Que (Cat#R006828) with a purity of HPLC ≥97% was purchased from Shanghai Rawhn Chemical Technology Co., Ltd. (Shanghai, China). RU.521 (Cat#M9447), a cGAS inhibitor, from AbMole (Houston, United States), DSS (Cat#160110) from MP Biomedicals (San Diego, CA, United States), FITC-Dextran (Cat#HY-128868D) from MedChemExpress (Monmouth Junction, NJ, United States), M-CSF (Cat#315-02) from PeproTech (Rocky Hill, NJ, United States). CCK-8 (Cat#BS350A) from Biosharp (Hefei, China), Lipofectamine™ 3000 (Lip3000, Cat#L3000001) and APC CD206 antibody (REF#17-2061-82) from Invitrogen (Carlsbad, CA, United States). iNOS antibody (Cat#18985-1-AP), Arg1 antibody (Cat#16001-1-AP), α-Tublin (Cat#66031-1-lg), STING (Cat#19851-1-AP), IRF3 (Cat#11312-1-AP), p-IRF3 (Cat#29528-1-AP), ZO1 (Cat#21773-1-AP) and Occludin (Cat#27260-1-AP) from Proteintech (Wuhan, China). Additionally, TBK1 (Cat#3504T) and p-TBK1 (Cat#5483S) were purchased from Cell Signaling Technology (Danvers, MA, United States), cGAS (Cat#ZRB1406) from Merck Millipore (Billerica, MA, United States), ELISA kits for IL10, CCL17, CXCL10, TNF-α and IFN-β from Adsbio (Yancheng, China), β-actin (Cat#GB12001), RIPA lysis buffer (Cat#G2002-100ML) and Bicinchoninic Acid (BCA) protein assay kit (Cat# G2026-200T) from Servicebio (Wuhan, China), and the following antibodies BV510 anti-FVD (Cat# 564406) and APC-Cy7 anti-CD45 (Cat# 557659), PE F4/80 (Cat#565410), PC-CY5.5 CD11b (Cat#101227) and BV421 CD11C (Cat#117329) from BD Biosciences (Franklin Lakes, NJ, United States). Furthermore, Trizol was bought from TaKaRa (Tokyo, Japan), HiScript^® III RT SuperMix for quantitative polymerase chain reaction (qPCR) (+gDNA wiper) and ChamQ SYBR qPCR Master Mix from Vazyme (Nanjing, China), blood urea nitrogen (BUN) assay kit (Cat#C013-2-1), creatinine (CRE) assay kit (Cat#C011-2-1), alanine aminotransferase (ALT) assay kit (Cat#C009-2-1) and aspartate aminotransferase (AST) assay kit (Cat# C010-2-1) from Jiancheng Bioengineering Institute (Nanjing, China).

ISD was generated by heating equimolar amounts of sense and antisense DNA oligonucleotides to 95°C for 5 min, followed by cooling to room temperature (Gui et al., 2019). Si-cGAS was synthesized by Paivi Biotechnology Co., Ltd. (Wuhan, China) and had the following sequences: si-cGAS (sense, 5′-CAG​CUG​AAC​ACU​GGC​AGC​UAC​UAU​G-3′, antisense, 5′-CAU​AGU​AGC​UGC​CAG​UGU​UCA​GCU​G-3′). All oligonucleotides for ISD and qPCR primers used in this study were provided by Beijing Tsingke Biotech Co., Ltd.

Male C57BL/6 J mice (weighing 20–22 g and aged 6–8 weeks) were purchased from SPF Biotechnology Co., LTD. (Beijing) (Quality certification: SCXK (jing) 2019-0010). All animal-related experiments were conducted in strict adherence to the guidelines of the Animal Research Institute Committee of HUST and were approved by the HUST Institutional Animal Care and Use Committee (IACUC).

The mice were provided unrestricted access to food and water and housed under specific pathogen-free (SPF) conditions, which included a 12-h light/dark cycle, a temperature maintained at 22 ± 1°C, and humidity levels ranging from 45% to 55%. They were allowed to acclimate for 5 days, after which they were divided into four groups (n = 8): the normal control group (Normal), the model group (DSS), the RU.521 group (RU.521, administered at a dosage of 10 mg/kg, serving as the positive control) (Ma et al., 2020), and the Que group (QUE, administered at a dosage of 30 mg/kg) (Yang et al., 2014). Except for the control group, acute colitis was induced by the administration of 3% DSS for 7 days. RU.521 and Que were administered via intraperitoneal injections over the same seven-day period, starting at the onset of colitis induction. Meanwhile, the model group received an equivalent volume of drinking water. On the eighth day, the mice were euthanized, and the tissues were collected for analysis.

RAW264.7 cells (Cat#SNL-112) were purchased from SUNNCELL (Wuhan, China) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose containing 5% fetal bovine serum (FBS). The culture was maintained in a humidified incubator with 5% CO2 and 95% air at 37°C. Bone marrow mesenchymal cells were isolated from the tibiofibula of mice and were stimulated with M-CSF at a concentration of 30 ng/mL in 5% FBS-1640 RPMI medium. The medium was replaced with fresh medium containing 20 ng/mL M-CSF on the third and fifth days. On the seventh day, the cell morphology was observed and confirmed through flow cytometry.

To determine the appropriate concentration of Que for intervention, the viability of both cell types was assessed after exposure to different concentrations of Que for 24 h, following the instructions provided with the CCK8 kit.

The cell experiment involved four distinct groups: the control group, the ISD (2 μg/mL) group, the si-cGAS + ISD group, and the Que (50 μmol/L) + ISD group. RAW264.7 cells and BMDMs were seeded in 9.6 cm² culture plates at a density of 5 × 10⁵ cells per well and allowed to grow for 12 h in a humidified incubator. Subsequently, cells in all groups, except the control group, were transfected with ISD for 6 h. Following this transfection, the culture medium for all groups was replaced with 2 mL of fresh medium. Que was administered for an additional 24 h, and si-cGAS was transfected during the final 8 h of this period. To confirm the efficiency of si-cGAS transfection, quantitative real-time polymerase chain reaction analysis (qRT-PCR) and western blotting (WB) were performed. The transfection of ISD and si-cGAS was performed following the instructions provided for Lipofectamine™ 3000.

Throughout the experimental period, the weight of each mouse was consistently monitored. On the eighth day following drug intervention, the entire colon tissue was excised, photographed to record its length, and collected after mice were humanely euthanized via cervical dislocation under anesthesia induced by 1% pentobarbital sodium (50 mg/kg body weight). The intestines, specifically a segment approximately 0.5 cm proximal to the anus, were rinsed, circumferentially cut, and then preserved in 1 mL of 4% paraformaldehyde for the following experiments. To assess disease activity and calculate the disease activity index (DAI), weight loss, stool consistency and fecal occult blood were evaluated daily (Sann et al., 2013). Tissue sections were subjected to hematoxylin and eosin (H&E) staining for histological examination, while Alcian Blue/Phosphoric Acid Schiff (AB/PAS) staining was employed to visualize goblet cells, as previously described by Ma et al. (2020). Additionally, H&E staining was performed on the mice heart, liver, spleen, lung, and kidney tissues. The levels of blood urea nitrogen (BUN), creatinine (CRE), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum were measured using assay kits to assess the safety of Que intervention in this study.

The procedure for obtaining a single-cell suspension from the mouse spleen and mesenteric lymph nodes was performed as previously described (Gao et al., 2022). Upon collection of the spleen and mesenteric lymph nodes, they were promptly immersed in pre-chilled 3% BSA in PBS. The tissues were finely chopped and softly homogenized. Following filtration through a 70 μm sieve, the cellular suspension was harvested. For spleen samples, erythrocytes were lysed prior to centrifugation and subsequent resuspension.

Flow cytometry was utilized to identify BMDMs and assess the M2/M1 cell ratio in the spleen, and MLNs post-intervention for macrophage polarization analysis. On the seventh day of M-CSF stimulation, bone marrow mesenchymal cells were collected and labeled with antibodies, including anti-FVD, APC-Cy7-conjugated anti-CD45, PE-conjugated F4/80, and PC-CY5.5-conjugated CD11b. For macrophage identification, cultured BMDMs received BV421-conjugated CD11C and APC-conjugated CD206 antibodies. Macrophages were characterized as F4/80⁺CD11b⁺ cells, with M1 and M2 subsets defined as F4/80⁺CD11b⁺CD11C⁺CD206^- and F4/80⁺CD11b⁺CD11C⁻CD206⁺, respectively. The same flow cytometry protocol was applied to assess macrophage polarization in tissues (Ying et al., 2013; Ding et al., 2022). Cells and other tissues were analyzed using a BD LSR flow cytometer (BD Biosciences, San Jose, CA, United States), and the data were analyzed using the CytExpert software.

Total RNA was extracted from both clones and cells using Trizol. Subsequently, reverse transcription was carried out using HiScript^® III RT SuperMix for qPCR (+gDNA wiper). Following reverse transcription, qPCR was conducted using ChamQ SYBR qPCR Master Mix. The procedures were conducted following the instructions of the respective reagents. The relative mRNA expression levels were calculated using the 2^−ΔΔCT method, with β-actin serving as the endogenous control. The primer sequences are provided in Table 1.

The cells or intestinal tissues were homogenized in RIPA lysis buffer using a grinder (50Hz, 120s, LUKA, Guangzhou). After centrifugation, the supernatant was collected, and protein concentrations were determined using a BCA protein assay kit. Subsequently, the samples were loaded onto either 8% or 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels with 15 wells and a thickness of 1.0 mm for rapid electrophoresis (200V, 30min). The target proteins were then transferred to a polyvinylidene (PVDF) membrane (Millipore, Billerica, MA, United States) (400mA, 30 min). Following a 20-min blocking step with a fast-blocking solution at room temperature, the protein bands were washed with 1 × TBST for 10 min and subsequently incubated with primary antibodies including cGAS, STING, TBK1, p-TBK1, iNOS, p-IRF3, β-actin, α-Tubulin (at a dilution of 1:1000), IRF3, and Arg1 (at a dilution of 1:5000) overnight at 4°C. The next day, the protein bands were washed three times with 1×TBST for 10 min each time and incubated with a secondary antibody (dilution, 1:5000) at room temperature for 1 h, followed by additional washing. Lastly, the bands were briefly immersed in an ECL exposure solution and placed in an exposure apparatus for visualization. Image analysis was performed using ImageJ software.

The expression levels of IL10, CCL17, CXCL10, TNF-α and IFN-β in the colon homogenate supernatant were assessed using ELISA kits. Following the manufacturer’s instructions, the standard product was initially diluted in a gradient, then both the samples and biotin-labeled antibodies were introduced to the enzyme labeling plate. Absorbance was measured at 450 nm using a microplate reader.

Intestinal sections stained with H&E were deparaffinized, while RAW264.7 cells were fixed using 4% paraformaldehyde. After rehydration, both were washed with 1% TWEEN-PBS and blocked with 3% goat serum. For IF experiments, specific primary antibodies (anti-iNOS, anti-ZO1, anti-Occludin at 1:200 dilution, and anti-Arg1 at 1:500 dilution) were incubated overnight at 4°C after blocking. On the following day, the sections and cells were washed and subsequently incubated with secondary antibodies labeled with fluorescein isothiocyanate (FITC) or Cy3. Finally, they were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min, and IF images were captured using a confocal laser scanning microscope (Olympus-FV1000).

The abdominal hairs of the mice were removed, and they underwent a 24-h fasting. Next, they were orally administered with FITC-Dex (average molecular weight: 4000, 0.6 mg/g) while being kept away from light for 4 h. Prior to the assay, the mice were anesthetized using 1% pentobarbital. To measure fluorescence intensity (excitation 490 nm/emission 535 nm), serum samples were diluted 1:1 with a PBS solution. The quantification of Fluorescein isothiocyanate-dextran (FITC-Dex) in the serum was performed using a fluorescent enzyme label kit, following the manufacturer’s instructions.

Statistical analysis was conducted using GraphPad Prism 8.0 (Graph Pad Software Inc., San Diego, CA, United States). Data are presented as mean ± standard deviation (SD). Two-tailed unpaired t-tests were employed to compare two groups, while a two-way analysis of variance (ANOVA) was utilized to analyze multiple groups. The significance levels were denoted as *p < 0.05, **p < 0.01, and ***p < 0.001.

The DSS-induced UC method in mice is well-established. First, we verified that Que at a concentration of 30 mg/kg was safe for mice after 7 days of intervention, The results of H&E staining showed that no obvious changes in the heart, liver, spleen, lung, or kidney tissues between the normal and Que-treated groups of mice. In addition, there were no significant deviations in serum levels of BUN, CRE, ALT and AST between groups (Figure 1). As shown in Figures 2A, B, mice treated with the cGAS inhibitor (RU.521) and Que exhibited significant improvements in weight loss, fecal characteristics and overall wellbeing compared to those receiving only DSS. Moreover, the interventions led to significant improvements in intestinal morphology and pathology, including reduced intestinal shortening, decreased infiltration of inflammatory cells, preserved crypts, and increased goblet cell counts in DSS-treated mice (Figures 2C–G). These results highlight the effectiveness of cGAS inhibition in reducing intestinal damage and improving UC and support the therapeutic potential of Que.

To investigate the changes in M2 and M1 subpopulations in mice following Que intervention and cGAS inhibition, we isolated the spleen and mesenteric lymph nodes for flow analysis. Compared to the model group, the intervention groups exhibited significant suppression of the M1 subpopulation and an increase in the M2 subpopulation (Figures 3A–C). Furthermore, the levels of Arg1 and iNOS in intestinal tissues were assessed using WB and qPCR. It was observed that iNOS levels increased while Arg1 levels decreased during colitis. However, these changes were reversed following intervention with RU.521 and Que (Figures 3D–H). Thus, these findings indicate that Que and cGAS inhibition contribute to the restoration of the M2/M1 ratio in mice.

BMDMs and RAW264.7 cells were used to assess macrophage polarization in vitro. Initially, we observed the morphology of RAW264.7 cells with round shape, and the M-CSF-induced cells which displayed a flattened appearance with numerous protrusions (Figures 4A, B). Subsequently, flow cytometry analysis revealed that F4/80⁺CD11b⁺ cells constituted 93.66% of the total cells, confirming successful macrophage induction (Figure 4C). BMDMs and RAW264.7 cells were subjected to different concentrations of Que for 24 h, and 50 μmol/L was determined as the optimal intervention concentration for Que, as it did not affect the viability of both cell types (Figures 4D, E).

To investigate the impact of Que and cGAS inhibition on macrophage polarization in vitro, we used ISD, an exogenous stimulator known to activate cGAS expression, to intervene with macrophages. We assessed the state of macrophage polarization after inhibiting cGAS expression and administering Que. Following ISD intervention, there was a significant decrease in the fluorescence expression of Arg1 and an increase in iNOS in RAW264.7 cells. However, when compared to the ISD group, the utilization of si-cGAS and Que led to a significant enhancement in Arg1 fluorescence expression, accompanied by a reduction in iNOS levels (Figure 5A). Similar changes were observed in the mRNA levels of Arg1 and iNOS in RAW264.7 cells (Figures 5B, C). Flow cytometry analysis provided further insights, demonstrating that ISD intervention resulted in an increased proportion of M1 cells and a reduced population of M2 cells. However, si-cGAS and Que effectively reversed this trend, redirecting macrophages away from the inflammatory phenotype (M1) towards M2 cells with anti-inflammatory properties (Figures 5D, E).

To investigate the role of the cGAS-STING pathway in the polarization of intestinal macrophages, we conducted double fluorescent labeling of intestinal F4/80 and cGAS proteins, which helped identify macrophages in the intestine. We observed that both Que and RU.521 reduced the fluorescence expression of F4/80 compared to the model group, indicating a decrease in intestinal macrophage infiltration. Under a 400-fold fluorescence microscope, we observed that the fluorescence expression of cGAS in intestinal macrophages was elevated in the intestinal tissues of mice in the model group. However, this expression was significantly reduced after the intervention of Que and RU.521, suggesting that both Que and RU.521 inhibited cGAS in intestinal macrophages (Figure 6A). Furthermore, we examined the changes in the cGAS-STING pathway in the colon and found that the expression of cGAS-STING pathway-related proteins (cGAS, STING, p-TBK, p-IRF3 and IFN-β) in mice significantly increased after modeling. Both Que and RU.521 reduced the levels of these proteins (Figures 6B, C, E, G–I). Additionally, mRNA levels of cGAS, STING, and IFN-β were detected by qPCR in both in vitro and in vivo experiments (Figures 6D, F, J). Based on findings regarding the regulation of macrophage polarization by Que and cGAS inhibition, it is evident that Que can inhibit the cGAS-STING pathway in gut macrophages, thereby influencing macrophage polarization.

We found that quercetin inhibited the ISD-activated cGAS-STING pathway in macrophages like RU.521 by detecting protein content and mRNA levels related to cGAS-STING pathway in BMDMs and raw264.7 cells (Figure 7). Combined with animal experiments’ results, it was indicated that quercetin’s regulation of macrophage polarization in UC mice was related to this pathway.

To assess the impact of Que on intestinal inflammation through the inhibition of the cGAS-STING pathway, we measured the protein levels of anti-inflammatory factors CXCL10 and TNF-α, as well as the inflammatory factors CCL17 and IL10, using ELISA. Following DSS-induced enteritis, there was a significant increase in the protein levels of CXCL10 and TNF-α, coupled with a notable decrease in IL10 and CCL17. Both Que and RU.521 could significantly reduce the protein levels of CXCL10 and TNF-α while increasing the protein levels of IL10 and CCL17 following the induction of enteritis (Figures 8A–D). Consistent results were obtained when measuring mRNA levels in intestinal tissues and macrophages (Figures 8E–L). Thus, Que could mitigate intestinal inflammation by inhibiting the cGAS-STING pathway.

ZO1 and Occludin are important components of TJs that are responsible for maintaining the integrity of intestinal epithelial and endothelial cells. The structural integrity of the intestine can be visually assessed through ultrastructure analysis. In DSS-induced mice, the fluorescence expression of ZO1 and Occludin in intestinal tissues was notably weakened. However, both Que and RU.521 significantly enhanced the fluorescence expression (Figure 9A). Consistent trends were observed in the mRNA levels of intestinal ZO1 and Occludin (Figures 9B, C). In the ultrastructure analysis, normal intestinal tissues exhibited long and well-arranged microvilli, with intact TJs located directly beneath them. In contrast, the model group displayed shorter, sparser or even shed microvilli, along with interrupted TJs. Both Que and RU.521 could repair the intestinal microvilli and TJs (Figure 10A). Intestinal permeability was assessed by tracking the distribution of FITC-labeled dextran, a macromolecular compound, in the intestine and its absorption into the bloodstream. After the induction of colitis, the fluorescence distribution of FITC-Dex significantly increased, as did the content of FITC-Dex in the serum. Treatment with Que and RU.521 alleviated this situation and improved intestinal permeability (Figures 10B, C). Collectively, Que demonstrated the ability to repair the damaged intestinal structure and enhance intestinal barrier function in UC mice by inhibiting cGAS.