Non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disease in both humans and animals. Low dose of aflatoxin B1 (AFB1), a potent hepatotoxin, aggravated NAFLD. However, whether gut microbiome and its metabolites are involved in the aggravating effects of AFB1 on NAFLD remains unclear. The study aims to investigate the role of gut microbiome and metabolites in the aggravating effects of AFB1 on NAFLD. The results showed that AFB1 at 20, 40 and 80 μg/kg.bw for 4 weeks aggravated NAFLD as demonstrated by increasing lipid accumulation, inflammation and fibrosis. AFB1 at 40 μg/kg.bw aggravated gut microbiota disorders and intestinal barrier damage, elevated serum and liver sphingomyelin (SM) and ceramide (Cer) levels and necroptosis in CDAHFD-fed mice. Fecal microbiota from AFB1-treated mice aggravated NAFLD and necroptosis, and increased SM and Cer levels in CDAHFD-fed mice. SM treatment aggravated NAFLD and necroptosis just like that of AFB1. Myriocin, inhibitor of SM synthesis, alleviated the AFB1-aggravated NAFLD and necroptosis. Collectively, these results indicate that low dose of AFB1 induced gut microbiota dysbiosis, thereby aggravated SM accumulation, which in turn, aggravated NAFLD and necroptosis. Future studies will focus on identifying the key gut microbe responsible for SM accumulation.

Fumonisin B1 (FB1) and Ochratoxin A (OTA) are two nephrotoxic mycotoxins that frequently co-occur in feed and food. Ferroptosis, an iron-dependent form of programmed cell death, is involved in kidney injury. Hippo/YAP signalling pathway is involved in cell proliferation and ferroptosis. This study aims to investigate the role of the Hippo/YAP signalling pathway and ferroptosis in the nephrotoxicity induced by co-exposure to FB1 and OTA in mice and PK-15 cells. Results showed that co-exposure to FB1 and OTA significantly induced kidney injury as demonstrated by increasing histopathological lesions, kidney index, serum BUN, CRE and UA levels, decreasing PK-15 cell viabilities, increasing PK-15 cell LDH release and kidney injury molecule and inflammatory cytokine expression levels of kidney and PK-15 cells. RNA-seq analysis of PK-15 cells revealed that OTA and FB1 co-exposure identified 664 DEGs and enriched ferroptosis and Hippo signalling pathway of KEGG. FB1 and OTA co-exposure induced ROS production, decreased GSH content, increased MDA level, iron content, and 4-HNE expression, and increased ACSL4, LPCAT3 and HO-1 expressions, and decreased GPX4, SLC7A11 and FTH expressions. FB1 and OTA co-exposure increased YAP1, TEAD1, and LAST1 expressions, while concurrently reduced YAP1 and LAST1 phosphorylation levels. Knock-down ASCL4 attenuated nephrotoxicity induced by OTA and FB1 co-exposure. Knock-down YAP1 rescued the MDA and iron accumulation and GSH decreasing, inhibited ferroptosis, and alleviated OTA and FB1-induced nephrotoxicity. This study elucidates that YAP1 played a key role in OTA and FB1 co-exposure induced nephrotoxicity through regulating ferroptosis, providing targets against combination of OTA and FB1 exposure-induced nephrotoxicity.

Arsenic is an environmental contaminant with potent renal toxicity. Accumulating evidence has indicated that gut microbiota dysbiosis plays an important role in kidney disease. However, the role of gut microbiota in arsenic-exposure-induced renal injury remains unclear. In our study, chronic exposure to NaAsO2 and fecal microbiota transplantation (FMT) from NaAsO2-exposed mice increased intestinal permeability and elevated renal indoxyl sulfate (IS), a key metabolite of gut microbiota, which promoted renal fibrosis and activated the AhR/NLRP3 inflammasome signaling pathway. Moreover, AST-120, a non-specific adsorbent, alleviated chronic NaAsO2 exposure-induced renal fibrosis by reducing the IS level in mice. In HK-2 cells, IS mediated NaAsO2-induced fibrosis via the AhR/NLRP3 inflammasome signaling pathway. Collectively, we confirmed that chronic exposure to NaAsO2 caused renal fibrosis and intestinal barrier dysfunction. Meanwhile, the gut-kidney axis plays a significant role in the mechanism of NaAsO2-induced renal fibrosis, providing a new therapeutic target for the prevention and treatment of arseniasis.

BACKGROUND:Aflatoxin B1 (AFB1) is a potent hepatotoxin that poses significant health risks to both livestock and humans. β-Nicotinamide mononucleotide (NMN) has potential therapeutic benefits for liver diseases. However, its mechanism against AFB1-induced liver injury remains unclear. OBJECTIVE:This study investigates whether NMN supplementation reduces AFB1-induced liver injury through the modulation of the gut-liver axis and elucidates the underlying molecular mechanism. METHODS:Mice were exposed to AFB1 (0.75 mg·kg-1, p.o.) for 2 weeks to induce liver injury, with or without NMN (300 mg·kg-1, p.o.). Changes in gut microbiota were assessed via 16S rRNA sequencing, while bile acids (BAs) profiles were quantified using targeted metabolomics. The farnesoid X receptor (FXR/NR1H4) pathway was analysed using qPCR, western blot and immunofluorescence. To establish causality, antibiotic depletion, faecal microbiota transplantation (FMT) and intestine-specific FXR knockout (FXRΔIE) mice were utilized. RESULTS:NMN supplementation attenuated AFB1-induced liver injury, inflammation and oxidative stress, while restoring intestinal barrier integrity and reducing hepatic bile acids accumulation. Mechanistically, NMN reshaped the gut microbiota, increased bile salt hydrolase (BSH) activity and lowered intestinal conjugated bile acids, which correlated with activation of intestinal FXR/fibroblast growth factor 15 (FGF-15) signalling and suppression of hepatic Cyp7a1 expression. Importantly, antibiotic depletion of gut microbiota abolished NMN protection, whereas FMT from NMN-treated donors conferred resistance. Importantly, NMN failed to protect FXRΔIE mice, demonstrating that intestinal FXR is essential. CONCLUSION:NMN alleviates AFB1-induced liver injury via a gut microbiota-bile acid-FXR axis, highlighting a novel mechanism for its hepatoprotective effects.

Infection with porcine circovirus type 2 (PCV2) can increase oxidative stress, impair immune function and reduce growth performance in weaned piglets. Selenium-enriched probiotics (SeP), as a novel form of organic selenium (Se), have attracted increasing attention due to their combined nutritional and immunomodulatory properties. However, the effects of SeP on weaned piglets under PCV2 infection conditions remain unclear. This study focused on weaned piglets from a PCV2-positive pig farm in Jiangsu Province. A total of 60 piglets were randomly divided into 5 groups, with 3 replicates per group and 4 piglets per replicate. The control group was fed a basal diet, while the experimental groups received basal diets supplemented with sodium selenite (Na2SeO3) or SeP at levels of 0.3 mg/kg and 0.6 mg/kg, respectively. This study systematically evaluated thegrowth performance, antioxidant capacity, immune function, and PCV2 infection levels in weaned piglets. Compared with the control group, Se supplementation significantly increased daily weight gain and feed-to-gain ratio (F/G) while decreasing the feed-to-gain ratio in weaned piglets (P < 0.05). Se supplementation significantly increased serum glutathione peroxidase (GPX) and superoxide dismutase activity while decreasing malondialdehyde content. It also significantly upregulated mRNA expression levels of selenoprotein-related genes including GPX1, GPX4, and Thioredoxin Reductase 1 (TR1) in blood (P < 0.05). In terms of immune regulation, supplementation with Na2SeO3 or SeP significantly increased serum levels of interleukin-2 and tumor necrosis factor-α, while enhancing the proliferative capacity of peripheral blood lymphocytes (P < 0.05). Additionally, Se supplementation significantly reduced the copy number of PCV2 DNA in the blood of PCV2-infected weaned piglets (P < 0.05). Comprehensive analysis revealed that SeP demonstrated superior overall efficacy compared to Na2SeO3 in enhancing growth performance, boosting antioxidant and immune functions, and suppressing PCV2 replication. This study provides a theoretical basis for employing SeP as a precise nutritional strategy in pig populations affected by PCV2.

Gut-liver axis disturbance is the unifying pathogenesis of cholestatic liver diseases. The purpose of this study was to explore the underlying mechanisms of the probiotic Lactobacillus amylovorus (LA) and its secreted extracellular vesicles (EVs) on liver damage and fibrosis in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed and multidrug resistance protein 2 knockout (Mdr2-/-) mice. Direct replenishment of LA is sufficient to correct the DDC-fed and Mdr2-/--induced liver damage and fibrosis. Mechanistic studies show that the secretion of EVs is required for the LA-induced liver protective effects. RNA sequencing results demonstrated that the enrichment of differentially expressed genes was associated with glutathione metabolism, microbial metabolism in diverse environments and inflammatory mediator regulation of TRP channels in DDC-fed mice. Our findings revealed that LAEVs reshaped the gut microbiota, which was associated with increased bile acids (BAs) deconjugation and fecal BAs excretion, repaired gut barrier function, activated intestinal Farnesoid X receptor/Fibroblast growth factor 15 (FXR/FGF-15) axis, reduced liver BAs and oxidative stress level, which ultimately mitigated liver damage and fibrosis in both DDC-fed and Mdr2-/- mice. Notably, LAEVs did not ameliorate DDC-induced liver damage or fibrosis in antibiotic-treated mice. Furthermore, LAEVs provided protection against DDC-induced liver injury and fibrosis in fecal microbiota transplantation mice. LAEVs did not ameliorate DDC-induced liver damage or fibrosis in BSH inhibitor (CAPE)-treated mice. LAEVs also failed to improve liver damage and fibrosis in DDC-induced intestinal epithelial cell-specific FXR knockout (Fxr△IE) mice. This study revealed that LAEVs mitigated cholestatic liver fibrosis via regulating gut microbiota-bile acid-ROS axis in mice.

Cadmium (Cd) is a persistent environmental pollutant that poses a significant health risk to humans and animals, with acute exposure known to induce kidney injury. Fucoidan (Fc), a natural bioactive polysaccharide derived from brown algae, exhibits diverse biological activities; however, its potential to protect against Cd-induced kidney damage and the underlying mechanisms remain unclear. In this study, we investigated the effects of Fc on Cd-induced renal injury in vitro and further explored the role of transcription factor EB (TFEB) in regulating autophagy in its protective mechanism. Our results demonstrate that in Cd-exposed porcine kidney cells (PK-15), Fc suppressed the expression of renal inflammatory factors (TNF-α, IL-1β) and kidney injury markers (NGAL, NTN-1, KIM-1), reduced reactive oxygen species (ROS) production, and downregulated apoptosis-related proteins (cleaved caspase-3 and cleaved caspase-9). Mechanistically, Fc upregulated TFEB protein expression, enhanced the levels of lysosomal function-related proteins (Cathepsin B, CTSB; Cathepsin D, CTSD), and reversed Cd-induced autophagic flux blockade. Importantly, TFEB silencing abolished the protective effects of Fc. Collectively, these findings suggest that Fc exerts renoprotective effects against Cd-induced injury by restoring autophagic flux, a process that involves TFEB.

The prevalence and spread of canine influenza virus (CIV) pose a threat to the health of dogs and humans. Some studies have shown that autophagy is closely related to virus replication, but the exact relationship between CIV replication and autophagy is still unclear. Therefore, this study investigated the effects of autophagy on CIV replication in vitro and in vivo. The data showed that CIV infection significantly caused respiratory tract damage in mice, upregulated the mRNA/protein levels of CIV replication-related genes and autophagy-related genes. In addition, the activation of autophagy by rapamycin (Rapa) significantly intensified the CIV replication and the respiratory tract damage of mice, while the inhibition of autophagy by 3-Methyladenine (3-MA) significantly alleviated these effects. Data of MDCK cells also demonstrated that CIV promoted self-replication through activating autophagy, and the upregulation of AKT/mTOR by insulin significantly inhibited the CIV replication. In summary, this study showed that CIV could promote self-replication by activating AKT/mTOR mediated autophagy, which provides new ideas for the prevention and treatment of canine influenza.

Cholestatic liver disease is a type of severe liver disease that results mainly from the retention of bile acids (BAs). Our study aimed to explore the protective mechanism of Lactobacillus rhamnosus GG-derived extracellular vesicles (LGG-EVs) on hepatic BA synthesis, liver injury and fibrosis in multidrug resistance protein 2 knockout (Mdr2-/-) mice. LGG-EVs markedly attenuated liver injury and fibrosis, concomitant with a substantial reduction in hepatic BA retention in Mdr2-/- mice. The RNA sequencing results demonstrated that the enrichment of differentially expressed genes was associated with lipid metabolism, cholesterol biosynthesis, bile acid metabolism, and the PPAR signalling pathway. LGG-EVs reprogrammed the intestinal flora and enhanced intestinal BA deconjugation. Targeted bile acid metabolomics revealed a pronounced shift: ileal conjugated BAs (TDCA, TUDCA, and THDCA, especially TCA and T alpha/ beta MCA) were extensively reduced, whereas unconjugated species (UDCA, HDCA, CA, alpha/beta/omega MCA and especially DCA) exhibited widespread elevation, which prominently activated the intestinal Farnesoid X receptor (FXR)/Fibroblast growth factor 15 (FGF-15) axis and inhibited hepatic BA synthesis. However, LGG-EVs failed to ameliorate BA synthesis and liver fibrosis in antibiotic-treated Mdr2-/- mice. Furthermore, these alterations were mitigated by intestine-specific FXR inhibitors in Mdr2-/- mice. In conclusion, LGG-EVs ameliorate cholestatic liver disease by reprogramming hepatic metabolism, reshaping the gut microbiota, increasing BSH-mediated bile acid deconjugation and activating the intestinal FXR/FGF-15 axis. These collective findings establish LGG-EVs as a novel and effective therapeutic strategy for cholestatic liver diseases.

Nonalcoholic fatty liver disease (NAFLD) represents an increasing public health concern. The underlying pathophysiological mechanisms of NAFLD remains unclear, and as a result, there is currently no specific therapy for this condition. However, recent studies focus on extracellular vesicles (EVs) as a novelty in their role in cellular communication. An imbalance in the gut microbiota composition may contribute to the progression of NAFLD, making the gut-liver axis a promising target for therapeutic strategies. This review aims to provide a comprehensive overview of EVs in NAFLD. Additionally, exosome-like nanovesicles derived from plants (PELNs) and probiotics-derived extracellular vesicles (postbiotics) have demonstrated the potential to re-establish intestinal equilibrium and modulate gut microbiota, thus offering the potential to alleviate NAFLD via the gut-liver axis. Further research is needed using multiple omics approaches to comprehensively characterize the cargo including protein, metabolites, genetic material packaged, and biological activities of extracellular vesicles derived from diverse microbes and plants.

Mycotoxins are toxic metabolites produced by fungal species, commonly exist in animal feeds, and pose a serious risk to human as well as animal health. But limited studies have focused on combined effects of no-observed adverse effect levels. In vivo study, 6 weeks old twenty-four mice were individually exposed to Deoxynivalenol (DON) at 0.1 mg/kg BW, Aflatoxin B1 (AFB1) at 0.01 mg/kg BW, and mixture of DON and AFB1 (0.1 mg/kg BW and 0.01 mg/kg BW, respectively) for 28 days. Then, DON at 0.5 μg/mL, AFB1 at 0.04 μg /mL, and mixtures of DON and AFB1 (0.5 μg /mL, 0.04 μg /mL, respectively) were applied to porcine alveolar macrophages (PAMs) in vitro study. Our in vivo results revealed that the combined no-observed adverse effect levels of DON and AFB1 administration decreased IgA and IgG levels in the serum, the splenic TNF-α, IFN-γ, IL-2 and IL-6 mRNA expression and T-lymphocyte subset levels (CD4+ and CD8+) in the spleen. Additionally, the combined administration increased caspase-3, caspase-9, Bax, Cyt-c, and decreased Bcl-2 protein expression. Taken together, the combined no-observed adverse effect levels of DON and AFB1 could induce immunosuppression, which may be related to apoptosis. This study provides new insights into the combined immune toxicity (DON and AFB1).

To investigate the effects of B. subtilis on the specific immune response of lactating sows to E. coli and the diarrhea rate in suckling piglets, thirty large white sows with similar farrowing dates were randomly divided into two groups: a feedback feeding (i.e., feeding a homogenate of intestinal contents and tissues from E. coli-infected piglets to sows; FB) group and a feedback feeding with B. subtilis (FB + BS) group. Serum, colostrum, and intestinal tissues from sows and piglets were collected to assess the immune response and intestinal barrier function at weaning. T and B cells from Peyer’s patches (PPs) and mesenteric lymph nodes (MLNs) in lactating mice (with treatments consistent with the sows’) were isolated to explore the underlying mechanism. The results showed that, compared with the FB group, the reproductive performance of sows and the growth performance of their offspring were effectively improved in the FB + BS group. Moreover, the levels of IgG/IgA and those of IgG/IgA against E. coli in the serum and colostrum of sows in the FB+BS group were increased (p < 0.05). Meanwhile, the ratio of CD4+/CD8+, CD4+CXCR5+PD1+, and B220+IgA+ cells in MLNs and PPs, and the IgA levels in the mammary glands of mice, were also increased in the FB + BS group (p < 0.05). Notably, in suckling piglets in the FB + BS group, the diarrhea rate was decreased (p < 0.05), and the intestinal barrier function and intestinal flora composition at weaning were significantly improved. Overall, these results indicated that B. subtilis feed supplementation combined with feedback feeding in pregnant and lactating sows can reduce diarrhea in suckling piglets by enhancing the maternal immune response against E. coli and intestinal barrier function in their offspring, improving survival rates and pre-weaning growth.

Fumonisin B1 (FB1), one of the most widely distributed mycotoxins found in grains and feeds as contaminants, affects many organs including the kidney once ingested. However, the nephrotoxicity of FB1 remains to be further uncovered. The connection between necroptosis and nephrotoxicity of FB1 has been investigated in this study. The results showed that mice exposed to high doses of FB1 (2.25 mg/kg b.w.) developed kidney damage, with significant increases in proinflammatory cytokines (Il-6, Il-1β), kidney injury-related markers (Ngal, Ntn-1), and gene expressions linked to necroptosis (Ripk1, Ripk3, Mlkl). The concentration-dependent damage effects of FB1 on PK-15 cells contain cytotoxicity, cellular inflammatory response, and necroptosis. These FB1-induced effects can be neutralized by pretreatment with the necroptosis inhibitor Nec-1. Additionally, FB1 caused mitochondrial damage and mitophagy in vivo and in vitro, whereas Mdivi-1, a mitophagy inhibitor, prevented these effects on PK-15 cells as well as, more crucially, necroptosis. In conclusion, the RIPK1/RIPK3/MLKL signal route of necroptosis, which may be controlled by mitophagy, mediated nephrotoxicity of FB1. Our findings clarify the underlying molecular pathways of FB1-induced nephrotoxicity.

Cadmium (Cd) is a widespread environmental contaminant with high toxicity to human health. Melatonin has been shown to improve Cd-induced liver damage. However, its mechanism has not yet been elucidated. In this study, we aimed to investigate the effects of melatonin on Cd-induced liver damage and fibrosis. A combination of 16S rRNA gene sequencing and mass spectrometry-based metabolomics was adopted to investigate changes in the gut microbiome and its metabolites on the regulation of melatonin in Cd-induced liver injury and fibrosis of mice. Further, nonabsorbable antibiotics, a fecal microbiota transplantation (FMT) program and intestine-specific farnesoid X receptor (FXR) knockout mice were employed to explore the mechanism of melatonin (MT) on liver injury and fibrosis in Cd treated mice. MT significantly improved hepatic inflammation, bile duct hyperplasia, liver damage, and liver fibrosis, with a notable decrease in liver bile acid levels in Cd-exposed mice. MT treatment remodeled the gut microbiota, improved gut barrier function, and reduced the production of gut-derived lipopolysaccharide (LPS). MT significantly decreased the intestinal tauro-β-muricholic acid levels, which are known as FXR antagonists. Notably, MT prominently activated the intestinal FXR signaling, subsequently inhibiting liver bile acid synthesis and decreasing hepatic inflammation in Cd-exposed mice. However, MT could not ameliorate Cd-induced liver damage and fibrosis in Abx-treated mice. Conversely, MT still exerted a protective effect on Cd-induced liver damage and fibrosis in FMT mice. Interestingly, MT failed to reverse liver damage and fibrosis in Cd-exposed intestinal epithelial cell-specific FXR gene knockout mice, indicating that intestinal FXR signaling mediated the protective effect of MT treatment. MT improves Cd-induced liver damage and fibrosis through reshaping the intestinal flora, activating the intestinal FXR-mediated suppression of liver bile acid synthesis and reducing LPS leakage in mice.

Ochratoxin A (OTA) induces kidney damage in animals and humans. Ferroptosis is an iron-dependent form of regulated cell death that is involved in OTA-induced kidney injury. Quercetin (QCT), which is commonly found in numerous fruit and vegetables, has extensive pharmacological properties, such as anti-oxidant and anti-inflammatory. The present study aimed to evaluate the effects of QCT on OTA-induced kidney damage and the associated ferroptosis mechanism in mice. The results showed that OTA induced kidney damage, as demonstrated by the presence of kidney histopathological lesions, increased serum BUN and CRE levels, mRNA levels of Ntn1, Kim1, Tnfa, Ilb and Il6, and immunofluorescence of TNFα. OTA induced lipid peroxidation and ferroptosis by increasing the MDA level, 4-HNE production, and the iron concentration, decreasing the GSH content, increasing ACSL4 and HO-1 mRNA and protein levels, and decreasing GPX4 mRNA and protein levels. QCT supplementation alleviated OTA-induced kidney damage and inhibited OTA-induced lipid peroxidation and ferroptosis by reversing the OTA-induced above changes. Erastin weakened the protective effects of QCT on the histopathological damage, renal function, and inflammation induced by OTA. These findings indicated that QCT alleviated OTA-induced kidney injury through ferroptosis, suggesting that QCT might serve as a feed additive in mycotoxin contamination environments.

Cholestatic liver disease is characterized by disturbances in the intestinal microbiota and excessive accumulation of toxic bile acids (BA) in the liver. Melatonin (MT) can improve liver diseases. However, the underlying mechanism remains unclear. This study aimed to explore the mechanism of MT on hepatic BA synthesis, liver injury, and fibrosis in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed and Mdr2-/- mice. MT significantly improved hepatic injury and fibrosis with a significant decrease in hepatic BA accumulation in DDC-fed and Mdr2-/- mice. MT reprogramed gut microbiota and augmented fecal bile salt hydrolase activity, which was related to increasing intestinal BA deconjugation and fecal BA excretion in both DDC-fed and Mdr2-/- mice. MT significantly activated the intestinal farnesoid X receptor (FXR)/fibroblast growth factor 15 (FGF-15) axis and subsequently inhibited hepatic BA synthesis in DDC-fed and Mdr2-/- mice. MT failed to improve DDC-induced liver fibrosis and BA synthesis in antibiotic-treated mice. Furthermore, MT provided protection against DDC-induced liver injury and fibrosis in fecal microbiota transplantation mice. MT did not decrease liver injury and fibrosis in DDC-fed intestinal epithelial cell-specific FXR knockout mice, suggesting that the intestinal FXR mediated the anti-fibrosis effect of MT. In conclusion, MT ameliorates cholestatic liver diseases by remodeling gut microbiota and activating intestinal FXR/FGF-15 axis-mediated inhibition of hepatic BA synthesis and promotion of BA excretion in mice.

Fumonisin B1 (FB1), a water-soluble mycotoxin released by Fusarium moniliforme Sheld, is widely present in corn and its derivative products, and seriously endangers human life and health. Recent studies have reported that FB1 can lead to pyroptosis, however, the mechanisms by which FB1-induced pyroptosis remain indistinct. In the present study, we aim to investigate the mechanisms of pyroptosis in intestinal porcine epithelial cells (IPEC-J2) and the relationship between FB1-induced endoplasmic reticulum stress (ERS) and pyroptosis. Our experimental results showed that the pyroptosis protein indicators in IPEC-J2 were significantly increased after exposure to FB1. The ERS markers, including glucose-regulated Protein 78 (GRP78), PKR-like ER kinase protein (PERK), and preprotein translocation factor (Sec62) were also significantly increased. Using small interfering RNA silencing of PERK or Sec62, the results demonstrated that upregulation of Sec62 activates the PERK pathway, and activation of the PERK signaling pathway is upstream of FB1-induced pyroptosis. After using the ERS inhibitor 4-PBA reduced the FB1-triggered intestinal injury by the Sec62-PERK pathway. In conclusion, we found that FB1 induced pyroptosis by upregulating Sec62 to activate the PERK pathway, and mild ERS alleviates FB1-triggered damage. It all boils down to one fact, the study provides a new perspective for further, and improving the toxicological mechanism of FB1.

Aflatoxin B1 (AFB1) is a mycotoxin that widely found in the environment and mouldy foods. AFB1 initially targets the intestine, and AFB1-induced intestinal injury cannot be ignored. Lactobacillus amylovorus (LA), a predominant species of Lactobacillus, plays a role in carbohydrate metabolism. Extracellular vesicles (EVs), small lipid membrane vesicles, are widely involved in diverse cellular processes. However, the mechanism by which Lactobacillus amylovorus-QC1H-derived EVs (LA.EVs) protect against AFB1-induced intestinal injury remains unclear. In our study, a new strain named Lactobacillus amylovorus-QC1H (LA-QC1H) was isolated from pig faeces. Then, EVs derived from LA-QC1H were extracted via ultracentrifugation. Our results showed that LA.EVs significantly alleviated AFB1-induced intestinal injury by inhibiting the production of proinflammatory cytokines, decreasing intestinal permeability and increasing the expression of tight junction proteins. Moreover, 16 S rRNA analysis revealed that LA.EVs modulated AFB1-induced gut dysbiosis in mice. However, LA.EVs did not exert beneficial effects in antibiotic-treated mice. LA.EVs treatment increased intestinal levels of indole-3-acetic acid (IAA) and activated intestinal aryl hydrocarbon receptor (AHR)/interleukin-22 (IL-22) signalling in AFB1-exposed mice. Inhibition of intestinal AHR signalling markedly weakened the protective effect of LA.EVs in AFB1-exposed mice. LA.EVs alleviated AFB1-induced intestinal injury by modulating the gut microbiota, activating the intestinal AHR/IL-22 signalling, reducing the inflammatory response and promoting intestinal barrier repair in mice.

Aflatoxin B1 (AFB1), a worldwide mycotoxin found in food and foodstuffs, is a potent hepatotoxin in humans and animals. Non-alcoholic fatty liver disease (NAFLD), a widespread disease, could progress from simple steatosis to non-alcoholic steatohepatitis (NASH), hepatic cirrhosis, and even hepatocellular carcinoma (HCC). To date, little is known concerning the relationship between AFB1 and the progression of NAFLD. The effects of low doses of AFB1 on the development of NASH and their mechanism were investigated in vivo and in vitro. The results in vivo showed that AFB1 at 20 and 40 μg/kg.bw aggravated CDAHFD-induced NASH in mice as demonstrated by increasing the serum and liver lipid accumulation, liver inflammation and injury. The results in vitro showed that AFB1 at 1.0 μM aggravated FFA-induced lipid accumulation, inflammation and cell damage in HepG2 cells. In addition, RNA-seq indicated that necroptosis, Toll like receptor signaling and TNF-α signaling showed a significant change in KEGG pathway enrichment in AFB1 at 40 μg/kg.bw. AFB1 significantly upregulated the mRNA and protein levels of TLR4, RIPK3, p-RIPK3, MLKL and p-MLKL, and increased TUNEL positive cells. Also, immunofluorescence results showed that TUNEL, TLR4, TNF-α had co-localized with RIPK3, respectively. Necroptosis inhibitor (GSK-872) attenuated the aggravating effects of AFB1 on NASH. Knockout of TLR4 inhibited necroptosis and rescued the aggravating effects of AFB1 on NASH. These data indicate that low dose of AFB1 aggravated NASH via TLR4-mediated necroptosis. This suggests that low dose of AFB1 is potentially harmful to animals and humans, as they exacerbate NASH.