Female BALB/c mice measuring 18–25 g at 6–8 weeks of age were acquired from Ningxia Medical University’s Experimental Animal Centre. The mice were kept at 22°C in a specialized pathogen-free (SPF) environment. All experimental protocols, including euthanasia (Sodium pentobarbital (≥100 ug/kg IP) combo followed by exsanguination), were strictly carried out in accordance with the Ningxia Medical University’s Animal Welfare Guidelines.

Our laboratory was able to effectively synthesize and recombine the Eg.P29 gene from Echinococcus granulosus obtained from Escherichia coli (16). The recombinant strain harboring the Eg.P29 gene was initially streaked onto LB agar plates supplemented with kanamycin and incubated overnight at 37°C to obtain single colonies. A single colony was inoculated into kanamycin-containing LB broth and cultured at 37°C with shaking at 200 rpm for approximately 12 h until visible turbidity was observed. The culture was scaled up until the bacterial cells reached the logarithmic growth phase, as indicated by an OD600 value of 0.6, at which point isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce Eg.P29 expression. Following induction, the cells were harvested by centrifugation, treated with lysozyme and phenylmethylsulfonyl fluoride (PMSF), and lysed by sonication. A pre-equilibrated resin suspension was combined with the supernatant and incubated overnight at 4 °C under mild agitation. The recombinant Eg.P29 protein was purified using affinity chromatography. Subsequently, endotoxins were eliminated from purified protein and used in animal immunization experiments.

In the rEg.P29 vaccination experiment, 21 female BALB/c mice (6–8 weeks old) were randomly assigned to three groups: phosphate-buffered saline (PBS), Freund’s complete adjuvant (FCA), and rEg.P29+FCA. The mice in the PBS group received 100 uL of PBS, the FCA group received 20 μg of FCA diluted with 100 uL of PBS, and the rEg.P29+FCA group received 20 μg rEg.P29 and 20 μg of FCA in 100 uL of PBS. To minimize protein degradation during emulsification, the adjuvant and PBS were pre-emulsified briefly before adding the proteins, and the protocol was conducted on ice. The emulsified mixture was then loaded into a 1 mL syringe for immunization. The immunization protocol consisted of two stages, primary and booster immunizations. For booster immunization, Freund’s incomplete adjuvant (FIA) was used instead of FCA. Both primary and booster immunizations were administered via a subcutaneous three-point injection.

Mice were euthanized by cervical dislocation and soaked in 75% alcohol. Sterile spleens were then obtained by removing the epidermis. Lymphocytes were isolated from the spleen using a mouse spleen lymphocyte separation medium kit (Tianjin HaoYang Biological Manufacture Co., Ltd., China). Subsequently, CD4⁺T cells were purified from the lymphocytes using a mouse Cells isolation kit (Miltenyi Biotec, Inc., Cologne, Germany), briefly, lymphocytes (1×10⁷ cells) were labeled with Biotin-Antibody Cocktail in 40 μL MACS buffer (4°C, 5 min), followed by incubation with Anti-Biotin MicroBeads (20 μL in 20 μL buffer; 4°C, 10 min). Cells were separated through pre-wetted MS columns mounted on a magnetic stand over ice. After centrifugation (350 × g, 10 min, 4°C), purified cells were resuspended in complete RPMI-1640 medium for subsequent experiments. Naive CD4⁺T cells were further purified using a mouse naive CD4⁺T cells isolation kit (Miltenyi Biotec, Inc., Cologne, Germany), specifically, lymphocytes (1×10⁷ cells) were first labeled with Naive CD4⁺ T Cell Biotin-Antibody Cocktail in 40 μL MACS buffer (4°C, 5 min). Subsequently, 20 μL Anti-Biotin MicroBeads and 10 μL CD44 MicroBeads were added in 20 μL buffer (4°C, 12 min). Cells were separated through ice-cooled MS columns pre-rinsed with MACS buffer, with sequential loading of cell suspension interrupted by buffer washes. Target cells were collected by centrifugation (350 × g, 10 min, 4°C) and resuspended in complete RPMI-1640 medium”.

The naive CD4⁺T cells were cultured in 24-well plates with 5 × 10⁵ cells, (precoated with anti-CD3, 1 μg/ml, Thermo Fisher Scientific Co., Ltd, Xian,USA) and were then transfected with miR-378a-5p mimics (60 nM, Shanghai GenePharma Co.,Ltd) and inhibitor (150 nM, QIAGEN, Shanghai GenePharma Co.,Ltd) using HiPerFect transfection reagent (QIAGEN, USA). After 6 h, soluble anti-CD28 (0.2 μg/ml, Thermo Fisher Scientific Co., Ltd, USA) was added along with cytokines that differentiate naive CD4⁺T cells into different T-cell subtypes. For Th1 cells, IL-2 (20 ng/ml, BioLegend, Inc, California, USA.), IL-12 (50 ng/ml, BioLegend, Inc, California, USA.), and anti-IL-4 antibody (10 ng/ml, BioLegend, Inc, California, USA) were added to the well plates for 48 h. To differentiate the cells into Th2 cells, IL-2 (20 ng/ml, BioLegend, Inc, California, USA), IL-4 (10 ng/ml, BioLegend, Inc, California, USA.), and anti-IL-IFN-γ antibody (10 ng/ml, BioLegend, Inc, California, USA.) were added to the well plates for 48 h.

Three miRNA target prediction databases were used, namely miRWALK (version 3.0), miRDB (http://mirdb.org/miRDB/), and TargetScan (version 8.0).The analysis was conducted with the species parameter set to Mus musculus (mouse) and the miRNA sequence of interest was used as the query. The predicted target genes from each database were subsequently analyzed for common targets using the Venn diagram approach implemented in the Draw Venn Diagram tool. The overlapping target genes identified by all three prediction platforms were considered high-confidence candidates and visualized using a Venn diagram representation.

293T (Key Laboratory of Common Infectious Disease Prevention and Control in Ningxia) cells were reconstituted and cultivated in full media. Wild-type and mutant BRAF plasmids, in conjunction with miR-378a-5p mimics or negative controls (NC), were prepared for transfection. At 70% cellular confluence, transfection complexes were formulated by combining 10 μL DMEM with 0.3 μL transfection reagent and incubated at room temperature for 5 minutes. Concurrently, 10 μL of DMEM was combined with 0.1 μg of plasmid DNA (either wild-type or mutant BRAF) and miR-378a-5p mimics/negative control, followed by a 5-minute incubation at ambient temperature. The transfection reagent was amalgamated with the DNA solution, gently agitated by tapping the tube, and incubated for 10 minutes at an ambient temperature. The completed transfection complexes were uniformly placed into 293T cell culture plates and incubated in a humidified environment (37°C, 5% CO₂) for 48 hours. After incubation, cells were lysed by the addition of 100 μL of Passive Lysis Buffer (PLB) each well in a 96-well plate. Following careful pipetting, the plates were agitated at an ambient temperature for 15 minutes. Cell lysates were aliquoted into 1.5 mL microcentrifuge tubes and subjected to centrifugation at 13,400 ×g for 8 minutes at 4 °C. Supernatants were obtained for further analysis. To quantify luciferase activity, 100 μL of Luciferase Assay Reagent II was mixed with 20 μL of cell lysate in each well. The activity of firefly luciferase was quantified as an internal reference. Subsequently, 100 μL of Stop & Glo^® Reagent was administered to each well for the assessment of Renilla luciferase activity. Relative luciferase activity was determined by the ratio of Renilla luciferase activity to firefly luciferase activity.

Log in to the STRING(https://cn.string-db.org/) online database and enter the target gene and species source to obtain the proteins or signaling pathways interacting with the target gene.

TRIzol (Thermo Fisher Scientific, USA) was used to extract total RNA from CD4⁺T cells and naïve CD4⁺T cells. The First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) was used to synthesis cDNA for mRNA analysis. An ABI 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, USA) was used to conduct quantitative real-time PCR (qPCR). The Bestar^® Sybr Green qPCR Master Mix (DBI^® Bioscience) was used to assess the levels of mRNA expression. Both mRNA and miRNA were normalized using U6 and GAPDH as endogenous controls. The 2−ΔΔCT technique was used to determine the relative expression levels of genes. Table 1 lists the primer sequences utilized in this investigation.

Whole-cell lysis buffer (KeyGen Biotech, Jiangsu, China) was used to extract the total protein from the treated cells. A BCA protein quantification assay (KeyGen Biotech, Jiangsu, China) was used to measure the protein content. Protein samples were denatured for eight minutes at 100°C after being combined with 5X SDS-PAGE loading buffer (Shanghai Epizyme Biomedical Technology Co., Ltd). After being separated using 12% SDS-PAGE, the denatured proteins were put onto 0.2 μm PVDF membranes. For two hours at room temperature, the membranes were blocked using 5% skim milk in PBST. The membranes were incubated with primary antibodies against Phospho-B-Raf (Ser445), Phospho-MEK1/2, Phospho-p44/42(Erk1) (Tyr204)/(Erk2) (Tyr187) (D1H6G) Mouse mAb, (Cell Signaling Technology, USA)(1:1000), and β-actin, IFN-γ, IL-4 (Bioworld Biotech, Nanjing, China)(1:1000) overnight at 4°C. Following PBST washing, membranes were incubated for two hours at room temperature with secondary antibodies conjugated to horseradish peroxidase (HRP) (Cell Signaling Technology, Danvers, MA, USA)(1:10000). The ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Shanghai, China) and an ECL detection kit (KeyGen Biotech, Jiangsu, China) were used to observe protein bands. To normalize the results, β-actin was employed as an internal loading control.

To evaluate the purity of magnetically isolated naive CD4⁺ T cells, surface staining was performed using the following antibodies: FITC-anti-CD4, PE-anti-CD44, APC-anti-CD25, and PerCP-Cy5.5-anti-CD62L antibodies (all from BioLegend, San Diego, CA, USA).

The data was analyzed using GraphPad Prism software and given as mean ± SD. Student’s t-test was used to compare two groups. P-values < 0.05 were deemed statistically significant.

A His-tag purification kit was used to purify the rEg.P29 protein, and endotoxins were then eliminated ( Figure 1A ). The purified protein solution was sterilized by filtration through a 0.22-μm filter to meet experimental standards. Spleen lymphocytes were isolated from mice, and CD4⁺T cells. with >95% purity were obtained via magnetic-activated cell sorting (MACS). ( Supplementary Figure 1A ). Quantitative reverse transcription PCR (qRT-PCR) quantified miR-378a-5p expression in purified CD4⁺T cells. Comparative analysis revealed that rEg.P29, when administered with FCA or FIA, significantly upregulated miR-378a-5p expression in mouse splenic CD4⁺T cells compared with the PBS and adjuvant-only control groups ( Figure 1B ). These results suggest that miR-378a-5p was may involved in the differentiation of CD4+T cells in mouse spleen induced by rEg.P29.

To elucidate the specific mechanism by which rEg.P29-induced miR-378a-5p modulates CD4⁺T cells differentiation into Th1 cells, we used three databases (miRWalk, miRDB, and TargetScan) to predict miR-378a-5p target genes. Intersecting the target genes from these databases identified 108 overlapping genes ( Figure 1C ). Dual luciferase results showed that co-transfection of miR-378a-5p mimics in 293T cells significantly inhibited the luciferase activity of Braf wild-type 3’-UTR, validated BRAF as a target gene regulated by miR-378a-5p ( Figures 1D, E ). Using the STRING online database, we analyzed BRAF-interacting proteins and found a significant association between BRAF and the MAPK pathway ( Figure 1F ). Extensive research emphasizes BRAF’s crucial function in regulating the MAPK/ERK signaling pathway, which is critical for cell division and differentiation in many disorders (43–50). One week after using rEg.P29 immunized mice, spleen lymphocytes were isolated from mice, and CD4⁺T cells with >95% purity were obtained using magnetic bead sorting. mRNA and protein expression levels of BRAF, MEK1/2, and ERK1/2 in CD4⁺T cells were quantified by qRT-PCR and western blotting, respectively. The results demonstrated that rEg.P29 combined with FCA/FIA significantly downregulated BRAF, MEK1/2, and ERK1/2 expression in mouse splenic CD4⁺T cells compared to the PBS and adjuvant groups ( Figures 1G, H ). These findings suggest that the BRAF and MAPK/ERK signaling pathways are may involved in miR-378a-5p-mediated regulation of CD4⁺T cells differentiation induced by rEg.P29.

First, the optimal concentration of miR-378a-5p mimics or miR-378a-5p inhibitors were screened. ( Supplementary Figure 1B ). Then, Magnetic bead sorting was used to separate naïve CD4⁺T cells from mouse splenic lymphocytes, Naïve CD4⁺T cells were transfected with miR-378a-5p mimics, miR-mimics negative control (miR-mimics-NC), miR-378a-5p inhibitors, or miR-inhibitor negative control (miR-inhibitor-NC),following transfection, cells were polarized towards either the Th1 or Th2 lineage using lineage-specific cytokine cocktails. After 48 hours of stimulation, total RNA and total protein were extracted for downstream analyses. We found that miR-378a-5p mimics substantially increased Th1-associated cytokine IFN-γ and transcription factor T-bet expression compared to the control group, according to qRT-PCR analysis. In contrast, miR-378a-5p inhibitors drastically decreased these indicators. In contrast, miR-378a-5p mimics decreased the production of the Th2-associated cytokine IL-4 and transcription factor GATA-3, whereas miR-378a-5p inhibitors boosted their levels. ( Figures 2A, B ). Western blot analyses confirmed these findings, showing that miR-378a-5p mimics increased IFN-γ levels and decreased IL-4 levels, whereas miR-378a-5p inhibition had the opposite effects ( Figures 2C, D ).

To determine the mechanism by which rEg.P29 induces miR-378a-5p to regulate CD4⁺T cells differentiation toward the Th1 phenotype, we examined the expression of key molecules in the BRAF and MAPK/ERK pathways at the mRNA and protein levels using qRT-PCR and Western blot analyses. The results demonstrated that overexpression of miR-378a-5p significantly decreased the mRNA and protein expression levels of BRAF, MEK1/2, and ERK1/2. Conversely, miR-378a-5p inhibition significantly increased the expression of these molecules ( Figures 3A–D ). These findings suggest that under the induction of rEg.P29, miR-378a-5p targeted to BRAF regulation and initiated the differentiation of CD4⁺T cells in mouse spleen to Th1 direction, and MAPK/ERK pathway may be involved in this process.

Magnetic bead-based cell sorting was used to extract naïve CD4⁺T cells from mouse splenic lymphocytes. To elucidate the regulatory function of BRAF in CD4⁺T cells differentiation, we first Screened of OE-BRAF (overexpression - BRAF) via Recombinant Lentivirus Packaging ( Supplementary Figure 2A ),.and synthesized siRNA-BRAF (siRNA-induced BRAF silencing) by GenePharma company, then, determined the optimal transfection about OE-BRAF and siRNA-BRAF. we performed qRT-PCR to assess the effects of OE-BRAF and siRNA-BRAF. Our findings revealed that OE-BRAF significantly suppressed the expression of Th1-associated cytokine IFN-γ and its key transcription factor T-bet relative to control groups. In contrast, siRNA-BRAF significantly enhanced the expression of Th1 markers ( Figures 4A ). Conversely, OE-BRAF promoted the expression of the Th2-associated cytokine IL-4 and its transcription factor GATA-3, whereas siRNA-BRAF significantly attenuated their expression ( Figures 4B ). These observations were further corroborated by Western blot analysis, which demonstrated that OE-BRAF reduced IFN-γ production while augmenting IL-4 expression ( Figures 4C, D ). Taken together, these data indicate that OE-BRAF inhibits Th1 differentiation in primary CD4⁺T cells, whereas siRNA-BRAF promote Th1 differentiation.

Using qRT-PCR, the impact of co-transfection BRAF and miR-378a-5p on Th1/Th2 differentiation was evaluated. Th1-associated cytokine IFN-γ and transcription factor T-bet levels in CD4⁺T cells co-transfected with OE-BRAF and miR-378a-5p mimics were greater than those in cells transfected with OE-BRAF alone, but considerably lower than those in cells transfected with miR-378a-5p mimics alone. Contrastingly, when cells were co-transfected with the miR-378a-5p inhibitor and siRNA-BRAF, Th1 cytokineTh1-associated cytokine IFN-γ and transcription factor T-bet levels significantly decreased in comparison to cells transfected with siRNA-BRAF alone, but they did significantly rise in comparison to cells transfected with the miR-378a-5p inhibitor alone. ( Figures 5A–D ). Moreover, Th2-associated cytokine IL-4 and transcription factor GATA-3 levels were considerably increased by co-transfection with miR-378a-5p mimics and OE-BRAF in comparison to cells transfected with miR-378a-5p mimics alone but decreased in comparison to cells transfected with OE-BRAF alone. Conversely, Th2-associated cytokine IL-4 and transcription factor GATA-3 levels were significantly lower after co-transfection with the miR-378a-5p inhibitor and siRNA-BRAF, than after cells transfected with the miR-378a-5p inhibitor alone, but higher after co-transfection with siRNA-BRAF ( Figures 5E–H ).