Effects of effective microorganisms on the physiological status, intestinal microbiome, and serum metabolites of Eriocheir sinensis.

The compound known as effective microorganisms (EMs) is widely used in aquaculture to improve water quality, but how they affect the health of Chinese mitten crab (Eriocheir sinensis) is unclear, especially in terms of intestinal microbiota and serum metabolites. In this study, we fed juvenile crabs with an EM-containing diet to explore the effects of EM on the physiological status, intestinal microbiome, and metabolites of E. sinensis. The activities of alanine aminotransferase and alkaline phosphatase were significantly enhanced by EM, indicating that EM supplementation effectively enhanced the antioxidant capacity of E. sinensis. Proteobacteria, Tenericutes, Firmicutes, Bacteroidetes, and Actinobacteria were the main intestinal microbes in both the control and EM groups. Linear discriminant effect size analysis showed that Fusobacteriaceae, Desulfovibrio, and Morganella were biomarkers in the control group, and Exiguobacterium and Rhodobacteraceae were biomarkers in the EM group. Metabolomics analysis revealed that EM supplementation increased cellular energy sources and decreased protein consumption, and oxidative stress. Together, these results indicate that EM can optimize the intestinal microbiome and serum metabolites, thereby benefiting the health of E. sinensis.

Whole exome sequencing with a focus on cardiac disease-associated genes in families of sudden unexplained deaths in Yunnan, southwest of China.

OBJECTIVES: To explore the causes of sudden unexpected death (SUD) and to search for high-risk people, whole exome sequencing (WES) was performed in families with SUDs.  METHODS: Whole exome sequencing of 25 people from 14 SUD families were screened based on cardiac disease-associated gene variants, and their echocardiograms and electrocardiograms (ECG) were also examined. The protein function of mutated genes was predicted by SIFT, PolyPhen2 and Mutation Assessor. RESULTS: In the group of 25 people from 14 SUD families, 49 single nucleotide variants (SNVs) of cardiac disease-associated genes were found and verified by Sanger sequencing. 29 SNVs of 14 cardiac disorder-related genes were predicted as pathogens by software. Among them, 7 SNVs carried by two or more members were found in 5 families, including SCN5A (c.3577C > T), IRX4 (c.230A > G), LDB3 (c.2104 T > G), MYH6 (c.3G > A), MYH6 (c.3928 T > C), TTN (c.80987C > T) and TTN (c.8069C > T). 25 ECGs were collected. In summary, 4 people had J-point elevation, 2 people had long QT syndrome (LQTS), 4 people had prolonged QT interval, 3 people had T-wave changes, 3 people had sinus tachycardia, 4 people had sinus bradycardia, 4 people had left side of QRS electrical axis, and 3 people had P wave broadening. Echocardiographic results showed that 1 person had atrial septal defect, 1 person had tricuspid regurgitation, and 2 people had left ventricular diastolic dysfunction. CONCLUSIONS: Of the 14 heart disease-associated genes in 14 SUDs families, there are 7 possible pathological SNVS may be associated with SUDs. Our results indicate that people with ECG abnormalities, such as prolonged QT interval, ST segment changes, T-wave change and carrying the above 7 SNVs, should be the focus of prevention of sudden death.

Microcystin-LR-induced autophagy via miR-282-5p/PIK3R1 pathway in Eriocheir sinensis hepatopancreas.

With the intensifying climate warming, blue-green algae blooms have become more frequent and severe, releasing environmental hazards such as microcystin that pose potential threats to human and animal health. Autophagy has been shown to play a crucial role in regulating immune responses induced by environmental hazards, enabling cells to adapt to stress and protect against damage. Although microcystin-LR (MC-LR) has been identified to affect autophagy in mammalian, its impact on aquatic animals has been poorly studied. To investigate the toxicological effects of MC-LR in aquatic ecosystems, we constructed a microRNA profile of acute MC-LR stress in the hepatopancreas of the Chinese mitten crab. Interestingly, we found the MC-LR exposure activated autophagy in the hepatopancreas based on the following evidence. Specifically, mRNA expression level of ATG7, Beclin1 and Gabarap was significantly up-regulated, autophagy regulatory pathways were significantly enriched, and numerous autolysosomes and autophagosomes were observed. Additionally, we found that miR-282-5p and its target gene PIK3R1 played important regulatory roles in autophagy by in vivo and in vitro experiments. Overexpression of miR-282-5p mimicked MC-LR-induced autophagy by inhibiting PIK3R1 expression, while miR-282-5p silencing inhibited autophagy by promoting PIK3R1 expression. Altogether, our findings suggest that MC-LR increases miR-282-5p, which then targets inhibition of PIK3R1 to stimulate autophagy. This study focused on the stress response regulatory mechanisms of juvenile crabs to toxic pollutants in water, offering a potential target for alleviating the toxicity of MC-LR. These findings lay a foundation for reducing the toxicity of MC-LR and environmental hazards in organisms.

Effects of prometryn on oxidative stress, immune response and apoptosis in the hepatopancreas of Eriocheir sinensis (Crustacea: Decapoda).

Prometryn, a triazine pesticide product used to control weed growth, poses a high risk to aquatic organisms in the environment. Several toxicological evaluations have been performed on bony fish and shrimp exposed to prometryn. However, there have been no reports conducted on the toxic mechanism of prometryn with regard to Eriocheir sinensis. In this study, our research evaluated the toxic effects of prometryn via in vitro and in vivo toxicity tests on E. sinensis. Firstly, we estimated the exposure toxicity of prometryn to E. sinensis, and then we constructed a 6 h transcriptional profile and conducted an enrichment analysis. To further reveal the toxicity of prometryn, the hepatopancreas (hepatopancreatic cells) was analyzed for antioxidant, immune and lipid-metabolism-related enzymes, antioxidant- and apoptosis-related gene expression, histopathology and TUNEL. From the results, we determined that the 96 h-LD50 was 70.059 mg/kg, and using RNA-seq, we identified 933 differentially expressed genes (DEGs), which were mainly enriched in the amino and fatty acid metabolism and the cell-fate-determination-related signaling pathway. The results of the biochemical assays showed that prometryn could significantly decrease the activities/levels of CAT, SOD, GSH, AKP and ACP, reduce the levels of T-AOC, TG, TCH, C3 and C4, and increase the MDA content. In addition, the expression levels of Nrf2, GSTs and HO-1 were first upregulated and then downregulated with increasing time. Histopathology showed that prometryn damaged the structure of the hepatopancreas cells and induced apoptosis, suggesting that the PI3K-Akt signaling pathway may be involved in the damage process of hepatopancreas cells (PI3K, PDK and Akt were downregulated whereas Bax was upregulated), leading to their apoptosis. The above results indicated that prometryn could cause injury of the hepatopancreas through oxidative stress, induce cell apoptosis, disrupt the lipid metabolism and cause immune damage. This study provided useful data for understanding and evaluating the toxicity of prometryn to aquatic crustacea.

Integrated Analysis of Transcriptomics and Metabolomics Unveil the Novel Insight of One-Year-Old Precocious Mechanism in the Chinese Mitten Crab, Eriocheir sinensis.

Eriocheir sinensis is traditionally a native high-value crab that is widely distributed in eastern Asia, and the precocity is considered the bottleneck problem affecting the development of the industry. The precocious E. sinensis is defined as a crab that reaches complete sexual maturation during the first year of its lifespan rather than as normally in the second year. However, the exact regulatory mechanisms underlying the precocity are still unclear to date. This study is the first to explore the mechanism of precocity with transcriptome-metabolome association analysis between the precocious and normal sexually mature E. sinensis. Our results indicated that the phenylalanine metabolism (map00360) and neuroactive ligand-receptor interaction (map04080) pathways play an important role in the precocity in the ovary of E. sinensis. In map00360, the predicted aromatic-L-amino-acid decarboxylase and 4-hydroxyphenylpyruvate dioxygenase isoform X1 genes and the phenethylamine, phenylethyl alcohol, trans-2-hydroxycinnamate, and L-tyrosine metabolites were all down-regulated in the ovary of the precocious E. sinensis. The map04080 was the common KEGG pathway in the ovary and hepatopancreas between the precocious and normal crab. In the ovary, the predicted growth hormone secretagogue receptor type 1 gene was up-regulated, and the L-glutamate metabolite was down-regulated in the precocious E. sinensis. In the hepatopancreas, the predicted forkhead box protein I2 gene and taurine metabolite were up-regulated and the the L-glutamate metabolite was down-regulated in the precocious crab. There was no common pathway in the testis. Numerous common pathways in the hepatopancreas between male precocious and normal crab were identified. The specific amino acids, fatty acids and flavorful nucleotide (inosine monophosphate (MP), cytidine MP, adenosine MP, uridine MP, and guanosine MP) contents in the hepatopancreas and gonads further confirmed the above omics results. Our results suggest that the phenylalanine metabolism may affect the ovarian development by changing the contents of the neurotransmitter and tyrosine. The neuroactive ligand-receptor interaction pathway may affect the growth by changing the expressions of related genes and affect the umami taste of the gonads and hepatopancreas through the differences of L-glutamate metabolite in the precocious E. sinensis. The results provided valuable and novel insights on the precocious mechanism and may have a significant impact on the development of the E. sinensis aquaculture industry.

Cytotoxicity of 4 Wild Mushrooms in a Case of Yunnan Sudden Unexplained Death.

OBJECTIVES: To explore the cytotoxicity of four wild mushrooms involved in a case of Yunnan sudden unexplained death (YNSUD), to provide the experimental basis for prevention and treatment of YNSUD. METHODS: Four kinds of wild mushrooms that were eaten by family members in this YNSUD incident were collected and identified by expert identification and gene sequencing. Raw extracts from four wild mushrooms were extracted by ultrasonic extraction to intervene HEK293 cells, and the mushrooms with obvious cytotoxicity were screened by Cell Counting Kit-8 (CCK-8). The selected wild mushrooms were prepared into three kinds of extracts, which were raw, boiled, and boiled followed by enzymolysis. HEK293 cells were intervened with these three extracts at different concentrations. The cytotoxicity was detected by CCK-8 combined with lactate dehydrogenase (LDH) Assay Kit, and the morphological changes of HEK293 cells were observed under an inverted phase contrast microscope. RESULTS: Species identification indicated that the four wild mushrooms were Butyriboletus roseoflavus, Boletus edulis, Russula virescens and Amanita manginiana. Cytotoxicity was found only in Amanita manginiana. The raw extracts showed cytotoxicity at the mass concentration of 0.1 mg/mL, while the boiled extracts and the boiled followed by enzymolysis extracts showed obvious cytotoxicity at the mass concentration of 0.4 mg/mL and 0.7 mg/mL, respectively. In addition to the obvious decrease in the number of HEK293 cells, the number of synapses increased and the refraction of HEK293 cells was poor after the intervention of Amanita manginiana extracts. CONCLUSIONS: The extracts of Amanita manginiana involved in this YNSUD case has obvious cytotoxicity, and some of its toxicity can be reduced by boiled and enzymolysis, but cannot be completely detoxicated. Therefore, the consumption of Amanita manginiana is potentially dangerous, and it may be one of the causes of the YNSUD.

Lactobacillus plantarum 17-5 attenuates Escherichia coli-induced inflammatory responses via inhibiting the activation of the NF-κB and MAPK signalling pathways in bovine mammary epithelial cells.

BACKGROUND: Mastitis is one of the most prevalent diseases and causes considerable economic losses in the dairy farming sector and dairy industry. Presently, antibiotic treatment is still the main method to control this disease, but it also brings bacterial resistance and drug residue problems. Lactobacillus plantarum (L. plantarum) is a multifunctional probiotic that exists widely in nature. Due to its anti-inflammatory potential, L. plantarum has recently been widely researched in complementary therapies for various inflammatory diseases. In this study, the apoptotic ratio, the expression levels of various inflammatory mediators and key signalling pathway proteins in Escherichia coli-induced bovine mammary epithelial cells (BMECs) under different doses of L. plantarum 17-5 intervention were evaluated. RESULTS: The data showed that L. plantarum 17-5 reduced the apoptotic ratio, downregulated the mRNA expression levels of TLR2, TLR4, MyD88, IL1ß, IL6, IL8, TNFα, COX2, iNOS, CXCL2 and CXCL10, and inhibited the activation of the NF-κB and MAPK signalling pathways by suppressing the phosphorylation levels of p65, IκBα, p38, ERK and JNK. CONCLUSIONS: The results proved that L. plantarum 17-5 exerted alleviative effects in Escherichia coli-induced inflammatory responses of BMECs.

Microcystin-LR induces apoptosis in Juvenile Eriocheir sinensis via the mitochondrial pathway.

Microcystin-LR (MC-LR), the toxic substance of cyanobacteria secondary metabolism, widely exists in water environments and poses great risks to living organisms. Some toxicological assessments of MC-LR have performed at physiological and biochemical levels. However, plenty of blanks about the potential mechanism in aquatic crustacean remains. In this study, we firstly assessed the exposure toxicity of MC-LR to juvenile E. sinensis and clarified that the 96 h LD50 of MC-LR was 73.23 µg/kg. Then, hepatopancreas transcriptome profiles of MC-LR stressed crabs were constructed at 6 h post-injection and 37 differential expressed genes (DEGs) were identified. These DEGs were enriched in cytoskeleton, peroxisome and apoptosis pathways. To further reveal the toxicity of MC-LR, oxidative stress parameters (SOD, CAT, GSH-px and MDA), apoptosis genes (caspase 3, bcl-2 and bax) and apoptotic cells were detected. Significant accumulated MDA and rise-fall enzyme activities verified the oxidative stress caused by MC-LR. It is noteworthy that quantitative real-time PCR and TUNEL assay indicated that MC-LR stress-induced apoptosis via the mitochondrial pathway. Interestingly, activator protein-1 may play a crucial role in mediating the hepatotoxicity of MC-LR by regulating apoptosis and oxidative stress. Taken together, our study investigated the toxic effects and the potential molecular mechanisms of MC-LR on juvenile E. sinensis. It provided useful data for exploring the toxicity of MC-LR to aquatic crustaceans at molecular levels.

The pathogenic potential and genetic attributes of Escherichia coli in milk from dairy cows with subclinical mastitis.

The centrality of milk and dairy products to the human diet allows potential pathogens to pose a threat to human health. Pathogenic Escherichia coli is a zoonotic foodborne pathogen with many virulence genes which cause variations in its pathogenicity. The current study aimed to investigate the pathogenic potential of E. coli from milk of dairy cows with subclinical mastitis and evaluate the genetic relatedness to E. coli from human sources. The majority of the E. coli isolates belonged to the A (55.0%) and B2 (22.5%) phylogenetic groups and the most prevalent virulence genes were colV (90.0%), fyuA (75.0%) and vat (42.5%). Mice injected with G4-BD23 (P < 0.05) and G5-BD3 had lower survival rates than controls and visible pathological changes to lung and kidney. Nineteen MLST types were identified in 40 dairy E. coli isolates and three STs (ST10, ST48 and ST942) were shared with those from human sources. Some dairy E. coli isolates were phylogenetically related to human E. coli isolates indicating pathogenic potential.

Angelica polysaccharide attenuates LPS-induced inflammation response of primary dairy cow claw dermal cells via NF-κB and MAPK signaling pathways.

BACKGROUND: Laminitis, an inflammation of the claw laminae, is one of the major causes of bovine lameness, which can lead to enormous economic losses and animal welfare problems in dairy farms. Angelica polysaccharide (AP) is proved to possess anti-inflammatory properties. But the role of AP on inflammatory response of the claw dermal cells has not been reported. The aim of this study was to investigate the anti-inflammatory effects of AP on lipopolysaccharide (LPS)-induced primary claw dermal cells of dairy cow and clarify the potential mechanisms. In the current research, the primary claw dermal cells were exposed to gradient concentrations of AP (10, 50, 100 µg/mL) in the presence of 10 µg/mL LPS. The levels of cytokines and nitric oxide (NO) were detected with ELISA and Griess colorimetric method. The mRNA expressions of TLR4, MyD88 and chemokines were measured with qPCR. The activation of NF-κB and MAPK signaling pathways was detected with western blotting. RESULTS: The results indicated that AP reduced the production of inflammatory mediators (TNF-α, IL-1ß, IL-6 and NO), downregulated the mRNA expression of TLR4, MyD88 and some pro-inflammatory chemokines (CCL2, CCL20, CXCL2, CXCL8, CXCL10), and suppressed the NF-κB and MAPK signaling pathways evidenced by inhibition of the phosphorylation of IκBα, p65 and ERK, JNK, p38. CONCLUSIONS: Our results demonstrated that AP may exert its anti-inflammatory effects on claw dermal cells of dairy cow by regulating the NF-κB and MAPK signaling pathways.

Alteration of lipid metabolism, autophagy, apoptosis and immune response in the liver of common carp (Cyprinus carpio) after long-term exposure to bisphenol A.

Bisphenol A (BPA), as a phenolic compound, is harmful to human health, and its residue in the aquatic environment also threatens the health of aquatic animals. In this research, the toxicity effects of BPA on liver tissues were evaluated in common carp (Cyprinus carpio) after long-term exposure. Fish were exposed to five concentrations of BPA (0, 0.01, 0.1, 0.5 and 2 mg/L) for 30 days. The blood and liver tissues were gathered to analyze biochemical indices and genes transcription levels. The data related to lipid metabolism showed that BPA exposure increased serum total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) levels, upregulated the expressions of fatp1, pparγ, fas, atgl, hsl, pparα, cpt1b, acox-1, and downregulated the expression of dgat1 in liver. Antioxidative parameters displayed a reduced antioxidant ability and increased lipid peroxidation after BPA exposure. Meanwhile, the upregulations of nrf2, ho-1, cyp1a and cyp1b genes revealed an adaptive response mechanism against oxidative stress-induced adverse effects. After 30 days of exposure, BPA induced apoptosis and endoplasmic reticulum stress (ERS) via upregulating the expression levels of apoptosis and ERS-related genes and increasing Ca2+ concentration in liver. Moreover, the downregulation of mtor and the upregulation of atg3, atg7, tfeb, uvrag and mcoln1 indicated that BPA could influence the normal process of autophagy. Furthermore, BPA exposure activated toll like receptors (TLRs) pathway to mediate the inflammatory response. Our results demonstrated that BPA exposure disturbed lipid metabolism, and induced oxidative stress, ERS, apoptosis, autophagy and inflammatory response in the liver of common carp. These findings contributed to the understanding of hepatotoxicity mechanism induced by BPA in fish.

Pneumonia, Multiple Pulmonary Infarction and Abscess Caused by a Bamboo Stick Accidentally Piercing into Chest: a Case Misdiagnosed as Pulmonary Tuberculosis.

Computed tomography (CT) examination is the major measure for detecting and diagnosis of foreign bodies in human body. Although CT has high sensitivity in diagnosis of foreign body, some interference factors may still lead to missed diagnosis or misdiagnosis. Here we report a rare case that a bamboo stick accidentally pierced into the left chest of a young man who was drunk and unware of this hurt. The patient experienced cough, chest pain, fever, hemoptysis, and was misdiagnosed as primary and secondary tuberculosis based on chest CT examinations at a local hospital, although no tubercular bacillus detected by sputum smear. He subsequently received anti-tuberculous treatments in the following three years, but no improvement of his symptoms was observed. Until one month before his death, the bamboo stick was detected by spiral CT examination as well as three-dimensional image reconstruction at another hospital. Postmortem examination revealed pneumonia, pulmonary infarction, and abscess as the causes of his death. We analyze the potential reasons of misdiagnosis in this case, aiming to provide reference for the diagnosis and treatment of pulmonary inflammation associated with foreign body in the future.

Effects of high-fat diet on antioxidative status, apoptosis and inflammation in liver of tilapia (Oreochromis niloticus) via Nrf2, TLRs and JNK pathways.

Fatty liver injury (or disease) is a common disease in farmed fish, but its pathogenic mechanism is not fully understood. Therefore the present study aims to investigate high-fat diet (HFD)-induced liver injury and explore the underlying mechanism in fish. The tilapia were fed on control diet and HFD for 90 days, and then the blood and liver tissues were collected to determine biochemical parameter, gene expression and protein level. The results showed that HFD feeding signally increased the levels of plasma aminotransferases and pro-inflammatory factors after 60 days. In liver and plasma, HFD feeding significantly suppressed antioxidant ability, but enhanced lipid peroxidation formation, protein oxidation and DNA damage after 60 or 90 days. Further, the Nrf2 pathway and antioxidative function-related genes were adversely changed in liver of HFD-fed tilapia after 60 and/or 90 days. Meanwhile, HFD treatment induced apoptosis via initiating mitochondrial pathway in liver after 90 days. Furthermore, after 90 days of feeding, the expression of genes or proteins related to JNK pathway and TLRs-Myd88-NF-κB pathway was clearly upregulated in HFD treatment. Similarly, the mRNA levels of inflammatory factors including tumor necrosis factor (TNF-α), interleukin-1ß (IL-1ß), IL-6, IL-8 and IL-10 were also upregulated in liver of HFD-fed tilapia after 60 and/or 90 days. In conclusion, the current study suggested that HFD feeding impaired antioxidant defense system, induced apoptosis, enhanced inflammation and led to liver injury. The adverse influences of HFD in the liver might be due to the variation of Nrf2, JNK and TLRs-Myd88-NF-κB signaling pathways.

Anti-oxidative, anti-inflammatory and hepatoprotective effects of Radix Bupleuri extract against oxidative damage in tilapia (Oreochromis niloticus) via Nrf2 and TLRs signaling pathway.

Radix Bupleuri extract (RBE) is one of the most popular oriental herbal medicines, which has anti-oxidative and anti-inflammatory properties. However, its protective effects and underlying molecular mechanisms on oxidative damage in tilapia are still unclear. The aims of the study were to explore the anti-oxidative, anti-inflammatory and hepatoprotective effects of RBE against oxidative damage, and to elucidate underlying molecular mechanisms in fish. Tilapia received diet containing three doses of RBE (0, 1 and 3 g/kg diet) for 60 days, and then were given an intraperitoneal injection of H2O2 or saline. Before injection, RBE treatments improved growth performance and partial anti-oxidative capacity in tilapia. After oxidative damage, RBE pretreatments were able to signally reduce the higher serum aminotransferases, alkaline phosphatase (AKP) and liver necrosis. In serum and liver, the abnormal lipid peroxidation level and antioxidant status induced by H2O2 injection were restored by RBE treatments. Furthermore, RBE treatments activated erythroid 2-related factor 2 (Nrf2) signaling pathway, which promoted the gene expression of heme oxygenase 1 (HO-1), NAD(P) H:quinone oxidoreductase 1 (NQO-1), glutathione-S-transferase (GST) and catalase (CAT). Meanwhile, RBE treatments reduced inflammatory response by inhibiting TLRs-MyD88-NF-κB signaling pathway, accompanied by the lower interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and IL-8 mRNA levels. In addition, RBE treatments upregulated complement (C3) gene expression and downregulated heat shock protein (HSP70) gene expression. In conclusion, the current study suggested that RBE pretreatments protected against H2O2-induced oxidative damage in tilapia. The beneficial activity of RBE may be due to the modulation of Nrf2/ARE and TLRs-Myd88-NF-κB signaling pathway.

Antioxidative, inflammatory and immune responses in hydrogen peroxide-induced liver injury of tilapia (GIFT, Oreochromis niloticus).

Oxidative stress has been implicated in the pathogenesis of many liver diseases in fish, but the molecular mechanism is still obscure. Here, we used hydrogen peroxide (H2O2) as a reactive oxygen species (ROS) to induce liver injury and assess underlying molecular mechanism linking oxidative stress and liver injury in fish. Tilapia were injected with various concentrations of H2O2 (0, 40, 120, 200, 300 and 400 mM) for 72 h. The blood and liver were collected to assay biochemical parameters and genes expression after 24, 48 and 72 h of injection. The results showed that treatments with higher H2O2 levels (300 and/or 400 mM) significantly increased the levels of GPT, GOT, AKP and MDA, and apparently decreased the levels of TP, ALB, SOD, GSH, CAT, GST and T-AOC throughout of the 72 h. The gene expression data showed that treatments with 200, 300 and/or 400 H2O2 suppressed Nrf2/keap1 pathway and its downstream genes including ho-1, nqo1 and gsta, activated inflammatory response via enhancing the mRNA levels of nf-κb, tnf-α, il-1ß and il-8, and attenuating il-10 mRNA level, and caused immunotoxicity through downregulating the genes expression of c3, hep, lzm and Igm for 24, 48 and/or 72 h. Additionally, there was a mild or strong increase in levels of nrf2 and its subsequent antioxidant genes or enzymes such as ho-1, nqo1, gst, CAT and SOD in treatments with lower concentrations of H2O2 (40 or 120 mM) for 24 and/or 48 h. Overall results suggested that H2O2 hepatotoxicity was mainly concerned with lipid peroxidation, impairment antioxidant defense systems, inflammatory response and immunotoxicity, and Nrf2/Keap1 and NF-κB signaling pathways played important roles in oxidative stress-induced liver injury in fish.

Effects of Rhizoma Alismatis extract on biochemical indices and adipose gene expression in oleic acid-induced hepatocyte injury in Jian carp (Cyprinus carpio var. Jian).

Fatty liver is an increasingly serious disease of fish in aquaculture. However, the mechanisms responsible for the occurrence of fatty liver remain unclear, and no effective methods for the prevention and treatment of this disease have yet been found. In the present study, we aimed to develop an in vitro model of hepatocyte injury using oleic acid as hepatotoxicant and evaluate the protective effects of Rhizoma Alismatis extract (RAE) in Jian carp using this model. Primary hepatocytes from Jian carp were isolated and purified and cultured in vitro. The result indicated that 0.4 mmol L-1 oleic acid and 48 h could be the optimal conditions to induce hepatocyte injury model in cultured hepatocytes. Hepatocytes were exposed to oleic acid, followed by the addition of RAE at 0, 1, 5, 10, 20, or 50 µg mL-1. The hepatocytes and supernatant were then analyzed. RAE suppressed oleic acid-induced elevations in aspartate aminotransferase, alanine aminotransferase, triglycerides, total cholesterol, lactate dehydrogenase, alkaline phosphatase, cholinesterase, malondialdehyde, γ-glutamyl transferase, cytochrome P450 1A, cytochrome P450 2E1, liver-type fatty acid binding protein, free fatty acid, fatty acid synthetase, and tumor necrosis factor-α (P < 0.01 or P < 0.05); reduced protein levels of cytochrome P450 1A, nuclear factor (NF)-κB p65, and NF-κB c-Rel; and inhibited cytochrome P4503A, NF-κB c-Rel, nuclear factor erythroid-related factor 2, peroxisome proliferator-activated receptor-α, and cytochrome P4501A mRNA levels. In conclusion, RAE exhibited a protective effect against hepatocyte injury in Jian carp. Further in vivo studies are needed to provide more evidence for the use of RAE as a hepatoprotective agent for the treatment of hepatocyte injury.

Protective effect of Ganoderma lucidum polysaccharide against carbon tetrachloride-induced hepatic damage in precision-cut carp liver slices.

The aim of the present study was to investigate the protective effects of Ganoderma lucidum polysaccharide (GLPS) against carbon tetrachloride (CCl4)-induced hepatotoxicity in vitro in common carp. Precision-cut liver slices (PCLSs), which closely resemble the organ from which they are derived, were employed as an in vitro model system. GLPS (0.1, 0.3, and 0.6 mg/ml) was added to PCLS culture system before the exposure to 12 mM CCl4. The supernatants and slices were collected to detect molecular and biochemical responses to CCl4 and PCLS treatments. The levels of CYP1A, CYP3A, and CYP2E1 were measured by ELISA; the mRNA expressions of TNF-α, IL-1ß, IL-6, and iNOS were determined by RT-PCR; and the relative protein expressions of c-Rel and p65 were analyzed by western blotting. Results showed that GLPS inhibited the elevations of the marker enzymes (GOT, GPT, LDH) and MDA induced by CCl4; it also enhanced the suppressed activity of antioxidant enzymes (SOD, CAT, GSH-Px, T-AOC). The treatment with GLPS resulted in significant downregulation of NF-κB and inflammatory cytokine mRNA levels and significant decreases in the hepatic protein levels of CYP1A, CYP3A, and CYP2E1. These results suggest that GLPS can protect CCl4-induced PCLS injury through inhibiting lipid peroxidation, elevating antioxidant enzyme activity, and suppressing immune inflammatory response.

Hepatoprotective and antioxidant effects of dietary Glycyrrhiza polysaccharide against TCDD-induced hepatic injury and RT-PCR quantification of AHR2, ARNT2, CYP1A mRNA in Jian Carp (Cyprinus carpio var. Jian).

To evaluate the protective effects of Glycyrrhiza polysaccharide (GPS) against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced hepatotoxicity in Jian carp, the fish were fed diets containing GPS at doses of 0.1, 0.5 and 1.0g/kg for 60days before an intraperitoneal injection of 0.6µg/kg TCDD at a volume of 0.05mL/10g body weight. At 72hr post-injection, blood and liver samples were taken for biochemical analysis and the fish liver samples were used for the preparation of pathological slices. The results showed that increases in alanine aminotransferase (GPT), aspartate aminotransferase (GOT), lactate dehydrogenase (LDH), and alkaline phosphatase (AKP) in serum induced by TCDD were significantly inhibited by pre-treatment with 1.0g/kg GPS. Following the 1.0g/kg GPS pre-treatment, total protein (TP), albumin (Alb), catalase (CAT), glutathione peroxidase (GPx), total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activities in liver tissue increased significantly, malondialdehyde (MDA) formation (P<0.05 or P<0.01) was significantly inhibited, and the expression of cytochrome P4501A (CYP1A), aryl hydrocarbon receptor 2 (AHR2) and aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) mRNA (P<0.05) was significantly enhanced. Histological observations on fish liver were obtained by preparing paraffin tissue sections via HE staining, and the results showed that histological changes were obviously reduced by 0.5 and 1.0g/kg GPS. GPS significantly reduced liver tissue damage caused by TCDD. Overall, these results proved the hepatoprotective effect of GPS in protecting against fish liver injury induced by TCDD, and supported the use of GPS (1.0g/kg) as a hepatoprotective and antioxidant agent in fish.

A Study of 2,3,7,8-Tetrachlorodibenzo-p-dioxin Induced Liver Injury in Jian Carp (Cyprinus carpio var. Jian) Using Precision-Cut Liver Slices.

The aim of this study was to establish a model for the study of liver injury induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in Jian carp using precision-cut liver slices (PCLS). PCLS were treated with TCDD at concentrations of 0, 0.05, 0.1, 0.3, and 0.6 µg/L for 6 h, followed by collection of the culture supernatant and PCLS for analysis. Several biochemical indices were analyzed, including glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA). Expression of mRNA was also estimated for cytochrome P4501A (CYP1A), aryl hydrocarbon receptor2 (AhR2), and aryl hydrocarbon receptor nuclear translocator2 (ARNT2). Results showed that some significant effects (p < 0.05) in MDA, GSH-Px and PCLS viability were observed at a TCDD concentration as low as 0.05 µg/L, and the observed effects increased with exposure concentration. Following exposure to TCDD for 6 h at a concentration of 0.3 µg/L, significant increases (p < 0.01) in the content of GPT, GOT, MDA, and LDH were observed, while SOD activity, GSH-Px activity, and PCLS viability were decreased (p < 0.01 or p < 0.05). Exposure to 0.3 µg/L TCDD also resulted in increased expression of mRNA for CYP1A, AhR2, and ARNT2. Overall, these results provide evidence of TCDD-induced liver injury and oxidative stress in Jian carp. These results also support the use of PCLS as an in vitro model for the evaluation of hepatotoxicity in Jian carp.

Prometryn exposure disrupts the intestinal health of Eriocheir sinensis: Physiological responses and underlying mechanism.

Most toxicity studies of prometryn in non-target aquatic animals have focused on hepatotoxicity, cardiotoxicity, embryonic developmental and growth toxicity, while studies on the molecular mechanisms of intestinal toxicity of prometryn are still unknown. In the current study, the intestinal tissues of the Chinese mitten crab (Eriocheir sinensis) were used to uncover the underlying molecular mechanisms of stress by 96-h acute in vivo exposure to prometryn. The results showed that prometryn activated the Nrf2-Keap1 pathway and up-regulated the expression of downstream antioxidant genes. Prometryn induced the expression of genes associated with non-specific immunity and autophagy, and induced apoptosis through the MAPK pathway. Interestingly, the significant up-or down-regulation of the above genes mainly occurred at 12 h- 24 h after exposure. Intestinal flora sequencing revealed that prometryn disrupted the intestinal normal barrier function mainly by reducing beneficial bacteria abundance, which further weakened the intestinal resistance to exogenous toxicants and caused an inflammatory response. Correlation analyses found that differential flora at the genus level had potential associations with gut stress-related genes. In conclusion, our study contributes to understanding the molecular mechanisms behind the intestinal stress caused by herbicides on aquatic crustaceans.