Bile acid and short chain fatty acid metabolism of gut microbiota mediate high-fat diet induced intestinal barrier damage in Macrobrachium rosenbergii.

The limited tolerance of crustacean tissue physiology to a high-fat diet has captured the attention of researchers. Yet, investigations into the physiological response mechanisms of the crustacean intestinal barrier system to a high-fat diet are progressing slowly. Elucidating potential physiological mechanisms and determining the precise regulatory targets would be of great physiological and nutritional significance. This study established a high-fat diet-induced intestinal barrier damage model in Macrobrachium rosenbergii, and systematically investigated the functions of gut microbiota and its functional metabolites. The study achieved this by monitoring phenotypic indicators, conducting 16S rDNA sequencing, targeted metabolomics, and in vitro anaerobic fermentation of intestinal contents. Feeding prawns with control and high-fat diets for 8 weeks, the lipid level of 7 % in the CON diet and 12 % in the HF diet. Results showed that high-fat intake impaired the intestinal epithelial cells, intestinal barrier structure, and permeability of M. rosenbergii, activated the tight junction signaling pathway inhibiting factor NF-κB transcription factor Relish/myosin light chain kinase (MLCK), and suppressed the expression of downstream tight junction proteins zona occludens protein 1 (ZO-1) and Claudin. High-fat intake resulted in a significant increase in abundance of Aeromonas, Enterobacter, and Clostridium sensu stricto 3 genera, while Lactobacillus, Lactococcus, Bacteroides, and Ruminococcaceae UCG-010 genera were significantly decreased. Targeted metabolomics results of bile acids and short-chain fatty acids in intestinal contents and in vitro anaerobic fermentation products showed a marked rise in the abundance of DCA, 12-KetoLCA, 7,12-diketoLCA, and Isovaleric acid, and a significant reduction in the abundance of HDCA, CDCA, and Acetate in the HF group. Pearson correlation analysis revealed a substantial correlation between various genera (Clostridium sensu stricto 3, Lactobacillus, Bacteroides) and secondary metabolites (DCA, HDCA, 12-KetoLCA, Acetate), and the latter was significantly correlated with intestinal barrier function related genes (Relish, ZO-1, MLCK, vitamin D receptor, and ecdysone receptor). These findings indicate that gut microorganisms and their specific bile acids and short-chain fatty acid secondary metabolites play a crucial role in the process of high-fat-induced intestinal barrier damage of M. rosenbergii. Moreover, identifying and targeting these factors could facilitate precise regulation of high-fat nutrition for crustaceans.

Promotion of improved intestinal barrier health by soybean-derived bioactive peptides in Chinese mitten crab (Eriocheir sinensis) fed a low fishmeal diet.

To alleviate the growth inhibition, and intestinal damage of Chinese mitten crab (Eriocheir sinensis) induced by low fishmeal diets (LF), an 8-week feeding trial was conducted to evaluate the addition of dietary soybean-derived bioactive peptides (SBP) in LF diets on the regulation of growth, digestion and intestinal health. The crabs were fed isonitrogenous and isoenergetic conventional diet and LF diets (10 % fishmeal replaced by soybean meal, LF) supplemented with 0, 1 %, 2 %, 4 % and 6 % SBP, respectively. The results showed that LF diet inhibited growth while inclusion of SBP quadratically remitted the growth inhibition induced by LF. For digestive function, increasing addition level of SBP quadratically improved the α-amylase and trypsin activities. For antioxidant function, LF group significantly increased the malondialdehyde content, while SBP linearly decreased the malondialdehyde level and cubically increased the anti-superoxide anion activity and total antioxidant capacity level. For intestinal health, the peritrophic membrane (PM) almost completely separated from the inner wall of the intestinal lumen, the epithelial cells reduced, the muscularis became thinner and the apoptotic signals increased in LF group; with SBP addition, the intestinal morphology was improved, with the PM adhering to the inner wall of the intestinal lumen, an increase in the number of epithelial cells and an increase in the thickness of the muscularis. Additionally, there was a decrease in apoptotic signals. Dietary SBP also increased the expression of PT and Crustin1 quadratically and decreased the expression of ALF1 linearly, ALF3 and ILF2 quadratically.

Tea Tree Oil Improves Energy Metabolism, Non-Specific Immunity, and Microbiota Diversity via the Intestine-Hepatopancreas Axis in Macrobrachium rosenbergii under Low Fish Meal Diet Administration.

Tea tree oil (TTO) is an essential plant oil with diverse antibacterial and antioxidant properties; however, whether the role played by TTO in low fish meal (LF) diets induced the observed effects in the farmed crustaceans remains unclear. Therefore, this study used Macrobrachium rosenbergii as the model crustacean, and an 8-week feeding experiment with NF (normal fish meal), LF (soybean meal replacing 40% fish meal), and LFT (LF with 200 mg/kg TTO) diets was conducted to evaluate the positive effects of TTO under the LF diet. Compared to the NF diet, the LF diet reduced hemolymph antioxidant capacity and non-specific immunity, and induced hepatopancreas apoptosis and damage. However, in comparison with LF, LTF significantly ameliorated morphological impairment in the hepatopancreas, improved hepatopancreas energy metabolism by upregulating the Bcl-2/Bax and Akt/mTOR pathways, and enhanced antioxidant and non-specific immune capacity by activating the NF-κB/NO pathway. In addition, LFT repaired intestinal barrier injury and the imbalance of intestinal microbiota induced by the LF diet. Moreover, the Pearson correlation revealed the variations of the above indicators, which were related to the abundance changes of Klebsiella, Clostridium sensu stricto 12, Thermobifida, Bifidobacterium, and Alistipes, indicating that these microbes might serve as prospective targets for the intestine-hepatopancreas axis to affect hepatopancreas apoptosis, metabolism, and non-specific immunity. In summary, 200 mg/kg TTO supplementation mediated gut microbiota and positively improved energy metabolism and non-specific immunity, thereby alleviating hepatopancreas dysplasia and damage induced by the LF diet in M. rosenbergii.

M2 Macrophage-Derived Extracellular Vesicles Containing MicroRNA-501-3p Promote Colon Cancer Progression Through the SETD7/DNMT1/SOCS3 Axis.

BACKGROUND: Macrophage-derived extracellular vesicles with microRNAs can cause and develop colon cancer. OBJECTIVE: To investigate M2 macrophage-derived extracellular vesicles and colon cancer. DESIGN: A prospective and experimental study of M2 macrophage-derived extracellular vesicles in colon cancer. SETTING: This study was completed at the Fourth Hospital of Hebei Medical University. PATIENTS: Patients with colon cancer who had undergone surgical resection. MAIN OUTCOME MEASURES: Suppressor of cytokine signaling 3, miR-501-3p, SET domain containing 7, and DNA methyltransferase 1 were measured in colon cancer samples. Multiple experiments determined suppressor of cytokine signaling 3, miR-501-3p, SET domain containing 7, and DNA methyltransferase 1 binding affinity. M2 macrophages were cultivated from M0 macrophages isolated from peripheral blood mononuclear cells of a healthy donor and polarized to produce extracellular vesicles. Gain- or loss-of-function tests using colon cancer cells and M2 macrophage-derived extracellular vesicles revealed cell biological processes. Finally, animal models were created to test how miR-501-3p from M2-extracellular vesicles affects tumor growth via the SET domain containing 7/DNA methyltransferase 1/suppressor of cytokine signaling 3. RESULTS: Colon cancer increased miR-501-3p and DNA methyltransferase 1 and downregulated suppressor of cytokine signaling 3 and SET domain containing 7. miR-151-3p inhibited SET domain containing 7, upregulating DNA methyltransferase 1. Increased promoter methylation by DNA methyltransferase 1 decreased suppressor of cytokine signaling 3 expression. M2-EVs with miR-501-3p regulated the SET domain containing 7/DNA methyltransferase 1/suppressor of cytokine signaling 3 axis to induce apoptosis and colon cancer cell growth, invasion, and migration. M2-EV-delivered miR-501-3p also regulated the SET domain containing 7/DNA methyltransferase 1/suppressor of cytokine signaling 3 axis to promote tumor growth in animals. LIMITATIONS: Further research is needed in clinical application of M2 macrophage-derived extracellular vesicles containing miR-501-3p as a biomarker of colon cancer. CONCLUSIONS: M2 macrophage-derived extracellular vesicles with miR-501-3p regulate the SET domain containing 7/DNA methyltransferase 1/suppressor of cytokine signaling 3 axis to promote colon cancer. LAS VESCULAS EXTRACELULARES DERIVADAS DE MACRFAGOS M QUE CONTIENEN MICROARNP PROMUEVEN LA PROGRESIN DEL CNCER DE COLON A TRAVS DEL EJE SETD/DNMT/SOCS: ANTECEDENTES:Las vesículas extracelulares derivadas de macrófagos con microARN pueden causar y desarrollar cáncer de colon.OBJETIVO:Investigamos las vesículas extracelulares derivadas de macrófagos M2 y el cáncer de colon.DISEÑO:Un estudio prospectivo y experimental de vesículas extracelulares derivadas de macrófagos M2 en el cáncer de colon.ESCENARIO:Este estudio se completó en el Cuarto Hospital de la Universidad Médica de Hebei.PACIENTES:Pacientes con cáncer de colon sometidos a resección quirúrgica.PRINCIPALES MEDIDAS DE RESULTADO:Se midieron el supresor de la señalización de citoquinas 3, miR-501-3p, SETD7 y la ADN metiltransferasa 1 en muestras de cáncer de colon. Múltiples experimentos determinaron la afinidad de unión del supresor de la señalización de citoquinas 3, de miR-501-3p, de SETD7 y de la ADN metiltransferasa 1. Los macrófagos M2 se cultivaron a partir de macrófagos M0 aislados de células mononucleares de sangre periférica de donantes sanos y se polarizaron para producir vesículas extracelulares. Las pruebas de ganancia o pérdida de función utilizando células de cáncer de colon y vesículas extracelulares derivadas de macrófagos M2 revelaron procesos biológicos celulares. Finalmente, se crearon modelos animales para probar cómo miR-501-3p de vesículas extracelulares M2 afecta el crecimiento tumoral a través del SETD7/ADN metiltransferasa 1/supresor de la señalización de citocinas 3.RESULTADOS:El cáncer de colon aumentó el miR-501-3p y la ADN metiltransferasa 1 y reguló negativamente el supresor de la señalización de citoquinas 3 y SETD7. miR-151-3p inhibió SETD7, regulando positivamente la ADN metiltransferasa 1. El aumento de la metilación del promotor por la ADN metiltransferasa 1 produjo disminución de la expresión del supresor de señalización de citocinas 3. Los M2-EV con miR-501-3p regularon el eje SETD7/ADN metiltransferasa 1/supresor de la señalización de citocinas 3 para inducir apoptosis y crecimiento, invasión y migración de células de cáncer de colon. El miR-501-3p administrado por M2-EV también reguló el eje SETD7/ADN metiltransferasa 1/supresor de la señalización de citocinas 3 para promover el crecimiento tumoral en animales.LIMITACIONES:Se necesita más investigación en la aplicación clínica de vesículas extracelulares derivadas de macrófagos M2 que contienen miR-501-3p como biomarcador de cáncer de colon.CONCLUSIONES:Las vesículas extracelulares derivadas de macrófagos M2 con miR-501-3p regulan el eje SETD7/ADN metiltransferasa 1/supresor de la señalización de citocinas 3 para promover el cáncer de colon. (Traducción-Dr. Felipe Bellolio ).

Recovery of methane and acetate during ex-situ biogas upgrading via novel dual-membrane aerated biofilm reactor.

Biological biogas upgrading has been well-proven to be a promising approach for renewable bioenergy recovery, but hydrogen (H2)-assisted ex-situ biogas upgrading is hindered by a large solubility discrepancy between H2 and carbon dioxide (CO2). This study established a new dual-membrane aerated biofilm reactor (dMBfR) to improve the upgrading efficiency. Results showed that dMBfR operated at 1.25 atm H2 partial pressure, 1.5 atm biogas partial pressure, and 1.0 d hydraulic retention time could significantly improve the efficiency. The maximum methane purity of 97.6%, acetate production rate of 34.5 mmol L-1d-1, and H2 and CO2 utilization ratios of 96.5% and 96.3% were achieved. Further analysis showed that the improved performances of biogas upgrading and acetate recovery were positively correlated with the total abundances of functional microorganisms. Taken together, these results suggest that the dMBfR, which facilitates the precise CO2 and H2 supply, is an ideal approach for efficient biological biogas upgrading.

Low fish meal diet supplemented with probiotics ameliorates intestinal barrier and immunological function of Macrobrachium rosenbergii via the targeted modulation of gut microbes and derived secondary metabolites.

The unsuitable substitution ratio of fish meal by plant protein will reshape the intestinal microbial composition and intestine immunity. However, previous studies were mostly limited to investigating how different feed or probiotics characterized the microbial composition but ignored the biological interactions between bacteria and host physiology through secondary metabolites. Therefore, this study integrates the apparent indicators monitoring, 16S rDNA sequencing, and metabonomics to systematically investigate the effects of cottonseed protein concentrate (CPC) substitution of fish meal and Bacillus coagulans intervention on gut microbes, secondary metabolites, and intestinal immunity of Macrobrachium rosenbergii. Prawns were fed with three diets for 70 days: HF diets contained 25% fish meal, CPC in LF diets were replaced with 10% fish meal, and LF diets supplemented with 2 × 108 CFU/g diet B. coagulans were designated as BC diets. Results showed that CPC substitution induced a significant decrease in digestive enzyme activities (trypsin and lipase) and gut barrier protein PT-1 expression and a significant increase in γ-GT enzyme activity and inflammatory-related factors (Relish and Toll) expression. B. coagulans treatment mitigated the negative changes of the above indicators. Meanwhile, it significantly improved the expression levels of the barrier factor PT-1, the reparative cytokine IL-22, and Cu/Zn-SOD. CPC substitution resulted in a remarkable downregulated abundance of Firmicutes phyla, Flavobacterium spp., and Bacillus spp. B. coagulans treatment induced the callback of Firmicutes abundance and improved the relative abundance of Sphingomonas, Bacillus, and Ralstonia. Functional prediction indicated that CPC substitution resulted in elevated potential pathogenicity of microbial flora, and B. coagulans reduces the pathogenesis risk. Pearson's correlation analysis established a significant positive correlation between differential genera (Sphingomonas, Bacillus, and Ralstonia) and secondary metabolites (including sphingosine, dehydrophytosphingosine, amino acid metabolites, etc.). Meanwhile, the latter were significantly associated with intestinal immunoregulation-related genes (Cu/Zn-SOD, IL-22, PT-1, Toll, and Relish). This study indicated that B. coagulans could mediate specific gut microbes and the combined action of multiple functional secondary metabolites to affect intestinal barrier function, digestion, and inflammation. Our study revealed the decisive role of gut microbes and derived secondary metabolites in the model of dietary composition-induced intestinal injury and probiotic treatment from a new perspective.

Tea Tree Oil Mediates Antioxidant Factors Relish and Nrf2-Autophagy Axis Regulating the Lipid Metabolism of Macrobrachium rosenbergii.

Both oxidative stress and autophagy refer to regulating fat metabolism, and the former affects autophagy, but the role and mechanism of the antioxidant-autophagy axis in regulating lipid metabolism remains unclear. As an antioxidant, tea tree oil (TTO) has little research on the regulatory mechanism of lipid metabolism in crustaceans. This study investigated whether TTO could alter hepatopancreatic lipid metabolism by affecting the antioxidant-autophagy axis. Feed Macrobrachium rosenbergii with three different levels of TTO diets for 8 weeks: CT (0 mg/kg TTO), 100TTO (100 mg/kg TTO), and 1000TTO (1000 mg/kg TTO). The results showed that 100TTO treatment reduced the hemolymph lipids level and hepatopancreatic lipid deposition compared to CT. In contrast, 1000TTO treatment increased hepatopancreatic lipid deposition, damaging both morphology and function in the hepatopancreas. The 100TTO treatment promoted lipolysis and reduced liposynthesis at the transcriptional level compared to the CT group. Meanwhile, it improved the hepatopancreas antioxidant capacity and maintained mitochondrial structural and ROS homeostasis. In addition, it simultaneously activated the expression of transcription factors Keap1-Nrf2 and Imd-Relish. By contrast, the 1000TTO group significantly enhanced the ROS level, which considerably activated the Keap1-Nrf2 signaling expression but had no significant effects on the expression of Imd-Relish. The 100TTO group supplementation significantly enhanced lipid droplet breakdown and autophagy-related genes and protein expression. On the contrary, the 1000TTO group significantly inhibited the expression of genes and proteins related to autophagy. Pearson analysis revealed that Nrf2 has a positive correlation to lipid anabolism-related genes (Fasn, Srebp1, Pparγ) and autophagy regulators (mtor, akt, p62), and were negatively correlated with lipolysis-related genes (Cpt1, Hsl, Ampkα) and autophagy markers (Ulk1, Lc3). Relish was positively correlated with Atgl, Cpt1, Ampkα, Ulk1, and Lc3, and negatively correlated with Pparγ and p62. Moreover, Keap1 and Imd were negatively correlated with p62 and mtor, respectively. In sum, 100 mg/kg TTO enhanced antioxidant activity and increased autophagy intensity through the Relish-Imd pathway to enhance lipid droplet breakdown, while 1000 mg/kg TTO overexpressed Nrf2, thus inhibiting autophagy and ultimately causing excessive lipid deposition and peroxidation. Our study gives a fresh perspective for deciphering the bidirectional regulation mechanism of lipid metabolism by different doses of TTO based on the antioxidant-autophagy axis.

Amelioration of ammonia-induced intestinal oxidative stress by dietary Clostridium butyricum in giant freshwater prawn (Macrobrachium rosenbergii).

The use of Clostridium butyricum in crustacean aquaculture for anti-abiotic stress is yet unknown. Feeds were formulated containing 0, 125, 250, 500, and 1000 mg/kg Clostridium butyricum (2 × 107 CFU/g), respectively. The giant freshwater prawns (Macrobrachium rosenbergii) were fed for 8 weeks in triplicate. The results showed that C. butyricum-supplemented groups improved growth performance significantly with the optimum level at 610 mg/kg. Ammonia stress reduced hemolymph glucose, total protein, total cholesterol, and triglyceride concentrations while dietary C. butyricum significantly increased hemolymph glucose and total protein levels after the ammonia challenge. Ammonia stress increased inducible nitric oxide synthase (iNOS) and nitric oxide (NO) levels, and the treatments supplemented with C. butyricum had considerably enhanced levels of iNOS and NO after stress. Treatment with C. butyricum increased the level of superoxide dismutase (SOD), and decreased the level of malondialdehyde (MDA) and superoxide anion, with the 125 mg/kg treated groups having the extreme value. Furthermore, C. butyricum-treated groups reduced the expression of HSPs after ammonia stress while the ammonia stress induced the expression of HSP60, HSP70, and HSP90. Dietary C. butyricum elevated the expression of peroxiredoxin-5 and toll in response to ammonia stress. The results indicate that dietary supplementation with 125-500 mg/kg of C. butyricum (2 × 107 CFU/g) improved biochemical and antioxidant features as well as intestinal immunity of M. rosenbergii under ammonia challenge by activating the toll signal pathway.

Effects of high fat in the diet on growth, antioxidant, immunity and fat deposition of Macrobrachium rosenbergii post-larvae.

Lipids are essential nutrients for organisms, and high-fat feeds for shrimp may cause oxidative stress. This study evaluated the effects of feeding high fat in the diet on the growth, antioxidant, immunity, and liver fat accumulation of Macrobrachium rosenbergii post-larvae. Five groups with an initial body weight of 0.0084 ± 0.001 g were fed five isonitrogenous and isoenergetic diets (47.01% crude protein and 18.40 kJ/g gross energy) containing 8%, 10%, 12%, 14% and 16% (named L8, L10, L12, L14 and L16) lipid for 8 weeks, respectively. The results showed that the weight gain rate (WGR) and specific growth rate (SGR) of L8 group were significantly higher than those of L10, L12, L14 and L16 group (P < 0.05), and the feed coefficient (FCR) of L8 group was significantly lower than that of other groups (P < 0.05). With the increase of dietary fat level, the content of MDA and the activity of SOD increased significantly, and the activities of T-AOC and CAT decreased significantly (P < 0.05). H&E staining clearly revealed the occurrence of hepatocyte swelling, hepatocyte vacuolization and nucleus displacement to the peripheral cell vacuolization in the L16 group, and hepatic lipid accumulation was further observed in the L14 and L16 group by Oil red O staining. In addition, high-fat diet significantly upregulated the expression of Dorsal, Relish and IκBα mRNA, and also upregulated the expression of fat synthesis-related genes FAS, ACC, DGAT and fat transport-related gene FABP (P < 0.05), and significantly downregulated the expression of fat metabolism-related genes AMPK and CPT-1 (P < 0.05) compared to that of the L8 group. In conclusion, this study showed that feeding a high-fat diet could induce oxidative stress, inhibit growth performance, alter antioxidant capacity, cause hepatic fat deposition and affect the immune system of M. rosenbergii post-larvae.

Effects of dietary threonine on growth and immune response of oriental river prawn (Macrobrachium nipponense).

A 70-day feeding trial was conducted to ascertain the effects of threonine on immune response of juvenile oriental river prawn (Macrobrachium nipponense). Six isonitrogen and isolipidic feeds were formulated according to levels of dietary threonine (0.35%, 0.79%, 1.18%, 1.67%, 2.08% and 2.48% respectively). The juvenile prawns were divided into six groups with four replicates, and stocked into 24 tanks with 50 prawns per tank (initial weight 0.20 ± 0.02 g). The results showed a significant increasing trend of final body weight, specific growth rate, protein efficiency ratio, and weight gain rate when threonine levels increased to 1.67% (P < 0.05). However, feed intake, feed conversion ratio, and whole-body lipid composition significantly decreased as threonine levels in the feed increased up to 1.67% (P < 0.05). Moreover, haemolymph N-urea content was significantly lowest at 1.67% threonine level (P < 0.05), whereas glucose was highest at 0.79% followed by 1.67% of threonine levels in the feeds. Aspartate aminotransferase (AST) enzyme activities were significantly decreased by an imbalance (except 1.67%) of threonine in the feed (P < 0.05). Activities of Alanine aminotransferase (ALT) and albumen (ALB) were not significantly affected by threonine in the feed (P > 0.05). Excessive dietary threonine level (2.48%) significantly activated haemolymph catalase (CAT) activity (P < 0.05), whereas malondialdehyde (MDA) content was significantly affected by deficient (0.35% and 0.79%) dietary threonine levels (P < 0.05). Inducible nitric oxide synthase (iNOS) activity and haemolymph complement component 4 (C4) content were significantly decreased by deficient levels of threonine in the feed (P < 0.05). Excess threonine concentration significantly down-regulated Toll, Dorsal, Relish, and heat shock protein 60 (Hsp60) gene expressions in the hepatopancreas of M. nipponense (P < 0.05), while all genes were significantly up-regulated by the optimal (1.67%) threonine level (P < 0.05). The threonine level at which maximum specific growth rate of M. nipponense occurred was estimated by second degree polynomial regression analysis as 1.65% of threonine level, equivalent to 4.44% dry weight bases of protein in the feed.

Response of microbiota and immune function to different hypotonic stress levels in giant freshwater prawn Macrobrachium rosenbergii post-larvae.

This study explored the effects of different hypotonic stress levels on antioxidant capacity, microbial composition, and gene expression of Macrobrachium rosenbergii post-larvae. The salinity of the control group was 15 ‰ (S15), and the hypotonic stress groups included three levels of 10 ‰ (S10), 8 ‰ (S8), and 6 ‰ (S6). Different hypotonic stress levels caused oxidative damage in post-larvae, evidenced by decreased superoxide dismutase (SOD) and anti-superoxide anion free radical (ASAFR). They increased malondialdehyde (MDA), nitric oxide (NO), and inducible nitric oxide synthase (iNOS) levels. Microbiological analysis exhibited that different hypotonic stress levels significantly changed microbial composition and diversity. The microbial composition in the water environment where post-larvae living was different from post-larvae. The pathogenic bacteria, including Vibrio and Flavobacterium, were abundant in S6. Transcriptome analysis showed 2, 7967, 297 DEGs, including 1, 3564, 27 up-regulated genes and 1, 4403, 270 down-regulated genes in S10, S8, and S6 groups, respectively. KEGG enrichment results showed that immune and glucose metabolism-related pathways were enriched significantly. Correlation network analysis demonstrated close interactions among antioxidant parameters, microbes, and differentially-expressed genes. In conclusion, hypotonic stress reduced the antioxidant capacity, caused oxidative damage, and altered microbial composition in M. rosenbergii post-larvae. Moreover, when the salinity is below 8 ‰, hypotonic stress impairs the immune system of M. rosenbergii post-larvae.

Comparative Proteomic Analysis Revealed the Mechanism of Tea Tree Oil Targeting Lipid Metabolism and Antioxidant System to Protect Hepatopancreatic Health in Macrobrachium rosenbergii.

Tea tree oil (TTO) is a pure natural plant essential oil. The studies evaluated the hepatopancreas lipid metabolism and antioxidant efficacy of Macrobrachium rosenbergii fed with 0 (CT group) and 100 mg/kg TTO (TT group) by label-free quantification proteomic analysis. Compared to the CT group, the TT group improved growth performance and increased the survival rate after stress. Dietary TTO also decreased hemolymph AST and ALT activities and decreased hepatopancreatic vacuolation. At the same time, hepatopancreas lipids droplets and hemolymph lipids (TG, TC, LDL-C) were decreased, and the peroxidation products content (MDA, LPO, 4-HNE) was also decreased. In addition, the levels of hepatopancreas antioxidant enzymes (T-AOC, CAT, and SOD) were increased in the TT group. With proteomic analysis, a total of 151 differentially expressed proteins (DEPs) (99 up-regulated and 52 down-regulated) were identified in the hepatopancreas. Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction analysis showed that the 16 DEPs have interactions, which are mainly involved in the pathways related to lipid metabolism (fatty acid biosynthesis, fatty acid metabolism, glycerophospholipid metabolism) and redox reaction (cytochrome P450 enzyme systems). Furthermore, the mRNA expression of 15 proteins followed the proteomic analysis with qRT-PCR validation. Pearson correlation analysis showed that fatty acids and glycerophospholipid metabolism-related proteins were highly correlated to peroxide content, glycerophospholipid metabolism, and cytochrome P450 system-related proteins (CYP1A1, GSTT1, GPX4) were highly correlated to AST and ALT. Additionally, GPX4 is closely related to peroxide content and antioxidant enzyme activity. Our results revealed that TTO plays a protective role in the hepatopancreas targeting the critical enzymes and antioxidant reactions in lipid metabolism. Provides a new perspective to elucidate the action path of TTO in protecting invertebrate hepatopancreas, highlights the influence of lipid metabolism on hepatopancreas health and the interaction between lipid metabolism and antioxidant system in the regulation of TTO.

High-Fat-Diet-Induced Oxidative Stress in Giant Freshwater Prawn (Macrobrachium rosenbergii) via NF-κB/NO Signal Pathway and the Amelioration of Vitamin E.

Lipids work as essential energy sources for organisms. However, prawns fed on high-fat diets suffer from oxidative stress, whose potential mechanisms are poorly understood. The present study aimed to explore the regulation mechanism of oxidative stress induced by high fat and the amelioration by vitamin E (VE) of oxidative stress. Macrobrachium rosenbergii were fed with two dietary fat levels (LF 9% and HF 13%) and two VE levels (200 mg/kg and 600 mg/kg) for 8 weeks. The results showed that the HF diet decreased the growth performance, survival rate and antioxidant capacity of M. rosenbergii, as well as inducing hypertrophied lipid droplets, lipophagy and apoptosis. A total of 600 mg/kg of VE in the HF diet alleviated the negative effects induced by HF. In addition, the HF diet suppressed the expression of toll-dorsal and imd-relish signal pathways. After the relish and dorsal pathways were knocked down, the downstream iNOS and NO levels decreased and the MDA level increased. The results indicated that M. rosenbergii fed with a high-fat diet could cause oxidative damage. Its molecular mechanism may be attributed to the fact that high fat suppresses the NF-κB/NO signaling pathway mediating pro-oxidant and antioxidant targets for regulation of oxidative stress. Dietary VE in an HF diet alleviated hepatopancreas oxidative stress and apoptosis.

Protective effects of dietary icariin on lipopolysaccharide-induced acute oxidative stress and hepatopancreas injury in Chinese mitten crab, Eriocheir sinensis.

To investigate the effects of dietary icariin (ICA) supplementation on acute oxidative stress and hepatopancreatic injury induced by lipopolysaccharide (LPS) injection in Eriocheir sinensis, an 8-week feeding trial of crabs was conducted using 4 diets with different supplementation levels of ICA (0, 50, 100, and 200 mg/kg diet weight, respectively), and then challenged with LPS of 400 µg/kg body weight for 6 h. Results showed that 100 mg/kg ICA supplementation increased the antioxidant capacity, reduced the stress-related indicators in haemolymph, strengthen the mitochondrial membrane potential, and reduce apoptosis compared to the single LPS-treated crabs. The expressions of apoptosis-related genes and proteins were also evaluated to further understand the effects of dietary ICA pretreatment on LPS-induced cell apoptosis. As a result, dietary 100 mg/kg diet weight ICA pre-addition significantly down-regulated the expression of HSP60, HSP70, Caspase 3c, Caspase 8, Caspase 3, Caspase 9, P38, and Bax (P < 0.05), and alleviated the suppressed expression of PI3K, AKT, MEK, and Bcl-2 (P < 0.05) in crabs challenged with LPS. Overall, this research reveals that ICA supplementation of 100 mg/kg diet weight could enhance the resistance to oxidative damage and apoptosis in E. sinensis facing LPS challenge.

Effects of dietary tea tree oil on the growth, physiological and non-specific immunity response in the giant freshwater prawn (Macrobrachium rosenbergii) under high ammonia stress.

This study aimed to investigate the effects of dietary tea tree oil (TTO) on the performance, intestinal antioxidant capacity, and non-specific immunity after ammonia nitrogen stress in Macrobrachium rosenbergii. Six experimental diets were formulated with 0, 25, 50, 100, 200, 400 mg/kg TTO, respectively. A total of 900 prawns (average initial weight, 0.39 ± 0.01 g) were randomly assigned to 6 groups in triplicate in 18 tanks. After an 8-week feeding trial, 20 prawns from each tank were changed with 20 mg/L ammonia stress for 24 h. The results showed that 100 mg/kg TTO significantly increased prawns performance and survival rate compared with the control group. Moreover, 100 and 200 mg/kg TTO significantly improved intestinal antioxidant capabilities by increasing SOD enzyme activities and decreasing MDA levels. In addition, the prawns fed with 100 mg/kg TTO diet showed the highest survival rate under ammonia stress. After ammonia stress, the group of 100 mg/kg TTO significantly improved antioxidant capacity by increasing hemolymph respiratory burst activity, as well as intestinal anti-superoxide anion activity and SOD. Coincidentally, 100 mg/kg TTO significantly upregulated the intestinal relative expression of antioxidant-related genes (peroxiredoxin-5). Further, it was found that 100 mg/kg TTO activated the toll-dorsal pathway in prawns, which performed the similar function as the classic NF-κB pathway by upregulating the TNF-α and IL-1. Finally, 100 mg/kg TTO increased the levels of iNOS activities and NO contents after ammonia stress and enhanced non-specific immunity. The results indicated that 100 mg/kg TTO could significantly improve the M. rosenbergii performance, antioxidant capacity and ammonia stress resistance. We suggested that the mechanisms may be attributed to that TTO enhanced the antioxidant capacity and non-specific immunity of M. rosenbergii via the NF-κB signal pathway.

Plasminogen activator, urokinase enhances the migration, invasion, and proliferation of colorectal cancer cells by activating the Src/ERK pathway.

Background: This paper aims to explore the effects of plasminogen activator, urokinase (PLAU) expression on the migration, invasion, and proliferation of colorectal cancer (CRC) cells and to preliminarily analyze its possible mechanism, thereby laying a foundation for the research on potential biological targets of CRC. Methods: CRC-related mRNA was screened in Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/gds/). Differentially expressed genes (DEGs) were obtained for functional enrichment analysis. The enriched pathway and key involved functional gene were screened for further in vitro and in vivo analysis CRC cells were transfected with PLAU-NC (negative control), PLAU-mimic, and PLAU-inhibitor for 48 h and divided into the above groups for later studies. The migration, invasion, and proliferation capacities of CRC cells were detected using wound healing, Transwell, and colony formation assays, respectively. The Src inhibitor saracatinib (AZD0530) was added to the PLAU-NC and PLAU-mimic groups, and the expression levels of Src/extracellular signal-regulated kinase (ERK) pathway-, migration-, invasion-, and proliferation-related proteins were detected by Western blotting. Results: The results showed that after upregulation of PLAU, the number of CRC cells (SW480) that migrated to the center of the wound significantly increased, the number of cells that migrated and invaded through the basement membrane increased in the PLAU-mimic group, and the number of colonies also increased. These results suggest that increasing PLAU expression promotes the migration, invasion, and proliferation of CRC cells. At the same time, the molecular mechanism of PLAU in CRC cells was investigated by downregulating the protein expression of Src combined with the results of the bioinformatics analysis. Western blotting revealed that the protein expressions of phosphorylated Src (p-Src) and phosphorylated ERK (p-ERK) in SW480 and SW620 cells increased significantly in the PLAU-mimic group compared with the PLAU-NC group, while the results were the opposite in the PLAU-inhibitor group. After being treated with saracatinib, we observed significantly decreased protein levels of p-ERK, matrix metallopeptidase 2 (MMP-2), MMP-3, MMP-9, Cyclin D1, and Cyclin A2 in the SW480 cells. Conclusions: In conclusion, PLAU affects the migration, invasion, and proliferation of CRC cells by activating the Src/ERK pathway.

Carbonyl cyanide 3-chlorophenylhydrazone induced the imbalance of mitochondrial homeostasis in the liver of Megalobrama amblycephala: A dynamic study.

Carbonylcyanide-3-chlorophenylhydrazone (CCCP) is a protonophore, which causes uncoupling of proton gradient in the inner mitochondrial membrane, thus inhibiting the rate of ATP synthesis. However, this information is manly derived from mammals, while its effects on the mitochondrial homeostasis of aquatic animals are largely unknown. In this study, the mitochondrial homeostasis of a carp fish Megalobrama amblycephala was investigated systematically in a time-course manner by using CCCP. Fish was injected intraperitoneally with CCCP (1.8 mg/kg per body weight) and DMSO (control), respectively. The results showed that CCCP treatment induced hepatic mitochondrial oxidative stress, as was evidenced by the significantly increased MDA and PC contents coupled with the decreased SOD and MnSOD activities. Meanwhile, mitochondrial fission was up-regulated remarkably characterized by the increased transcriptions of Drp-1, Fis-1 and Mff. However, the opposite was true for mitochondrial fusion, as was indicative of the decreased transcriptions of Mfn-1, Mfn-2 and Opa-1. This consequently triggered mitophagy, as was supported by the accumulated mitochondrial autophagosomes and the increased protein levels of PINK1, Parkin, LC3-II and P62 accompanied by the increased LC3-II/LC3-I ratio. Mitochondrial biogenesis and function both decreased significantly addressed by the decreased activities of CS, SDH and complex I, IV and V, as well as the protein levels of PGC-1ß coupled with the decreased transcriptions of TFAM, COX-1, COX-2 and ATP-6. Unlikely, DMSO treatment exerted little influence. Overall, CCCP treatment resulted in the imbalance of mitochondrial homeostasis in Megalobrama amblycephala by promoting mitochondrial oxidative stress, fission and mitophagy, but depressing mitochondrial fusion, biogenesis and function.

Effects of dietary icariin supplementation on the ovary development-related transcriptome of Chinese mitten crab (Eriocheir sinensis).

The Chinese mitten crab (Eriocheir sinensis) is an economically important aquaculture species in China, with distinct differences in ovarian maturation status between crabs fed with natural diets and artificial diets during the listing period, thus, leading to selling price differentiation. Our previous study showed that dietary supplementation with 100 mg/kg icariin can effectively promote ovarian development of E. sinensis. However, the internal molecular mechanism has not yet been elucidated because of a lack of comprehensive genome sequence information. We compared the ovary transcriptomes of E. sinensis fed with two diets containing 0 and 100 mg/kg ICA using the BGISEQ-500 platform. This yielded 12.54 Gb clean bases and 54,794 unigenes, 13,832 of which were found to be differentially expressed after icariin exposure. Twenty pathways closely related to gonadal development were selected through KEGG analysis. Seven differentially expressed genes relevant to vitellogenesis and oocyte maturation (serine/threonine-protein kinase mos-like, Eg2, cytoplasmic polyadenylation element-binding protein, cyclin B, vitellogenin 1, cathepsin D, and juvenile hormone esterase-like carboxylesterase 1) were validated by qRT-PCR, and four proteins (MEK1/2, ERK1/2, Cyclin B and Cdc2) associated with the progesterone mediated oocyte maturation pathway (i.e., MAPK/MPF pathway) were analyzed by western-blot. The results showed that icariin could promote the synthesis, processing and deposition of vitellogenin in oocytes, and that it also has the potential to promote oocyte maturation (resumption of Meiosis I) by altering the expression of the relevant genes and proteins.

Protective effects of dietary arginine against oxidative damage and hepatopancreas immune responses induced by T-2 toxin in Chinese mitten crab (Eriocheir sinensis).

T-2 toxin is a secondary metabolite produced by Fusarium spp. that is a major cereal and animal feed contaminant. T-2 toxin has numerous adverse effects on animals, including hepatotoxicity. Arginine (Arg) is closely associated with the regulation of immune responses and antioxidant activity in tissues. The objective of the present study was to evaluate the protective effects of dietary Arg against oxidative damage and immune responses of the hepatopancreas induced by T-2 toxin in Chinese mitten crab. According to the results, 3.17% Arg in the diet decreased alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activity in the haemolymph significantly, when compared with the levels of activity in the T-2 toxin group. Arg supplementation also increased superoxide dismutase and glutathione peroxidase activity, while decreasing malondialdehyde concentrations in the hepatopancreas, when compared with the levels in the T-2 toxin group. In addition, 3.17% Arg in the diet increased acid phosphatase and alkaline phosphatase activity in the hepatopancreas, as well as albumin concentrations in the haemolymph, when compared with the T-2 toxin group. Dietary Arg also regulated the expression of antioxidant enzyme-related genes (mitochondrial manganese superoxide dismutase, cytosolic manganese superoxide dismutase, and catalase) and immune related genes (prophenoloxidase, NF-κB-like transcription factor Relish, and lipopolysaccharide-induced TNF-α factor) to alleviate the damage associated with the T-2 toxin. Furthermore, Arg ameliorated damage to the hepatopancreas microstructure in the crabs. The results of the present study indicate that dietary Arg could enhance the antioxidant and immune capacity of Chinese mitten crab against oxidative damage and immune injury to the hepatopancreas induced by T-2 toxin.

Dietary reduced glutathione supplementation can improve growth, antioxidant capacity, and immunity on Chinese mitten crab, Eriocheir sinensis.

Eriocheir sinensis is an important aquaculture species in China, and its yield and quality are threatened by oxidative stress caused by deteriorating water conditions. Reduced glutathione (GSH) is an endogenous antioxidant, but whether dietary GSH can increase the resistance of E. sinensis to environmental stress remains unclear. Therefore, in this study, crabs were fed with dietary GSH (0, 300, 600, 900, and 1200 mg/kg diet weight) for up to 10 weeks to determine the effects of different dietary GSH concentrations on growth, antioxidant capacity, and immunity of E. sinensis. The results showed that the weight gain rate and survival rate increased significantly as dietary GSH levels increased from 0 to 900 mg/kg, but decreased at 1200 mg/kg. Compared with the control group, the diet supplemented with 900 mg/kg GSH not only increased the concentration of GSH in the haemolymph and hepatopancreas, but also enhanced the activity of glutathione peroxidase (GSH-Px) (p < 0.05). Diets supplemented with 600 or 900 mg/kg GSH significantly increased the enzymes activities of superoxide dismutase (SOD), lysozyme (LZM), alkaline phosphatase, and acid phosphatase, and significantly decreased the content of malondialdehyde. To understand the changes in the activity of these enzymes further, the expression of related genes was detected. Diets supplemented with 600 or 900 mg/kg GSH significantly upregulated the genes expressions of cytosolic manganese SOD, mitochondrial manganese SOD, copper, zinc-SOD, GSH-Px, LZM, and prophenoloxidase activating factor, and significantly down regulated the expression of Toll-like receptor 1, Toll-like receptor 2, Dorsal, and the myeloid differentiation factor 88. However, a diet supplemented with 1200 mg/kg GSH decreased those positive indicators. Overall, our results demonstrated that an appropriate diet supplemented with 600 or 900 mg/kg GSH enhances antioxidant capacity and immunity, which will enhance the general health of E. sinensis.