was given to the mice through their drinking water for a duration of 7 days. Mice in the DSS + NAR and DSS + 5ASA groups were administered by gavage (dissolved in 1% CMC) on days 0–7, and equal doses of 1% CMC were administered to the normal and DSS groups until day 7. The mice were weighed every 3 days. Upon completion of the study, all mice received an intraperitoneal injection of tribromoethanol (0.2 mL/kg) to induce systemic anesthesia. The colon’s weight and length were then measured, and blood was extracted from the inferior vena cava. The animal protocols used in this study were approved by the Institutional Animal Care and Use Committee of Guilin Medical University (IACUC-GMU, approval number: GLMG-IACUG-20241019).

2.3. Disease Activity Index (DAI)

The DAI was utilized to determine the extent and severity of bowel inflammation. Daily monitoring of body weight (BW), fecal blood levels, and stool consistency were the measures of DAI, as in our previous investigation.

2.4. Histopathological Evaluation

Colon tissue (1–2 cm) was fixed in 4% paraformaldehyde and paraffin-embedded. Histopathological observations were carried out utilizing H&E staining, Alcian Blue (AB) and Periodic Acid-Schiff (PAS) staining (for goblet cells and Mucin2), and Masson’s staining (for collagen deposition) in accordance with the guidelines provided with the corresponding kits. Eventually, the sections were passed through a Leica DM4B microscope (Leica Microsystems Inc., Buffalo Grove, USA) for microscopic imaging to observe pathological changes in the colonic tissue.

2.5. Proinflammatory Mediator and Oxidative Stress Assays

Blood was extracted from the inferior vena cava of each mouse and centrifuged in a DLAB DM0412 centrifuge (900g, 4 °C, 15 min) to prepare serum samples. The serum levels of IL-1β (MM-0040), IL-18 (MM-0169), IL-6 (MM-0163), LPS (MM-0634) and TNF-α (MM-0132) were measured in accordance with the ELISA kits’ manufacturer’s instructions (Jiangsu Meimian Industrial Co. Ltd., China). D-LA (ADS-W-T017) was obtained from Addison Biotechnology Co. Ltd. (Jiangsu, China). The levels of GSH-PX (A005-1-2), SOD (A001-1-2) and MDA (A003-1-2) in the colon were determined by biochemical kits (Nanjing Jiancheng Bioengineering Institute, China) as described in the guidelines.

2.6. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Colon tissues were washed with ice saline and total RNA extracted using RNAeasy animal RNA isolation kit (R0024, Beyotime). The FastQuant reverse transcription system (Solarbio) was used for reverse transcription. The mRNA levels of α-Sma, Muc2, Asc, Collagen-I, Nlrp3, Caspase-1, Il-1β, Sod1, Sod2, Cat and Mgst1 (for primer sequences, see Supporting Information Table S1) were detected by using the SYBR Green Quantitative PCR Kit (Solarbio) on an Applied Biosystems StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The 2–ΔΔCt method was utilized to calculate relative gene expression levels, with β-actin used as a reference value.

2.7. Western Blot Analysis

The cooled PBS washed colon tissue (25 mg) was extracted with RIPA buffer (0.3 mL) using a KZ-III-F high-speed tissue homogenizer, and the mixture was centrifuged (12,000g, 4 °C) for 10 min. The boiling protein samples were separated on sodium dodecyl sulfate polyacrylamide gels and then transferred to PVDF membranes. Following the transfer and sealing of the membrane with skim milk (5%) at 4 °C for 24 h, the appropriate primary antibody (Supporting Information Table S2) was incubated overnight at 4 °C. All membranes were then washed with 1× TBST buffer, followed by incubation with HRP-conjugated goat antirabbit IgG secondary antibody (1:20,000) for 1 h at 4 °C, and finally washed with TBST three times. Enhanced chemiluminescence (ECL) and a FluorChem MSystem (Protein Simple, California, USA) were used to observe the protein bands. Utilizing ImageJ (https://imagej.net/ij), the band density was examined.

2.8. Immunohistochemical (IHC) Analysis

The paraffin sections underwent deparaffinization in xylene and were subsequently rehydrated using varying concentrations of ethanol. The antigenic repair solution should be prepared by boiling it in a pot at 96–98 °C. Next, place the slices in the hot solution for 15 min and allow them to cool naturally to room temperature. Blocking of endogenous peroxidase activity was achieved by incubating the sample with 3%H2O2 for 20 min. The slices were preincubated with blocking buffer (P0260, Beyotime) for 30 min, and then incubated with the corresponding antibodies overnight at 4 °C for 8 h. Subsequently, streptavidin-horseradish peroxidase (SP) was carefully dripped over a 20 min period at room temperature. Diaminobenzidine (DAB) was added to develop the color, restained with hematoxylin, and finally recorded under the Leica DM4B microscope.

2.9. Statistical Analysis

With the assistance of SPSS 25.0 (SPSS, Inc., Chicago, IL, USA), the data were examined, and the mean ± standard deviation was given. The statistical significance of the data was determined through the use of one-way analysis of variance (ANOVA) and Duncan’s multiple range test. In the case of p < 0.05, a statistically significant difference was deemed to be present.

3. Results

3.1. Naringenin Ameliorates Clinical Symptoms in Mice with DSS-Induced Colitis

As observed in Figure B, the BW of colitis mice was significantly lowered by 2.5% DSS commencing on day 5 (p < 0.05). Significant weight loss (9.48%) was observed in the DSS group at the end of the experiment (p < 0.05). The BW of the DSS + 5ASA and DSS + NAR groups increased by 3.66% and 1.40%, respectively, in contrast to the DSS group. The DAI is an indicator that provides an accurate representation of the clinical symptoms of UC. In contrast to the normal group, the DAI score in the DSS group rose from day 3. Nonetheless, the DSS + 5ASA and DSS + NAR groups’ DAI scores increased more slowly than those of the DSS group (p < 0.05; Figure C). The severity of inflammation can be objectively measured by the length of the colon, with more severe cases of colitis exhibiting shorter colon lengths. Mice in the DSS group had colon lengths that were 19.38% shorter than those of normal mice (p < 0.05). Following treatment with 5ASA and NAR, the colon length increased to 6.50 cm (∼1.07 times that of the DSS group) and 7.30 cm (∼1.20 times that of the DSS group). Here, we also observed that 5ASA and NAR resulted in a significantly lower ratio of colon weight to colon length compared to the DSS group (24.79% and 7.18%, respectively).

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Naringenin ameliorates the clinical symptoms in DSS-induced colitis mice. (A) Schematic diagram of the colitis modeling process. (B) Body weight changes in the individual groups, (C) DAI index in the individual groups (n = 8). (D) Representative pictures of colons. (E) Colon length of the mice, (F) Ratio of weight to length of the mouse colon (n = 8). a,bBars with different superscript letters differ significantly (p < 0.05), as determined by Duncan’s multiple range test.

3.2. Naringenin Ameliorates Colonic Inflammatory Responses in DSS-Induced Colitis Mice

As can be seen in Figure A, the colon of the normal mice exhibited a healthy structure according to H&E staining. The DSS-treated colitis mice exhibited disintegration of the colonic tissue structure, breach of the colonic epithelial barrier, considerable diminishment of crypts, and infiltration of cells into the colonic tissue. When the experiment ended, HE scores in the colon of DSS mice were significantly lower after 5ASA and NAR interventions than in DSS mice (p < 0.05; Figure B). In the DSS + 5ASA group and DSS + NAR group, the colonic crypts were regularly aligned, and the ulcers were significantly recovered.

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Naringenin ameliorates colonic inflammatory responses in DSS-induced colitis mice. (A) H&E staining of colon sections in mice at day 14 (ruler 200 μm). (B) Histological scoring of the respective groups on day 14. (C) Expression of F4/80 and MPO proteins in colon by IHC. (D) Positive area of F4/80, (E) Positive area of MPO. (F–I) Serum levels of IL-1β, IL-6, IL-18 and TNF-α level (n = 8). (J) Protein immunoblotting for protein expression of NLRP3, ASC, Caspase-1, IL-1β. (K–N) Protein levels of NLRP3, Caspase-1, IL-1β, and ASC (n = 3). (O-R) mRNA levels of Nlrp3, Caspase-1, Asc, and IL-1β (n = 4). a,bBars with different superscript letters differ significantly (p < 0.05), as determined by Duncan’s multiple range test.

In the colitis model, a significant number of macrophages expressing the F4/80 proteins are present in the intestinal lamina propria. The DSS group had more F4/80 positive areas than the NOR group in both the mucosal and submucosal layers (Figure C). The proportion of F4/80-positive regions was significantly lower after 5ASA intervention than in model mice (32.59%; p < 0.05). F4/80 expression was also reduced by 2.23% in the DSS + NAR group versus the DSS group. But there was no statistical significance (Figure D). Myeloperoxidase (MPO) is one of the key indicators of oxidative stress and colonic inflammation. MPO was significantly greater (39.21%) in the colitis mice than in the normal mice. In contrast, 5ASA and NAR reduced the DSS-induced elevation of MPO expression in the colon of colitis-affected mice by 36.29% and 32.33%, respectively (p < 0.05; Figure E).

In contrast to normal mice, serum levels of TNF-α, IL-1β, IL-18 and IL-6 were elevated in the DSS group by 30.92%, 83.42%, 23.20% and 41.60% respectively (p < 0.05). Following intervention with 5ASA and NAR, the levels of pro-inflammatory factors in the serum of colitis mice were significantly reduced (p < 0.05). The DSS + NAR group exhibited a 25.40%, 36.12%, 15.39%, and 40.17% reduction in the concentrations of TNF-α, IL-1β, IL-18 and IL-6 in the serum respectively, in comparison with the DSS group (Figure F–I).

The expression at protein level of NLRP3, Caspase-1, IL-1β and ASC was significantly upregulated in the colonic tissues of mice in the DSS group; these levels were 1.99, 1.29, 1.43, and 2.64 times greater than those in the healthy mice, respectively (p < 0.05; Figure K–N). Following intervention with 5ASA, the protein levels of NLRP3, caspase-1, IL-1β and ASC were decreased by 85.17%, 33.82%, 59.22% and 13.73%, respectively, and the mRNA expression was decreased by 34.34%, 74.85%, 93.30% and 81.13%, respectively, in the colonic tissue (p < 0.05). We found that the oral administration of NAR resulted in a reduction of NLRP3 inflammasome protein expression by 62.34%, 73.68%, 25.67%, and 53.58%, and mRNA expression by 78.28%, 43.35%, 89.29%, and 92.19%, respectively (p < 0.05; Figure O–R). In conclusion, NAR attenuates DSS-induced intestinal inflammation via a mechanism that may inhibit the activity of the NLRP3 inflammasome (NLPR3, Caspase-1, ASC) and the secretion of IL-1β and IL-18.

3.3. Naringenin Reduced Oxidative Stress in Mice with DSS-Induced Colitis

As is revealed in Figure A–C, MDA levels induced by lipid peroxidation were higher in the colons of mice consuming water containing DSS compared to healthy mice (∼1.95 times, p < 0.05), meanwhile the SOD and GSH-PX levels decreased by 53.80% and 53.09%, respectively. Following intervention with 5ASA or NAR, there was a significant decrease in colonic MDA levels 27.94% and 38.98%, respectively (p < 0.05). Both 5ASA and NAR were able to increase the colonic levels of SOD (by 76.49% and 71.89%, respectively) and GSH-PX (by 84.54% and 42.68%, respectively) more than DSS mice. We also investigated the mRNA levels of Sod1, Mgst1 and Cat in 5ASA- and NAR-treated colitis mice (Figure D–F). Intervention with 5ASA and NAR could promote the expression of colonic Cat, Sod1 and Mgst1 mRNA compared to the DSS group. Cat, Sod1, and Mgst1 mRNA levels in the DSS + NAR group were 3.65, 2.08, and 6.68 times higher than the levels seen in model mice, correspondingly (p < 0.05).

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Naringenin reduced the oxidative stress in DSS-induced colitis mice. (A–C) SOD, GSH-PX and MDA in colon (n = 8). (D–F) mRNA levels of Sod1, Mgst1 and Cat (n = 4). (G) Protein immunoblotting assay for Nrf2, p-Nrf2, and Keap1. (H–I) Protein expression levels of Keap1 and p-Nrf2 (n = 3). The relative mRNA levels of each gene were normalized to the mRNA level of the control group using β-actin as a control. a–eBars with different superscript letters differ significantly (p < 0.05), as determined by Duncan’s multiple range test.

The development and course of colitis are strongly correlated with disruption of the Nrf2/Keap1 pathway. Stimulating the Nrf2/Keap1 pathway can augment the antioxidant response. In this study, we found elevated levels of Keap1 and decreased levels of p-Nrf2 the colons of colitis-affected mice. NAR exhibited markedly greater activity in reducing the protein levels of Keap1 than did 5ASA in colitis mice. NAR can alleviate oxidative stress by reducing Keap1 expression and activating Nrf2 phosphorylation. 5ASA significantly induced the phosphorylation of Nrf2, and this effect was also demonstrated in the DSS + NAR group, in which the p-Nrf2 level was increased to 1.19 times that of the model mice (p < 0.05).

3.4. Naringenin Treatment Maintained Intestinal Chemical Barrier and Reduced Mucosal Damage in Mice with DSS-Induced Colitis

The colonic chemical barrier can prevent direct contact between intestinal epithelial cells and the vast number of microbes in the intestinal cavity, thus preserving the health of the intestinal epithelium. The disappearance of the intestinal chemical barrier is a major indication of colitis. An important morphological foundation for diagnosing changes in intestinal functions and structure is the study of variations in goblet cell numbers and mucopolysaccharide concentrations. After treatment with 5-ASA and NAR, the colitis mice showed an increase in MUC2 production and goblet cell numbers (Figure A,B). When the colons of NAR-treated mice were compared with those of DSS-treated animals, there was a 1.61 times elevation in the MUC2-positive area and a 3.26 times elevation in Muc2 mRNA levels (p < 0.05; Figure E,F).

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Naringenin treatment maintained the intestinal chemical barrier structure and reduced the mucosal damage in DSS-induced colitis mice. (A) Distribution of colonic goblet cells stained with AB staining. (B) PAS staining (ruler 200 μm). (C) IHC observation of the colonic MUC2 in mice (ruler 200 μm). (D) Numbers of goblet cells. (E) Positive levels of MUC2, and (F) mRNA levels of Muc2 (n = 4). a–eBars with different superscript letters differ significantly (p < 0.05), as determined by Duncan’s multiple range test.

3.5. Naringenin Enhanced the Intestinal Mechanical Barrier of Mice with DSS-Induced Colitis

The integrity of the gut mechanical barrier was indirectly reflected by the serum D-LA and LPS levels. Exposure to DSS resulted in highly significant increases (2.59-fold and 1.07-fold, respectively; p < 0.05) in the serum concentrations of D-LA and LPS in colitis mice, indicating that intestinal mucosal barrier function was disrupted. We discovered that 5ASA intervention reduced serum D-LA and LPS concentrations in colitis mice by 68.96% and 9.04%, respectively, while NAR reduced serum D-LA and LPS concentrations by 45.09% and 12.42% (p < 0.05; Figure A,B). Accordingly, we speculated that NAR may be able to attenuate the increase in intestinal permeability and promote the integrity of the intestinal barrier structure.

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Naringenin enhanced the intestinal mechanical barrier of mice with DSS-induced colitis. (A) Serum levels of LPS, (B) Serum levels of D-LA (n = 8). (C) IHC assay to detect the expression of ZO-1, Occludin, and Claudin-1 proteins in the colon (ruler 200 μm). (D) Positive levels of ZO-1, (E) Positive levels of Claudin-1, (F) Positive levels of Occludin. (G) WB detection of ZO-1, Occludin, Claudin-1, Claudin-7 expression in the colon. (H) Protein expression of ZO-1, (I) Occludin, (J) Claudin-1, (K) Claudin-7 (n = 3). (L) mRNA expression of ZO-1, (M) mRNA expression of Occludin, (N) mRNA expression of Claudin-1, and (O) mRNA expression of Claudin-7 (n = 4). The relative mRNA levels of each gene were normalized to the control mRNA level using β-actin as a control. The values marked with different letters (a,b) were significantly different at p < 0.05.

IHC analysis also revealed that the expression of the colonic tight junction (TJ) proteins located between epithelial cells was suppressed in the DSS group, and the absence of TJ proteins was reversed by administration of 5ASA and NAR (Figure C). Consistent with the histochemical results, DSS significantly reduced the concentrations of colonic TJ proteins (p < 0.05). The DSS-induced deficiency in TJ protein expression described above was ameliorated by NAR treatment (p < 0.05; Figure D–F). Additionally, we also observed that the colonic protein expression concentrations of ZO-1, Claudin-1, Occludin and Claudin-7 in the DSS + NAR group were 1.81, 1.36, 4.64, and 3.10 times higher than those in the DSS group (p < 0.05; Figure G–K). Moreover, the mRNA levels of Zo-1, Occludin, Claudin-1, and Claudin-7 in the DSS + NAR group of mice were increased by 5.19, 3.15, 11.17, and 4.86-fold, respectively, when compared with the DSS mice (Figure L–O).

3.6. Naringenin Ameliorates Colonic Fibrosis in Mice with DSS-Induced Colitis

The fiber concentration of fibers in the lamina propria of the colon and fibers in the muscularis mucosae of the colon was more pronounced in DSS-induced colitis mice, as shown by the Masson’s staining method (Figure A). In mice with colitis, administration of 5ASA and NAR reduced fiber aggregation in the colon. Activated fibroblasts produce α-SMA and Collagen-I, which in turn lead to the development of fibrosis in the tissue. Immunohistochemical staining revealed that the colitis mice had higher concentrations of Collagen-I (18.42%) and α-SMA (27.37%) than the normal mice (Figure B). Furthermore, in 5ASA-treated mice compared to mice with DSS-induced colitis, there was a significant decrease of 26.2% and 20.6% in the proportions of α-SMA- and Collagen-I-positive cells, respectively. NAR-treated mice exhibited a decrease of 31.22% in the expression levels of α-SMA, as well as a reduction of 44.96% in the levels of Collagen-I. In this study, we also examined the protein and mRNA levels of genes involved in fibrosis in the colons of mice with colitis. Relative to normal mice, the colons of DSS-induced colitis mice exhibited increases in the levels of α-SMA protein (∼1.47 times) and Collagen-I (∼1.31 times), as well as increased mRNA levels up to 1.41 and 1.55 times, respectively. Nevertheless, the protein levels of α-SMA in the 5ASA- and NAR-treated DSS-colitis mice were significantly downregulated by 18.70% and 51.70%, respectively (p < 0.05), and the protein levels of Collagen-I were also downregulated by 36.96% and 52.32%, respectively (p < 0.05). In addition, qRT-PCR results showed that 5ASA and NAR treatments significantly suppressed α-SMA and Collagen-I mRNA levels in the colon of DSS mice. Our research demonstrated that NAR effectively suppressed the synthesis of α-SMA and collagen-I in the colons of colitis-affected mice.

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Naringenin ameliorates the colonic fibrosis in the DSS-induced colitis mice. (A) Masson’s staining assay (ruler 200 μm). (B) Immunohistochemistry assay for α-SMA and Collagen-I in the colon tissue of colitis mice (ruler 200 μm). (C,D) positive levels of α-SMA and Collagen-I. (E,F) mRNA levels of α-SMA and Collagen-I (n = 4). (G–I) α-SMA and Collagen-I protein expression in the colon (n = 3). The relative mRNA levels of each gene were normalized to the mRNA level of the control group using β-actin as a control. a–eBars with different superscript letters differ significantly (p < 0.05), as determined by Duncan’s multiple range test.

3.7. Naringenin Modulated the AMPK-Akt/mTOR Pathway to Enhance Autophagy in Mice with Colitis Induced by DSS

In colitis model mice, LC3-II/I and Beclin-1 protein levels were decreased by 21.31% and 27.16%, respectively, and p62 expression was elevated by 67.56% in colon tissue. NAR resulted in elevated protein levels of Beclin-1 (1.66 times) and LC3II/LC3I (2.79 times) and a reduction in the protein level of p62 (13.79%) in the colon of colitis mice (p < 0.05; Figure B–D). In Figure E–G, we also discovered that, compared with DSS, DSS induced a 73.75% reduction in colonic p-AMPK protein expression in colitis mice and a 2.22-times and 1.67-times elevation in p-mTOR and p-Akt protein expression, respectively. In the DSS + 5ASA group, the colonic levels of p-AMPK were 1.87 times more than in the DSS group. Additionally, the levels of p-Akt and p-mTOR were reduced by 33.06% and 83.96%, respectively (p < 0.05). The level of the p-AMPK protein in the colon was elevated 1.48-fold in NAR-treated mice (p < 0.05). In the DSS + NAR group, colonic p-Akt protein expression was markedly lowered by 63.29% and p-mTOR level was lowered by 82.57% versus the DSS group (p < 0.05).

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Naringenin modulated the AMPK-Akt/mTOR pathway to promote autophagy in DSS-induced colitis mice. (A) Protein expressions of LC3, Beclin-1, p62, AMPK, p-AMPK, Akt, p-Akt, mTOR, p-mTOR by protein immunoblotting (n = 3). (B) Protein expression of Beclin-1. (C) Protein expression of LC3II/I. (D) Protein expression of p62. (E) Protein expression of p-AMPK. (F) Protein expression of p-Akt (n = 3). (G) Protein expression of p-mTOR. The values marked with different letters (a,b) were significantly different at p < 0.05.

4. Discussion

Due to long-term chronic inflammation of the bowel, patients with UC develop intestinal fibrosis. The development of intestinal fibrosis can delay wound healing and interfere with normal tissue remodeling. Major concerns for patients are the development of organ fibrosis and the significant financial burden associated with its treatment. NAR is a citrus flavonoid with proven biological activity that reduces the incidence of fibrosis in various organs. The study aimed to investigate whether NAR intervention could improve the development of colonic fibrosis in a DSS-induced colitis mouse.

Patients with UC typically experience recurrent episodes of diarrhea, mucopurulent bloody stools, and abdominal pain, which significantly affects their quality of life. The colitis mice exhibited persistent BW loss, diarrhea, rectal bleeding, and increased DAI scores, which were significantly reduced by the administration of NAR. Inflammatory mediators and proinflammatory cytokines secreted by macrophages and neutrophils can cause intestinal damage. The examination revealed that the colonic tissues of colitis mice exhibited mucosal defects and disorganized, atrophied, or even lost glandular arrangement. F4/80 is a primary marker of mature macrophages. Increased expression of F4/80 and MPO and elevated concentrations of serum pro-inflammatory factors were found in the intestinal tissues of colitis mice. Histological damage to the colon is associated with increased MPO activity and the secretion of proinflammatory cytokines. Preventing the DSS-induced increase in MPO levels blocks the resulting increase in neutrophil aggregation in the submucosal layer. Administration of NAR restored intestinal tissue structure damage in colitis model mice, inhibited the expression of F4/80 and MPO in intestinal tissue, and significantly reduced the serum levels of inflammatory cytokines. This is consistent with what Cao et al. found.

Upon exposure to internal and external stimuli, such as LPS on the bacterial surface or ATP released when cells are damaged, NLRP3 aggregates with ASC and pro-caspase-1 to form the NLRP3 inflammasome. Activated NLRP3, which cleaves pro-caspase-1 to active caspase-1 and facilitates the release of IL-1β and IL-18, accelerates the course of DSS-induced colitis. In this research, NAR inhibited the binding of NLRP3, ASC and caspase-1, hence reducing DSS-induced high levels of IL-18, TNF-α, IL-1β and IL-6. The ability of sinapic acid and phloretin to suppress the NLRP3 inflammasome in DSS-treated mice has been suggested in several studies. ,, Naringin, a flavanone glycoside composed of naringenin, could alleviates UC by suppressing the NLRP3 inflammasome through PPARγ activation, which in turn reduces subsequent NF-κB activation. Given that naringenin has a low molecular weight and lacks the sugar group that can impede absorption, it is absorbed more rapidly and efficiently in the gastrointestinal tract, thereby enabling it to exert its therapeutic effects more effectively.

Intestinal oxidative stress is closely correlated with the development of UC. Chronic inflammation in the gut can lead to an excessive production of ROS/RNS, resulting in oxidative stress injury. This process directly contributes to the excessive increase in MDA in intestinal tissue. Enzymatic CAT, SOD and GSH-Px, as well as nonenzymatic GSH effectively scavenge free radicals and ROS, protecting cells from lipid peroxidation and damage induced by oxidative stress. As previously reported by our group, sinapic acid can significantly increase the expression of Mgst1, Sod1, Sod2 and Cat in the colon of UC mice to alleviate oxidative stress through regulation of the Nrf2/Keap1 pathway. Nrf2 is present in the cytoplasm in a state bound to Keap1. Under oxidative stress, the Nrf2/Keap1 protein dissociates, and p-Nrf2 binds to the ARE and transcribes antioxidant genes such as Sod1, Mgst1 and Cat, which increase the expression of antioxidant proteases to antagonize oxidative stress-induced damage. Puerarin (a flavonoid structurally similar to naringenin) and sinapic acid have been found to exert antioxidant effects by regulating the Nrf2-related pathway and promoting the expression of antioxidant enzyme. , Here, our findings suggest that NAR upregulates the mRNA expression of antioxidant factors (Sod1, Mgst1, and Cat), leading to increased expression of p-Nrf2, downregulated expression of Keap1, and elevated levels of SOD and GSH. This resulted in reduced MDA levels and inhibited oxidative damage in the colons of UC mice.

One of the primary methods for treating UC is to reconstruct the intestinal barrier. Intestinal goblet cells secrete mucins, which are the primary components of the chemical barrier in the colon and play a critical role in protecting intestinal cells from certain pathogens, including microbes. DSS therapy damages the colon epithelium in mice, causing inflammatory cells to infiltrate, goblet cells to diminish, and mucin secretion to decrease. We also noticed that NAR enhanced the number of goblet cells and promoted the levels of Muc2 compared with those in the colon of mice with colitis. The tight junction (TJ) proteins form a mechanical barrier that blocks antigens and microorganisms from entering epithelial cells and causing damage. , Our research demonstrated that NAR substantially increases the synthesis of TJ proteins in the colon of colitis-ridden mice, contributing to the rebuilding of the intestinal mucosal barrier. It has been observed that dihydroquercetin supplementation can improve intestinal damage in colitis mice, as evidenced by the increased levels of ZO-1 and Occludin in the colon, similar to the results of our study. NAR plays a pivotal role in mitigating DSS-induced damage to the colonic mucosa by a mechanism that involves a rise in TJ protein levels in epithelial cells through an upregulation of mucin synthesis.

Overgrowth of myofibroblasts is responsible for the influx of collagen into tissues and organs, thus resulting in fibrosis. Patients with chronic IBD may experience intestinal fibrosis, which can eventually lead to intestinal sclerosis. Overactivated neutrophils can release profibrotic cytokines, chemokines, and ROS to induce the activation of myofibroblasts and result in excessive wound healing and intestinal fibrosis during the repair of colon epithelial injury. Elevated levels of α-SMA (a biomarker of activated myofibroblasts), collagen-I and collagen-II, and collagen-III, as well as fibronectin and other profibrotic factors (IL-13 and TGF-β1) were observed in the colitis model induced by DSS. In this study, we surveyed that NAR diminished the intestinal accumulation of fibroblasts and the levels of α-SMA and Collagen-I in DSS-induced colitis mice. Furthermore, other studies have indicated that curcumin administration can suppress the levels of Collagen-I and α-SMA in the intestinal tract and attenuate intestinal fibrosis in colitis rats. By virtue of its antioxidant and antiinflammatory properties, troxerutin impedes apoptosis in colonic tissue and mitigates colonic fibrosis. The present findings demonstrate that NAR can decrease the expression of fibrosis-related proteins such as Collagen-I and α-SMA in colon tissue, potentially facilitating the amelioration of intestinal fibrosis in mice with colitis.

Cellular autophagy is indispensable for regulating intestinal ecology, maintaining normal intestinal immunity and resisting bacterial infection. Excessive amounts of pro-inflammatory cytokines can result from aberrant activation of the NLRP3 inflammasome caused by autophagic dysfunction, which can then produce experimental colitis in mice. An LPS-induced mouse colitis model has demonstrated that restricting mTOR activity can improve intestinal inflammation by promoting autophagy and reducing intestinal oxidative stress damage (increasing T-AOC and SOD and decreasing MDA production). Rizzo et al. indicated that baicalin could upregulate the mRNA expression of Lc3, Atg and Beclin1 and promote Claudin-1 expression to reduce permeability in the LPS-treated human colonic epithelial cell line HT-29. Kang et al. also reported a new 3′,4′,5′-trihydroxyflavone that increases LC3 expression and reduces p62 activity in intestinal epithelial cells (HT-29 and HCT-116 cells). This compound also attenuated DSS-induced inflammation-associated intestinal damage by inhibiting the activation of p62 and p-mTOR and raising the ratio of LC3B-II to LC3B-I to promote autophagy in mice with colitis. In mice with colitis, NAR was discovered to enhance autophagy by stimulating the activation of Beclin-1 and LC3-II and reducing p62 protein levels.

Autophagy is a regulator of myofibroblast differentiation, tissue remodeling, and fiber formation. Cellular and animal experiments revealed that promoting autophagy has an antifibrotic effect on intestinal fibrosis and enhances collagen degradation in fibroblasts. In contrast, the inhibition of autophagy exacerbates fibrosis. The AMPK-related pathway is involved in regulating autophagy as well as cellular energy metabolism. Under glucose starvation, AMPK phosphorylates mammalian autophagy-initiating kinase (ULK1) at Ser317 and Ser77 while inhibiting mTOR to promote autophagy. mTOR inhibitors such as rapamycin were shown to inhibit the expression of intestinal inflammation-related factors in TNBS-induced fibrotic mice, thereby attenuating intestinal fibrosis. Studies have shown that promoting phosphate AKT (p-Akt), an effector of PI3K, and p-mTOR inhibits autophagy and exacerbates the severity of pulmonary fibrosis. Procyanidin A1 inhibits AMPK and its downstream signaling pathways, mTOR and p70S6K. This promotes autophagy in intestinal epithelial cells and has a protective effect on the colon. Furthermore, dihydromyricetin prevents the development of intestinal fibrosis in mice by regulating the PI3K/Akt/mTOR pathway to trigger autophagy and activating autophagy to inhibit TGF-β-induced activation of colon fibroblasts in vitro. Importantly, we observed that NAR enhances autophagy in intestinal epithelial cells by boosting p-AMPK and reducing p-Akt and p-mTOR levels. This inhibits colon fibroblast activation and lowers the expression of fibrosis markers like α-SMA and Collagen-I. In rats with liver fibrosis, naringenin suppressed the increase in hepatic TGF-β, α-SMA, CTGF, Collagen-I, and MMP-13 protein expression induced by CCl4. These results imply that naringenin might attenuate the high levels of fibrosis-related proteins expression in the DSS-induced colon by modulating the AMPK-Akt/mTOR pathway to promote autophagy, thereby alleviating intestinal fibrosis.

In conclusion, the present results indicate that NAR supplementation suppresses DSS-induced NLRP3 inflammasome activation and therefore mitigates the seriousness of colitis. Furthermore, NAR supplementation mitigates oxidative stress via the Nrf2-Keap1 pathway, facilitating the restoration of impaired intestinal epithelial barrier function. There is evidence suggesting that NAR may exert an antifibrotic effect by promoting autophagy via regulation of the AMPK-Akt/mTOR pathway. Dietary intake of naringenin-rich fruits like grapefruit and oranges provides a natural strategy for managing colitis and intestinal fibrosis. In this study, a naringenin dose of 40 mg/kg (307.3 mg for a 70 kg adult) was targeted, requiring approximately 646.5 mL of combined grapefruit and orange juice (each contributing half of the naringenin) to meet the daily intake. Naringin is readily available as a dietary supplement, but the superior colonic bioavailability of naringenin compared to naringin highlights its enhanced therapeutic potential for mitigating DSS-induced intestinal fibrosis. Our studies have focused on animal models, highlighting the need for future clinical trials to assess the efficacy and safety of naringenin-rich fruits like grapefruit and oranges as dietary supplements for inflammatory bowel disease patients. Future studies will be instrumental in elucidating the role of naringenin in the prevention and treatment of intestinal fibrosis, while also assessing the systemic implications critical to its therapeutic potential.

Supplementary Material

ao5c03232_si_001.pdf (107.7KB, pdf)

Acknowledgments

We thank AJE (http://www.aje.cn) for its linguistic assistance during the preparation of this manuscript. We thank Figdraw (https://figdraw.com/) for providing the graphical materials used in this work.

The data is available throughout the manuscript and supporting files.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c03232.

  • Contains author information, and the tables of primers and primary antibodies used in this study (Supplementary Tables S1 and S2) (PDF)

#.

Ke-Ying Wang, Chun-Xiang Huang and Xiao-Jia Hu contributed equally to this work. The investigation was performed by Ke-Ying Wang (K.-Y. W.), Chun-Xiang Huang (C.-X. H.), Jun-Yang Liu (J.-Y. L.), Xiao-Hui Qin (X.-H. Q.), Chen-Xi Tong (C.-X. T.), and Zhi-Qiang Liu (Z.-Q. L.). The experimental data were analyzed by Xiao-Jia Hu (X.-J. H.) and J.-Y. L. All graphs were drawn by C. X. T., X.-H. Q. and Z.-Q. L. The manuscript was drafted by K.-Y. W. and X.-J. H. Wen He (W. H.) and Tie-Min Jiang (T.-M. J.) supervised the manuscript. Jia-Le Song (J.-L. S.) designed the entire investigation and reviewed the final manuscript. K.-Y. W. and C.-X. H. collaborated on this investigation with equal participation.

The present investigation was supported by the Special Funds for Guiding Local Scientific and Technological Development by the Central Government (Grant No. Guike ZY22096025 to J.-L. S. and T.-M. J.), National Natural Science Foundation of China (Grant Nos. 81560530, 81760589, 81960590, and 82273630 to J.-L. S.), the Science and Technology Projects of Guilin (Grant Nos. 20220139-8-2, 20230127-2 and 20230135-4-1 to J.-L. S.), the Bagui Outstanding Younger Scholar for Guangxi (Grant No. 2023 to J.-L. S.), the Funding Scheme for High-Level Overseas Chinese Students’ Return of Ministry of Human Resources and Social Security (Grant No. Renshetinghan[2019]­160 to J.-L. S.), China.

The authors declare no competing financial interest.

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