BIECs-21, previously immortalized by our laboratory (17), and Vero cells (African green monkey kidney epithelium, kindly provided by Prof. Lei He, Henan University of Science and Technology) were utilized. NC tachyzoites were obtained from Prof. Qun Liu at China Agricultural University.

BIECs-21 were maintained in DMEM (Gibco, USA) supplemented with 10% FBS (Cegrogen, Germany) and 500 μg/mL G418 (Beyotime, China) at 37 °C under 5% CO₂. Vero cells were cultured in DMEM with 10% FBS for NC propagation. Infection models were established by inoculating BIECs-21 with NC tachyzoites at a 3:1 parasite-to-host cell ratio. For pretreatment experiments, BIECs-21 were incubated with the optimal dose of GCP (1,000 μg/mL, Supplementary Figure 1) for 12 h prior to NC exposure.

BIECs-21 were seeded in 96-well plates and divided into four groups: control (C), GCP-treated (GCP), NC-infected (NC), GCP-pretreated + NC-infected (GNC). After 12 h GCP incubation and 4 h NC infection (MOI = 3:1), cell viability was assessed using CCK-8 (Solarbio, China). Following reagent addition (10 μL CCK-8 + 90 μL DMEM), plates were incubated at 37 °C for 1.5 h. The absorbance of the supernatant was measured at 450 nm using a microplate reader (Thermo, USA).

BIECs-21 cells were seeded in a 96-well plate and the lactate dehydrogenase (LDH) activity in the culture supernatant was quantified using a Lactate Dehydrogenase Assay Kit (Nanjing Jiancheng, China). After incubation at 37 °C for 1 h, the absorbance of the supernatant was measured at 490 nm to assess membrane integrity.

Total RNA from four experimental groups (C, GCP, NC, GNC) was extracted with TRIzol (Ambion, USA). RNA libraries were prepared using NEBNext Ultra II reagents (New England Biolabs) and sequenced on Illumina NovaSeq 6,000 (150-bp paired-end) by Personalbio (Shanghai, China). Differentially expressed genes (DEGs) were identified with |log₂FC| > 1 and p < 0.05. Functional enrichment analyses were performed using topGO (v2.40.0) for Gene Ontology and ClusterProfiler (v3.16.1) for KEGG pathways.

Total RNA was extracted from cells or tissues using TRIzol (Ambion). Specific primers were designed and synthesized by Sangon Biotech (Shanghai, China), and the primer sequences can be found in the Supplementary Table 1. cDNA was synthesized using a reverse transcription kit (Vazyme, China). SYBR Green-based qPCR (Vazyme, China) was conducted on a Bio-Rad system(Bio-Rad, USA) with β-actin as endogenous control. Relative expression was calculated via 2^^(-ΔΔCt) method.

Fifty female Kunming mice (6 ~ 8 weeks old) were housed under controlled conditions (20 ~ 24 °C, 40 ~ 70% humidity, 12 h light/dark cycle) with ad libitum access to food and water. Mice were randomized into five groups (n = 10/group): control (no treatment), NC-infected (NC), low-dose GCP (100 mg/kg) + NC infected (NC + L), medium-dose GCP (200 mg/kg) + NC infected (NC + M), High-dose GCP (400 mg/kg) + NC infected (NC + H). GCP was administered via drinking water for 25 days pre-infection. All groups except controls were intraperitoneally inoculated with 1 × 10⁶ tachyzoites/mouse. GCP supplementation continued for 8 days post-infection.

Brain and duodenal tissues collected 8 days post-infection were homogenized for genomic DNA extraction. Absolute qPCR was performed using standardized DNA (200 ng/μL) to quantify parasite load. Primer sequences for NC: F: 5’-ACTGGAGGCACGCTGAACAC-3′, R: 5’-AACAATGCTTCGCAAGAGGAA-3′.

Total proteins extracted with RIPA buffer (Solarbio, China) were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% BSA, membranes were probed with primary antibodies followed by HRP-conjugated secondary antibodies. Signals were detected using ECL substrate (Millipore, USA) and analyzed with Image J.

Data are presented as mean ± SEM. Group comparisons employed Student’s t-test (pairwise) or one-way ANOVA with Duncan’s post hoc test (SPSS v19.0). Graphical outputs were generated using GraphPad Prism 8. Significance thresholds: *p < 0.05, **p < 0.01, ***p < 0.001.

No morphological changes were observed in BIECs-21 among control, NC group, GCP group or GNC group (Figure 1A). However, cell viability significantly increased in the GCP group and decreased in the NC group compared to controls. GCP pretreatment markedly inhibited NC-induced viability reduction (Figure 1B). Furthermore, LDH activity was significantly lower in the GCP group than in controls, while the NC group exhibited a trend toward elevated LDH. Notably, GCP pretreatment (GNC group) substantially reduced LDH activity relative to the NC group (Figure 1C).

Transcriptomic analysis revealed high reproducibility and intergroup correlation (Figure 2A). Comparative DEG analysis identified significant differences between G (GCP-treated) vs. C (control), NC vs. C, and GNC vs. NC groups, with pronounced changes in G vs. C and GNC vs. NC (Figure 2B). Specifically, 688 DEGs (226 upregulated, 462 downregulated) were detected in G vs. C, 115 DEGs (69 upregulated, 46 downregulated) in NC vs. C, and 575 DEGs (216 upregulated, 359 downregulated) in GNC vs. NC (Figures 2C–G).

GO enrichment analysis indicated that NC altered immune-related processes in BIECs-21, including chemokine-mediated signaling, neutrophil chemotaxis and inflammatory response. GCP exerted protective effects by modulating stimulus response regulation, signal transduction and cell proliferation (Figures 3A–C). KEGG pathway analysis highlighted significant enrichment of DEGs in IL-17 and TNF signaling pathways across groups (Figures 3D–F). Cross-comparison of these pathways revealed upregulated inflammatory genes (FOSB) in NC vs. C and downregulated CCAAT/enhancer-binding protein β(C/EBPβ) and FOS in GNC vs. NC.

Cluster heatmaps of top 50 DEGs (Figures 4A–C) and qRT-PCR validation confirmed transcriptomic data consistency. Compared to controls, GCP downregulated IL1RAP, IL1RL1, IL18R1, IL4R, IL33, IL6, ACKR3, and NR4A1 mRNA (Figure 4D), and NC upregulated CXCL1, CXCL2, and CXCL3 mRNA (Figure 4E). GNC downregulated IL1RL1, IL6, and NR4A1 mRNA versus NC (Figure 4F). Additionally, FOSB mRNA was elevated in NC vs. C, while C/EBPβ and FOS mRNA were reduced in GNC vs. NC (Figures 4G,H). These results suggest that NC exacerbates inflammation via FOSB upregulation, whereas GCP attenuates damage by suppressing C/EBPβ and FOS expression.

To validate the in vivo efficacy of GCP, this study established a NC-infected mouse model. In vivo, low-dose GCP (50 mg/kg) significantly increased body weight gain pre-infection and reduced post-infection weight loss (8 days post-infection) compared to untreated controls (Figure 5A). GCP improved survival rates (Supplementary Figure 2) and mitigated intestinal hemorrhage and swelling (Figure 5B). Further analysis of parasite load in the duodenum and brain tissues of mice across groups revealed that, compared with the NC group, the low-dose GCP treatment significantly reduced parasite load in these two tissues (Figure 5C).

To investigate the mechanism by which GCP alleviates NC-induced intestinal damage in mice, this study detected relevant biomarkers in duodenal tissues based on transcriptome sequencing. qRT-PCR analysis revealed significantly higher FOSB mRNA levels in the NC group compared with uninfected controls. C/EBPβ and FOS mRNA levels were significantly lower in GCP-treated groups versus the NC group. However, no significant differences in IL-6 or NF-κB mRNA levels were observed between the high-dose GCP group and the NC group (Figures 6A–C). Western blot analysis of proteins associated with the IL-17 and TNF signaling pathways revealed that the expression levels of TNF-α, p-NF-κB/NF-κB, IL-17RA, and p-C/EBPβ in the NC group were significantly higher than those in the control group. These elevated protein levels were effectively attenuated by low-dose GCP intervention, whereas high-dose GCP exhibited no significant regulatory effects (Figures 6D–I).