Curcumin Alleviates Aflatoxin B1-Induced Liver Pyroptosis and Fibrosis by Regulating the JAK2/NLRP3 Signaling Pathway in Ducks

Aflatoxin B1 (AFB1) is a serious pollutant in feed and food which causes liver inflammation, fibrosis, and even cirrhosis. The Janus kinase 2 (JAK2)/signal transducers and activators of the transcription 3 (STAT3) signaling pathway is widely involved in inflammatory response and promotes the activation of nod-like receptor protein 3 (NLRP3) inflammasome, thus leading to pyroptosis and fibrosis. Curcumin is a natural compound with anti-inflammatory and anti-cancer properties. However, whether AFB1 exposure leads to the activation of the JAK2/NLRP3 signaling pathway in the liver and whether curcumin can regulate this pathway to influence pyroptosis and fibrosis in the liver remains unclear. In order to clarify these problems, we first treated ducklings with 0, 30, or 60 µg/kg AFB1 for 21 days. We found that AFB1 exposure caused growth inhibition, liver structural and functional damage, and activated JAK2/NLRP3-mediated liver pyroptosis and fibrosis in ducks. Secondly, ducklings were divided into a control group, 60 µg/kg AFB1 group, and 60 µg/kg AFB1 + 500 mg/kg curcumin group. We found that curcumin significantly inhibited the activation of the JAK2/STAT3 pathway and NLRP3 inflammasome, as well as the occurrence of pyroptosis and fibrosis in AFB1-exposed duck livers. These results suggested that curcumin alleviated AFB1-induced liver pyroptosis and fibrosis by regulating the JAK2/NLRP3 signaling pathway in ducks. Curcumin is a potential agent for preventing and treating liver toxicity of AFB1.


Introduction
Aflatoxin B 1 (AFB 1 ) is one of the most harmful mycotoxins in feed and food [1]. In 1996, the World Health Organization set the upper limit of AFB 1 in cereals entering the market at 20 µg/kg [2]. However, many parts of the world have remained heavily contaminated with AFB 1 for many years, posing a threat to animal and human health. In 2014, Iram et al. found that the average content of AFB 1 in 487 poultry feeds and raw materials in Pakistan was 37.62 µg/kg and 23.75 µg/kg, respectively [3]. In 2019, Akinmusire et al. found that 101 poultry feeds and raw materials in Nigeria contained an average of 74 µg/kg AFB 1 [4]. In 2019, Mahuku et al. found that the average AFB 1 content in maize samples from eastern and southwestern Kenya was 67.8 µg/kg and 22.3 µg/kg, respectively [5]. Long-term ingestion of food or feed contaminated with AFB 1 can lead to chronic poisoning in humans and animals, with symptoms such as dizziness, anorexia, convulsions, and loss of memory function [6]. Chronic AFB 1 poisoning can cause damage to the liver, kidney, spleen, testis, and other parenchymal organs, resulting in growth disorder, immunosuppression, hematopoietic damage, and reduced reproductive Figure 1. Schematic overview of experimental program. Firstly, ducks were exposed to a gradient dose of AFB1 for 21 days. The effects of AFB1 on fibrosis, JAK2/NLRP3 signaling pathway, and pyroptosis of duck livers were analyzed. Then, the dose of AFB1, which can cause significant liver fibrosis in ducks, was selected and treated with curcumin for 21 days. The effects of curcumin on fibrosis, JAK2/NLRP3 signaling pathway, and pyroptosis of duck livers caused by AFB1 were analyzed.

Animals and Treatment
In the first part of the experiment, one-day-old healthy male Jinding ducks (Anas platyrhyncha) were randomly divided into three groups (n = 6). The dose of AFB1 was determined according to previous studies [28,29] and the median lethal dose in ducks (LD50 = 300 µ g/kg body weight) [30]. AFB1 (≥99.8%, Qingdao Pribolab Pte. Ltd., Qingdao, China) was dissolved in corn oil. The low-dose group (LG) and high-dose group (HG) were given 30 µ g/kg (1/10 LD50) and 60 µ g/kg (1/5 LD50) body weight of AFB1 by intragastric administration daily, respectively. The control group (CG) was given the same volume of solvent. The experiment lasted three weeks.
We found that 60 µ g/kg AFB1 caused more significant fibrotic lesions in the duck liver, so this dose was selected for the second part of the experiment. One-day-old healthy male ducks were divided into three groups (n = 6): control group (CG), AFB1 exposure group (AG, 60 µ g/kg AFB1), and AFB1 + curcumin group (ACG, 60 µ g/kg AFB1 and 500 mg/kg curcumin). Curcumin (≥99.8%, Nanjing NutriHerb BioTech Co., Ltd., Nanjing, China) was added to the base diet at doses determined by previous studies [27,31]. The experiment lasted three weeks.
All experimental routines and protocols have been approved by the Animal Ethics Committee of the Northeast Agricultural University (NEAUEC20230332). The ducks were kept in the Biomedical Research Center of Northeast Agricultural University and had free access to water and pellet feed formulated according to the National Research Council. The temperature in the room was 32 °C for the first week, 30 °C for the second week, and 28 °C for the third week, guaranteeing 18 h of incandescent light a day. No animals died during the experiment. Firstly, ducks were exposed to a gradient dose of AFB 1 for 21 days. The effects of AFB 1 on fibrosis, JAK2/NLRP3 signaling pathway, and pyroptosis of duck livers were analyzed. Then, the dose of AFB 1 , which can cause significant liver fibrosis in ducks, was selected and treated with curcumin for 21 days. The effects of curcumin on fibrosis, JAK2/NLRP3 signaling pathway, and pyroptosis of duck livers caused by AFB 1 were analyzed.
We found that 60 µg/kg AFB 1 caused more significant fibrotic lesions in the duck liver, so this dose was selected for the second part of the experiment. One-day-old healthy male ducks were divided into three groups (n = 6): control group (CG), AFB 1 exposure group (AG, 60 µg/kg AFB 1 ), and AFB 1 + curcumin group (ACG, 60 µg/kg AFB 1 and 500 mg/kg curcumin). Curcumin (≥99.8%, Nanjing NutriHerb BioTech Co., Ltd., Nanjing, China) was added to the base diet at doses determined by previous studies [27,31]. The experiment lasted three weeks.
All experimental routines and protocols have been approved by the Animal Ethics Committee of the Northeast Agricultural University (NEAUEC20230332). The ducks were kept in the Biomedical Research Center of Northeast Agricultural University and had free access to water and pellet feed formulated according to the National Research Council. The temperature in the room was 32 • C for the first week, 30 • C for the second week, and 28 • C for the third week, guaranteeing 18 h of incandescent light a day. No animals died during the experiment.

Sample Collection
After stopping feeding overnight (12 h) on the 21st day, the ducks were weighed; then, the blood was collected through the wing vein and serum was collected after centrifugation at 1500× g for 10 min. The ducks were anesthetized by intravenous injection of sodium pentobarbital (50 mg/kg body weight). Livers were quickly collected, one part was fixed with 4% formaldehyde for histopathological examination, and the rest was stored at −80 • C for other studies.

Measurement of Serum ALT and AST Activities
Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using an automatic hematology analyzer.

Histopathological Observation
The liver was fixed in 4% paraformaldehyde for 24 h, then embedded in paraffin and sliced. Hematoxylin eosin (HE) staining and sirius red staining were performed, respectively, according to previous studies [32]. The sections were scanned and then observed with CaseViewer software (3Dhistech, Budapest, Hungary) to evaluate the degree of histopathological damage and fibrosis.

TUNEL Analysis
According to the instruction of the biochemical kit (Beyotime, Shanghai, China), the above liver sections were stained using terminal deoxynucleoside transferase (TdT) dUTP nick end labeling (TUNEL). The sections were scanned and then observed with CaseViewer software (3Dhistech, Budapest, Hungary) to evaluate the liver pyroptosis level.

Detection of IL-1β and IL-18 Levels in the Liver and Serum
The 50 mg liver sample was added to 1 mL phosphate-buffered saline, fully ground with homogenizer, and then centrifuged at 12,000× g for 10 min to collect the supernatant. The contents of IL-1β and IL-18 in the supernatant and serum were determined using the ELISA kits (Nanjing Jiancheng, Nanjing, China).

RT-qPCR Analysis
The total RNA of duck liver was extracted according to the instruction of Trizol reagent (Invitrogen, Carlsbad, CA, USA), and then the cDNA was synthesized using the reverse transcription kit (Roche, Basel, Switzerland). The mRNA expression was detected with an ABI PCR system (Thermo Fisher, Waltham, MA, USA). β-actin was selected as the internal reference gene, and the relative mRNA expression was calculated using the 2 -∆∆Ct method. All the information on primers is shown in Table S1.

Western Blot Analysis
The 100 mg liver sample was added to 1 mL RIPA lysate (Beyotime, Shanghai, China) with 10 µL PMSF (Beyotime, Shanghai, China), fully ground with homogenizer, then centrifuged at 12,000× g for 10 min at 4 • C to collect the supernatant. A BCA kit (Solarbio, Beijing, China) was used to detect the protein concentration, and 30 µg protein samples were selected for 5−12% SDS-PAGE gel electrophoresis. The gel was then transferred to the PVDF membrane, incubated overnight with the required primary antibody at 4 • C, and then incubated with the corresponding secondary antibody for 1 h at 37 • C. Images were collected in a gel imaging system (General Electric, Fairfield, CT, USA) using an ECL luminescence solution and analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA). All the information on antibodies is shown in Table S2.

Statistical Analysis
Data were analyzed using SPSS 25.0 software (SPSS Incorporated, Chicago, IL, USA) and expressed as mean ± standard deviation (mean ± SD). * and ** represent p < 0.05 and p < 0.01 vs. the CG, respectively; # and ## represent p < 0.05 and p < 0.01 vs. the AG, respectively.

AFB 1 Exposure Caused Liver Damage in Ducks
Compared with the CG, the body weight decreased significantly by 11% and 24.5%, the serum ALT activities increased significantly by 41% and 87%, and the serum AST activities increased significantly by 39% and 76% in the LG and HG, respectively ( staining showed that the CG liver had normal microscopic structure, while the LG and HG livers showed hepatic cord disorders, inflammatory cell infiltration, and even hepatic cell disintegration ( Figure 2D). < 0.01 vs. the CG, respectively; # and ## represent p < 0.05 and p < 0.01 vs. the AG, re-spectively.

AFB1 Exposure Caused Liver Damage in Ducks
Compared with the CG, the body weight decreased significantly by 11% and 24.5%, the serum ALT activities increased significantly by 41% and 87%, and the serum AST activities increased significantly by 39% and 76% in the LG and HG, respectively ( Figure  2A-C). HE staining showed that the CG liver had normal microscopic structure, while the LG and HG livers showed hepatic cord disorders, inflammatory cell infiltration, and even hepatic cell disintegration ( Figure 2D).

AFB1 Exposure Activated JAK2/NLRP3-Mediated Pyroptosis in Duck Livers
The mRNA expressions of JAK2 and STAT3 in the LG were 1.74 and 2 times that in the CG, respectively, and the mRNA expressions of JAK2 and STAT3 in the HG were 2.74 and 3.04 times that in the CG, respectively ( Figure 3A). The protein expressions of pJAK2/JAK2 and pSTAT3/STAT3 in the LG were 1.76 and 2.41 times that in the CG, respectively, and the protein expressions of pJAK2/JAK2 and pSTAT3/STAT3 in the HG were 3.06 and 4.59 times that in the CG, respectively ( Figure 3B). Compared with the CG, TUNEL staining showed that the positive rate of the LG and HG increased from 1.43% to 10.6% and 20%, respectively ( Figure 3C,D). The mRNA expressions of NLRP3, ASC, and Caspase-1 in the LG were 2.87, 2.56, and 1.95 times that in the CG, respectively, and the mRNA expressions of NLRP3, ASC, and Caspase-1 in the HG were 4.37, 4.23, and 4.43 times that in the CG, respectively ( Figure 3E). The protein expressions of NLRP3, ASC, Caspase-1, GSDMD, and GSDMD-N in the LG were 1.36, 2.19, 1.59, 1.89, and 2.58 times that in the CG, respectively, and the protein expressions of NLRP3, ASC, Caspase-1, GSDMD, and GSDMD-N in the HG were 2.56, 3.86, 2.5, 4.15, and 8.32 times that in the CG, respectively ( Figure 3F).

AFB 1 Exposure Activated JAK2/NLRP3-Mediated Pyroptosis in Duck Livers
The mRNA expressions of JAK2 and STAT3 in the LG were 1.74 and 2 times that in the CG, respectively, and the mRNA expressions of JAK2 and STAT3 in the HG were 2.74 and 3.04 times that in the CG, respectively ( Figure 3A). The protein expressions of pJAK2/JAK2 and pSTAT3/STAT3 in the LG were 1.76 and 2.41 times that in the CG, respectively, and the protein expressions of pJAK2/JAK2 and pSTAT3/STAT3 in the HG were 3.06 and 4.59 times that in the CG, respectively ( Figure 3B). Compared with the CG, TUNEL staining showed that the positive rate of the LG and HG increased from 1.43% to 10.6% and 20%, respectively ( Figure 3C,D). The mRNA expressions of NLRP3, ASC, and Caspase-1 in the LG were 2.87, 2.56, and 1.95 times that in the CG, respectively, and the mRNA expressions of NLRP3, ASC, and Caspase-1 in the HG were 4.37, 4.23, and 4.43 times that in the CG, respectively ( Figure 3E). The protein expressions of NLRP3, ASC, Caspase-1, GSDMD, and GSDMD-N in the LG were 1.36, 2.19, 1.59, 1.89, and 2.58 times that in the CG, respectively, and the protein expressions of NLRP3, ASC, Caspase-1, GSDMD, and GSDMD-N in the HG were 2.56, 3.86, 2.5, 4.15, and 8.32 times that in the CG, respectively ( Figure 3F).

AFB 1 Exposure Caused Liver Fibrosis in Ducks
Compared with the CG, the liver IL-1β levels increased significantly by 22% and 62%, the liver IL-18 levels increased significantly by 14% and 38%, the serum IL-1β levels increased significantly by 22% and 61%, and the serum IL-18 levels increased significantly by 15% and 34% in the LG and HG, respectively ( Figure 4A,B). The mRNA expressions of IL-1β and IL-18 in the LG were 2.82 and 1.85 times that in the CG, and the mRNA expressions of IL-1β and IL-18 in the HG were 4.4 and 2.67 times that in the CG, respectively ( Figure 4C). Sirius red staining showed that the liver fibrosis of ducks became more and more obvious with the gradual increase in AFB 1 exposure dose ( Figure 4D). The mRNA expressions of α-SMA, Col-I, and TGF-β in the LG were 2.39, 2.22, and 1.79 times that in the CG, and the mRNA expressions of α-SMA, Col-I, and TGF-β in the HG were 4.08, 4.33, and 3.33 times that in the CG, respectively ( Figure 4E). The protein expressions of α-SMA, Col-I, and TGF-β in the LG were 2.5, 2.12, and 1.53 times that in the CG, and the protein  came more and more obvious with the gradual increase in AFB1 exposure dose ( Figure  4D). The mRNA expressions of α-SMA, Col-Ⅰ, and TGF-β in the LG were 2.39, 2.22, and 1.79 times that in the CG, and the mRNA expressions of α-SMA, Col-Ⅰ, and TGF-β in the HG were 4.08, 4.33, and 3.33 times that in the CG, respectively ( Figure 4E). The protein expressions of α-SMA, Col-Ⅰ, and TGF-β in the LG were 2.5, 2.12, and 1.53 times that in the CG, and the protein expressions of α-SMA, Col-Ⅰ, and TGF-β in the HG were 4.15, 3.6, and 2.33 times that in the CG, respectively ( Figure 4F).

Curcumin Alleviated Liver Damage in Ducks Caused by AFB 1 Exposure
Compared with the AG, the body weight increased significantly by 19%, the serum ALT activity decreased significantly by 24%, and the serum AST activity decreased significantly by 21% in the ACG, respectively ( Figure 5A-C). Compared with the AG, HE staining showed significant remission of liver lesions in the ACG ( Figure 5D).

Curcumin Alleviated Liver Damage in Ducks Caused by AFB1 Exposure
Compared with the AG, the body weight increased significantly by 19%, the serum ALT activity decreased significantly by 24%, and the serum AST activity decreased significantly by 21% in the ACG, respectively ( Figure 5A-C). Compared with the AG, HE staining showed significant remission of liver lesions in the ACG ( Figure 5D).

Curcumin Alleviated JAK2/NLRP3-Mediated Pyroptosis in the Liver of Ducks Exposed to AFB1
The mRNA expressions of JAK2 and STAT3 in the ACG were 0.61 and 0.58 times that in the AG, respectively ( Figure 6A). The protein expressions of pJAK2/JAK2 and pSTAT3/STAT3 in the ACG were 0.74 and 0.71 times that in the AG, respectively ( Figure  6B). Compared with the AG, TUNEL staining showed that the positive rate of ACG decreased from 18% to 9.4% ( Figure 6C,D). The mRNA expressions of NLRP3, ASC, and Caspase-1 in the ACG were 0.71, 0.53, and 0.6 times that in the AG, respectively ( Figure  6E). The protein expressions of NLRP3, ASC, Caspase-1, GSDMD, and GSDMD-N in the ACG were 0.79, 0.68, 0.63, 0.65, and 0.65 times that in the AG, respectively ( Figure 6F).

Curcumin Alleviated JAK2/NLRP3-Mediated Pyroptosis in the Liver of Ducks Exposed to AFB 1
The mRNA expressions of JAK2 and STAT3 in the ACG were 0.61 and 0.58 times that in the AG, respectively ( Figure 6A). The protein expressions of pJAK2/JAK2 and pSTAT3/STAT3 in the ACG were 0.74 and 0.71 times that in the AG, respectively ( Figure 6B). Compared with the AG, TUNEL staining showed that the positive rate of ACG decreased from 18% to 9.4% ( Figure 6C,D). The mRNA expressions of NLRP3, ASC, and Caspase-1 in the ACG were 0.71, 0.53, and 0.6 times that in the AG, respectively ( Figure 6E). The protein expressions of NLRP3, ASC, Caspase-1, GSDMD, and GSDMD-N in the ACG were 0.79, 0.68, 0.63, 0.65, and 0.65 times that in the AG, respectively ( Figure 6F).

Curcumin Alleviated Liver Fibrosis in Ducks Caused by AFB 1 Exposure
Compared with the AG, the liver IL-1β and IL-18 levels decreased significantly by 24% and 17%, respectively, and the serum IL-1β and IL-18 levels decreased significantly by 27% and 13% in the ACG, respectively ( Figure 7A,B). The mRNA expressions of IL-1β and IL-18 in the ACG were 0.63 and 0.62 times that in the AG, respectively ( Figure 7C). Compared with the AG, sirius red staining showed significant remission of liver fibrosis in the ACG ( Figure 7D). The mRNA expressions of α-SMA, Col-I, and TGF-β in the ACG were 0.76, 0.54, and 0.67 times that in the AG, respectively ( Figure 7E). The protein expressions of α-SMA, Col-I, and TGF-β in the ACG were 0.85, 0.73, and 0.67 times that in the AG, respectively ( Figure 7F).  by 27% and 13% in the ACG, respectively ( Figure 7A,B). The mRNA expressions of IL-1β and IL-18 in the ACG were 0.63 and 0.62 times that in the AG, respectively ( Figure 7C). Compared with the AG, sirius red staining showed significant remission of liver fibrosis in the ACG ( Figure 7D). The mRNA expressions of α-SMA, Col-Ⅰ, and TGF-β in the ACG were 0.76, 0.54, and 0.67 times that in the AG, respectively ( Figure 7E). The protein expressions of α-SMA, Col-Ⅰ, and TGF-β in the ACG were 0.85, 0.73, and 0.67 times that in the AG, respectively ( Figure 7F).

Discussion
In this study, we first found that AFB1 exposure resulted in slow growth, liver structural and functional impairment, and caused the JAK2/NLRP3-mediated liver pyroptosis and fibrosis in ducks. Secondly, we found that curcumin alleviated AFB1-induced liver pyroptosis and fibrosis by regulating the JAK2/NLRP3 signaling pathway in ducks. These results provide a new understanding for exploring the mecha-

Discussion
In this study, we first found that AFB 1 exposure resulted in slow growth, liver structural and functional impairment, and caused the JAK2/NLRP3-mediated liver pyroptosis and fibrosis in ducks. Secondly, we found that curcumin alleviated AFB 1 -induced liver pyroptosis and fibrosis by regulating the JAK2/NLRP3 signaling pathway in ducks. These results provide a new understanding for exploring the mechanism of hepatotoxicity of AFB 1 and provide an experimental basis for curcumin to alleviate the toxicity of AFB 1 . AFB 1 has the highest accumulation in liver after entering the body [33]. Meissonnier et al. found that AFB 1 exposure resulted in decreased weight gain and dysfunction of liver structure and function in pigs [34]. Gao et al. found that AFB 1 exposure resulted in liver damage in chickens, including hepatocyte destruction, swelling, and inflammatory cell infiltration, as well as increased ALT and AST activities [35]. Consistent with these results, we also demonstrated that AFB 1 exposure caused slow growth and liver histopathological damage and dysfunction in ducks ( Figure 2). These studies indicate that AFB 1 is harmful to a variety of species, but the specific mechanism of liver toxicity of AFB 1 still needs to be further explored.
Liver fibrosis is a repair reaction after liver injury which can lead to cirrhosis or even liver cancer if it continues to develop excessively [36]. During liver inflammation, hepatic stellate cells are activated and converted into myofibroblasts, which produced large amounts of extracellular matrix leading to fibrosis [37]. The increased expression of α-SMA and Col-I is the main characteristic of hepatic stellate cell activation, and TGF-β can promote myofibroblast proliferation [38]. In this study, we first found that AFB 1 exposure caused significant fibrosis in duck livers through sirius red staining ( Figure 4D). RT-qPCR and Western blot detection revealed that the expression levels of α-SMA, Col-I, and TGF-β were significantly increased, which further confirmed the occurrence of fibrosis ( Figure 4E,F). AFB 1 exposure has been shown to cause liver fibrosis in Oncorhynchus mykiss and rats in previous studies [39,40]. However, there are few studies on the specific mechanism of liver fibrosis induced by AFB 1 .
Inflammation is undoubtedly a key factor leading to fibrosis. As an intracellular protein complex, the NLRP3 inflammasome is a major inflammatory factor [41]. The activation of the NLRP3 inflammasome, on the one hand, produces IL-1β and IL-18, leading to liver fibrosis. On the other hand, it activates Caspase-1-mediated pyroptosis to promote fibrosis development [42]. In this study, we found that AFB 1 exposure led to increased expressions of NLRP3, ASC, Caspase-1, GSDMD, and GSDMD-N in duck livers, and combined with TUNEL staining analysis, this confirmed the occurrence of pyroptosis ( Figure 3C-F). In addition, the JAK2/STAT3 signaling pathway has been reported to not only activate the NLRP3 inflammasome and lead to pyroptosis, but also to promote the proliferation of hepatic stellate cells and promote fibrosis [43]. Therefore, we continued to detect the JAK2/STAT3 signaling pathway, and the results showed that this pathway was activated in the liver of AFB 1 -exposed ducks ( Figure 3A,B). In conclusion, AFB 1 exposure leads to JAK2/NLRP3-mediated pyroptosis and fibrosis in duck livers and may have a critical effect on liver damage.
The activation of the NLRP3 inflammasome directly promotes the progression of liver pyroptosis and fibrosis [44,45]. Therefore, reducing the activation of the NLRP3 inflammasome is a reliable method to reverse the development of liver pyroptosis and fibrosis and effectively alleviates liver injury. Curcumin, a naturally occurring polyphenol in turmeric, has been shown to be effective in inhibiting inflammation and pyroptosis. Yu et al. found that curcumin alleviated doxorubicin-induced cardiac injury by inhibiting NLRP3 inflammasome activation and myocardial pyroptosis [46]. In a recent study, Gan et al. found that curcumin mitigated arsenic trioxidation-induced hypothalamic damage in ducks by inhibiting neuronal pyroptosis mediated by the NF-κB/NLRP3 signaling pathway [47]. In addition, curcumin has been shown to alleviate inflammation by inhibiting the JAK/STAT signaling pathway [48,49]. Therefore, we hypothesized that curcumin could alleviate AFB 1 -induced pyroptosis and fibrosis by regulating the JAK2/NLRP3 signaling pathway in duck livers. AFB 1 -exposed ducklings were fed a diet supplemented with curcumin for 21 days. These ducks showed improved growth rates compared to ducks exposed only to AFB 1 ( Figure 5A). HE staining showed that the damage of the liver microstructure was significantly alleviated, and the activities of ALT and AST were also decreased ( Figure 5B-D). Similarly, Jin et al. demonstrated that dietary curcumin could improve the growth performance, brisket quality, and antioxidant capacity of multiple organs, as well as the damage of liver structure and function in ducks [28,50]. Moreover, we found that curcumin alleviated AFB 1 -induced duck liver fibrosis by sirius red staining and detection of α-SMA, Col-I, and TGF-β expression ( Figure 7D-F). The alleviative effect of curcumin on duck liver fibrosis induced by AFB 1 may be the key factor for curcumin to protect the liver, and we further explored the molecular mechanism.
We first detected factors related to the JAK2/STAT3 pathway and found that curcumin inhibited the activation of this pathway in the liver of AFB 1 -exposed ducks ( Figure 6A,B). We then found that curcumin inhibited the increased expressions of NLRP3, ASC, Caspase-1, GSDMD, and GSDMD-N in the liver of AFB 1 -exposed ducks ( Figure 6E,F). Combined with TUNEL staining analysis ( Figure 6C,D), these results demonstrated that curcumin inhibited AFB 1 -induced JAK2/NLRP3-mediated pyroptosis in duck livers. It should be noted that there was no significant difference between AG in the curcumin treatment experiment and HG in the toxicity experiment in this study. These reflect the stability of the test method and the effectiveness of curcumin therapy in this study. On the whole, JAK2/NLRP3-mediated pyroptosis and fibrosis may be a key target for the prevention and treatment of liver damage induced by AFB 1 exposure, and curcumin can regulate this pathway and alleviate the liver toxicity of AFB 1 .

Conclusions
Curcumin can alleviate AFB 1 -induced liver pyroptosis and fibrosis by regulating the JAK2/NLRP3 signaling pathway in ducks. Therefore, curcumin as a natural plant extract shows potential in preventing AFB 1 -induced liver fibrosis.