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. 2026 Feb 18;105(6):106652. doi: 10.1016/j.psj.2026.106652

Curcumin ameliorates aflatoxin B1-induced brain toxicity in ducks by attenuating inflammation and PANoptosis

Limeng Zhou a,b,1, Ying He c,d,e,1, Yueyang Li a,b, Xudong Han a,b, Yanli You f, Ziqing Li a,b, Xuanliang Li a,b, Renbin chen a,b, Hang Pan a,b, Jianzhao Liao a,b, Zhaoxin Tang a,b, Zhiwen Wu a,⁎, Lianmei Hu a,b,⁎
PMCID: PMC13018940  PMID: 41861631

Abstract

Background

Aflatoxin B1 (AFB1) is a highly toxic mycotoxin that poses significant risks to poultry health, causing neurotoxicity and economic losses. However, the precise mechanisms underlying AFB1-induced brain injury, particularly regarding the role of PANoptosis—a coordinated cell death pathway—remain largely unexplored.

Aim

This study aimed to investigate the mechanisms of AFB1-induced neurotoxicity in ducks, focusing on the inflammatory response and PANoptosis, and to evaluate the potential neuroprotective effects of curcumin.

Methodology

A duck model of AFB1 intoxication was established. Ducks were divided into four groups: Control, Curcumin (Cur), AFB1, and AFB1+Cur. We assessed body weight, brain coefficients, and histopathological changes. Oxidative stress was measured via SOD activity and MDA content. The integrity of the blood-brain barrier (BBB) and the expression of genes and proteins related to the TLR4/NF-κB signaling pathway and PANoptosis were analyzed using Western blot, RT-qPCR, and immunofluorescence.

Results

AFB1 exposure resulted in significant growth retardation, degenerative brain damage (such as neuronal necrosis, cytoplasmic vacuolization, and nuclear shrinkage), reduced SOD activity by approximately 53%, and MDA levels increased by approximately 1.88-fold. It also compromised the BBB by downregulating ZO-1 and Occludin expression. Furthermore, AFB1 significantly upregulated the TLR4/NF-κB signaling pathway and key PANoptosis markers (ZBP1, RIPK1/3, Caspase-8, NLRP3, GSDMD). Curcumin attenuated the pathological damage of AFB1-intoxicated duck brain and was able to mitigate AFB1-induced neurotoxicity by inhibiting the phosphorylation of NF-κB and IκB, preventing IκB degradation, and downregulating the expression of key PANoptosis mediators.

Conclusion

AFB1 could trigger PANoptosis and activate the TLR4/NF-κB signaling pathway, leading to brain damage in ducks. Curcumin exerted a significant protective effect by inhibiting these pathways, suggesting its potential as a therapeutic agent for alleviating mycotoxin-induced neurological damage.

Keywords: AFB1, curcumin, duck brain, TLR4/NF-κB signaling pathway, PANoptosis

Introduction

The very lethal mycotoxin metabolite Aflatoxin B1 (AFB1) is generated by Aspergillus flavus (Wang et al., 2022). AFB1 is chemically stable and widely found in soil, animal feed, corn, peanuts, soybeans, nuts and other food crops (Sanchis et al., 1986), and therefore has become an important threat to global food safety. The latest survey findings indicate that the overall contamination rate of AFB1 in staple grains such as maize, groundnuts and wheat worldwide is approximately 29% (Goda et al., 2025). AFB1 not only directly jeopardizes the health of animals, but also remains in food of animal origin through the food chain, and eventually threatens human health (Wan et al., 2022). The toxicity mechanism of AFB1 is complex, and current research on AFB1 focuses on the inhibition of protein synthesis, suppression of the immune system, energy metabolism and oxidative stress, etc (Liu et al., 2023; Jiang et al., 2024). Poultry have been found to be extremely sensitive to AFB1. Ducks, in particular, exhibit extreme sensitivity to AFB1 compared to other species (Wu et al., 2021), making them a critical model for investigating mycotoxin-induced toxicity. Once AFB1-contaminated food is ingested, AFB1 accumulates in the liver, kidney and other organs of poultry, leading to serious biochemical and structural changes in these organs (Amminikutty et al., 2023; Liu et al., 2023). Despite the protective function of the blood-brain barrier, AFB1 can still accumulate within the brain and induce severe neurological deficits. Studies have found that AFB1-poisoned animals have obvious neurological symptoms (Aytekin et al., 2022; Ibrahim et al., 2022; Ranjbar et al., 2025). Animals suffering from neurotoxicity will significantly reduce their feed intake and lose weight, which will impair their ability to grow and cause financial losses (Qing et al., 2022; Stefanović et al., 2023). However, more research is needed to determine the precise mechanism by which AFB1 causes brain injury in ducks.

Exposure to environmental toxicants is frequently linked to neurodevelopmental injury driven by convergent mechanisms involving oxidative stress and inflammation (Fan et al., 2021; Nabi et al., 2024). Consistent with this, AFB1 exposure has been found to be associated with anxiety, depression, and learning disabilities, as well as various histopathological defects in brain tissue, and AFB1 could increase oxidative stress and inflammatory responses, which could have harmful effects on the brain (Souto et al., 2018; Wang et al., 2023). Superoxide dismutase (SOD) is a key antioxidant enzyme in living organisms (Saxena et al., 2022), while malondialdehyde (MDA) is a by-product of lipid peroxidation (Busch and Binder, 2017); both serve as important biomarkers for assessing oxidative stress.

Research has demonstrated that nuclear factor-κB (NF-κB) plays an important role in inflammation (Cartwright et al., 2016). An upstream regulator of NF-κB, Toll-like receptor 4 (TLR4) is crucial for inducing immunological responses (Capitani et al., 2023). Inflammation is significantly influenced by the TLR4/NF-κB pathway (Capitani et al., 2023), which when activated produces and releases pro-inflammatory cytokines (Pan et al., 2024). Recent evidence further highlights that various plant-derived bioactive compounds can exert potent anti-inflammatory effects by specifically targeting and inhibiting the NF-κB signaling cascade (Wang et al., 2024). Furthermore, it was shown that TNF-α and IL-1β directly cause endothelial cell damage and breakdown of the blood-brain barrier (Mantle and Lee, 2018).

A unique type of programmed cell death (PCD), which encompasses apoptosis (Caspase-3/7/8 mediated), cellular pyroptosis(GSDMD/NLRP3 mediated), and necroptosis (RIPK1/RIPK3/MLKL mediated), is PANoptosis (Pandian and Kanneganti, 2022). PANoptosis represents not merely the synchronous occurrence of these pathways, but rather a coordinated inflammatory cell death mechanism regulated by the PANoptosome complex, particularly the ZBP1-PANoptosome complex which incorporates components from multiple programmed cell death pathways (Samir et al., 2020). ZBP1 was originally reported to be an IFN-induced tumor-associated protein that has a Z-form and a RHIM (Schwartz et al., 2001). According to recent research, ZBP1 is an intrinsic indicator of viral infection and a target of viral evasion tactics that control pro-inflammatory reactions, inflammatory vesicle activation, and cell death (Karki et al., 2022; Gao et al., 2024). ZBP1 regulates inflammation through activation of NF-κB and IRF3 signaling pathways, after exposure to various noxious stimuli (Kaiser et al., 2008). Additionally, activation of ZBP1 promotes the assembly of the ZBP1-PANoptosome, which in turn triggers PANoptosis (Song et al., 2024). This process involves initiating Caspase-3-mediated apoptosis, driving NLRP3 and CSDMD-mediated cellular pyroptosis, and activating RIPK1, MLKL-mediated necroptosis (Cui et al., 2024).

Finding ways to prevent and treat AFB1 poisoning is essential since it poses a major risk to human health as well as animal life. Several studies have been conducted recently on the use of natural antioxidants to combat AFB1. Recent research has highlighted that natural antioxidants can protect the brain by regulating distinct cell death pathways such as ferroptosis, supporting the broad potential of phytochemicals in neuroprotection (Cheng et al., 2024). A naturally occurring monomer that is extracted from plants, curcumin has gained popularity in recent decades and is used extensively in animal husbandry. It has been shown to have therapeutic effects on a variety of diseases and to affect a wide range of biological targets (Zheng et al., 2020). More and more evidence suggests that curcumin's health effects are mediated by its antioxidant and anti-inflammatory qualities (Llano et al., 2019; Hong et al., 2022). Recent studies have further confirmed curcumin's efficacy in mitigating AFB1-induced toxicity in various duck organs, including the liver and intestines (Nabi et al., 2024; Pan et al., 2024; Wang et al., 2025). Although curcumin has low systemic bioavailability, its lipophilic nature allows it to effectively cross the blood-brain barrier and accumulate in brain tissue (Donadio et al., 2022). However, it is unknown if curcumin can be utilized as a neuroprotective medication to lessen AFB1-induced neurotoxicity.

Therefore, we hypothesized that AFB1 induces neurotoxicity in ducks by triggering TLR4/NF-κB-mediated inflammation and activating PANoptosis, and that curcumin administration can ameliorate this damage. The aim of this study was to elucidate these specific molecular mechanisms and evaluate the neuroprotective efficacy of curcumin in an avian model. To our knowledge, this is the first study to investigate the specific role of PANoptosis in AFB1-induced avian neurotoxicity and to further examine how curcumin protects duck brains.

Materials and methods

Animal model

Following their purchase from Guangzhou Huimin Poultry Co., Ltd, eighty one-day-old Sanshui white ducks were assigned to four groups at random using a random number table method, each consisting of twenty ducks: the control group, the Cur group, the AFB1 group and the AFB1+Cur group. Every duckling received drinking water and a standard feed. AFB1 was procured from Chengdu Aifeng Biotechnology Co., Ltd., China, with a purity of 98%. AFB1 powder was gavaged after being dissolved in 1% dimethyl sulfoxide (DMSO) solution, whereas curcumin powder (Nuzhui Traditional Chinese Medicine Bio-technology Co., Ltd.) was thoroughly mixed into the basal diet to achieve a final concentration of 400 mg/kg. The control group was maintained on standard feed, whereas the Cur group was given this curcumin-supplemented feed daily starting from day 1 of the experiment. The AFB1+Cur group received the same curcumin-supplemented diet (400 mg/kg) concurrently with the daily AFB1 gavage. The doses of curcumin and AFB1 were determined based on previous laboratory studies (Jiang et al., 2024; Su et al., 2025). The 400 mg/kg dose was selected based on previous studies in poultry which demonstrated this concentration to be safe and effective in mitigating oxidative stress and inflammation, without causing toxicity to the ducks. Body weights were measured every day during the feeding phase of the 21-day experiment. The Animal Protection and Ethics Committee of South China Agricultural University has approved the animal experimentation protocol for this study. (License number: 2020A004).

Analysis of body weight changes and brain coefficients in animals

A graph of weight change was generated based on daily recorded weight data. Brain tissue coefficient = brain weight/body weight * 100%.

Histopathological studies

Brain tissues were taken and stored in a 4% paraformaldehyde solution, which was replaced a day later. After xylene clearing and ethanol dehydration, the tissue underwent paraffin embedding and sectioning. The paraffin slices were stained using H&E staining and Nysted staining solution (Beijing Biyun Tian). Pathological changes in the brain were observed under an optical microscope (Leica, Germany) and photographed for documentation. To evaluate the extent of brain injury quantitatively, a semi-quantitative scoring system was applied. Histological damage (including neuronal degeneration, nuclear shrinkage, cytoplasmic vacuolization, and inflammatory cell infiltration) was graded on a scale of 0 to 3: 0 = normal (no damage); 1 = mild (<25% of the field affected); 2 = moderate (25–50% of the field affected); and 3 = severe (>50% of the field affected). The scores were assessed in five randomly selected fields (400 ×) per section.

Determination of SOD activity and MDA content in brain tissue

After the brain tissues stored at -80°C were thoroughly ground, SOD activity and MDA content in duck brains were determined using a kit (Nanjing Jiancheng Bioengineering Institute).

Western blotting analysis

Specific methods for protein immunoblot analysis were referenced to previous research methods (Zhong et al., 2021). Following extraction of total brain tissue protein, the sample was separated by SDS-PAGE and subsequently transferred onto a 0.45-micron polyvinylidene fluoride (PVDF) membrane. PVDF membranes were blocked with 5% non-fat milk for 1 hour at room temperature. The membranes were then incubated with specific primary antibodies overnight at 4°C (detailed information on antibody sources and dilutions is provided in Supplementary Table S2). After three washes with TBST, membranes were incubated with HRP-conjugated secondary antibodies (1:5000 dilution) for 1 hour at room temperature. Protein bands were visualized using an ECL detection system and quantified using ImageJ software. The relative protein expression levels were normalized to the density of the internal control (GAPDH or β-Tubulin). Each Western blot experiment was performed in triplicate independent biological replicates.

Real-time fluorescent quantitative PCR assay

The protocol for real-time quantitative PCR detection draws upon previous research (Pan et al., 2024). Total RNA was extracted from brain tissue using Trizol reagent, reverse transcribed into cDNA, and subsequently analysed for mRNA expression of key genes. The qPCR reaction was performed using the SYBR Green system with the following cycling conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Melt curve analysis was performed immediately after the amplification to validate the specificity of the PCR products. Relative gene expression was calculated using the $2^{-\Delta\Delta Ct}$ method, with GAPDH serving as the internal reference. The primer sequences for the target genes are listed in Table S1 (supplementary materials).

Immunofluorescence staining analysis

ZO-1, NF-κB, IL-1β, Caspase-3, GSDMD, and MLKL expression levels in the brain were evaluated using immunofluorescence. Images were captured using a fluorescence microscope (Leica, Germany). The fluorescence intensity was quantified using ImageJ software (NIH, USA). Three randomly selected fields of view per section were analyzed to determine the Mean Fluorescence Intensity (MFI), and background fluorescence was subtracted to ensure accuracy. Quantitative analysis results for the fluorescence intensities of ZO-1, NF-κB, IL-1β, caspase-3, GSDMD, and MLKL are presented in the Supplementary Materials.

Immunohistochemical analysis

Immunohistochemistry was used to analyze the brain's GFAP expression levels.

Statistical analyses

Every experiment was conducted in at least three independent replicates. The data results are presented as mean ± SD. Prior to analysis, data distribution normality was verified using the Shapiro-Wilk test, and homogeneity of variance was assessed using Levene's test. Data meeting these assumptions were analyzed using One-way Analysis of Variance (ANOVA) followed by Tukey’s post-hoc test for multiple comparisons. Statistical analysis of the experimental data was performed using GraphPad Prism 8.0 software. A p-value < 0.05 is considered statistically significant. Significant differences between the control and AFB1 groups are denoted by "*," while significant differences between the AFB1 and AFB1+CUR groups are denoted by #.

Results

AFB1 hinders growth and development in ducks

Daily measurements of the ducks' body weights were made during the experiment and it was found that the body weights of all ducks increased over time, but at different rates (Fig. 1 B, P < 0.01). The AFB1 group began to gain weight at a noticeably slower rate than the other groups on day 13. On day 21, body weights in the AFB1 group were markedly lower than those in the control and Cur groups, whereas body weights in the AFB1 and Cur combination treatment group were higher than those in the AFB1 alone treatment group. This suggests that AFB1 influences the ducks' normal growth and that curcumin can counteract AFB1′s growth-inhibiting effects. At the conclusion of the experiment, the brains from all ducks were collected and weighed. It was observed that AFB1-treated ducks' brains showed no obvious pathological changes in appearance, but their weights were markedly lower and the cerebral coefficient was raised compared with the control group, while curcumin treatment could attenuate this trend (Fig. 1 A, C and D, P < 0.01). The increase in the cerebral coefficient (brain/body weight ratio) was primarily driven by the severe retardation in body weight gain caused by AFB1, which outweighed the reduction in brain mass. This indicates growth inhibition rather than brain hypertrophy. The results suggest that AFB1 affects the normal growth and development of ducks and is cerebrotoxic, while curcumin use has a therapeutic effect.

Fig. 1.

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Effects of AFB1 exposure on changes in growth and brain coefficients as well as on the structure and function of duck brain tissues and the protective effects of curcumin. (A) Pathological changes in the brains. (B) Changes in body weight. (C) Brain weight differences between groups. (D) Differences in brain coefficients across groups. (E) SOD activity in brain tissue (U/mgprot). (F) MDA content in brain tissue (nmol/g). (G) Hematoxylin-eosin (H&E) staining of brain tissues, blue triangles indicate swollen or even ruptured cells;yellow triangles indicate contracted nuclei; red triangles indicate cytoplasmic vacuoles. (H) Nissl body staining of brain tissues. (I) Immunohistochemical staining of GFAP. All data were expressed as mean ± SD; “*” indicates a significant difference between the control group and the AFB1 group (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001); “#” indicates a significant difference between the AFB1 group and the AFB1 + Cur group (#P < 0.05, ##P < 0.01, ###P < 0.01 and ####P < 0.0001).

Curcumin alleviates AFB1-induced histopathological damage in duck brain tissue

The control and Cur-treated groups' brain tissues displayed normal and well-organized cellular morphology, according to HE staining. In contrast, the AFB1 group displayed varying degrees of cellular damage, as evidenced by disorganized cellular arrangement, cellular swelling and even rupture, cellular nuclear shrinkage, and cytoplasmic vacuoles. The standardised histological grading results in neuropathology revealed that brain tissue damage following AFB1 exposure progressed from grade 0 to grade 3 (Supplementary material, Fig. 1). However, the AFB1+Cur group displayed a considerable improvement in these pathologic alterations (Fig. 1 G). Additionally, the AFB1+Cur group's Nissl body count was equivalent to that of the control group, whereas the AFB1 group showed greater neuronal necrosis and fewer Nissl bodies, according to the results of Nissl staining (Fig. 1 H). GFAP is a marker of astrocyte activation. Astrocyte proliferation is usually accompanied by increased GFAP expression. Therefore, GFAP can be used as a biomarker for astrocyte proliferation in CNS injury. According to immunohistochemical results, the AFB1 group had higher GFAP expression than the control group, while the curcumin-treated group had lower GFAP expression than the AFB1 group (Fig. 1 I). This implies that exposure to AFB1 damages the duck brain neurologically, which is attenuated by curcumin treatment. In conclusion, AFB1 exposure causes histopathological damage to duck brain, and curcumin has a protective effect against AFB1 poisoning.

Curcumin alleviates AFB1-induced abnormalities in indicators of oxidative stress

Oxidative stress-related indices were subsequently investigated to see if AFB1 may cause oxidative damage in duck brain tissue. The findings demonstrated that the AFB1 alone treatment group's SOD activity in duck brain tissue was considerably lower than that of the control group (Fig. 1 E, P < 0.01). Specifically, SOD activity decreased from 403.5 ± 3.6 U/mgprot in the control group to 263.4 ± 2.7 U/mgprot in the AFB1 group, representing a 53% reduction. Conversely, the content of MDA was significantly increased (Fig. 1 F, P < 0.05), rising from 47.2 ± 8.5 nmol/g in the control group to 88.6 ± 38.4 nmol/g in the AFB1 group, an approximate 1.88-fold increase. In contrast, the AFB1+Cur group's brain tissue had significantly lower MDA concentration and significantly higher SOD activity than the AFB1 group. This proves that AFB1 exposure induces oxidative damage in duck brain tissues and curcumin reduces this damage and has a protective effect.

Curcumin repairs AFB1-induced blood-brain barrier damage

To further explore whether the blood-brain barrier was impaired, Western blotting and RT-qPCR were performed to detect the expression of ZO-1 and Occludin in the brain. The findings demonstrated that following AFB1 exposure, ZO-1 and Occludin's protein expression levels were significantly lower (Fig. 2 B-D, P < 0.01), and their mRNA expression levels were correspondingly reduced (Fig. 2 A, P < 0.05). When compared to the AFB1 group, the curcumin plus AFB1 combination therapy significantly increased the expression levels of both ZO-1 and occludin. Additionally, immunofluorescence labeling revealed that the AFB1 group's red fluorescence of ZO-1 was much lower than that of the control group, suggesting that ducks exposed to AFB1 may have damaged blood-brain barriers (Fig. 2 E). Furthermore, the AFB1+Cur group's fluorescence intensity was significantly higher than the AFB1 group's, suggesting that curcumin may be helpful in restoring the blood-brain barrier. It is hypothesized that exposure to AFB1 causes the duck brain's expression of ZO-1 and Occludin to decrease, impairing the blood-brain barrier. In contrast, curcumin promotes the expression of these tight junction proteins and serves to repair the blood-brain barrier.

Fig. 2.

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Effects of exposure to AFB1 on blood-brain barrier and protective effects of curcumin in ducks. (A) Relative mRNA expression levels of ZO-1 and Occludin genes. (B) Western blot analysis of ZO-1 and Occludin proteins. (C-D) Protein expression levels of ZO-1 and Occludin. (E) Immunofluorescence images of ZO-1. All data were expressed as mean ± SD; “*” indicates a significant difference between the control group and the AFB1 group (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001); “#” indicates a significant difference between the AFB1 group and the AFB1 + Cur group (#P < 0.05, ##P < 0.01, ###P < 0.01 and ####P < 0.0001).

Curcumin inhibits TLR4/NF-κB-mediated inflammatory response induced by AFB1 exposure

The alterations in TLR4/NF-κB signaling pathway markers and associated inflammatory variables were subsequently investigated to see if AFB1 may cause inflammatory responses in duck brains. Based on the experimental results, TLR4, Myd88, NF-κB, IL-1β, IL-6 and TNF-α mRNA expression levels sharply increased after AFB1 exposure, while IκB mRNA expression levels sharply declined (Fig. 3 H, P < 0.01). The aforementioned findings were supported by the Western blot results, which also showed a significant increase in the phosphorylation levels of NF-κB and IκB (Figure 3 A-G, I-L, P < 0.01). The AFB1+Cur group exhibited considerably lower levels of TLR4, Myd88, NF-κB, IL-1β, IL-6 and TNF-α protein and gene expression, greater levels of IκB protein and gene expression, and significantly lower levels of phosphorylation of both NF-κB and IκB compared to the AFB1 group. Furthermore, using immunofluorescence, we discovered that the AFB1 group's green fluorescence intensity of NF-κB was markedly stronger than the control group's, whereas the AFB1+Cur group's fluorescence intensity was weaker than the AFB1 group's (Fig. 3 M). The immunofluorescence results of IL-1β were the same as those of NF-κB (Fig. 3 N). These results suggest that AFB1 can activate the TLR4/NF-κB-mediated inflammatory response, while curcumin inhibits the process.

Fig. 3.

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Effects of AFB1 exposure on TLR4/NF-κB-mediated inflammation in ducks and protective effects of curcumin. (A) Western blot analysis of proteins related to the TLR4/NF-κB signaling pathway. (B-G) Protein expression levels of genes related to the TLR4/NF-κB signaling pathway. (H) Relative mRNA expression levels of genes associated with inflammation. (I) Western blot analysis of inflammatory factor proteins. (J-L) Protein expression levels of inflammatory factor genes. (M) Immunofluorescence images of NF-κB. (N) Immunofluorescence images of IL-1β. All data were expressed as mean ± SD; “*” indicates a significant difference between the control group and the AFB1 group (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001); “#” indicates a significant difference between the AFB1 group and the AFB1 + Cur group (#P < 0.05, ##P < 0.01, ###P < 0.01 and ####P < 0.0001).

Curcumin inhibits AFB1-induced apoptosis

The expression levels of genes and proteins associated with apoptosis were assessed using Western blot and RT-qPCR. The findings demonstrated that whereas curcumin and AFB1 treatment significantly reduced the expression levels of the genes Caspase-8, cleaved-Caspase-8, Caspase-3, cleaved-Caspase-3, Caspase-7, and BAX, AFB1 significantly raised the relative mRNA expression levels and protein expression levels of these genes. The curcumin-only therapy group's outcomes were almost the same as the control group's (Fig. 4 A-I, P < 0.01). Additionally, the fluorescence intensity of caspase-3 in the AFB1-treated group was not considerably stronger than that of the control group, as determined by immunofluorescence; however, the fluorescence intensity of the AFB1 and Cur co-treatment group was significantly less than that of the AFB1 group (Fig. 4 J). These results suggest that AFB1 intoxication induces excessive apoptosis in duck brain cells, and curcumin can inhibit this process.

Fig. 4.

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Effects of AFB1 exposure on apoptosis and protective effects of curcumin in duck brain tissues. (A) Western blot analysis of apoptosis-related proteins. (B-G) Protein expression levels of apoptosis-related genes. (H-I) Relative mRNA expression levels of apoptosis-related genes. (J) Immunofluorescence images of Caspase 3. All data were expressed as mean ± SD; “*” indicates a significant difference between the control group and the AFB1 group (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001); “#” indicates a significant difference between the AFB1 group and the AFB1 + Cur group (#P < 0.05, ##P < 0.01, ###P < 0.01 and ####P < 0.0001).

Curcumin inhibits AFB1-induced cellular pyroptosis

To find out how curcumin affected cellular pyroptosis in AFB1-intoxicated ducks, we examined proteins and genes related to cellular pyroptosis. The protein expression levels of NLRP3, ASC, GSDMD, Caspase-1, and IL-18, as well as the relative mRNA expression levels of the genes, were considerably enhanced in the AFB1 group, but these expressions were lowered by the co-treatment of curcumin and AFB1 (Fig. 5 A-H, P < 0.01). Immunofluorescence results showed that while the AFB1+Cur group had less positive expression of GSDMD than the AFB1 group, the AFB1-treated group had much more than the control group (Fig. 5 I). These findings imply that curcumin prevents cellular pyroptosis in the ducks' brains that have been exposed to AFB1.

Fig. 5.

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Effects of AFB1 exposure on cellular pyroptosis in duck brain tissues and protective effects of curcumin. (A) Western blot analysis of cellular pyroptosis-associated proteins. (B-F) Protein expression levels of cellular pyroptosis-associated genes. (G-H) Relative mRNA expression levels of cellular pyroptosis-associated genes. (I) Immunofluorescence images of GSDMD. All data were expressed as mean ± SD; “*” indicates a significant difference between the control group and the AFB1 group (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001); “#” indicates a significant difference between the AFB1 group and the AFB1 + Cur group (#P < 0.05, ##P < 0.01, ###P < 0.01 and ####P < 0.0001).

Curcumin inhibits AFB1-induced necroptosis

After that, we explored whether AFB1 could induce necroptosis in duck brain and whether curcumin had a protective effect. The results showed that the AFB1 group had considerably higher levels of protein expression of RIPK1, RIPK3, MLKL, and ZBP1, as well as higher levels of mRNA expression of the genes, as compared to the control group, while the AFB1+Cur group had lower levels of these expressions (Fig. 6 A-G, P < 0.01). The immunofluorescence positive expression of MLKL was markedly raised in the AFB1 group, while its positive expression was reduced in the curcumin and AFB1 co-treatment group (Fig. 6 H). These results suggest that AFB1 intoxication induces necroptosis in duck brain cells, and curcumin can inhibit this process.

Fig. 6.

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Effects of AFB1 exposure on necrotic apoptosis in duck brain tissues and protective effects of curcumin. (A-B) Relative mRNA expression levels of necrotic apoptosis-related genes. (C) Western blot analysis of necrotic apoptosis-related proteins. (D-G) Protein expression levels of genes related to necrotic apoptosis. (H) Immunofluorescence images of MLKL. All data were expressed as mean ± SD; “*” indicates a significant difference between the control group and the AFB1 group (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001); “#” indicates a significant difference between the AFB1 group and the AFB1 + Cur group (#P < 0.05, ##P < 0.01, ###P < 0.01 and ####P < 0.0001).

Discussion

AFB1 exhibits extremely potent toxicity. Animals exposed to AFB1 have been shown to display abnormal behavioral alterations, such as anxiety, lethargy, and depressive-like behaviors. Nevertheless, the precise mechanism via which AFB1 induces neurotoxicity in ducks remains unclear. It has been demonstrated that AFB1 causes oxidative stress in the body and dramatically lowers serum levels of antioxidant enzymes as SOD, T-AOC, CAT, and GSH-Px. Lipid peroxidation's byproduct, MDA, is likewise elevated (Cao et al., 2022; Wu et al., 2022). Our previous research (Jiang et al., 2024) and the present study have both uncovered similar findings. This oxidative imbalance is not merely a byproduct of toxicity but a critical upstream driver of neurodegeneration. Reactive oxygen species (ROS) accumulation can directly damage the mitochondrial membrane, releasing mtDNA that acts as a DAMP to activate the TLR4/NF-κB inflammatory cascade (Pan et al., 2024) and nucleate the ZBP1-PANoptosome (Pan et al., 2026). Thus, the oxidative stress induced by AFB1 likely creates the permissive environment for the subsequent activation of inflammation and PANoptosis observed in our study.

The most prevalent type of cells in the animal brain are called astrocytes, and they help to establish the blood-brain barrier as well as support and separate neuronal cells (Macdonald et al., 2019). GFAP is a marker of astrocyte activation, and up-regulation of GFAP expression represents astrocyte proliferation (Gogishvili et al., 2025), which is a typical response of astrocytes to neuronal injury and CNS inflammation. It has been demonstrated that by adversely influencing astrocytes and endothelial cells, AFB1 can interfere with the blood-brain barrier's ability to function (Aytekin et al., 2022). A study has also demonstrated that persistent inflammatory signaling can directly degrade tight junction proteins and increase blood-brain barrier permeability, thereby leading to neurofunctional deterioration (Huang et al., 2024). This aligns with our observation that AFB1-induced inflammation parallels the disruption of barrier integrity and the appearance of neuronal degeneration and necrosis. These evidence further suggest that AFB1 may breach the blood-brain barrier, directly contacting astrocytes and neurons to cause cellular damage, while the subsequent parenchymal inflammation further degrades barrier integrity.

As a neurotoxic substance, AFB1 and its metabolites can directly or indirectly bind to TLR4 receptors as endogenous danger signals (DAMPs), thereby triggering downstream MyD88-dependent signaling cascades (Sakai et al., 2017) that ultimately result in NF-κB nuclear translocation (Zhou et al., 2022). Consistent with previous findings in microglia (Mehrzad et al., 2017), our data confirmed that following exposure to AFB1, the TLR4/NF-κB signaling pathway is activated, subsequently triggering an inflammatory response and resulting in brain damage. Recent research has further highlighted the critical role of molecular regulators in the resolution of such inflammatory responses (Wang et al., 2024), suggesting that pharmacological agents like curcumin may exert their protective effects by modulating these key regulatory networks.

Research on PANoptosis has yielded substantial findings (Pandian and Kanneganti, 2022; Qi et al., 2023). Emerging evidence has implicated PANoptosis in various CNS pathologies, such as ischemic stroke and viral encephalitis (Li and Qu, 2025). Consistent with these CNS disease models, numerous studies suggest a similar mechanism in mycotoxin-induced neurotoxicity. In IMR-32 neuronal cells, exposure to AFB1 was observed to cause apoptosis (Huang et al., 2020). It has also been shown that AFB1 can lead to inflammation by inducing GSDMD-mediated microglial cell pyroptosis (Zhang et al., 2022). AFB1 also induced necrotic apoptosis in the hippocampus of mice (Naeini et al., 2025). AFB1-induced neurotoxicity has been linked to apoptosis, cellular pyroptosis, and necroptosis, according to these investigations. However, no research has examined the connection between AFB1-induced neurotoxicity and PANoptosis (Li and Qu, 2025). To find out if AFB1 could trigger PANoptosis and harm duck brains, we looked at genes and proteins linked to necroptosis, cellular pyroptosis, and apoptosis independently. Our data reveal a synchronized upregulation of key effectors across apoptosis, pyroptosis, and necroptosis signaling pathways. Furthermore, we discovered that the ZBP1 gene's expression level was likewise markedly elevated. This simultaneous activation provides robust evidence that AFB1 triggers PANoptosis in avian neuronal tissue, in which ZBP1, RIPK1, RIPK3 and Caspase-8, NLRP3, ASC and Caspase-1 form the ZBP1-PANoptosome complex (Qi et al., 2023). However, it should be noted that this study only detected the presence of key components of this complex simultaneously; direct protein-protein interactions remain to be confirmed. Mechanistically, we propose that AFB1-induced genotoxicity and oxidative stress may stabilize Z-form DNA structures or trigger the leakage of mitochondrial DNA (mtDNA) into the cytosol, serving as endogenous ligands that activate ZBP1 in this sterile inflammatory context. Research has also demonstrated that fumonisin B1 can induce damage to chicken hepatocytes by activating PANoptosis, whilst ochratoxin A can induce PANoptosis in pulmonary cells (Wang et al., 2025; Xie et al., 2025). These studies indicate that toxins or pathogens can induce similar PANoptosis-like changes across cell types. This suggests that PANoptosis represents a conserved mechanism of innate cell death in various species under stress conditions.

Our research revealed that AFB1 might trigger an inflammatory response in the duck brain and activate the TLR4/NF-κB signaling pathway. Additionally, AFB1 had the ability to harm cells and trigger PANoptosis. Although PANoptosis and the TLR4/NF-κB signaling pathway seemed to have separate roles in AFB1-induced neurotoxicity, they closely interacted and are closely related. ZBP1, the primary gene for PANoptosis, has been demonstrated to activate NF-κB and promote the synthesis of inflammatory cytokines (Song et al., 2024). Endogenous RIPK1 can also induce the activation of NF-κB after binding with ZBP1 (Kondylis et al., 2017). NLRP3 is a sensor of inflammatory vesicles that selectively interacts with ASC to form the NLRP3-ASC complex (Nagar et al., 2023), which in turn regulates NF-κB activity. At the same time, NLRP3 also inhibits the transcriptional activity of RelA (Yang et al., 2021), thus finely regulating the inflammatory response at the transcriptional level. Necroptosis is triggered primarily by TNFR and TLR3/4, cytoplasmic nucleic acid sensors, and other mediators that induce the production of IFN-I and TNF-α, which promotes necroptosis in an autocrine feedback loop (Li and Beg, 2000). These mediators also trigger NFκB-dependent inflammatory signaling (Seo et al., 2021). All of the above studies suggest that there is a close link between the TLR4/NF-κB signaling pathway and PANoptosis, and that both play a common role in AFB1-induced neurotoxicity, as confirmed in our study. However, we acknowledge that this study did not utilize specific pathway inhibitors or gene-knockout models to delineate the precise causal hierarchy. Future studies are needed to determine whether the inhibition of TLR4/NF-κB directly prevents the assembly of the PANoptosome or if these pathways operate in parallel.

Knowing that AFB1 is neurotoxic, it is important to find effective neuroprotective agents against AFB1. Curcumin is a powerful antioxidant that is used to treat a variety of illnesses (Llano et al., 2019; Zheng et al., 2020). Although curcumin's clinical application has been historically limited by poor bioavailability, recent advancements in formulation technologies have significantly improved its stability and absorption, thereby enhancing its translational potential for neuroprotection (Wang et al., 2025). Our findings demonstrated that curcumin treatment attenuated the effects of AFB1 exposure, which reduced SOD activity and raised MDA levels in the duck brain. Recent studies emphasize that the TLR4/NF-κB axis is a core driver of neuroinflammation (Zhou et al., 2025), and inhibiting this pathway is a key mechanism for achieving neuroprotection. Curcumin can directly block IKKβ phosphorylation and inhibit NF-κB nuclear translocation (Jobin et al., 1999). Curcumin can also prevent cell death by reducing pyroptosis and apoptosis (Yin et al., 2018; Muhmood et al., 2024). This is consistent with our findings. We hypothesized that curcumin primarily exerted its effects upstream. By blocking TLR4/NF-κB activation, curcumin downregulated the abundance of key structural components (ZBP1, RIPK1, RIPK3, Caspase-8) required for PANoptosome formation, thereby mitigating PANoptosis. However, the precise molecular interaction between curcumin and the ZBP1-PANoptosome assembly dynamics remains to be elucidated in future studies using Co-Immunoprecipitation assays.

Additionally, it is important to note that due to interspecies differences in metabolic pathways and blood-brain barrier physiology, caution should be exercised when translating these avian findings to mammals or humans.

Conclusions

In conclusion, the current study demonstrated that AFB1 caused brain damage in ducks and that exposure to AFB1 triggered PANoptosis and the TLR4/NF-κB signaling pathway. Curcumin may also function as a neuroprotectant to attenuate the effects of AFB1-induced brain damage. This study provides a theoretical basis for developing countermeasures against AFB1 neurotoxicity. However, as this study relied on expression profiling, future in vitro studies using gene silencing or pharmacological inhibitors are warranted to confirm these direct interactions.

Funding Declaration

This research was funded by the National Key R&D Program of China, grant number(2023YFD1801100), Supported by the earmarked fund for GARSIT-Swine Industry (nycytxgxcxtd-2023-15-02).

Author Agreement

We declare that this manuscript is original, has not been published before and is not currently being considered for publication elsewhere. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We understand that the Corresponding Author is the sole contact for the Editorial process. She is responsible for communicating with the other authors about progress, submissions of revisions, and final approval of proofs.

CRediT authorship contribution statement

Limeng Zhou: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization. Ying He: Resources. Yueyang Li: Methodology, Investigation, Formal analysis. Xudong Han: Methodology, Investigation, Formal analysis. Yanli You: Resources. Ziqing Li: Investigation, Formal analysis. Xuanliang Li: Writing – review & editing. Renbin chen: Writing – review & editing. Hang Pan: Writing – review & editing, Writing – original draft. Jianzhao Liao: Resources. Zhaoxin Tang: Resources. Zhiwen Wu: Resources. Lianmei Hu: Supervision, Resources, Funding acquisition.

Disclosures

We declare that there is no conflict of interest in this work

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2026.106652.

Contributor Information

Zhiwen Wu, Email: scauwuzhiwen@scau.edu.cn.

Lianmei Hu, Email: hulianmei@scau.edu.cn.

Appendix. Supplementary materials

mmc1.docx (16.5KB, docx)
mmc2.docx (4.5MB, docx)

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