Anti-Inflammatory Effects of Chrysanthemum coronarium L. on Dextran Sodium Sulfate-Induced Intestinal Inflammation in a Drosophila Model

Abstract

Intestinal inflammation is closely linked to aging, metabolic disorders, and immune dysregulation. Maintaining epithelial homeostasis and regulating immune responses in the gut are critical for systemic health. Natural bioactive compounds are currently garnering attention as potential agents for controlling intestinal inflammation. Among them, Chrysanthemum coronarium has emerged as a promising candidate owing to its antioxidant and anti-inflammatory properties. In this study, we investigated the protective effects of C. coronarium on intestinal homeostasis under inflammatory conditions using a Drosophila model. Intestinal inflammation was induced by feeding Drosophila dextran sodium sulfate (DSS), and C. coronarium’s efficacy was assessed across parallel treatment groups. We quantitatively analyzed stem cell proliferation, gut length, intestinal barrier integrity (using the Smurf assay), Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway activation, and cell death. DSS treatment resulted in increased intestinal stem cell (ISC) proliferation, shortened gut length, impaired barrier function, and elevated STAT signaling, all of which were significantly mitigated by cotreatment with C. coronarium. Notably, C. coronarium also restored the compromised gut barrier in DSS-treated Drosophila and suppressed STAT activation, indicating modulation of inflammatory signaling. These findings show that C. coronarium supports intestinal tissue homeostasis by suppressing DSS-induced ISC hyperproliferation, restoring barrier integrity, inhibiting STAT pathway activation, reducing cell death, and improving lifespan under inflammatory conditions. Our results offer experimental evidence supporting C. coronarium as a promising functional food ingredient for preventing inflammatory bowel disease and management of metabolic disorders.

INTRODUCTION

Gut health is closely linked to systemic health, with intestinal inflammation playing a key role in the development of various conditions, including aging, metabolic disorders, and immune dysfunction. Intestinal epithelial homeostasis and immune responses are precisely regulated through complex interactions with gut microbiota. Disrupting this balance can result in chronic inflammatory bowel diseases (IBDs) (Zhao et al., 2023). Thus, identifying and developing natural compounds that can regulate inflammatory responses have become central to advancing functional foods and preventive therapies.

Drosophila has long served as a model organism in genetics and physiology research, and its use has now expanded to intestinal disease studies (Piper and Partridge, 2018; Mirzoyan et al., 2019; Funk et al., 2020). Although structurally simple, the Drosophila gut shares key cellular and functional traits with the mammalian intestine (Mannino et al., 2022). Core physiological mechanisms, such as stem cell-driven epithelial maintenance, immune activity, and microbiota interactions, are highly conserved, making Drosophila an effective model for investigating intestinal health and disease (Mirzoyan et al., 2019; Di Tommaso et al., 2021).

The Drosophila gut includes intestinal stem cells (ISCs), enterocytes (absorptive), enteroendocrine cells (secretory), and enteroblasts (progenitors) (Micchelli and Perrimon, 2006). These cell types adapt dynamically to stress, pathogens, and aging (Biteau et al., 2008). Thus, Drosophila is a robust model for quantitatively and genetically assessing gut pathologies, such as inflammation, barrier dysfunction, and aging, as well as intestinal regeneration and immune responses due to microbiota changes (Jiang and Edgar, 2011).

Chrysanthemum coronarium is used in traditional herbal medicine for its antipyretic, detoxifying, and anti-inflammatory properties. In recent years, studies have highlighted its antioxidant and anti-inflammatory effects, supported by identification of its various bioactive compounds (Lee et al., 2023). In particular, abundant flavonoids and polyphenols in C. coronarium may regulate inflammation-related pathways, including the NF-κB and MAPK pathways, potentially suppressing immune responses (Kim et al., 2023). The species’ key components include gentisic glucoside, chlorogenic acid, quercetin-glucosyl-rhamnosyl-galactoside, patuletin-3-(4”-acetyl-rhamnopyranosyl)-7-(2”-acetyl-rhamnopyranoside), rutin, cynarine, and 3,5-dicaffeoyl-4-succinoylquinic acid (Kim et al., 2023). However, its therapeutic effects on intestinal inflammation remain untested in Drosophila.

This study investigated the anti-inflammatory effects of C. coronarium on intestinal inflammation using Drosophila as a model organism. This model is widely used to study gut inflammation owing to its conserved gut architecture and innate immune pathways related to mammals. Its short life cycle and genetic accessibility also support functional compound screening. Using this approach, we sought to validate C. coronarium’s inflammation-alleviating effects and explore its potential as a functional food for preventing metabolic and age-related intestinal disorders.

MATERIALS AND METHODS

Drosophila husbandry and genetic lines

All Drosophila strains were maintained on a standard diet at 25°C under a 12/12-h light/dark cycle. The diet comprised 15.8 g of yeast, 9 g of soy flour, 5.2 g of agar, 67 g of cornmeal, and 0.5% propionic acid. To avoid larval overcrowding, fewer than 30 adult flies were kept per vial and transferred to vials with fresh food every 2－3 days. The fly lines used were esg-Gal4, UAS-GFP/CyO (from the Bruce Edgar lab; Jin et al., 2015), 10XStat92E-GFP (#26197, Bloomington Drosophila Resource Center), and Oregon-R (#5, Bloomington Drosophila Resource Center). Flies were raised at 22°C until adulthood, with conditions subsequently shifted to 29°C for midgut dissection and analysis.

Preparation of C. coronarium extract

Dried C. coronarium (1 kg) was crushed and extracted twice using 10 L of 50% ethanol at 25°C for 24 h. The extract was filtered, concentrated using a rotary evaporator, and freeze-dried at −70°C, yielding 8% (w/w). For analysis, 10 mg of C. coronarium was dissolved in 10 mL of 50% methanol and analyzed using UPLC-tunable UV (Waters) with an Acquity UPLC BEH C18 column (100×2.1 mm, 1.7 µm, Waters). All procedures involving C. coronarium complied with institutional, national, and international guidelines and regulations.

Dextran sodium sulfate (DSS) treatment

Flies were fed diets containing 0.5% or 1.0% DSS at 29°C for 4 days. Post-treatment, midguts were dissected for analysis.

C. coronarium treatment

Flies were fed diets supplemented with 1% C. coronarium (500 µg/mL from a 50 mg/mL stock) at 29°C for 4 days. Midguts were dissected for analysis after treatment.

Survival assay

Each experimental group included 25－30 flies. DSS and C. coronarium were dissolved in 5% sucrose solution and administered under identical conditions. All experiments were performed in triplicate. Survival was recorded daily, and fresh media were provided daily to reduce environmental stress.

Smurf assay

Intestinal barrier integrity was assessed using a modified Smurf assay (Salazar et al., 2023). Adult Drosophila (typically 20－40 days old) were placed in vials containing Whatman filter paper soaked with 2.5% (w/v) blue dye (FD&C Blue No. 1) dissolved in 5% sucrose. Before dye exposure, flies were starved for 2 h on moist tissue to promote ingestion. Flies were fed on the dye solution for 16 h at 25°C in the dark, after which they were then scored under a stereomicroscope. Flies showing systemic blue coloration, indicating dye leakage into the hemocoel, were classified as Smurf-positive, reflecting loss of intestinal barrier function. The Smurf index was calculated as follows:

Smurf index (%)=(Number of Smurf-positive flies/Total number of flies)×100

Immunohistochemistry

Adult fly midguts were dissected and fixed in 4% paraformaldehyde for 1 h at room temperature. Samples were washed with phosphate-buffered saline containing 0.1% Tween-20 and incubated first with primary antibodies overnight at 4°C and then with secondary antibodies for 2 h at room temperature. After washing, samples were mounted using Vectashield (Thermo Fisher Scientific) and imaged using an Olympus FV3000 confocal.

Quantification of phospho-histone H3 (PH3)-positive cells

PH3-positive cells in whole guts were counted to quantify cell proliferation.

Statistical analysis

GraphPad Prism (GraphPad Software, Inc.) was used for data visualization and statistical analysis. Unpaired t-tests were used for pairwise comparisons, and one-way ANOVA was used followed by Tukey’s post hoc multiple comparisons test, and multiplicity-adjusted P values are reported. All experiments were repeated at least three times. N denotes the number of midguts analyzed.

RESULTS

C. coronarium suppresses ISC hyperproliferation and restores gut length under DSS treatment

DSS is widely used to chemically induce colitis in model organisms, including mice and Drosophila, by disrupting the intestinal epithelium and triggering inflammation (Amcheslavsky et al., 2009; Keshav et al., 2022; Zhang et al., 2022). In the present study, DSS significantly increased the number of ISCs and enteroblasts (Fig. 1A). In contrast, cotreatment with C. coronarium suppressed this proliferation, indicating that it mitigates inflammation-induced ISC hyperactivity. Levels of PH3, a mitosis marker, were also elevated in the DSS group but declined following C. coronarium cotreatment (Fig. 1C). Additionally, DSS-induced gut shortening was partially reversed by C. coronarium treatment (Fig. 1B and 1D). These findings suggest that C. coronarium supports intestinal homeostasis by limiting ISC overproliferation and restoring gut morphology under inflammatory stress.

Fig. 1.

Chrysanthemum coronarium suppresses intestinal stem cell hyperproliferation and restores gut length under inflammatory conditions. (A) Immunofluorescence staining images of esg-GFP (green) in midguts from control (con), 0.5% dextran sodium sulfate (DSS)-treated (4 days), and 0.5% DSS+1% C. coronarium (CC)-treated (4 days) flies. (B) Representative images of gut length in each treatment group. (C) Quantification of phospho-histone H3 (PH3)-positive cells (mitotic marker) in fly midguts (con, N=18; 0.5% DSS, N=17; 0.5% DSS+1% CC, N=18). (D) Quantification of gut length (con, N=10; 0.5% DSS, N=10; 0.5% DSS+1% CC, N=10). N indicates the number of guts analyzed. Values are presented as mean±standard deviation. ***P<0.001 and ****P<0.0001.

C. coronarium improves DSS-induced intestinal barrier dysfunction

To assess whether C. coronarium alleviates DSS-induced intestinal barrier dysfunction, we performed a Smurf assay in Drosophila. DSS is known to increase gut permeability and impair barrier function (Salazar et al., 2023). The Smurf assay uses Brilliant Blue dye ingestion to detect leaky gut phenotypes: in flies with intact barriers, the dye remains confined to the gut; in barrier-compromised flies, it spreads throughout the body (“Smurf phenotype”). Results revealed that 0.5% DSS significantly increased the percentage of Smurf phenotypes compared with controls. However, cotreatment with 500 µg/mL C. coronarium significantly reduced the proportion of Smurf flies (Fig. 2A and 2B), suggesting that C. coronarium preserves gut barrier integrity during inflammation.

Fig. 2.

Chrysanthemum coronarium mitigates DSS-induced intestinal barrier dysfunction, signal transducer and activator of transcription (STAT) activation, and apoptosis. (A) Representative images of Smurf phenotypes in control (con), 1% dextran sodium sulfate (DSS)-treated (4 days), and 1% DSS+1% C. coronarium (CC)-treated (4 days) flies. (B) Quantification of Smurf phenotypes. Each group contained 8−9 flies; the experiment was repeated three times, and results are shown as percentages. (C) STAT-GFP (green) immunofluorescence in midguts from con, 0.5% DSS-treated, and 0.5% DSS+1% CC-treated flies. (D) Distribution of STAT-GFP patterns (normal=yellow, mild=blue, and severe=red) in each treatment group from (C). Experiments were repeated three times, and the results are shown as percentages (20× magnification). (E) Immunofluorescence staining of esg-GFP (green) and cleaved-Caspase-3 (cCaspase3) (red) in the midguts of con, 1% DSS-treated, and 1% DSS+1% CC-treated flies. (F) Quantification of cCaspase3-positive cells (con, N=21; 0.5% DSS, N=23; 0.5% DSS+1% CC, N=24). N indicates the number of guts analyzed. Values are presented as mean±standard deviation. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

C. coronarium attenuates DSS-induced signal transducer and activator of transcription (STAT) activation and apoptosis

The Janus kinase (JAK)/STAT pathway regulates ISC proliferation, tissue regeneration, immune responses, and inflammation (Jiang et al., 2009; Xu et al., 2011). DSS-induced tissue damage increases the production of inflammatory cytokines, such as Upd3, which activate JAK/STAT signaling and promote ISC proliferation (Amcheslavsky et al., 2009). Chronic activation of this pathway disrupts tissue homeostasis and drives pathological changes (Sarapultsev et al., 2023). To determine whether C. coronarium modulates STAT activation, we employed the 10XSTAT-GFP reporter line. DSS robustly increased STAT-GFP expression, which was significantly suppressed by C. coronarium cotreatment (Fig. 2C). We categorized STAT-GFP expression in the Drosophila midgut into three groups: normal (yellow), mild (blue), and severe (red). In the control group, 86.3% of midguts showed a normal pattern. Under 0.5% DSS treatment, 77.6% and 22.4% exhibited severe and mild patterns, respectively. In flies treated with both 0.5% DSS and 1% C. coronarium, severe cases decreased to 8.25%, whereas mild and normal cases occurred in 30.78% and 60.97% of specimens, respectively (Fig. 2D). We also evaluated apoptosis by staining for cleaved-Caspase-3 (cCaspase3). DSS significantly increased cCaspase3 levels, indicating elevated cell death, whereas cotreatment with C. coronarium markedly reduced this effect (Fig. 2E). Additionally, cCaspase3-positive cell abundance was elevated by 1% DSS but significantly reduced by C. coronarium treatment (Fig. 2F). These findings indicate that C. coronarium suppresses DSS-induced JAK/STAT signaling and apoptosis, thereby supporting gut tissue homeostasis.

C. coronarium extends lifespan under DSS-induced inflammatory stress

We also examined whether C. coronarium affects Drosophila lifespan under inflammatory stress. Flies were fed 1% DSS alone or combined with 500 µg/mL C. coronarium, and survival was monitored. DSS significantly shortened lifespan and increased early mortality compared with controls. However, cotreatment with C. coronarium partially rescued survival and extended lifespan (Fig. 3). These findings suggest that C. coronarium reduces inflammation-induced physiological stress and improves lifespan under inflammatory conditions.

Fig. 3.

Chrysanthemum coronarium extends lifespan under dextran sodium sulfate (DSS)-induced inflammatory stress. Survival curve of flies treated with 1% DSS alone (4 days) or in combination with 1% C. coronarium (CC) [control (con), N=78; 0.5% DSS, N=115; 0.5% DSS+1% CC, N=96]. Con (A), 1% DSS (B), and 1% DSS+1% CC (C). A vs. B, P=0.0012 (**). B vs. C, P=0.0033 (**). A vs. C, P=0.144 (ns). Values are presented as mean±standard deviation. **P<0.01 and ns, non-significant.

DISCUSSION

This study demonstrated that C. coronarium alleviates intestinal inflammation and preserves tissue homeostasis in a Drosophila model of DSS-induced colitis. Specifically, we showed that C. coronarium modulates ISC proliferation, improves gut barrier function, and suppresses JAK/STAT signaling, providing molecular evidence of its anti-inflammatory and gut-protective effects. Additionally, C. coronarium mitigates inflammation-induced lifespan reduction, highlighting its physiological benefits under conditions involving inflammatory stress.

DSS induces intestinal inflammation by damaging the gut epithelium and activating immune responses, leading to ISC hyperproliferation, gut shortening, and increased intestinal permeability in Drosophila (Feng et al., 2024). In this model, C. coronarium cotreatment significantly reversed these pathological features. It restored gut length, suppressed ISC overproliferation, and improved barrier integrity, as shown via a Smurf assay, suggesting that C. coronarium promotes gut homeostasis through its bioactive components.

Aberrant immune signaling, particularly through the JAK/STAT pathway, is central to IBD pathogenesis and other gut-related disorders (Cui et al., 2024). Using a STAT-GFP reporter system, we found that DSS-induced STAT activation was markedly reduced by C. coronarium. Similarly, cCaspase3 staining showed that DSS-induced apoptosis was attenuated by C. coronarium treatment. These results indicate that C. coronarium helps maintain immune homeostasis by attenuating STAT signaling and inflammation-associated cell death.

C. coronarium is rich in anti-inflammatory and antioxidant phytochemicals, including flavonoids, phenolic acids, and vitamins, known to modulate signaling pathways such as NF-κB, MAPK, and STAT (Kim et al., 2023). Our findings provide organism-level evidence that these compounds exert protective effects under inflammatory conditions. This complements previous in vitro or biochemical studies and supports the potential application of C. coronarium as a functional food.

Although the Drosophila model offers valuable insights, further validation in mammalian systems is required. Future studies should identify and quantify the active compounds and employ integrative omics approaches (e.g., transcriptomics and metabolomics) to elucidate systemic mechanisms. Given the importance of gut microbiota in intestinal inflammation and immunity, it will also be essential to investigate how C. coronarium influences microbial composition and metabolism.

In conclusion, we found that C. coronarium restores intestinal homeostasis in a DSS-induced colitis model by suppressing ISC hyperproliferation, enhancing barrier integrity, and inhibiting STAT activation. These findings support its potential as a functional food ingredient for preventing IBD and managing metabolic disorders.