Vitamin D3/VDR inhibits inflammation through NF-κB pathway accompanied by resisting apoptosis and inducing autophagy in abalone Haliotis discus hannai.

Vitamin D3 is believed to be a contributing factor to innate immunity. Vitamin D receptor (VDR) has a positive effect on inhibiting nuclear factor κB (NF-κB)-mediated inflammation. The underlying molecular mechanisms remain unclear, particularly in mollusks. Consequently, this study will investigate the process of vitamin D3/VDR regulating NF-κB pathway and further explore their functions on inflammation, autophagy, and apoptosis in abalone Haliotis discus hannai. Results showed that knockdown of VDR by using siRNA and dsRNA of VDR in vitro and in vivo led to more intense response of NF-κB signaling to lipopolysaccharide and higher level of apoptosis and autophagy. In addition, 1,25(OH)2D3 stimulation after VDR silencing could partially alleviate apoptosis and induce autophagy. Overexpression of VDR restricted the K48-polyubiquitin chain-dependent inhibitor of κB (IκB) ubiquitination and apoptosis-associated speck-like protein containing CARD (ASC) oligomerization. Besides, VDR silencing resulted in increase of ASC speck formation. In further mechanistic studies, we showed that VDR can directly bind to IκB and IKK1 in vitro and in vivo. In the feeding trial, H&E staining, TUNEL, and electron microscope results showed that vitamin D3 deficiency (0 IU/kg) could recruit more basophilic cells and increase more TUNEL-positive apoptotic cells and lipid droplets (LDs) than vitamin D3 supplement (1000 IU/kg and 5000 IU/kg). In summary, abalone VDR plays a negative regulator role in NF-κB-mediated inflammation via interacting with IκB and inhibiting ubiquitin-dependent degradation of IκB. Vitamin D3 in combination with VDR is essential to establish a delicate balance between autophagy and apoptosis in response to inflammation.

Arginine Regulates TOR Signaling Pathway through SLC38A9 in Abalone Haliotis discus hannai.

Arginine plays an important role in the regulation of the target of the rapamycin (TOR) signaling pathway, and Solute Carrier Family 38 Member 9 (SLC38A9) was identified to participate in the amino acid-dependent activation of TOR in humans. However, the regulations of arginine on the TOR signaling pathway in abalone are still unclear. In this study, slc38a9 of abalone was cloned, and the slc38a9 was knocked down and overexpressed to explore its function in the regulation of the TOR signaling pathway. The results showed that knockdown of slc38a9 decreased the expression of tor, ribosomal s6 protein kinase (s6k) and eukaryotic translation initiation factor 4e (eif4e) and inhibited the activation of the TOR signaling pathway by arginine. Overexpression of slc38a9 up-regulated the expression of TOR-related genes. In addition, hemocytes of abalone were treated with 0, 0.2, 0.5, 1, 2 and 4 mmol/L of arginine, and abalones were fed diets with 1.17%, 1.68% and 3.43% of arginine, respectively, for 120 days. Supplementation of arginine (0.5-4 mmol/L) increased the expressions of slc38a9, tor, s6k and eif4e in hemocytes, and abalone fed with 1.68% of dietary arginine showed higher mRNA levels of slc38a9, tor, s6k and eif4e and phosphorylation levels of TOR, S6 and 4E-BP. In conclusion, the TOR signaling pathway of abalone can be regulated by arginine, and SLC38A9 plays an essential role in this regulation.

The impaired immune function and structural integrity by dietary iron deficiency or excess in gill of fish after infection with Flavobacterium columnare: Regulation of NF-κB, TOR, JNK, p38MAPK, Nrf2 and MLCK signalling.

The aim of this study was to investigate the effects and potential mechanisms of dietary iron on immune function and structural integrity in gill of young grass carp (Ctenopharyngodon idella). A total of 630 grass carp (242.32 ±â€¯0.58 g) were fed diets containing graded levels of iron at 12.15 (basal diet), 35.38, 63.47, 86.43, 111.09, 136.37 and 73.50 mg/kg for 60 days. Subsequently, a challenge test was conducted by infection with Flavobacterium columnare to investigate the effects of dietary iron on gill immune function and structural integrity in young grass carp. First, the results indicated that compared with the optimal iron level, iron deficiency decreased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents, and down-regulated the mRNA levels of antibacterial peptides, anti-inflammatory cytokines (except IL-4/13B), inhibitor of κBα (IκBα), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1). In contrast, iron deficiency up-regulated the mRNA levels of pro-inflammatory cytokines (except IL-6 and IFN-γ2), nuclear factor κB p65 (NF-κBp65), IκB kinases α (IKK), IKKß, IKKγ, eIF4E-binding protein 1 (4E-BP1) and 4E-BP2 in gill of young grass carp, indicating that iron deficiency could impair immune function in fish gill. Second, iron deficiency down-regulated the mRNA levels of inhibitor of apoptosis protein (IAP) and myeloid cell leukemia 1 (Mcl-1), decreased activities and mRNA levels of antioxidant enzymes, down-regulated the mRNA levels of NF-E2-related factor 2 (Nrf2) and tight junction proteins (except claudin-12 and -15), and simultaneously increased malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) contents. Iron deficiency also up-regulated mRNA levels of cysteinyl aspartic acid-protease (caspase) -2, -7, -8, -9, Fas ligand (FasL), apoptotic protease activating factor-1 (Apaf-1), B-cell-lymphoma-2 associated X protein (Bax), p38 mitogen-activated protein kinase (p38MAPK), Kelch-like ECH-associating protein (Keap) 1a, Keap1b, claudin-12, -15 and MLCK, indicating that iron deficiency could disturb the structural integrity of gill in fish. Third, iron excess impaired immune function and structural integrity in gill of young grass carp. Forth, there was a better effect of ferrous fumarate than ferrous sulfate in young grass carp. Finally, the iron requirements based on ability against gill rot, ACP activity and MDA content in gill of young grass carp were estimated to be 76.52, 80.43 and 83.17 mg/kg, respectively.