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.

Identification and characterization of lipid metabolism-related microRNAs in the liver of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) by deep sequencing.

MicroRNAs (miRNAs) play vital roles in modulating diverse metabolic processes in the liver, including lipid metabolism. Genetically improved farmed tilapia (GIFT, Oreochromis niloticus), an important aquaculture species in China, is susceptible to hepatic steatosis when reared in intensive culture systems. To investigate the miRNAs involved in GIFT lipid metabolism, two hepatic small RNA libraries from high-fat diet-fed and normal-fat diet-fed GIFT were constructed and sequenced using high-throughput sequencing technology. A total of 204 known and 56 novel miRNAs were identified by aligning the sequencing data with known Danio rerio miRNAs listed in miRBase 21.0. Six known miRNAs (miR-30a-5p, miR-34a, miR-145-5p, miR-29a, miR-205-5p, and miR-23a-3p) that were differentially expressed between the high-fat diet and normal-fat diet groups were validated by quantitative real-time PCR. Bioinformatics tools were used to predict the potential target genes of these differentially expressed miRNAs, and Gene Ontology enrichment analysis indicated that these miRNAs may play important roles in diet-induced hepatic steatosis in GIFT. Our results provide a foundation for further studies of the role of miRNAs in tilapia lipid homeostasis regulation, and may help to identify novel targets for therapeutic interventions to reduce the occurrence of fatty liver disease in farmed tilapia.

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.

Effects of cyclophosphamide on antioxidative and immune functions of Nile tilapia (Oreochromis Niloticus) via the TLR-NF-κB signaling pathway.

Intensive aquaculture often results in immunosuppression in fish, which may cause a series of diseases. In this study, to investigate the immunosuppressive mechanisms in fish, tilapia were intrapleural injected cyclophosphamide (CTX) at the doses of 10, 25, 50, 75 and 100 mg·kg-1 to induce immunosuppression. We determined the viability of immune cells, the content of lysozyme (LZM) and immunoglobulin M (IgM), the levels of nitric oxide (NO) and antioxidant parameters. Meanwhile, the mRNA levels of complement C3 (c3), igm and the genes associated with the TLR-NF-κB signaling pathway in the head kidney (HK) and spleen were also determined. The results showed that CTX had a significant cytotoxic effect on peripheral blood leukocytes, HK macrophages and spleen cells in a dose-dependent manner. The protein and mRNA levels of C3 and IgM were down-regulated with the increase of CTX concentrations in serum, HK and/or spleen. The NO and LZM contents decreased significantly in HK and spleen after CTX treatments with 75 and 100 mg·kg-1. CTX treatments with 50, 75 and/or 100 mg·kg-1 markedly decreased the antioxidant ability and enhanced lipid peroxidation in HK and spleen. Furthermore, qPCR data showed that CTX treatments with 50-100 mg·kg-1 clearly down-regulated the mRNA levels of tlr2, myd88, irak1, traf6, nfκb1, nfκb2, il-6, il-10 and tnf-α in the HK and/or spleen. Overall results suggested that CTX treatment had a cytotoxic effect on immune cells, induced lipid peroxidation, decreased the antioxidant capacity and inhibited immune function. The immunosuppressive mechanisms of CTX may be associated with the TLR-NF-κB signaling pathway.

Changes in Physiological Parameters, Lipid Metabolism, and Expression of MicroRNAs in Genetically Improved Farmed Tilapia (Oreochromis niloticus) With Fatty Liver Induced by a High-Fat Diet.

Tilapia is susceptible to hepatic steatosis when grown in intensive farming systems. The aim of this study was to explore the mechanism of fatty liver induced by a high-fat diet (HFD) in genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Juvenile GIFT were fed with HFD or a normal-fat diet (NFD) for 60 days. Substantial fat deposition in the liver of HFD-fed GIFT on days 20, 40, and 60 was observed using hematoxylin - eosin staining and oil red O staining. The increased fat deposition was consistent with increased triglyceride (TG) and total cholesterol (TC) levels in the liver of HFD-fed GIFT. There were significant differences (P < 0.05) in serum biochemical indexes (TG, TC, low density lipoprotein-cholesterol, and insulin contents, and alanine aminotransferase activity) between GIFT fed a HFD and GIFT fed a NFD on days 20, 40, and 60. Furthermore, 60 days of a HFD significantly changed (P < 0.05) the hepatic fatty acid composition, and led to increased polyunsaturated fatty acid levels and decreased saturated fatty acid and monounsaturated fatty acid levels. Hepatic antioxidant enzyme activities increased by day 20 and then declined, which led to an increase in malondialdehyde contents in the liver of HFD-fed GIFT. Molecular analyses revealed that the microRNAs miR-122, miR-29a, and miR-145-5p were upregulated, whereas miR-34a was downregulated in HFD-fed GIFT. SCD, ELOVL6, and SRD5A2 encode three important enzymes in lipid metabolism, and were identified as potential targets of miRNAs. The transcript levels of hepatic SCD and ELOVL6 were decreased and that of hepatic SRD5A2 was increased in GIFT fed a HFD. Overall, the results of this study revealed a potential link between miRNAs and fatty liver induced by HFD, and suggest that a HFD could lead to excess fat deposition in the GIFT liver, which may disrupt hepatic lipid metabolism and reduce the antioxidant defense capacity.

miR-205-5p negatively regulates hepatic acetyl-CoA carboxylase ß mRNA in lipid metabolism of Oreochromis niloticus.

MicroRNAs (miRNAs) are non-coding RNAs that function as post-transcriptional gene regulators and that play vital roles controlling lipid metabolism. miR-205 is an important miRNA related to adipogenesis and lipid metabolism. However, little is known about the potential role of miR-205-5p in genetically improved farmed tilapia (GIFT, Oreochromis niloticus). In this study, we used miRanda software to search for potential miR-205-5p target genes and found a lipid-metabolism-related gene called acetyl-CoA carboxylase ß (ACACß). Quantitative real-time polymerase chain reaction data indicated that there may be a negative regulation relationship between miR-205-5p and ACACß gene expression under HFD rearing. Using luciferase reporter assays, we verified the binding site of miR-205-5p in the 3'-untranslated region of the ACACß mRNA. Furthermore, an in vivo functional analysis of miR-205-5p was performed by injecting GIFT juveniles with a miR-205-5p antagomir. Reduced levels of miR-205-5p in GIFT liver increased ACACß mRNA expression 12 h post-injection. miR-205-5p suppression also increased fatty acid synthase and peroxisome proliferator-activated receptor-α mRNA levels 48 h and 120 h post-injection, respectively. Taken together, our results indicate that miR-205-5p negative regulates hepatic ACACß mRNA expression, and may serve as an important regulator in controlling hepatic lipid metabolism in GIFT.

Protective action of the phyllanthin against carbon tetrachloride-induced hepatocyte damage in Cyprinus carpio.

The present study was to investigate the protective effect and possible mechanism of phyllanthin against carbon tetrachloride (CCl4)-induced hepatocyte damage in carp. Phyllanthin (5, 10, and 15 µg/ml) was added to carp primary hepatocytes before (pre-treatment) and after (post-treatment) incubation of the hepatocytes in medium containing CCl4 at 8 mM; supernatant and cell were collected for the analyses of cell viability, biochemical parameters, and gene expression. The results showed that phyllanthin at the concentration of 15 µg/ml significantly suppressed the elevation of glutamate pyruvate transaminase (GPT), glutamate oxalate transaminase (GOT), lactate dehydrogenase (LDH), and malondialdehyde (MDA), and the reduction of cell viability, superoxide dismutase (SOD) activity, cytochrome P450 1a (CYP1A), and cytochrome P450 3a (CYP3A) messenger RNA (mRNA) levels expect LDH in the post-treatment. The levels of GPT, GOT, and CYP1A mRNA were also effectively restored in the pretreatment with phyllanthin (10 µg/ml). Overall, our results suggested that phyllanthin may be used as a hepatoprotective agent to prevent liver diseases in fish.

Anti-inflammatory and hepatoprotective effects of Ganoderma lucidum polysaccharides on carbon tetrachloride-induced hepatocyte damage in common carp (Cyprinus carpio L.).

The aim of this study was to investigate the anti-inflammatory and hepatoprotective effects of Ganoderma lucidum polysaccharides (GLPS) on carbon tetrachloride (CCl4)-induced hepatocyte damage in common carp (Cyprinus carpio L.). GLPS (0.1, 0.3, 0.6mg/ml) were added to the primary hepatocytes before (pre-treatment), after (post-treatment) and both before and after (pre- and post-treatment) the incubation of the hepatocytes with CCl4 at the concentration of 8mM in the culture medium. The supernatants and cells were collected respectively to detect the biochemical indicators. The levels of TNF-α, IL-1ß, caspase-3 and caspase-8 were measured by ELISA, the mRNA expressions of CYP1A and CYP3A were determined by RT-PCR, and western blotting was used to assay the relative protein expressions of c-Rel and p65. Results showed that GLPS significantly improved cell viability and inhibited the elevations of the marker enzymes (GOT, GPT, LDH) and MDA induced by CCl4, and markedly increased the level of SOD. Treatments with GLPS resulted in a significant decrease in the expressions of CYP1A and CYP3A, and significantly down-regulated extrinsic apoptosis and immune inflammatory response. In brief, the present study showed that GLPS can protect hepatocyte injury induced by CCl4 through inhibiting lipid peroxidation, elevating antioxidant enzyme activity and suppressing apoptosis and immune inflammatory response.

Effects of carbon tetrachloride on oxidative stress, inflammatory response and hepatocyte apoptosis in common carp (Cyprinus carpio).

In the present study, the cellular and molecular mechanism of carbon tetrachloride (CCl4)-induced hepatotoxicity in fish was investigated by studying the effects of CCl4 on the oxidative stress, inflammatory response and hepatocyte apoptosis. Common carp were given an intraperitoneal injection of 30% CCl4 in arachis oil (0.5ml/kg body weight). At 72h post-injection, blood were collected to measure glutamate pyruvate transaminase (GPT), glutamate oxalate transaminase (GOT), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione (GSH), total antioxidant capacity (T-AOC) and malondialdehyde (MDA), liver samples were taken to analyze toll-like receptor 4 (TLR4), cytochrome P450 2E1 (CYP2E1) and gene expressions of inflammatory cytokines and nuclear factor-κB (NF-κB/cREL). Cell viability and apoptosis were analyzed after treatment of the primary hepatocytes with CCl4 at 8mM. The results showed that CCl4 significantly increased the levels of GPT, GOT, MDA, TLR4 and CYP2E1, reduced the levels of SOD, GPx, CAT, GSH and T-AOC, and up-regulated the gene expressions of NF-κB/cREL and inflammatory cytokines including tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), IL-1ß, IL-6 and IL-12. In vitro, CCl4 caused a dramatic loss in cell viability and induced hepatocyte apoptosis. Overall results suggest that oxidative stress lipid peroxidation, and TNF-α/NF-κB and TRL4/NF-κB signaling pathways play important roles in CCl4-induced hepatotoxicity in fish.

Grass carp reovirus induces apoptosis and oxidative stress in grass carp (Ctenopharyngodon idellus) kidney cell line.

Grass carp hemorrhage is an acute contagious disease caused by grass carp reovirus (GCRV). The pathogenesis of GCRV and the relationship between GCRV and the host cells remain unclear. The aim of the present study was to investigate the relations among apoptosis, intracellular oxidative stress and virus replication in GCRV infected-cells. The results showed that GCRV induced activation of caspase proteases as early as 12 h, and reached maximum activities at 24 h or 48 h post-infection in a grass carp kidney cell line (CIK cells). Meanwhile, the levels of tumor necrosis factor (TNF-α) and interleukin-1ß (IL-1ß) also were increased in GCRV-infected CIK cells and showed a statistically significant difference from 24 h to 96 h post-infection. The infection of GCRV caused the destruction of entire monolayer and the death of host cells. Accompanied by the infection, a severe oxidative stress occurred, which led to extensive loss of antioxidants and formation of lipid peroxidation after 48 h post-infection. These data suggested that the apoptosis which was triggered at an early stage (12-24 h) in the viral infection cycle, might be independent of virus replication, while the oxidative stress induced by GCRV was mostly related to the virus replication.