Dietary Bacillus velezensis R-71003 and sodium gluconate improve antioxidant capacity, immune response and resistance against Aeromonas hydrophila in common carp.

This study aimed to evaluate the effects of dietary supplementation with Bacillus velezensis R-71003 combined with sodium gluconate on antioxidant capacity, immune response and resistance against Aeromonas hydrophila in common carp. In addition, the biocontrol potential of the secondary metabolites of B. velezensis R-71003 was also evaluated to analyze the possible mechanism of B. velezensis R-71003 against A. hydrophila. The results indicated that the antibacterial crude extract of B. velezensis R-71003 can destroy the cell wall of A. hydrophila. Moreover, the results showed that dietary B. velezensis R-71003 could promote antioxidant capacity, which significantly increased the activities of CAT and SOD and decreased the content of MDA. Additionally, B. velezensis R-71003 supplementation significantly enhanced the immunity of common carp, as measured by the mRNA expression levels of cytokine-related genes (TNF-α, TGF-ß, IL-1ß and IL-10). In addition, dietary B. velezensis R-71003 exhibited an upregulation of IL-10 and a downregulation of IL-1ß, coupled with higher survival rates when challenged with A. hydrophila compared to the positive group. Furthermore, compared to prechallenge, the mRNA expression levels of TLR-4, MyD88, IRAK1, TRAF6, TRIF and NF-κB in the head kidney of common carp were significantly increased after challenge. The fish fed the B. velezensis R-71003 diet showed lower expression of TLR-4, MyD88, IRAK1, TRAF6, TRIF and NF-κB after the challenge than those fed the control diet. Thus, this study revealed that B. velezensis R-71003 can improve the resistance of common carp to pathogenic bacteria by destroying bacterial cell walls and improving fish immunity by activating the TLR4 signaling pathway. Importantly, this study indicated that sodium gluconate has a positive effect on B. velezensis R-71003 in enhancing the anti-infection ability of common carp. The results of this study will lay the foundation for the application of B. velezensis R-71003 in combination with sodium gluconate as an alternative to antibiotics in aquaculture.

Anti-miR33 therapy improved hepatopancreatic lipid and immune metabolism disorders in grass carp, Ctenopharyngodon idella.

Lipid metabolism disorders are found ubiquitously in farmed fish and occur as a result of excessive fat accumulation. Previous studies have found that miR-33 is involved in lipid metabolism; however, its role in fish lipid metabolism is unclear. We sought to clarify this relationship in grass carp in vivo and in vitro. Our findings revealed the length of miR-33 to be 65 bp. Phylogenetic tree analysis showed that grass carp miR-33 was most closely related to fish miR-33 (Siganus canaliculatus). Hepatocytes transfected with miR-33 mimic displayed markedly raised TG content (P < 0.05) as well as increased levels of lipid synthesis-related transcription factors (P < 0.05). Compared with blank and saline groups, total serum cholesterol, AST, and LDL levels were suppressed in groups treated with the miR-33 antagomir (P < 0.05). Moreover, the expression levels of PPARγ and SREBP-1c mRNA were significantly decreased in contrast to those found in the control group (P < 0.05). Similar findings were noted in the expression of immune-related proinflammatory molecules (TNFα, IL-1ß, IL-6, and NF-κB), which also demonstrated decreased levels (P < 0.05). Conversely, high expressions of anti-inflammatory factors (TGF-ß1 and IL-10) were noted (P < 0.05). This investigation strongly supports the role of miR-33 in hepatopancreas-based lipid metabolism and immunity. miR-33 may have been highly conserved in early vertebrates in order to facilitate liver-specific metabolic and immunomodulatory functions. Our findings provide a basis for further investigations exploring the mechanisms surrounding fish lipid metabolism and may aid in preventing and treating immunocompromised fish as well as fish with fatty hepatopancreas, and other metabolic diseases.

Grape seed proanthocyanidin extract ameliorates hepatic lipid accumulation and inflammation in grass carp (Ctenopharyngodon idella).

Hepatic lipid metabolism disorder due to excessive fat accumulation in fish is a significant problem in aquaculture. Studies have shown that grape seed procyanidin extract (GSPE) can regulate fish lipid metabolism and improve fish immunity. However, the mechanism is unclear. In this study, we used grass carp that stores excess fat in the liver as a model. In vitro, GSPE treatment of hepatocytes for 3 h significantly decreased TG content, accompanied with decreased expression of SREBP-1c, FAS, and ACC and increased expression of PPARα, ATGL, and LPL. GSPE treatment for 1 h significantly decreased expression of pro-inflammatory cytokines (TNFα, IL-6, IL-1ß, and NF-κB) and increased the expression of anti-inflammatory cytokines (IL-10 and TGF-ß1). In vivo, the administration of GSPE significantly reduced high-fat diet-induced increase of serum CHOL, TG, and HDL, but increased LDL content. GSPE treatment for 3 h increased expression of ATGL and LPL, and significantly decreased the expression of HFD-fed-induced SREBP-1c, ACC, FAS, PPARγ, PPARα, and H-FABP. GSPE treatment for 3 h also significantly decreased the expression of pro-inflammatory cytokines (TNFα, IL-6, and IL-1ß) and increased the expression of the anti-inflammatory cytokine IL-10. The expression levels of the lipogenic miRNAs, miR-33, and miR-122, were suppressed both in vivo and in vitro by GSPE. In summary, GSPE had hypolipidemic and potential anti-inflammatory effects in the liver, potentially mediated by miR-33 and miR-122.

Chinese yam peel enhances the immunity of the common carp (Cyprinus carpio L.) by improving the gut defence barrier and modulating the intestinal microflora.

The Chinese yam peel (CYP) is a by-product of yam processing that is rich in various nutrients and a good source for feed additives. This study investigated the effects of CYP on the intestinal microbiota and gut defence barrier of the common carp (Cyprinus carpio L.). Different groups of experimental fish were fed a normal control diet (NC), a low CYP diet (LYP) and a high CYP diet (HYP) for 8 weeks. After the feeding trial, the fish were assessed for intestinal enzyme activity, intestinal histology, immune-related gene expression, intestinal SCFAs and intestinal microbiota. Our results indicated that the intestinal integrity and antioxidant enzyme (CAT and SOD) activity in the common carp were enhanced following CYP supplementation. The mRNA levels of anti-inflammatory (TGF-ß), tight binding protein (occludin and ZO-1) and pathway factor genes (TLR4 and NF-κB) were significantly upregulated in the HYP group (P＜0.05), which was accompanied by an increase in the level of pro-inflammatory IL-1ß in the gut (P＜0.05). High-throughput sequencing revealed that Fusobacteria, Proteobacteria, and Bacteroidetes bacteria were most abundant in the microbial community in the gut of the common carp. The relative abundances of Bacteroides, Flavobacterium and Lactobacillus were increased, while the abundances of pathogenic microorganisms such as Enterobacteriaceae, Shewanella, Pseudomonas and Vibrio were reduced after treatment with CYP. Furthermore, the concentrations of acetic acid, propionic acid, butyric acid and total short-chain fatty acids (SCFAs) in the gut were also increased (P＜0.05). Finally, our results revealed correlations between gut microbiota, SCFAs, non-specific immunity and antioxidant enzymes in CYP-fed carp. These results suggest that CYP-supplemented feed could improve the immunity of the common carp by modulating the intestinal microflora and enhancing the gut defence barrier and has the potential to be used as an immunostimulating feed additive in aquaculture.

Characterization of a polysaccharide HP-02 from Honeysuckle flowers and its immunoregulatory and anti-Aeromonas hydrophila effects in Cyprinus carpio L.

Aquaculture systems are known to be major reservoirs of Aeromonas hydrophila. The use of Chinese medicinal polysaccharides has become a successful strategy to prevent bacterial diseases in aquaculture. A water-soluble polysaccharide, HP-02, was purified from honeysuckle flowers, and the present study was aimed to examine its immunomodulatory and anti-A. hydrophila effects in Cyprinus carpio L. The results showed that the molecular weight of HP-02 was estimated to be 38 kDa. Monosaccharide composition was determined to be arabinose, rhamnose, mannose, glucose, and galactose in a molar ratio of 2.5: 1.8: 3.6: 3.7: 1.9. HP-02 not only distinctly improved the proliferation and phagocytosis of head kidney cells, but also exerted significant immunoregulation activity by increasing the secretion of the pro-inflammatory cytokines TNF-α, IL-1ß, IL-6, and IL-12 and the anti-inflammatory cytokines IL-10 and TGF-ß at the protein level in head kidney cells and serum. Moreover, during pathogen infection in vivo, HP-02 significantly reduced the expression of pro-inflammatory cytokines and increased the expression of anti-inflammatory cytokines. These results indicated that HP-02 had immunomodulatory effects on common carp both in vitro and in vivo, and HP-02 can be applied as a common carp feed supplement to enhance fish immunity and disease resistance against A. hydrophila.

Regulation of growth performance and lipid metabolism in juvenile grass carp (Ctenopharyngodon idella) with honeysuckle (Lonicera japonica) extract.

This study investigated the effects of honeysuckle extract (Lonicera japonica, HE) on the growth performance and lipid metabolism of juvenile grass carp (Ctenopharyngodon idella). HE at doses of 10 g kg-1 (LHE), 20 g kg-1 (MHE), and 40 g kg-1 (HHE) were individually mixed with the basal diet and fed to grass carp for 10 weeks, and ginseng extract (20 g kg-1, GSE) was used as a positive control. The results showed that HE administration exerted no effect on growth performance, but the hepatosomatic index (HSI) and muscle and liver lipid contents were significantly decreased in the LHE and MHE groups. The serum levels of LDL-c, total triglyceride (TG) and total cholesterol (TC) also declined in the HE-treated groups. Moreover, the disordered vacuolization and nucleus migration in the liver were alleviated in the MHE and HHE groups, and mRNA expressions of lipogenesis-related genes, such as acc1, fas, srebp1, and pparγ decreased. Similarly, the expression of genes related to lipolysis, such as cpt1, atgl, lpl, and pparα, was found to be significantly increased in the MHE and HHE groups compared with the control. Taken together, HE can effectively improve the lipid metabolism and ameliorate the lipid deposition of grass carp and thus may be a promising feed additive in aquaculture.

Protective effects of osthole against inflammation induced by lipopolysaccharide in BV2 cells.

Inflammation and oxidative stress are implicated in the development of neurodegenerative diseases. Osthole is a compound that is extracted from She Chuang Zi, which is a type of traditional Chinese medicine. Osthole has previously been demonstrated to exhibit anticancer activities and has a low toxicity. However, to the best of our knowledge, the anti­inflammatory effects of osthole in microglial cells have not been investigated extensively. The aim of the present study was to investigate the potential protective effects of osthole against inflammation induced by lipopolysaccharide (LPS) in microglial cells. The present study employed LPS­stimulated BV2 mouse microglia to establish an inflammatory cell model and to investigate the anti­inflammatory effects of osthole. Cells were pretreated with osthole for 1 h prior to LPS (10 µg/ml) stimulation. At 6 h after the addition of LPS, alterations in the levels of inflammatory factors, including tumor necrosis factor (TNF)­α, interleukin (IL)­6 and IL­1ß, were determined by ELISA. Furthermore, at 24 h after the addition of LPS, western blot analysis was performed to analyze the alterations in the protein expression of nuclear factor­κB (NF­κB) p65, phosphorylated­NF­κB p65, nuclear factor erythroid 2­related factor 2 (Nrf2) and heme oxygenase (HO)­1. The results demonstrated that the secretion of the inflammatory cytokines TNF­α, IL­6 and IL­1ß by LPS­stimulated BV2 cells was significantly reduced by osthole treatment. Simultaneously, osthole treatment inhibited the LPS­induced activation of the NF­κB signaling pathway. In addition, osthole upregulated the expression of Nrf2 and HO­1 in a dose­dependent manner. Based on these results, osthole may exhibit anti­inflammatory effects via the NF­κB and Nrf2 pathways, indicating that osthole has the potential to be developed into an effective anti-inflammatory drug.

Molecular cloning of hemocyanin cDNA from Fenneropenaeus chinensis and antimicrobial analysis of two C-terminal fragments.

Peptides derived from shrimp hemocyanin have antimicrobial properties. This is the first report of hemocyanin cDNA (FCHc) cloned from Fenneropenaeus chinensis and recombinant expression of two C-terminal fragments. Based on sequence analysis of Fenneropenaeus chinensis hemocyanin FCHc, we subcloned two FCHc fragments by designing special primers. Two antimicrobial peptides (AMPs) were derived from FCHc (FCHc-C1 and FCHc-C2). The recombinant sequence of FCHc-C1 consisted of 207 bp encoding 69 amino acids and the recombinant sequence of FCHc-C2 consisted of 120 bp encoding 40 amino acids. The results of Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting indicated that recombinant FCHc-C1 and FCHc-C2 peptides (rFCHc-C1 and rFCHc-C2) were expressed successfully. An inhibition assay showed that FCHc-C1 and FCHc-C2 were anionic AMPs with antifungal and antibacterial activities.