Active constituents of ACZF were obtained from the TCM Systems Pharmacology (TCMSP) database (https://tcmspw.com/tcmsp.php), mandating an oral bioavailability (OB) ≥ 30% and drug likeness (DL) ≥ 0.18. The Swiss Targets Prediction platform (http://www.swisstargetprediction.ch/) was engaged to identify the targets of these components, specifically targeting “Homo sapiens.” Targets with a likelihood ≥0.11 were selected from these predictions. Should the effective components possess fewer than 15 targets meeting this probability, the top 15 targets by probability were chosen.

Targets linked to RE were retrieved from the GeneCards (https://www.genecards.org/) and OMIM (https://www.omim.org/) databases, using “ Radiation enteritis” or “radiation enteropathy” as search keywords and specifying “H. sapiens” as the species filter.

An online Venn diagram tool (http://bioinformatics.Psb.ugent.be/webtools/Venn/) was used to determine the intersection genes between genes related to RE and possible targets of ACZF. The “drug-compound-target-disease” network was created using Cytoscape 3.8.2 software. Nodes in the network stand for drugs and targets, while lines indicate the relationships between them. The network’s topological properties were analyzed using the Network Analyzer plugin, calculating the degree value of each node. The higher the degree value was, the more important active ingredients and target genes were.

The STRING database was queried for intersection targets with the species “H. sapiens” and a confidence level of ≥0.9 set. To build the interaction network, these results were imported into Cytoscape 3.8.2. In order to identify important targets, the Centiscape 2.2 plugin was used to measure the degree, betweenness, and proximity of every node (Zheng-Lan et al., 2023).

Using the DAVID database (https://david.ncifcrf.gov/), GO and KEGG pathway enrichment analyses were performed to investigate possible molecular pathways by which ACZF improves RE. The GO analysis applied a cutoff of p < 0.05, with the top 10 biological processes below this threshold visualized in a bar chart. Correspondingly, the KEGG pathway analysis adhered to a p < 0.05 significance threshold, with the top 30 pathways under this threshold illustrated in a bubble chart by the bioinformatics platform (https://www.bioinformatics.com.cn/).

ACZF, composed of Sheng Diyu (Sanguisorba officinalis L) (30 g), Baitouweng (Pulsatilla chinensis) (15 g), Huanglian (Coptis chinensis Franch.) (5 g), Baizhu (Atractylodes macrocephala) (15 g), Fuling (Poria cocos (Schw.) Wolf.) (15 g), Xianhecao (Agrimonia pilosa Ledeb.) (30 g), Muxiang (Aucklandia lappa Decne.) (9 g), Chao Baishao (Paeonia lactiflora Pall) (12 g), and Gancao (Glycyrrhiza uralensis Fisch.) (6 g), was sourced from Cangzhou Hospital of Integrated Traditional Chinese and Western of Hebei Province and accredited by a pharmacologist, Professor Baofen Li. More details are shown in Supplementary Table S1. The ACZF formula was soaked in distilled water for 30 min, decocted, and filtered twice. The filtrate was evaporated and concentrated into a decoction containing 4.2 g/mL crude drug and stored at 4°C (Supplementary Figure S1) (Gang, 1998). Masson’s trichrome staining kit was acquired from Solarbio (Batch No. G1340), and ELISA kits for TNF-α,IL-1β,IL-6, and IL-10 were provided by Shanghai Jianglai Biological Technology Co., Ltd. Antibodies for PI3K (No. 10131698) and p-PI3K (No. 10129672) were purchased from Abmart, while those for Akt (No. 10017913), p-Akt (No. 10020148), ZO-1 (No. 66452-1-Ig), and Claudin-1 (No. 13050-1-AP) came from Wuhan Proteintech Co., Ltd. The PI3K activator YS-49 (No. HY-15477) was obtained from MedChem Express.

Based on another study we are submitting, the analysis of ACZF was conducted using Ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The metabolites were quantified using multiple reaction monitoring (MRM) mode. Analysis results are available in the Supplementary Material (Supplementary Figure S2; Supplementary Table S2).

Healthy male C57BL/6 mice, aged 6–8 weeks and weighing 18–22 g, were procured from Henan Sike Best Biotechnology Co., Ltd. (SCXK (Yu) 2020-0005). These mice were housed in the Specific Pathogen-Free (SPF) facility at the Institute of Radiological Medicine, Chinese Academy of Medical Sciences. This study was approved by the Ethics Committee of the Cangzhou Hospital of Integrated Traditional Chinese and Western of Hebei Province (Ethical Approval Number: 2020018).

Following a week of acclimatization, the mice were randomly segregated into control, model, ACZF-treated, and ACZF + YS-49 groups. All groups, except the control, underwent X-ray exposure to induce RE. The mice received anesthesia with pentobarbital, were positioned on the operating board, and irradiated over the entire abdomen with 6 MV-X-rays. The irradiation area covered from the xiphoid process to the pubic symphysis, with an area of 3.0 cm × 3.0 cm, while areas not targeted were shielded with lead plates. The irradiation was performed from 100 cm, at 6 Gy/min for 2.5 min, totaling 15 Gy (Dai and Yang, 2023). Following radiation exposure, the ACZF group was administered 0.2 mL of the aforementioned decoction daily for a week. In the ACZF + YS-49 group, this regimen was supplemented with a daily 5 mg/kg intraperitoneal injection of YS-49. Control and model groups received an equivalent volume of saline. Monitoring was conducted daily, noting body weight, survival rates, fecal morphology and color for signs of diarrhea or hematochezia, and the Disease Activity Index (DAI) was maintained (Zhang et al., 2024). The mice were euthanized by cervical dislocation when the treatment was over. After that, their colons were measured, collected, and divided lengthwise; half of them were preserved in formalin, and the other half were stored at −80°C.

In accordance with predetermined procedures, the colon was fixed in 10% neutral buffered formalin over the night, embedded in paraffin, sectioned at 4 μm, and stained with H&E. The severity of colonic lesions was assessed by examining the extent of inflammatory cell infiltration, mucosal damage, and structural disruptions.

Following the same steps as for stained with H&E, the colon tissue was processed and sectioned. Afterwards, Masson’s trichrome was applied to the sections in accordance with established procedures. A light microscope was used to analyze the collagen fibers within the colon tissues, and their abundance was estimated using ImageJ software.

Levels of IL-1β, IL-6, IL-10, and TNF-α in the colonic mucosa were quantified using ELISA kits in accordance with the manufacturer’s guidelines.

Colonic tissues were fixed in 10% buffered neutral formalin, dehydrated, and paraffin embedded. Sections (5-μm-thick) were deparaffinised and rehydrated, processed by microwave antigen retrieval, and incubated overnight with primary antibodies against ZO-1 (1:500) and claudin-1 (1:500) at 4 °C. Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000) were then applied and incubated at room temperature for 25 min. Visualization was achieved using DAB chromogen, followed by hematoxylin counterstaining. The sections were dehydrated, mounted, and finally observed under an optical microscope. Image analysis was conducted using ImageJ software.

Total protein was extracted from colon tissues using RIPA lysate and then quantified using the BCA method. After separating by 10% SDS-PAGE, proteins were then transferred to PVDF membranes. This was followed by a 2-h room temperature blocking with 5% nonfat dry milk. After an overnight incubation at 4°C with primary antibodies against ZO-1 (1:1000), claudin-1 (1:1000), PI3K (1:1000), AKT (1:1000), p-PI3K (1:1000), p-AKT (1:1000), and GAPDH (1:5000), secondary antibodies were incubated and visualized using Enhanced Chemiluminescence (catalog no. P0018; Beyotime Institute of Biotechnology; Cat #ab276131; Abcam, Proteintech, 13409-1-AP; GeneTex, GTX108613). Quantity-One software version 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for densitometry analysis.

Data are expressed as mean ± standard deviation (SD). Statistical analysis involved one-way ANOVA for multi-group comparisons and Tukey’s multiple comparison tests for pairwise comparisons. A p-value <0.05 was considered indicative of statistical significance.

Active components for ACZF were screened from TCMSP database. A total of 159 components and 2124 targets were acquired, following duplicate removal, 782 unique gene targets remained. After merging and eliminating the duplicate targets, there were 2501 targets closely related to RE obtained from GeneCards and OMIM database. By using online Venn diagram tool, 376 intersection targets were obtained (Figures 1a). The drug, ingredients, targets and disease were imported into Cytoscape 3.8.2 to construct the drug-component-target-disease network diagram (Figures 1b), which included 545 nodes and 3,716 edges (Figures 1b). Network analysis indicated extensive interactions among chemical components, reflecting ACZF’s complex mechanism involving multiple pathways and targets. Notably, compounds like Quercetin, Kaempferol, Isorhamnetin, and Luteolin had prominent Degree values, highlighting their pivotal roles in ACZF’s efficacy against RE.

Target proteins were uploaded to the STRING database to establish a PPI network (Figures 2a), consisting of 301 nodes and 1,382 edges, with an average degree of 9.18. Using the Centiscape 2.2 plugin for topological analysis, 52 candidate targets with a degree ≥9.18 were initially identified. Further refinement based on a degree ≥11.15, betweenness ≥50.58, and closeness ≥0.01 isolated 15 core genes: SRC, STAT3, AKT1, HSP90AA1, EGFR, ESR1, EP300, CYNNB1, HRAS, MARK1, MARK3, JUN, BCL2, CCND1, and CASP3 (Figures 2b).

Based on the results of the DAVID database study, the molecular mechanisms by which ACZF may alleviate RE are mostly involved in the negative control of protein phosphorylation, apoptosis, and cell migration. Protein binding, identical protein binding, and ATP binding were the foci of molecular functions (MF), whereas membrane rafts, receptor complexes, cytoplasm, and the nucleus were the primary cellular components (CC) (Figures 3a). The primary related pathways were concentrated in the AGE-RAGE signaling pathway, PI3K/AKT pathway, RAP1 pathway, MAPK pathway, and FOXO signaling pathway (Figures 3b).

Radiation exposure significantly elevated DAI scores, confirming the effective induction of radiation colitis. In contrast, ACZF treatment markedly improved pathological conditions and reduced DAI scores, enhancing the survival rates of mice with RE and demonstrating its therapeutic efficacy (Figures 4a). Measurements of colon lengths, vital for assessing colon inflammation, showed recovery in ACZF-treated mice relative to those with radiation-induced colitis (Figures 4b). H&E staining evaluated morphological and histopathological changes, indicating that radiation caused substantial mucosal ulceration, inflammatory cell infiltration, crypt damage, and epithelial destruction, all significantly alleviated by ACZF. However, based on Masson staining, no significant differences in smooth muscle fibers were observed among the control group, model group, and ACZF-treated group. (Figures 4c). ELISA assessments confirmed that ACZF modulated the inflammatory cytokine profile, reducing pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and increasing anti-inflammatory cytokine IL-10 levels (Figures 4d). These results further validate ACZF’s capacity to mitigate radiation-induced colitis in mice.

To assess the impact of ACZF on epithelial barrier integrity, we examined the expression levels of tight junction (TJ) proteins, specifically ZO-1 and Occludin-1. As shown by the Western blot results, the expression of ZO-1 and Claudin-1 in the colon of RE model mice was significantly reduced compared to the Control group, indicating impaired intestinal barrier function (Figures 5a). Remarkably, the administration of ACZF effectively reversed this condition. Similar results were obtained using immunohistochemistry (Figures 5b). These results indicated that ACZF could effectively restore intestinal barrier function in mice with radiation enteritis.

Initial network pharmacology analysis suggested a possible interaction of ACZF with the PI3K/AKT pathway. To determine if ACZF influences intestinal barrier function via AKT and to evaluate its impact on AKT activation, we conducted a WB analysis. The results indicated that the levels of p-PI3K and p-AKT in the colon mucosal tissues of the model group were markedly elevated, implying that radiation may have activated the PI3K/AKT signaling pathway. Treatment with ACZF markedly reduced this activation (Figures 6a). Additional tests involving simultaneous treatment with ACZF and the PI3K activator YS-49 in a radiation colitis model demonstrated that ACZF’s suppression of the PI3K/AKT pathway, along with its impact on epithelial TJ protein expression and inflammatory marker levels in the colon mucosa, could be reversed (Figures 6b, c), confirming that ACZF ameliorates colitis by inhibiting the PI3K/AKT pathway.