Metabolites, gene expression, and gut microbiota profiles suggest the putative mechanisms via which dietary creatine increases the serum taurine and g-ABA contents in Megalobrama amblycephala.

A 90-day experiment was conducted to explore the effects of creatine on growth performance, liver health status, metabolites, and gut microbiota in Megalobrama amblycephala. There were 6 treatments as follows: control (CD, 29.41% carbohydrates), high carbohydrate (HCD, 38.14% carbohydrates), betaine (BET, 1.2% betaine + 39.76% carbohydrates), creatine 1 (CRE1, 0.5% creatine + 1.2% betaine + 39.29% carbohydrates), creatine 2 (CRE2, 1% creatine + 1.2% betaine + 39.50% carbohydrates), and creatine 3 (CRE3, 2% creatine + 1.2% betaine + 39.44% carbohydrates). The results showed that supplementing creatine and betaine together reduced the feed conversion ratio significantly (P < 0.05, compared to CD and HCD) and improved liver health (compared to HCD). Compared with the BET group, dietary creatine significantly increased the abundances of Firmicutes, Bacteroidota, ZOR0006, and Bacteroides and decreased the abundances of Proteobacteria, Fusobacteriota, Vibrio, Crenobacter, and Shewanella in the CRE1 group. Dietary creatine increased the content of taurine, arginine, ornithine, γ-aminobutyric acid (g-ABA), and creatine (CRE1 vs. BET group) and the expression of creatine kinase (ck), sulfinoalanine decarboxylase (csad), guanidinoacetate N-methyltransferase (gamt), glycine amidinotransferase (gatm), agmatinase (agmat), diamine oxidase1 (aoc1), and glutamate decarboxylase (gad) in the CRE1 group. Overall, these results suggested that dietary supplementation of creatine (0.5-2%) did not affect the growth performance, but it altered the gut microbial composition at the phylum and genus levels, which might be beneficial to the gut health of M. amblycephala; dietary creatine also increased the serum content of taurine by enhancing the expressions of ck and csad and increased the serum content of g-ABA by enhancing the arginine content and the expressions of gatm, agmat, gad, and aoc1.

Largemouth bass (Micropterus salmoides) exhibited better growth potential after adaptation to dietary cottonseed protein concentrate inclusion but experienced higher inflammatory risk during bacterial infection.

Cottonseed protein concentrate (CPC) has been proven to partially replace fishmeal without adverse effects on fish growth performance, while little information is known about the effects on liver health during bacterial infection. In the present study, 15% CPC was included into the diet of juvenile largemouth bass (32.12 ± 0.09g) to replace fishmeal for 8 weeks, with fish growth potential and hepatic inflammatory responses during Nocardia seriolae (N. seriolae) infection systemically evaluated. After adaptation to dietary CPC inclusion, largemouth bass even exhibited better growth potential with higher SGR and WGR during the last three weeks of whole feeding trial, which was accompanied with higher phosphorylation level of TOR signaling and higher mRNA expression level of myogenin (myog). At the end of 8-weeks feeding trial, the histological structure of largemouth bass liver was not significantly affected by dietary CPC inclusion, accompanied with the similar expression level of genes involved in innate and adaptive immunity and comparable abundance of T cells in bass liver. N.seriolae infection induced the pathological changes of bass liver, while such hepatic changes were more serious in CPC group than that in FM group. Additionally, RT-qPCR results also suggested that largemouth bass fed with CPC experienced much higher inflammatory potential both in liver and gill during N. seriolae infection, which was accompanied with higher expression level of genes involved in pyroptosis. Therefore, this study demonstrated that the application of CPC in largemouth bass diet should be careful, which may induce higher inflammatory potential during N. seriolae infection.

Dietary Glutamine Inclusion Regulates Immune and Antioxidant System, as Well as Programmed Cell Death in Fish to Protect against Flavobacterium columnare Infection.

The susceptibility of animals to pathogenic infection is significantly affected by nutritional status. The present study took yellow catfish (Pelteobagrus fulvidraco) as a model to test the hypothesis that the protective roles of glutamine during bacterial infection are largely related to its regulation on the immune and antioxidant system, apoptosis and autophagy. Dietary glutamine supplementation significantly improved fish growth performance and feed utilization. After a challenge with Flavobacterium columnare, glutamine supplementation promoted il-8 and il-1ß expression via NF-κB signaling in the head kidney and spleen, but inhibited the over-inflammation in the gut and gills. Additionally, dietary glutamine inclusion also enhanced the systematic antioxidant capacity. Histological analysis showed the protective role of glutamine in gill structures. Further study indicated that glutamine alleviated apoptosis during bacterial infection, along with the reduced protein levels of caspase-3 and the reduced expression of apoptosis-related genes. Moreover, glutamine also showed an inhibitory role in autophagy which was due to the increased activation of the mTOR signaling pathway. Thus, our study for the first time illustrated the regulatory roles of glutamine in the fish immune and antioxidant system, and reported its inhibitory effects on fish apoptosis and autophagy during bacterial infection.

Dietary Glycyrrhiza uralensis extracts supplementation elevated growth performance, immune responses and disease resistance against Flavobacterium columnare in yellow catfish (Pelteobagrus fulvidraco).

The present study was conducted to evaluate the influence of Glycyrrhiza uralensis (G. uralensis) extracts on the growth performance, histological structure, immune response and disease resistance against Flavobacterium columnare (F. columnare) of yellow catfish. Fish were fed with two different diets, i.e., basal diet as control group (CG) and diet containing G. uralensis extracts as experimental group (GG). After 60 days feeding, growth performance of GG fish was significantly improved, with increased WG and SGR but decreased FCR compared to CG fish. Fish were then challenged with F. columnare for two times, as fish showed rare mortality after the first infection. GG fish showed significantly lower cumulative mortality during F. cloumnare infection than CG fish after 21 days infection (dpi). Epithelial cell exfoliation and obvious cellular vacuolization in the skin and congestion of gill lamellae were detected in CG fish, while GG fish showed increased width of epidermis and mucous cells number in skin, and increased length of secondary lamina in gill. GG fish also exhibited higher enzyme activity of lysozyme in serum and mRNA expression of lysozyme in head kidney than CG fish at most time points post infection. G. uralensis extracts supplementation also induced earlier serum anti-oxidative responses, with increased superoxide dismutase activity and total antioxidant capacity in GG fish at 1 dpi. Compared to CG fish, GG fish showed increased expression level of genes involved in TLRs-NFκB signaling (TLR2, TLR3, TLR5, TLR9, Myd88, and p65NFκB), resulting in higher expression levels of pro-inflammatory cytokines (IL-1ß and IL-8) in the head kidney post infection. However, these genes showed deviation in the gill of GG fish, which increased at some time points but decreased at other time points. Moreover, G. uralensis extracts supplementation also significantly unregulated the expression levels of IgM and IgD in head kidney, and the expression levels of IgM in the gill of yellow catfish, suggesting the elevated humoral immune response during F. columnare infection. All these results contributed to the elevated disease resistance ability against F. cloumnare infection of yellow catfish after dietary G. uralensis extracts supplementation.

Promoting effect of baicalin on nitric oxide production in CMECs via activating the PI3K-AKT-eNOS pathway attenuates myocardial ischemia-reperfusion injury.

OBJECTIVE: Baicalin, which is isolated from Scutellariae Radix, has been shown to possess therapeutic potential for different diseases. Cardiac microvessel injury in myocardial ischemia-reperfusion (IR) has been extensively explored. However, there have been no studies investigating the physiological regulatory mechanisms of baicalin on nitric oxide production and the necroptosis of cardiac microvascular endothelial cells (CMECs) in myocardial IR injury. This study was designed to investigate the contribution of baicalin to repressing necroptosis and preventing IR-mediated CMEC dysfunction. MATERIALS AND METHODS: Indicators of ventricular structure and function were measured by an echocardiographic system. An MTT assay was performed to assess cell viability. Nitrite detection was performed to detect nitric oxide content, and cGMP content was determined using a commercially available cGMP complete ELISA kit. Morphology and molecular characteristics were detected by electron micrographs, quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. RESULT: Our results demonstrated that baicalin significantly improved cardiac function, decreased the myocardial infarction area, and inhibited myocardial cell apoptosis. Moreover, baicalin had a protective effect on cardiac microvessels and promoted the production of nitric oxide (NO) and the level of cGMP in rats that underwent myocardial IR injury. The results of the in vitro experiments showed that baicalin markedly improved cell activity and function in CMECs exposed to hypoxia-reoxygenation (HR). Further experiments indicated that baicalin supplementation suppressed the protein expression of RIP1, RIP3 and p-MLKL to interrupt CMEC necroptosis. In addition, baicalin promoted the production of NO via activating the PI3K-AKT-eNOS signaling pathway. Taken together, our results identified the PI3K-AKT-eNOS axis as a new pathway responsible for reperfusion-mediated microvascular damage. CONCLUSION: Baicalin protected CMECs in IR rats by promoting the release of NO via the PI3K-AKT-eNOS pathway and mitigated necroptosis by inhibiting the protein expression of RIP1, RIP3 and p-MLKL.

Glutamine protects against LPS-induced inflammation via adjusted NODs signaling and enhanced immunoglobulins secretion in rainbow trout leukocytes.

To evaluate effects of glutamine (GLN) on fish immune responses, leukocytes were isolated from head kidney of rainbow trout and cultured in GLN-free DMEM media supplemented with different combinations of lipopolysaccharide (LPS) and GLN. LPS significantly increased expression of pro-inflammatory cytokines, while GLN supplementation alleviated LPS-induced inflammation. Leukocytes in +GLN + LPS group showed more active GLN anabolism and catabolism, which signals could be sensed by O-GlcNAcylation, and then affected LPS binding to cell surface (LBP) and adjusted NODs signaling. The mRNA expression of immunoglobulins (Igs) and their receptor (pIgR) was also significantly increased after GLN supplementation. Further analysis showed that GLN increased the percentage of IgM+ B cells and IgT+ B cells, accompanied with the increased IgM and IgT secretion in culture media, which further increased complement C3 expression to perform effector functions. All these results illustrated the regulating mechanism of GLN against LPS-induced inflammation both via adjusted NODs signaling and increased Igs+ B cells to secrete Igs.

l-arginine inhibited apoptosis of fish leukocytes via regulation of NF-κB-mediated inflammation, NO synthesis, and anti-oxidant capacity.

The increased apoptosis plays an important role in bacterial invasion. In addition, LPS can induce inflammation and apoptosis of leukocytes via the production of reactive oxygen and nitrogen species. In the present study, we investigated the potential protective role of l-arginine (L-Arg) against the apoptosis of fish leukocytes in vitro. The results of Annexin V-FITC/PI staining and TUNEL assay indicated that L-Arg significantly alleviated the apoptosis of fish leukocytes induced by LPS at 24 h and 72 h post incubation (hpi). High caspase-3 activities induced by LPS at 72 hpi were significantly inhibited by L-Arg. Moreover, L-Arg supplementation also significantly decreased the mRNA expression levels of caspases at most time points, which contributed to the anti-apoptotic roles of L-Arg. Further analysis showed that L-Arg significantly inhibited the expression of several pro-inflammatory cytokines including IL-8 and TNF-α, partially via the down-regulation of the genes involved in NF-κB/MyD88 including NF-κB, IKKα and IKKγ. The down-regulation of these pro-inflammatory cytokines by L-Arg supplementation led to the further decrease in the expression of death receptor FasL, contributing to the anti-apoptotic effect of L-Arg. In addition, L-Arg supplementation increased both iNOS mRNA expression and NO production. The mRNA expressions of several anti-oxidant enzymes including SOD, CAT and GSHPx were also significantly increased after L-Arg supplementation, which accelerated the clearance of reactive oxygen species. In all, L-Arg inhibited apoptosis of fish leukocytes both via the increased NO production and antioxidant capacity and via the inhibition of inflammation mediated by NF-κB/MyD88 pathway.

Chronic rapamycin treatment on the nutrient utilization and metabolism of juvenile turbot (Psetta maxima).

High dietary protein inclusion is necessary in fish feeds and also represents a major cost in the aquaculture industry, which demands improved dietary conversion into body proteins in fish. In mammals, the target of rapamycin (TOR) is a key nutritionally responsive molecule governing postprandial anabolism. However, its physiological significance in teleosts has not been fully examined. In the present study, we examined the nutritional physiology of turbot after chronic rapamycin inhibition. Our results showed that a 6-week inhibition of TOR using dietary rapamycin inclusion (30 mg/kg diet) reduced growth performance and feed utilization. The rapamycin treatment inhibited TOR signaling and reduced expression of key enzymes in glycolysis, lipogenesis, cholesterol biosynthesis, while increasing the expression of enzymes involved in gluconeogenesis. Furthermore, rapamycin treatment increased intestinal goblet cell number in turbot, while the expressions of Notch and Hes1 were down regulated. It was possible that stimulated goblet cell differentiation by rapamycin was mediated through Notch-Hes1 pathway. Therefore, our results demonstrate the important role of TOR signaling in fish nutritional physiology.