The following Drosophila strains were used in this study: esg-GFP/CyO and UAS-lacZ lines were kindly provided by Dr. Allan Spradling; esg ^ts -Gal4 line was generously gifted from Dr. Benjamin Ohlstein; w ¹¹¹⁸ (BDSC #3605), Canton-S (BDSC #64349), UAS-bsk ^DN (BDSC #6409) and UAS-EGFR ^DN (BDSC #5364) were obtained from the Bloomington Drosophila Stock Center. All flies used in this study were mated females unless otherwise mentioned.

Flies were maintained on standard cornmeal-agar medium (the recipe is: 80 g sucrose, 50 g cornmeal, 20 g glucose, 18.75 g yeast, 5 g agar, 30 mL propionic acid, and 1 L water) at 25°C and 60% humidity under a 12/12 h light/dark cycle. Gene overexpression driven by the esg ^ts -Gal4 Drosophila line was repressed at 18°C and activated at 29°C.

Drosophila intestines were dissected in cold PBS (Phosphate Buffered Saline, Servicebio, #G0002), fixed in 4% paraformaldehyde for 30 min at room temperature, and then rinsed three times with 0.1% PBST (Triton X-100, BioFroxx, #1139ML100) for 10 min each. Tissues were immersed in the primary antibody solution and incubated overnight at 4°C. The next day, the intestines were rinsed three times with 0.1% PBST for 10 min each. Next, the tissues were incubated with the secondary antibody solution for 2 h at room temperature and then rinsed three times with 0.1% PBST for 10 min each. Finally, the intestines were soaked with the anti-fluorescence quenching agent and placed on slides for sealing and storage. Immunofluorescence images were taken with a Leica TCS-SP8 confocal microscope and processed with Application Suite X, Adobe Illustrator, and ImageJ software. Information on the antibodies used in this study is shown in Supplementary Table S1.

The smurf assay was performed to test the integrity of the Drosophila intestinal barrier (Rera et al., 2011). The blue food dye (Spectrum Chemical Manufacturing Corp, Shanghai, China, #FD110) was added to the standard medium to a concentration of 2.5% (wt/vol). Drosophila treated with different drugs (fed with or without SIL) were starved for 1 h and then cultured in the medium for 12 h. When the Drosophila intestinal barrier is intact, the blue food dye is confined to the digestive tract. We refer to flies that leak blue dye into tissues outside the intestine as smurf (+) Drosophila.

The bromophenol blue assay was performed to determine whether the acidic state of the copper cell region (CCR) in the Drosophila intestine is normal (Li et al., 2016). Briefly, 200 µL of 2% bromophenol blue solution (Sigma, #B5525, dissolved in 5% sucrose) was added to the surface of the standard medium and used after the solution was completely absorbed. The flies were starved for 1 h and cultured in food containing bromophenol blue solution for 24 h. The intestines were dissected and photographed immediately to prevent carbon dioxide from changing the color rendering results of bromophenol blue. When the CCR is acidic, the bromophenol blue appears yellow, which we called “Homeostasis”; if the CCR region is non-acidic, the bromophenol blue appears blue, which we called “Perturbed”.

The Drosophila excretion assay was performed to measure the excretory function of the Drosophila intestine (Cognigni et al., 2011). Add 200 µL of 2% bromophenol blue solution to the surface of the medium and leave to allow the food to fully absorb. Cut the chromatography paper into a size of 3.7 cm × 5.8 cm and placed it on the side wall of the food tube. Be careful that the paper cannot touch the food with the bromophenol blue solution. The flies were starved for 1 h and cultured in the food for 24 h. The paper was taken out, and the droppings of flies were imaged with a Leica M205 FA stereomicroscope.

For the lifespan assay under DSS application, put a 3.7 cm × 5.8 cm chromatography paper in the tube and completely wet the paper with 7% DSS (dissolved in 5% sucrose). A total of 100 flies hatched within 48 h were collected and randomly placed into four DSS tubes with or without SIL. The dead flies were recorded every day and the paper was replaced. Repeat the experiment three times.

For the lifespan assay under BLM application, the BLM was dissolved in the standard medium to a concentration of 5 µg/mL. A total of 100 flies hatched within 48 h were randomly placed into four BLM food tubes with or without SIL. The dead flies were recorded once a day and the food was replaced. The experiment was repeated three times.

The DHE staining was performed to detect the level of ROS in the Drosophila intestine (Hochmuth et al., 2011). The intestines were dissected and incubated with DHE (MKbio, Shanghai, China, #MX4812) for 20 min at room temperature, then rinsed 3 times with PBS. Tissues were fixed with 4% paraformaldehyde at room temperature for 30 min and rinsed 3 times with PBS, and finally soaked with the anti-fluorescence quench agent. Images were taken immediately after DHE staining, and ImageJ software was used to calculate fluorescence intensity.

The Drosophila guts were dissected, frozen with liquid nitrogen, and ground immediately. Total RNA was extracted with an RNA-easy Isolation Reagent (Vazyme, Nanjing, China, #R701-01) and reverse transcribed into cDNA with an Evo M-MLV RT Kit (Accurate Biology, #AG11711). RT-qPCR was performed with a CFX96TM Real-time PCR System (BIO-RAD, America) using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China, #Q711-02). The Rp49 gene was used as an internal control, and the relative mRNA expression of genes was calculated using the 2^−ΔΔCT method. The primer sequences used in this study are listed in Supplementary Table S2.

The corresponding targets of SIL were obtained from the PharmMapper database and screened the targets with Norm Fit >0.5 (Wei et al., 2023). The targets of IBD were searched by the OMIM, TTD, DrugBank, GeneCards, and DisGeNET databases using “inflammatory bowel disease” as the keyword (Zu et al., 2021). The gene names of these targets were obtained from the Uniprot database after removing the duplicates (Pei et al., 2023).

The common targets of SIL and IBD were analyzed by the Venny 2.1.0 database to predict the potential targets of SIL against IBD (He et al., 2023). These common targets were imported into the STRING database, the minimum required interaction score was set to 0.7 and the isolated targets were removed to obtain the protein-protein interaction (PPI) network (Wu et al., 2023).

Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the common targets was performed using the DAVID database (He et al., 2023). The enrichment results of KEGG pathways were visualized by the Bioinformatics platform (He et al., 2023).

The 3D structure of SIL in the SDF file was obtained from the PubChem database, converted to a mol2 file via Open Babel 3.1.1 software, used Autodock MGL Tools 1.5.7 to add hydrogen bonds, detect the root, and set rotatable bonds, then saved as PDBQT format (Li et al., 2023). The 3D structures of the key target proteins obtained from the PDB database were exported to the PDB file. The water molecules and excess inactive ligands were removed by PyMOL software. The proteins were hydrogenated and charged into AutoDockTools 1.5.7 software and exported to PDBQT format (Li et al., 2023). The molecular docking was performed using AutoDock Vina 1.1.2 software and the results were visualized by Discovery Studio 2016 Client software (Ersoy et al., 2023).

All experiments were performed with at least three replicates. All data are presented as means ± standard deviations (SD). The statistical significance was analyzed using GraphPad Prism version 8.0. The two-tailed Student’s t-test was performed to analyze differences between groups. The lifespan assays were tested for significance with a log-rank test. P < 0.05 was considered statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Non-significance (ns) represents p > 0.05.

When the Drosophila intestine was exposed to noxious stimuli, the barrier integrity was impaired, leading to intestinal inflammation along with an increased proliferation of ISCs (He et al., 2022; Yang et al., 2022). To investigate the effect of SIL on the proliferation of ISCs in Drosophila caused by DSS insults, we used a reporter line, esg-GFP/CyO, whose ISCs and progenitor cells could be detected by expressing green fluorescent protein (GFP). In addition, we observed the ISCs by Delta (Dl) staining and the dividing ISCs by phosphorylated-histone 3 (pH3) staining (Zhou et al., 2023). The results showed that when stimulated by DSS, the proliferation of ISCs was significantly enhanced, which was manifested by the increase in the number of esg-GFP⁺, Dl⁺, and pH3⁺ cells (Figures 1A, B,F–H). We found that SIL supplementation significantly reduced the DSS-induced increase in the number of esg-GFP⁺, Dl⁺, and pH3⁺ cells. Furthermore, of the three concentrations tested (0.1, 1, and 10 µM), 1 µM SIL was the most effective, showing the least esg-GFP⁺, Dl⁺, and pH3⁺ cells (Figures 1C–H). To enhance the persuasiveness and comprehensiveness of these findings, we also applied Bleomycin (BLM), another reagent that induces intestinal damage by causing DNA damage (Amcheslavsky et al., 2009), and obtained the same results (Supplementary Figures S1A–E, Figures 1I–K). Based on these results, we selected 1 µM SIL as the optimal concentration for conducting further experiments. A previous study reported that the intestinal damage caused by DSS or BLM would return to normal within 3 days (rec-3d) (Du et al., 2020). Our results suggest that SIL supplementation inhibits DSS-induced overproliferation of ISCs without affecting eventual intestinal repair (Supplementary Figures S1F, G). All these results suggested that SIL can inhibit the overproliferation of ISCs during intestinal inflammation in Drosophila.

The intestine is an important organism for the digestion and absorption of food, the metabolism and elimination of waste products, and the immune defense (Funk et al., 2020). When the intestine suffers from inflammation, these physiological functions are disrupted. As our results suggested that SIL can repress the overproliferation of ISCs induced by DSS or BLM, and ISCs play an important role in the maintenance of intestinal functions, we wanted to further investigate the effect of SIL on intestinal functions under inflammation. We performed Armadillo (Arm) staining and smurf assay to determine the barrier integrity of the Drosophila intestinal epithelium. When the intestine was damaged by inflammation caused by DSS or BLM, its barrier function was significantly impaired, as evidenced by the irregular shape of Arm staining and the increased proportion of smurf (+) Drosophila (fly that leaks blue dye into tissues outside the intestine) (Figures 2A, B, D–H, Supplementary Figures S2A, B). We found that SIL supplementation can relieve the impairment of barrier function due to inflammatory damage (Figures 2C–H, Supplementary Figure S2C).

The cells in the copper cell region (CCR) of the Drosophila intestine secrete acid to make the region acidic, and if the acidic state is disrupted, it indicates abnormal intestinal function (Dubreuil, 2004). We performed the bromophenol blue assay to detect whether the acidic state of the CCR is normal, if the region is acidic, bromophenol blue appears yellow, otherwise blue. The results revealed that SIL supplementation could protect the acidic state of the CCR from the damage of DSS or BLM (Figures 2I–L). Besides, we found that when the intestine is subjected to inflammation, its excretory function is impaired, and SIL supplementation protects the excretory function of the gut (Figures 2M–Q, Supplementary Figures S2D–F). Taken together, these results suggested that SIL supplementation protects Drosophila intestinal function from inflammatory impairment.

DSS causes inflammatory damage in the intestine, resulting in elevated levels of oxidative stress and activation of the immune response (Zhang G. et al., 2022; Dong et al., 2022). To investigate whether SIL can alleviate oxidative stress in DSS stimulated intestine, we used the fluorescent probe DHE to detect the ROS levels. We found that SIL supplementation significantly reduced the ROS induced by DSS (Figures 3A–D). In addition, RT-qPCR analyses revealed that SIL decreased the relative RNA expression level of antioxidant-related genes (Cat, SOD, and GstD1) in DSS-stimulated Drosophila midgut (Figure 3E). These results indicated that SIL supplementation decreased the high levels of oxidative stress in the Drosophila midgut induced by DSS.

Inflammatory damage to the intestinal epithelium activates the immune response and affects the expression of antimicrobial peptides (AMPs), a group of essential components of the intestinal epithelial barrier (Lazzaro et al., 2020). The results showed that when stimulated by DSS, the expression of AMPs (Attacin A, Cecropin C, Defensin, and Diptericin) and inflammatory cytokines [upd2 and upd3, inflammatory IL-6-like cytokines in Drosophila (Jiang et al., 2009)] was significantly increased in the Drosophila midgut and SIL supplementation decreased the expression of AMPs, upd2 and upd3 (Figures 3F,G). The lifespan of Drosophila is shortened when exposed to chronic deleterious stimuli (Du et al., 2021; Zhang G. et al., 2022). We found that SIL supplementation dramatically prolonged the lifespan under DSS or BLM stimulation (Figures 3H–J). In conclusion, these results suggested that SIL can alleviate DSS-induced intestinal inflammation and extend the lifespan of Drosophila.

Further, we investigated the mechanism by which SIL alleviates intestinal inflammation in Drosophila through network pharmacological analysis. 147 action targets for SIL were obtained from the PharmMapper and Uniprot databases and 2,115 targets for IBD were obtained from the OMIM, TTD, DrugBank, GeneCards, DisGeNET, and Uniprot databases (Figure 4B). Using the Venny database, we obtained 82 common targets for SIL and IBD (Figure 4B). These common targets were imported into the STRING database to obtain the protein-protein interaction (PIP) network (Figure 4C). We found that proteins (MAPK8, MAPK10, and MAPK14) involved in the JNK/p38 MAPK pathway, a classic stress signaling pathway (Hotamisligil and Davis, 2016), interact strongly with other proteins (Figure 4C). Besides, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed on the 82 common targets using the DAVID database to obtain the SIL-regulated signaling pathways, in which the genes related to the MAPK signaling pathway were significantly enriched (Figure 4D). To confirm the possible action targets and signaling pathways of SIL against IBD, molecular docking was performed to verify the accuracy of the network pharmacological predictions. The results showed that MAPK8 (JNK1), MAPK10 (JNK3), and MAPK14 (p38-α) possessed strong binding activity with SIL (Figure 4A, E–G). These results suggested that the JNK signaling pathway may be the mechanism by which SIL alleviates IBD.

To investigate whether SIL alleviates intestinal inflammation caused by DSS by regulating the JNK signaling pathway, we detected the activity of JNK signaling by staining the phosphorylated JNK (pJNK). We found that pJNK expression was significantly increased when the Drosophila intestine was subjected to DSS-induced injury (Figures 5A, B, D), and supplementation with SIL inhibited the activation of the JNK signaling pathway (Figures 5C, D). Besides, RT-qPCR analyses revealed that SIL administration decreased the expression of JNK signal-regulated genes (Mmp1, MtnA, and dpp) in DSS-stimulated Drosophila midgut (Figure 5E).

To further validate the effect of SIL on the JNK signaling pathway, we inhibited JNK signaling in ISCs by overexpressing the dominant-negative form of basket (bsk ^DN , bsk is a key component of the JNK signaling pathway (Tafesh-Edwards and Eleftherianos, 2020). In esg ^ts -driven bsk ^DN flies, we found that the number of esg-GFP⁺, Dl⁺, and pH3⁺ cells were all reduced when subjected to DSS injury compared to the control group (Figures 5F, G, I–K). In addition, SIL supplementation failed to further reduce the number of esg-GFP⁺, Dl⁺, and pH3⁺ cells based on inhibition of JNK signaling (Figures 5H–K), which suggested that SIL alleviates Drosophila intestinal inflammation by inhibiting the JNK signaling pathway.

EGFR signaling has been widely demonstrated to be involved in regulating the proliferation of ISCs and is downstream of JNK signaling (Jiang et al., 2011; Jiang et al., 2016). Therefore, we investigated whether SIL inhibits the DSS-induced overproliferation of ISCs by regulating the EGFR signaling pathway. Firstly, we determined the activation of EGFR signaling by detecting the expression of pErk (an indicator of EGFR signaling activation) in esg-GFP⁺ cells. We found that the expression of pErk was significantly elevated in esg-GFP⁺ cells when the Drosophila intestine was damaged by DSS (Figures 6A, B, D), and SIL supplementation can suppress the activation of EGFR signaling (Figures 6C, D). We performed RT-qPCR analyses and found that SIL administration decreased the expression of EGFR signal-regulated genes (pnt, Ets21C, stg and CycE) in DSS-stimulated Drosophila midgut (Figure 6E).

Next, we overexpressed the dominant-negative form of EGFR (EGFR ^DN ) to further verify whether SIL inhibits EGFR signaling. If SIL inhibits the DSS-induced ISC overproliferation by repressing the EGFR signaling pathway, then supplementing SIL in the esg ^ts -driven EGFR ^DN Drosophila would have no additional effect. As expected, the number of esg-GFP⁺, Dl⁺, and pH3⁺ cells in the EGFR ^DN Drosophila increased insignificantly when the intestine was damaged by inflammation (Figures 6F, G, I–K), and SIL supplementation did not further reduce the number of esg-GFP⁺, Dl⁺, and pH3⁺ cells (Figures 6H–K). These results demonstrated that SIL inhibits DSS-induced ISC overproliferation by suppressing the EGFR signaling pathway.