Lipopolysaccharide and caerulein Meilinbio (Dalian, China). CH223191, dexamethasone (DEX), and naringenin were purchased from MedChemExpress (New Jersey, United States). NLRP3, AhR, ZO-1, and occludin antibodies were purchased from Abcam (Cambridge, United Kingdom). Antibodies against β-actin and histone H3 were purchased from BOSTER (Wuhan, China). Goat anti-rabbit IgG was purchased from Absin (Wuhan, China).

Oral bioavailability (OB) is considered as the most crucial component of oral medications, which may assess how well they work when disseminated to the entire body after absorption. The TCMSP database’s guidelines and earlier studies state that an OB value of less than 30% is thought to have pharmacological activity (Ru et al., 2014). Drug-likeness (DL) is a concept to determine whether novel compounds fit the criteria to become a new drug, it is based on the chemical structure of existing drugs. According to earlier study, the DL threshold value is .18 (Zhang et al., 2020). Then we used the TCMSP database to predict the absorption, distribution metabolism, and excretion (ADME) pharmacokinetic characteristics information of naringenin.

Healthy male C57BL/6 mice were obtained from the Qingdao University Laboratory Animal Center (Qingdao, China). Three mice each were randomly assigned to one of five groups: the NS group (control group), the SAP group, the NAR group, the CH223191 group, and the DEX group. CH223191 is a specific inhibitor of AhR to block the transduction of AhR. Dexamethasone (DEX), a glucocorticoid commonly used to treat AP, was used as a positive control. Before the experiment, all mice spent 2 weeks acclimating to the new environment. The mice were fasted for approximately 18 h prior to the experiment. Drinking water was available without restriction. Mice were given intraperitoneal injections of caerulein at a weight ratio of 50 μg/kg once every hour for six straight hours to induce SAP. At the same time, intraperitoneal injections of lipopolysaccharide at a weight ratio of 10 mg/kg were given to induce SAP. The same volume of ordinary saline was similarly administered to the mice in the NS group. Naringenin (50 mg/kg) was administered by intraperitoneal injections to the mice in the NAR group (Li et al., 2018). Mice in the CH223191 group received intraperitoneal injections of CH223191 (10 mg/kg) and naringenin (50 mg/kg) 30 min later (Wang et al., 2018). The DEX group of mice received an intraperitoneal injection of dexamethasone (2.5 mg/kg) (Yu et al., 2019). Twelve hours after injection, animals were euthanized, and samples were collected for further analysis.

RAW264.7 cells were obtained from ATCC and cultured in DMEM (Procell, Wuhan, China) containing 10% fetal bovine serum (Procell) in an incubator at 37°C and 5% CO2.

RAW264.7 cells were seeded into 6-well plates the day before transfection at a confluency of 70%–80% for transfection experiments. After the cells had grown for 24 h, the medium was changed to serum-free medium. Then, 200 μL of DMEM (Procell) was incubated at room temperature for 20 min with 100 pmol of siAhR pool or siNC (GenePharma, Shanghai, China) and 8 μL of Lipofectamine 2000 (Invitrogen, California, United States) to facilitate complex formation. Subsequently, the cells were cultivated for 48 h at 37°C and 5% CO₂ in an incubator. The transfection mixture was gradually added to each well. The cells were then grown in an incubator for 48 h at 37°C with 5% CO₂.

The cells were divided into six groups: control, LPS, LPS + NAR, LPS + NAR + siAhR, LPS + NAR + siNC, and LPS + NAR + CH223191. The dosages administered were 1 μg/mL for LPS, 1 μM for CH223191 (Wang et al., 2018), and 50 μM for naringenin. CH223191 was added 30 min after naringenin was added. The treatments were conducted 24 h later.

The CCK8 assay was used to assess the viability of RAW264.7 cells. RAW264.7 cells were seeded into 96-well plates and incubated with naringenin (10, 20, 40, 50, and 80 μM) for 24 h. Then, each well received 10 μL of CCK-8 for 1 h, and the absorbance was measured at 450 nm. LPS (1 μg/mL) was added to the cells to establish inflammatory model, and gradient concentrations of naringenin (20 μM, 40 μM, and 50 μM) were treated with the cells for 12 and 24 h. Consequently, the viability of RAW264.7 was detected by CCK8 assay to find a concentration of naringenin that significantly inhibited the proliferation of inflammatory cells.

The levels of tumor necrosis factor (TNF), interleukin 1 beta (IL-1β) and interleukin 6 (IL-6) in the serum of mice and cell culture supernatant were detected using the corresponding ELISA kits (ABclonal, Wuhan, China) according to the kit instructions.

The intestines collected from mice were dehydrated using a gradient of ethanol after being fixed in 4% paraformaldehyde for 24 h at 4°C. The samples were subsequently cut into 5-μm-thick slices and fixed in paraffin. Following the recommended procedures, the sections were cleaned, rehydrated, and stained with hematoxylin and eosin (Solarbio, Beijing, China). The staining was observed under a microscope and captured in a ×200 magnification image. Pancreatitis histopathological changes were assed using the standards put forth by Schmidt et al. (1992). The grades of ileum damage were determined by Chiu’s description of the classification of gut injury (Chiu et al., 1970). There are five grades from mild to severe forms: Grade 0, normal mucosal villi; grade 1, Gruenhagen’s space development beneath the epithelium, typically at the villus’s tip; frequently accompanied by capillary congestion; grade 2, Extension of the subepithelial space with moderate lifting of epithelial layer from the lamina propria; grade 3, massive epithelial lifting down the sides of villi, possibly with a few denuded tips; grade 4, denuded villi with the lamina propria and dilated capillaries exposed, possibly with increased cellularity of the lamina propria; and grade 5, digestion and disintegration of the lamina propria, hemorrhage and ulceration.

The paraffin slices underwent standard dewaxing and hydration procedures. In sodium citrate buffer, 3% H₂O₂ was used to block non-specific antibody binding sites after the antigen was microwave-recovered. Then, the sections were incubated at 4°C overnight with the corresponding primary antibody (1:200) and goat-anti-rabbit IgG that had been HRP-labeled for 1 h at 37°C. Hematoxylin was used in conjunction with DAB to visualize the staining. The sections were examined at ×400 magnification and captured with a camera using a microscope.

RAW264.7 cells were cultured on coverslips, and bovine serum albumin (BSA) was used to block the tissue after it had been fixed with 4% paraformaldehyde for 30 min and permeabilized with .5% Triton X-100 for 15 min. The cells were then treated with an Alexa Fluor-labeled secondary antibody (1:100) for 2 hours after being immunostained with an AhR antibody (1:100) overnight at 4°C. The images were finally obtained using an Olympus 1X51 microscope.

Total RNA was extracted from the ileum tissue and cell samples using TRIzol reagent. An Evo M-MLV RT Mix Kit with gDNA clean for qPCR (AG, Changsha, China) was used to create cDNA from 2 μg of total RNA. Real-time PCR was carried out as instructed by the SYBR Green kit.

The 2^−ΔΔCT method was used to quantitatively evaluate the level of mRNA expression. The primer sequences are shown in Table 1.

Protein was isolated from intestinal tissue and cells, and the concentration of the protein was measured using a BCA quantitative kit (Meilinbio, Dalian, China). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was used to load the protein samples and transfer the protein samples onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with primary antibody at 4°C overnight after being blocked with 5% skim milk for 2 h. Following a TBST buffer wash, the PVDF membranes were incubated with IgG-HRP secondary antibody (1:5,000) for 1 h. Electrochemiluminescence substrate (Meilinbio) was used to visualize the proteins, and ImageJ was used to assess the gray value.

Molecular docking is able to anticipate the binding mechanism and affinity between small molecule ligands and biological macromolecular receptors by simulating their interaction (Udrea et al., 2021). The crystal structure of AhR was obtained from the PDB website, and the 3D structure of naringenin was retrieved from the PubChem database. Naringenin was the ligand, and AhR was the receptor. The receptor was added to AutoDock 4.2.6 and PyMol 2.4 for the removal of heteroatoms and water molecules as well as the addition of charges and hydrogen atoms. Then, AutoDockTools was used to predict the conformations of ligand‒receptor binding, and PyMol 2.4 was used to visualize the conformations.

GraphPad Prism 8.0.2 was used to perform the statistical analyses. To assess the significance of the variations between the groups, a standard one-way analysis of variance was performed. The data are expressed as the mean ± SD. Results were considered significant when p < .05.

First, the Pharmacokinetic Characteristics of Naringenin were showed in Table 2. Then, we assessed the effects of naringenin on SAP mouse pancreas. Pancreatic H&E staining of the SAP model mice revealed a clear focal necrotic region, lobular structural destruction, inflammatory cell infiltration, and hemorrhage (Figures 1A, B), and there were significant differences between these groups in the pancreas pathological scores (Figure 1D). In addition, the pancreatic damage was effectively reduced by naringenin administration. The impact of naringenin on pancreatic damage was eliminated by CH223191.

Next, we investigated how naringenin affected intestinal injury. H&E staining of the SAP group and CH223191 group showed intestinal villus rupture and mucosal injury, with a clearly increased inflammation score (Figure 1E). Naringenin effectively alleviated the pathological damage (Figure 1C).

The expression of inflammatory cytokines, including IL-1β, IL-6, and TNF, was increased in the SAP group compared with that in the NS group (Figures 2A, B). The NS and NAR groups showed less secretion of these cytokines. In addition, qRT‒PCR (Figure 2C) and western blot (Figure 2D) analyses revealed low levels of occludin and ZO-1 expression in the SAP group, while naringenin treatment inhibited the decrease in occludin and ZO-1. CH223191 consistently reversed the effect of naringenin on intestinal inflammation.

It is generally known that NLRP3 forms the NLRP3 inflammasome with ASC and caspase-1, which leads to inflammation by activating and producing IL-1β and IL-18 (Udrea et al., 2021). Immunohistochemistry, qRT‒PCR, and western blot analyses (Figure 3) showed significantly increased expression of NLRP3 in intestinal tissue in the SAP group, while the NAR group exhibited reduced levels of NLRP3. Similarly, CH223191 reversed the effect of naringenin.

We used AutoDock 4.2.6 for molecular docking, and the binding energy between AhR and naringenin was −7.36 kcal/mol. According to earlier research, a binding affinity of < −4.25 kcal/mol signifies that the two molecules could connect, <−5 kcal/mol indicates good binding, and <−7.0 kcal/mol suggests significant binding activity (Grosdidier et al., 2011). The docking result is displayed in a 3D graph (Figures 4A, B), and there were two hydrogen bonds (the yellow dotted lines represent hydrogen bonds) between AhR and naringenin. As shown, naringenin bound to the LEU-264 and ARG-267 binding sites of AhR. Naringenin administration promoted AhR nuclear translocation and increased its expression, as demonstrated by western blots, while CH223191 blocked the effect of naringenin on the AhR signaling pathway (Figure 4C).

We created an in vitro inflammatory cell model to better evaluate the therapeutic effect of naringenin on AP-related intestinal injury. An AhR-knockdown cell model was successfully created, as shown in Figures 5A, B. And the CCK8 assay (Figures 5C, D) showed that naringenin at a concentration of 50 μM significantly inhibited the proliferation of inflammatory cells. According to the ELISA results (Figure 5E), the expression of IL-1β, IL-6, and TNF in LPS-treated RAW264.7 cells was significantly upregulated, while naringenin reduced the expression of IL-1β, IL-6, and TNF. In addition, the effects of naringenin were eliminated by both CH223191 and AhR siRNA.

Western blot and qRT‒PCR analyses (Figures 6A, B) revealed that NLRP3 expression was considerably upregulated in LPS-treated RAW264.7 cells and downregulated in response to naringenin treatment. Similarly, CH223191 and AhR siRNA reversed the effect of naringenin.

AhR nuclear translocation was facilitated, and the expression of AhR was increased by naringenin therapy, as seen by immunofluorescence staining (Figures 6C, D). In contrast, the effect of naringenin on the AhR pathway was reversed by either CH223191 or AhR siRNA (Figure 6E).