All procedures involving animals in this research were conducted in accordance with the ethical standards of the institution or practice. The experiments were approved by the Ethics Committee for Animal Experiments of Foshan University (Ethical permit number: FOSU2008).

The highly pathogenic MDV Md5 strain and the MDV-transformed T-cell line MSB-1 (in suspension) were cultivated in our laboratory. The cells were nurtured in RPMI-1640 medium (Servicebio, China), supplemented with 10% FBS (Gibco, United States) and 1% antibiotic solution (penicillin 100 U/mL, streptomycin 100 g/mL; Beyotime, Jiangsu, China), and incubated at 37°C in a 5% CO₂ environment. Chicken embryonic fibroblasts (CEFs) were prepared from 9 to10-day-old specific pathogen-free (SPF) chicken embryos using conventional methods.

Cell Cytotoxicity/Viability Assay were performed using the CCK-8 Cell Counting Kit (Vazyme, Nanjing, China) in accordance with the manufacturer’s instructions as described previously (29). MSB-1 cells in the logarithmic growth phase were seeded in 96-well plates at a density of 5 × 10³ cells per well. Subsequently, the cells were exposed to various concentrations of NaB (0, 2, 4, 6, 8, and10 mM) for a duration of 24 h. The subsequent day, 10 μL of CCK-8 solution were introduced, followed by an additional incubation period of 2 h. Concurrently, the control cells were subjected to RPMI-1640 medium supplemented with 10% CCK-8.

Following the previously described method with some modifications (29, 30), plaque assays were performed. Once the monolayer of CEF cells in a 96-well tissue culture plate reached confluency, the treatment group was infected with 20 PFU of the Md5 virus per well for 1 h. After replacing the infection medium, NaB solution was added to a final concentration of 6 mM, and the cells were incubated for 24 h. Subsequently, the cells were covered with a 2% agarose gel containing an equal volume of 2× DMEM medium. For the prevention group, CEF cells were incubated with NaB solution (final concentration of 6 mM) for 24 h before viral infection, followed by the same infection protocol as the treatment group. The agarose gel was then applied. The number of cytopathic effect (CPE) lesions was observed daily and counted until no new CPEs were observed.

Local chicken Nanhai Ephedra chickens, devoid of any prior immunization, were procured from Nanhai Breeder Co. The chickens did not receive any vaccination procedures throughout the experiment. The NaB reagent was obtained from Sigma-Aldrich Chemical Company (303,410, Sigma Aldrich, Germany). Based on the drug dosage mentioned in the referenced literature (31) and subsequent conversions (32) of drug dosages, the approximate administration doses of the drug in the experiment were calculated using conversion factors for dose equivalence between chickens and other experimental animals (33). Building upon preliminary investigations, NaB was administered at a dose of 132 mg/kg.

To ensure adequate and consistent absorption of NaB in the chickens, intraperitoneal injection was employed during the experiment. In the three groups of NaB, prevention, and treatment, chickens of each group were injected with 100 μL of NaB solution. The groups of NaB and prevention were received the NaB injection 7 day in advance (day −7), the duration of NaB administration was 35 days in total. The group of treatment was received the NaB injection at the same time as MD5 infection (day 0), the duration of NaB administration was 28 days in total (Figure 1). While the two groups of Blank and Md5 were injected with an equal volume of PBS solution. The chickens in each group underwent bi-daily weighing, allowing for necessary adjustments in the injected amount of NaB based on their weights. A total of 120 1-day-old chickens were randomly allocated into five groups (n = 24) and housed in separate enclosures. Animals infected with non-human infectious viruses are isolated from healthy animals throughout the experiment to prevent transmission. Intraperitoneal injection of Md5, 2,000 PFU/chicken, was administered to the chickens, as outlined in previous literature (34). At each time point of dissection, five chickens were randomly selected from each group for sampling. Each spleen sample was tested with five replicates. Control groups included the Md5 group, NaB group, and blank group. Following Md5 infection, chickens were monitored daily for manifestations of MD, such as leg paralysis, head and neck rotation, blindness, tumor formation, and mortality. At the conclusion of the experimental period, surviving chickens were euthanized in a humane manner and subjected to dissection, focusing on organs like the liver, spleen, kidney, and heart, to assess the presence of internal tumors. The spleen index was calculated according to the formula, spleen index = weight of spleen (g) / body weight (g) × 100 (30). All chickens, whether deceased or alive, displaying MD symptoms or visibly detectable tumors were classified as MD cases. Cases that succumbed prematurely due to compromised autoimmunity or environmental factors within a week prior to the disease onset were excluded from analysis. Throughout the duration of animal experiments, restricted access, record keeping, animal isolation, sanitation and disinfection, sample collection, waste management, etc. have followed the relevant biosafety regulations, as detailed in Supplementary File 1.

On 7, 14, 28, and 50 days post-infections (dpi), Chicks from the respective experimental cohorts were humanely terminated through exsanguination via the ruptured jugular vein and used diazepam as a sedative drug. For each time point, a minimum of three chicks from each group were euthanized, and spleen tissues were meticulously procured. The spleens were carefully weighed, and the spleen index was ascertained by computing the ratio of spleen weight to body weight. The procured tissues were subsequently delicately placed within 1.5 mL sterile test tubes that were preloaded with 1 mL of RNA Keeper Tissue Stabilizer (R501, Vazyme, Jiangsu, China). According to the reagent instructions, the samples were incubated overnight at 4°C and stored at −80°C as previously described (35).

Spleen specimens were procured from chickens at various time intervals to facilitate the histopathological analysis. The specimens were meticulously preserved in a formaldehyde solution. Subsequently, the tissues underwent a process of alcohol dehydration, after which 5 μm sections of paraffin-embedded spleen tissue were meticulously stained with the classic hematoxylin and eosin (H&E) technique (36). Following staining, the sections were subjected to dehydration using anhydrous ethanol, and subsequently rinsed in xylene to enhance transparency. Ultimately, the transparent sections were meticulously embedded in a neutral resin medium and examined under a light microscope.

Total RNA was isolated from chicken spleen tissues utilizing TRIzol reagent (B511311, Sangon biotech, Shanghai, China). Subsequently, the RNA samples were subjected to reverse transcription using Hiscript® III All-in-One RT SuperMix Perfect for qPCR reverse transcriptase (Vazyme, Nanjing, China). Each sample was employed to evaluate the expression of MDV genes and host mitochondrial apoptosis pathway genes. The quantitative fluorescence detection instrument employed was the Thermo QuantStudio 5, and the qPCR reaction system consisted of a 20 μL volume. This system encompassed 10 μL of Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix, 0.5 μL each of upstream and downstream primers (10 μmol/L), 1 μL of cDNA, and the remaining volume was adjusted to 20 μL using ddH₂O. Following pre-denaturation at 95°C for 2 min, amplification was conducted for 40 cycles (95°C for 10 s, 60°C for 30 s). Each sample was tested in triplicate, and the data are presented as mean ± SD. Relative quantification of the genes was determined using the 2^−∆∆CT method. The primer sequences used are depicted in Table 1 (37, 38, 40).

Apoptosis was detected in the spleens of each group at 7 dpi using the TUNEL apoptosis detection kit (40306ES20, Yeasen, Shanghai, China) (39). Tissue sections were deparaffinized in xylene, followed by immersion in anhydrous ethanol for 5 min and sequential washes in gradient ethanol (90, 80, 70%) for 1 time each, with a duration of 3 min per wash. After rinsing the sections with PBS, proteinase K solution was added and incubated at room temperature for 20 min. Subsequently, the sections were washed with PBS and incubated with TDT enzyme, dUTP, and buffer solution. The sections were then incubated at 37°C without light for 60 min. After washing the samples three times with PBS, DAPI solution was applied to stain the cell nuclei for 10 min at room temperature. Following sample washing, fluorescence microscopy was used for examination and image acquisition. DAPI emits blue light with an excitation wavelength of 330–380 nm and an emission wavelength of 420 nm, while FITC emits green fluorescence with an excitation wavelength of 465–495 nm and an emission wavelength of 515–555 nm.

To assess the impact of NaB on cellular migration, the MSB-1 cells were cultivated in the upper compartment of 8 μm Transwell plates, with a density of 10⁵ cells per well, and immersed in 200 μL of serum-free medium. In the lower compartment, a 10% FBS solution was carefully introduced. NaB was then introduced into the medium to reach a final concentration of 6 mM. The wells were subsequently incubated under optimal conditions of 37°C and 5% CO₂ for a duration of 24 h. Following the incubation period, the cells were meticulously fixed with a formaldehyde solution for a period of 30 min. Subsequently, a 0.4% crystal violet solution was employed to stain the cells for a duration of 10 min at ambient temperature. To evaluate the efficacy of the treatment, five microscopic fields were randomly selected, and images were acquired utilizing a fluorescence microscope (Revolve FL, Echo Laboratories, United States) with a magnification of 100 × .

To assess the impact of NaB on MSB-1 clone formation, a colony formation assay in soft agar was conducted. The experiment was conducted according to the method described by Stanley Borowicz et al. (41). The procedure involved preparing the bottom gel layer by mixing 1.2% agar with 2× medium in equal volumes, which was then spread as the bottom layer in 6-well plates. The plates were incubated at 37°C for 30 min to allow the gel to solidify. Subsequently, 6 × 10³ MSB-1 cells were mixed with 0.7% agar and added to the plates as the upper gel layer. In the NaB group, a NaB solution was added to the upper gel layer to achieve a concentration of 6 mM. To prevent excessive drying, 100 μL of complete medium was added after solidification. After a two-week incubation period, the colonies were observed under a microscope, manually counted, and photographed for analysis.

To assess the impact of NaB on MSB-1 apoptosis, a flow cytometry assay was conducted. Cells were treated with or without NaB at a final concentration of 6 mM for 24 h. Subsequently, the cells were suspended and stained with Annexin V-FITC and propidium iodide (PI) according to the instructions provided with the Annexin V-FITC/PI Apoptosis Kit (Elabscience, China), as described by Han et al. (42). The resulting data were analyzed using Flow Jo V10 software for further interpretation.

The data from this experiment were presented as mean ± S.D. Statistical analysis was performed using SPSS 26.0 (SPSS Inc., Chicago, United States). The Morbidity and mortality were analyzed by SPSS Chi-Square comparing between each group and results are presented in Table 2. For normally distributed data, the experimental groups were compared using one-way ANOVA followed by the Tukey test. In the case of non-normally distributed data, the Dunnett’s T3 test was applied. The Dunnett’s T3 test was chosen because it is robust to the analysis of unequal variances (43). For the comparison of two treatments, the Student’s two-tailed t-test was employed. A significance level of p < 0.05 was considered statistically significant. Data visualization was accomplished by GraphPad software (CA, United States).

The safety concentration of NaB on CEF cells was evaluated using the CCK-8 assay as a preliminary step. As shown in Figure 2A, the cell viability was not affected in the experimental groups compared to the control group across different concentrations of NaB.

The impact of NaB on MDV replication was assessed using a plaque assay. As shown in Figure 2B, both the prevention and treatment groups exhibited a general trend where a higher concentration of NaB resulted in a lower number of CPE. In the prevention group, the lowest number of CPE was observed at the concentration of 8 mM, while in the treatment group, the lowest number of affected cells was observed at 6 mM.

The tumor tissues were collected from each group and subjected to histological examination. Cumulative morbidity, mortality, and tumor incidence were evaluated for all groups of birds, except for those that died prematurely due to non-specific causes prior to the Md5 challenge. Compared to the Md5 group, both the prevention group and the treatment group that received intraperitoneal injection of sodium butyrate had lower tumor incidence and mortality rates (Table 2).

Necropsy was performed on all chickens to assess the presence of tumors in internal organs, with the findings depicted in Figure 3. At 50 dpi, the Md5 group exhibited visually detectable tumor lesions in the spleen, which displayed significant enlargement compared to the other groups (Figure 3A). The presence of tumorous lesions in the spleen of the Md5 group results in an increase in both the weight and volume of the spleen. Figure 3K demonstrates a difference in splenic index in each group. No significant difference in spleen index was observed among the prevention group, treatment group, and blank group, which indicates the NaB in prevention and treatment groups did not cause splenomegaly.

The MDV immediate-early gene ICP4 is a transcription activator that is an essential factor for early and late promoter for viral gene expression (44), while another immediate-early gene ICP27 is known to be required for efficient expression of some viral DNA replication-related early genes and late viral genes as well as for virus growth (14). The quantification of ICP4 and ICP27, along with MDV oncogene Meq, was assessed in the spleen using qPCR. Regarding the ICP4 gene, treatment with NaB led to a substantial downregulation at all four time points in both the prevention and treatment groups, relative to the Md5 control group. Interestingly, at 28 dpi and 50 dpi, the prevention group displayed a significantly greater inhibition of ICP4 expression compared to the treatment group, as illustrated in Figure 4A. Regarding the ICP27 gene, no significant changes in expression were observed at 7 dpi among the three groups. However, a significant suppression was detected in both the prevention and treatment groups at 14 dpi, when compared to the Md5 control group. It is noteworthy that this suppression effect was reversed at 28 dpi and 50 dpi, as depicted in Figure 4B. In relation to the oncogene Meq, a notable reduction in relative expression was observed in both the prevention and treatment groups at 7, 14, 28, and 50 dpi, when compared to the Md5 control group. Furthermore, at 28 dpi, the treatment group exhibited a significantly more pronounced inhibition of Meq expression compared to the prevention group, as depicted in Figure 4C.

Effect of NaB on tumor-suppressor genes in MD5 infected chicken spleen, such as PTEN, TGF-β, and ARRDC3, within each experimental group are depicted in Figure 5. Notably, a profound upregulation of PTEN was observed in the prevention group at 14, 28, and 50 dpi, as compared to the Md5 control group (Figure 5A). Furthermore, at 50 dpi, the expression of TGF-β surpassed that of the other three groups within the prevention group, indicating a potential role in tumor suppression (Figure 5B). Interestingly, the expression of ARRDC3 exhibited a significant upregulation at both 7 and 50 dpi, highlighting its involvement in the prevention group when compared to the Md5 control group.

To explore the apoptosis in the spleen at 7 dpi after MDV infection, we conducted a TUNEL assay on spleen tissues. The results, as depicted in Figure 6, reveal a significant increase in green fluorescence signals within both the prevention and treatment groups, as compared to the Md5 group. Figure 6B further presents a quantitative representation of the extent of apoptosis in splenocytes. Remarkably, the treatment and prevention groups exhibited higher levels of apoptosis, followed by the Md5 group.

In order to ascertain the suppressive impact of NaB on cellular proliferation, a cohort of MSB-1 cells were subjected to incubation with varying concentrations of NaB for a duration of 24 h. The outcomes of this experimental endeavor are depicted in Figure 7. Upon administration of a 2 mM concentration of NaB, no substantial alteration in the vitality of MSB-1 cells was observed. However, once the concentration of NaB reached or surpassed 4 mM, a propensity to impede the proliferation of MSB-1 cells became readily apparent.

In order to assess the impact of NaB on cellular migration, we conducted a Transwell assay on MSB-1 cells. Following a 24-h migration period, cells were stained and subsequently examined under a fluorescence microscope. Our observations revealed a noteworthy suppression of cell migration in the experimental group, wherein an additional 6 mM NaB was administered (Figures 8A–C). To further explore the influence of NaB on cellular migration, a soft agar colony assay was performed. Remarkably, the treatment of MSB-1 cells with NaB resulted in a considerable reduction in both clone size and cell count when compared to the control group (Figures 8D–F).

In order to assess the occurrence of programmed cell death in MSB-1 cells, a flow cytometry assay was conducted subsequent to the administration of various concentrations of NaB for a duration of 24 h. The concentrations tested ranged from 0 to 10 mM (Figure 9A). The resulting data was analyzed and represented graphically in Figure 9B, highlighting the distribution of cells within the quadrants Q1, Q2, Q3, and Q4. These quadrants are indicative of the quantities of dead cells, late apoptotic cells, early apoptotic cells and lived cells, respectively.

To elucidate the molecular mechanism underlying the induction of apoptosis in MSB-1 cells by NaB, we assessed alterations in the transcriptional levels of cytokines associated with the mitochondrial apoptotic pathway. As a control, we incorporated Mdivi-1, a known inhibitor of mitochondrial outer membrane permeabilization that consequently retards apoptosis (45). The experimental framework comprised three distinct groups: NaB, mock, and Mdivi-1. The qPCR analysis revealed that NaB significantly enhanced the expression of TNF-α, FADD, and TRADD (Figures 10A–C). Additionally, we observed a decrease in the BCL2/Bax ratio in the NaB group, with the former being an anti-apoptotic protein and the latter a pro-apoptotic protein (Figure 10D). Furthermore, compared to the control group and Mdivi-1 group, the NaB group exhibited a significant increase in the activity of Bak, CytC, Apaf-1, Caspase-9, Caspase-8, Caspase-3, and decreased expression of PARP in MSB-1 cells (Figures 10E–K).

The expression of several tumor suppressive genes, such as PTEN, ARRDC3, and TGF-β, was investigated in MSB-1 cells after 24 h of NaB treatment. PTEN, an extensively studied tumor suppressor (46), plays a crucial role in tumor suppression. Down-regulation of ARRDC3 promotes tumor cell proliferation and inhibits apoptosis (47). In the early stages of tumor progression, TGF-β acts as a tumor suppressor (48). Our experimental results demonstrated that NaB enhances the expression of these three tumor suppressive genes mentioned above. Therefore, it was postulated that NaB may hinder tumor progression by stimulating the expression of anti-tumor genes (Figures 10L,N). The oncogene Meq, encoded by MDV, plays a critical role in lymphocyte transformation (49). Notably, our experimental findings indicate that NaB substantially inhibits Meq expression (Figure 10O).