Paeoniflorin recued hepatotoxicity under zinc oxide nanoparticles exposure via regulation on gut-liver axis and reversal of pyroptosis.

The risks of Zinc oxide nanoparticles (ZnO NPs) applications in biological medicine, food processing industry, agricultural production and the biotoxicity brought by environmental invasion of ZnO NPs both gradually troubled the public due to the lack of research on detoxification strategies. TFEB-regulated autophagy-pyroptosis pathways were found as the crux of the hepatotoxicity induced by ZnO NPs in our latest study. Here, our study served as a connecting link between preceding toxic target and the following protection mechanism of Paeoniflorin (PF). According to a combined analysis of network pharmacology/molecular docking-intestinal microbiota-metabolomics first developed in our study, PF alleviated the hepatotoxicity of ZnO NPs from multiple aspects. The hepatic inflammatory injury and hepatocyte pyroptosis in mice liver exposed to ZnO NPs was significantly inhibited by PF. And the intestinal microbiota disorder and liver metabolic disturbance were rescued. The targets predicted by bioinformatics and the signal trend in subacute toxicological model exhibited the protectiveness of PF related to the SIRT1-mTOR-TFEB pathway. These evidences clarified multiple protective mechanisms of PF which provided a novel detoxification approach against ZnO NPs, and further provided a strategy for the medicinal value development of PF.

TFEB coordinates autophagy and pyroptosis as hepatotoxicity responses to ZnO nanoparticles.

Zinc oxide nanoparticles (ZnO NPs) have drawn serious concerns about their biotoxicity due to their extensive applications in biological medicine, clinical therapeutic, daily chemical production, food and agricultural additives. In our present study, we clarified hepatotoxic mechanism of ZnO NPs through investigating the crosstalk between autophagy and pyroptosis, a remaining enigma in hepatocyte stimulated by ZnO NPs. Based on the effects of autophagy intervention by Rapamycin (Rap) and 3-Methyladenine (3-MA), and the observation of pyroptosis morphology and related indexes, the autophagy and pyroptosis simultaneously initiated by ZnO NPs were interrelated and the autophagy characterized by autophagosome production and increased expression of autophagy proteins was identified as a protective response of ZnO NPs against pyroptosis. According to the analysis of protein expression and fluorescence localization, the NLRP3 inflammasome assemble and the classical Caspase-1/GSDMD-dependent pyroptosis induced by ZnO NPs was modulated by autophagy. In this process, the adjustment of TFEB expression and nuclear translocation by gene knockout and gene overexpression, further altered the tendency of ZnO NPs-induced pyroptosis via the regulation of autophagy and lysosomal biogenesis. The knockout of TFEB gene exacerbated the pyroptosis via autophagy elimination and lysosome inhibition. While the alleviation of NLRP3 generation and pyroptosis activation was observed after treatment of TFEB gene overexpression. Additionally, the siRNA interference confirmed that TRAF-6 was involved in the TFEB-mediated global regulation of autophagy-lysosome-pyroptosis in response to ZnO NPs. Accordingly, pyroptosis induced by ZnO NPs in hepatocyte could be significantly avoided by TFEB-regulated autophagy and lysosome, further providing new insights for the risk assessment and therapeutic strategy.

Oxidative stress-related canonical pyroptosis pathway, as a target of liver toxicity triggered by zinc oxide nanoparticles.

Zinc oxide nanoparticles (ZnO NPs) have been widely used in the fields of daily necessities, clinical diagnosis, drug delivery and agricultural production. The improper use of ZnO NPs could pose a risk to ecological environment and public health. Liver has been known as a critical toxic target of ZnO NPs. However, the question whether ZnO NPs lead to hepatocyte death through pyroptosis has not been answered yet, and the effect of oxidative stress on ZnO NPs-induced pyroptosis remains a mystery. We revealed that ZnO NPs disrupted zinc homeostasis and induced oxidative stress impairment in rat liver. Meanwhile, ZnO NPs triggered the assembly of NLRP3-ASC-Caspase-1 inflammatory complex and pyroptosis in both rat liver and HepG2 cells, further causing the activation of GSDMD, promoting the leakage of inflammatory cytokines including IL-1ß and IL-18. Importantly, the inhibition of oxidative stress was found to provide protection against pyroptosis in hepatocyte exposed to ZnO NPs. We identified a novel mechanism of liver damage induced by ZnO NPs, demonstrating the activation of canonical Caspase-1-dependent pyroptosis pathway and clarifying the protection of antioxidation against pyroptosis damage. Our discovery provided a support for risk assessment of ZnO NPs and target exploration for clinical treatment related to pyroptosis.

Lethality of Zinc Oxide Nanoparticles Surpasses Conventional Zinc Oxide via Oxidative Stress, Mitochondrial Damage and Calcium Overload: A Comparative Hepatotoxicity Study.

Zinc oxide nanoparticles (ZnO NPs) with high bioavailability and excellent physicochemical properties are gradually becoming commonplace as a substitute for conventional ZnO materials. The present study aimed to investigate the hepatotoxicity mechanism of ZnO NPs and traditional non-nano ZnO particles, both in vivo and in vitro, and identify the differences in their toxic effects. The results showed that the extent and conditions of zinc ion release from ZnO NPs were inconsistent with those of ZnO. The RNA-seq results revealed that the expression quantity of differentially expressed genes (DEGs) and differentially expressed transcripts (DETs) affected by ZnO NPs was more than in ZnO, and the overall differences in genes or transcripts in the ZnO NPs group were more pronounced than in the ZnO group. Furthermore, the cell inactivation, oxidative stress, mitochondrial damage, and intracellular calcium overload induced by ZnO NPs were more serious than ZnO in HepG2 cells. Moreover, compared with traditional ZnO, the rat liver damage induced by ZnO NPs was more significant, with evidence of higher AST and ALT levels, weaker antioxidant capacity, and more serious histopathological damage (p < 0.05). In summary, the hepatotoxicity of ZnO NPs was more serious than that of conventional ZnO, which is helpful to understand the hepatotoxicity mechanism of Zn compounds in different states and improve the risk assessment of novel nano ZnO products in a variety of applications.

Food-Origin Mycotoxin-Induced Neurotoxicity: Intend to Break the Rules of Neuroglia Cells.

Mycotoxins are key risk factors in human food and animal feed. Most of food-origin mycotoxins could easily enter the organism and evoke systemic toxic effects, such as aflatoxin B1 (AFB1), ochratoxin A (OTA), T-2 toxin, deoxynivalenol (DON), zearalenone (ZEN), fumonisin B1 (FB1), and 3-nitropropionic acid (3-NPA). For the last decade, the researches have provided much evidences in vivo and in vitro that the brain is an important target organ on mycotoxin-mediated neurotoxic phenomenon and neurodegenerative diseases. As is known to all, glial cells are the best regulator and defender of neurons, and a few evaluations about the effects of mycotoxins on glial cells such as astrocytes or microglia have been conducted. The fact that mycotoxin contamination may be a key factor in neurotoxicity and glial dysfunction is exactly the reason why we reviewed the activation, oxidative stress, and mitochondrial function changes of glial cells under mycotoxin infection and summarized the mycotoxin-mediated glial cell proliferation disorders, death pathways, and inflammatory responses. The purpose of this paper is to analyze various pathways in which common food-derived mycotoxins can induce glial toxicity and provide a novel perspective for future research on the neurodegenerative diseases.

Targeting HMGB1 inhibits T-2 toxin-induced neurotoxicity via regulation of oxidative stress, neuroinflammation and neuronal apoptosis.

T-2 toxin, a food-derived mycotoxin, has been identified as a neurotoxin. Nonetheless, T-2 toxin-induced neuroinflammation has never been revealed. As an important therapeutic target for inflammatory diseases and cancers, the role of high mobility group B1 (HMGB1) in mycotoxin-mediated neurotoxicity remains a mystery. In current study, we found that PC12 cells were sensitive to trace amounts of T-2 toxin less than 12 ng/mL, distinguished by decreased cell viability and increased release of lactate dehydrogenase (LDH). Oxidative stress and mitochondrial damage were observed in PC12 cells, manifested as accumulation of oxidative stress products, up-regulation of Nrf2/HO-1 pathway and decrease of mitochondrial membrane potential (MMP), leading to mitochondria-dependent apoptosis. Meanwhile, we first discovered that tiny amounts of T-2 toxin triggered neuroinflammation directly, including raising the expression and translocation of NF-κB and promoting secretion of proinflammatory cytokines such as TNF-α, IL-6, IL-8 and IL-1ß. Most interestingly, the increased of HMGB1 was detected both inside and outside the cells. Conversely, HMGB1 siRNA reduced T-2 toxin-mediated oxidative stress, apoptosis and neuroinflammatory outbreak, accompanied by lessened caspase-3 and caspase-9, and decreased secretion of pro-inflammatory cytokines. Taken together, T-2 toxin-stimulated PC12 cells simultaneously displayed apoptosis and inflammation, whereas HMGB1 played a critical role in these neurotoxic processes.

Olaquindox-Induced Liver Damage Involved the Crosstalk of Oxidative Stress and p53 In Vivo and In Vitro.

Olaquindox (OLA), a member of the quinoxaline-N,N-dioxide family, has been widely used as a growth-promoting feed additive and treatment for bacterial infections. The toxicity has been a major concern, and the precise molecular mechanism remains poorly understood. The present study was aimed at investigating the roles of oxidative stress and p53 in OLA-caused liver damage. In a mouse model, OLA administration could markedly cause liver injury as well as the induction of oxidative stress and activation of p53. Antioxidant N-acetylcysteine (NAC) inhibited OLA-induced oxidative stress and p53 activation in vivo. Furthermore, knockout of the p53 gene could significantly inhibit OLA-induced liver damage by inhibiting oxidative stress and the mitochondria apoptotic pathway, compared to the p53 wild-type liver tissue. The cell model in vitro further demonstrated that p53 knockout or knockdown in the HCT116 cell and L02 cell significantly inhibited cell apoptosis and increased cell viability, presented by suppressing ROS production, oxidative stress, and the Nrf2/HO-1 pathway. Moreover, loss of p53 decreased OLA-induced mitochondrial dysfunction and caspase activations, with the evidence of inhibited activation of phosphorylation- (p-) p38 and p-JNK and upregulated cell autophagy via activation of the LC3 and Beclin1 pathway in HCT116 and L02 cells. Taken together, our findings provided a support that p53 primarily played a proapoptotic role in OLA-induced liver damage against oxidative stress and mitochondrial dysfunction, which were largely dependent on suppression of the JNK/p38 pathway and upregulation of the autophagy pathway via activation of LC3 and Beclin1.

Molecular mechanism of olaquindox-induced hepatotoxicity and the hepatic protective role of curcumin.

Olaquindox (OLA) is a chemosynthetic growth promoter, which could promote the treatment of bacterial infections and improve feed energy efficiency. Hepatotoxicity is still a poor feature associated with the adverse effects of OLA. The present study aimed to investigate the molecular mechanism of OLA-induced hepatotoxicity and the protective role of curcumin in mice and HepG2 cells. The result showed that representative biomarkers involved in mitochondrial pathway, p53 pathway, mitogen-activated protein kinase (MAPK) pathway, autophagy and antioxidant pathway were activated. Furthermore, curcumin attenuated OLA-induced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and liver damage in mice. In addition, cell viability of HepG2 was enhanced by curcumin pretreatment at 5, 10 and 20 µM. Meanwhile, curcumin markedly ameliorated OLA-induced oxidative stress, apoptosis and mitochondrial dysfunction. Moreover, curcumin pretreatment significantly up-regulated the expressions of nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1(HO-1) and down-regulated the expressions of nuclear factor-kappaB (NF-kB) and p53 through reduced the nuclear translocation of NF-kB induced by OLA. In summary, our findings indicated that OLA-induced hepatotoxicity involved in mitochondrial apoptosis, autophagy, p53 pathway, Nrf2/HO-1 pathways, and curcumin regulated OLA-induced liver damage, oxidative stress and apoptosis via activation of Nrf2/HO-1 pathway and suppression of p53 and NF-kB pathway.

Identification of Plant Soot as Novel Safe Feed Additive: Evaluation of 90-Day Oral Toxicity and Prenatal Developmental Toxicity in Rats.

Plant soot, as a novel feed additive, could not only improve digestive function but also adsorb mycotoxins and inhibit bacterial infections. The subchronic toxicity and prenatal developmental effects of plant soot were studied for the first time. Our results indicated that there was no subchronic toxicity in the range of 2,000-50,000 mg/kg plant soot added in the feed, and there was no significant difference in reproductive function, embryo development, and teratogenicity between the pregnant rats exposed to 312.5, 1,250, and 5,000 mg/kg plant soot and the control group. The maximum no-observed effect level (NOEL) of supplemental dosage in feed could be set to 50,000 mg/kg, and the maximum intragastric NOEL could be set to 5,000 mg/kg, which preliminarily provided guidance on daily additive amount or clinical protocols for plant soot, as well as promoting the development and application of this harmless antibiotic substitutes.

A duplex RT-PCR assay for detection of H9 subtype avian influenza viruses and infectious bronchitis viruses.

H9 subtype avian influenza virus (AIV) and infectious bronchitis virus (IBV) are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg production. As similar symptoms are elicited by the two pathogens, it is difficult for their differential diagnosis. So far, no reverse transcription-polymerase chain reaction (RT-PCR) assay has been found to differentiate between H9 AIV and IBV in one reaction. Therefore, developing a sensitive and specific method is of importance to simultaneously detect and differentiate H9 AIV and IBV. In this study, a duplex RT-PCR (dRT-PCR) was established. Two primer sets target the hemagglutinin (HA) gene of H9 AIV and the nucleocapsid (N) gene of IBV, respectively. Specific PCR products were obtained from all tested H9 AIVs and IBVs belonging to the major clades circulating in China, but not from AIVs of other subtypes or other infectious avian viruses. The sensitivity of the dRT-PCR assay corresponding to H9 AIV, IBV and mixture of H9 AIV and IBV were at a concentration of 1×101, 1.5×101 and 1.5×101 50% egg infective doses (EID50) mL-1, respectively. The concordance rates between the dRT-PCR and virus isolation were 99.1 and 98.2%, respectively, for detection of samples from H9N2 AIV or IBV infected chickens, while the concordance rate was 99.1% for detection of samples from H9N2 AIV and IBV co-infected chickens. Thus, the dRT-PCR assay reported herein is specific and sensitive, and suitable for the differential diagnosis of clinical infections and surveillance of H9 AIVs and IBVs.

Multi-residue fluorescent microspheres immunochromatographic assay for simultaneous determination of macrolides in raw milk.

A rapid, reliable, sensitive, and quantitative multi-residue fluorescent microspheres immunochromatographic assay (FMCA) was developed for simultaneous detection of four macrolides in raw milk. The IC50 value of the optimized FMCA was 1.36, 1.22, 1.01, and 1.39 ng/mL for erythromycin (ERY), spiramycin (SPI), tilmicosin (TIM), and tylosin (TYL), respectively. The limits of detection (LODs) for the four macrolides was 0.13 ng/mL. The recoveries of ERY, SPI, TIM, and TYL from spiked raw milk ranged from 91.8-109.2, 89.6-114.4, 84.8-111.6, and 85.8-115.2%, respectively, with coefficients of variation (CVs) of 5.4-11.3, 7.9-15.7, 6.2-13.7, and 3.2-14.9%, respectively. The whole testing process was completed within 20 min. The antibody-mixed labeled method was successfully applied to the FMCA, which greatly simplified the operation steps and saved a lot of time. Compared with the immunogold chromatographic assay (IGCA), the FMCA is more sensitive and stable and has less antibody consumption. A parallel analysis in blind raw milk samples was conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS); the results showed good correlation (r(2) = 0.99) between the two methods. Therefore, the developed multi-residue FMCA is reliable and can be easily applied to other antibiotics or other contaminants.

Rapid determination of phenylethanolamine A in biological samples by enzyme-linked immunosorbent assay and lateral-flow immunoassay.

Phenylethanolamine A (PA) is a ß-adrenergic agonist, which was first used in animal husbandry as a growth promoter in China in 2010. In this study, a monoclonal-antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) and lateral-flow immunoassay (LFA) for the detection of PA in swine urine and pork were developed. The immunogen was prepared by linking PA hapten with carrier protein via a diazotization method. The IC50 value of the optimized icELISA was 0.44 ng mL(-1). The limits of detection of the icELISA for PA in swine urine and pork were 0.13 ng mL(-1) and 0.39 ng g(-1), respectively. The recoveries of PA from spiked swine urine and pork were in the range 82.0-107.4 % and 81.8-113.3%, respectively, with the coefficients of variation in the range 4.1-16.2% and 1.2-6.3%, respectively. The mAbs had negligible cross reactivity with 10 other ß-agonists. In contrast, the LFA had a cut-off level of 5 ng mL(-1) (g) in swine urine and pork, and the results could be achieved within 5 min. Ten blind samples of swine urine were analyzed simultaneously by icELISA, LFA, and ultra-high-performance liquid chromatography-tandem mass spectrometry, and the results of the three methods agreed well. Therefore, the combination of two immunoassays provides an effective and rapid screening method for detection of PA residues in biological samples.

Preparation and characterization of an immunoaffinity column for the selective extraction of aflatoxin B1 in 13 kinds of foodstuffs.

A rapid and reliable immunoaffinity column (IAC) clean-up based ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of aflatoxin B1 (AFB1) in cereals, peanuts, vegetable oils and Chinese traditional food products like sufu and lobster sauce. The immunoaffinity column of AFB1 (AFB1-IAC) was prepared by coupling CNBr-activated Sepharose-4B with the anti-AFB1 monoclonal antibody. The column capacity of IAC was over 260ng/mL gel. Samples were extracted with methanol-water (60:40, v/v) and the extracts were then purified on an AFB1-IAC before UPLC-MS/MS analysis. The average recoveries of AFB1 in spiked samples at levels of 1.0, 5.0 and 10.0µg/kg ranged from 72% to 98%, with the relative standard deviations of 1.2-9.3% (n=6). The limits of qualification ranged from 0.07 to 0.23µg/kg, which were below the MRLs of AFB1 in the matrices evaluated. In this work, the developed method was suitable for the determination of trace AFB1 residues in 13 kinds of foodstuffs.