Mulberry Leaf Polysaccharides Attenuate Oxidative Stress Injury in Peripheral Blood Leukocytes by Regulating Endoplasmic Reticulum Stress.

The present study assessed the protective effects and underlying mechanisms of mulberry leaf polysaccharides (MLPs) against hydrogen peroxide (H2O2)-induced oxidative stress injury in the peripheral blood leukocytes (PBLs) of Megalobrama amblycephala. Five treatment groups were established in vitro: the NC group (PBLs incubated in an RPMI-1640 complete medium for 4 h), the HP group (PBLs incubated in an RPMI-1640 complete medium for 3 h, and then stimulated with 100 µM of H2O2 for 1 h), and the 50/100/200-MLP pre-treatment groups (PBLs were pre-treated with MLPs (50, 100, and 200 µg/mL) for 3 h, and then stimulated with 100 µM of H2O2 for 1 h). The results showed that MLP pre-treatment dose-dependently enhanced PBLs' antioxidant capacities. The 200 µg/mL MLP pre-treatment effectively protected the antioxidant system of PBLs from H2O2-induced oxidative damage by reducing the malondialdehyde content and lactic dehydrogenase cytotoxicity, and increasing catalase and superoxide dismutase activities (p < 0.05). The over-production of reactive oxygen species, depletion of nicotinamide adenine dinucleotide phosphate, and collapse of the mitochondrial membrane potential were significantly inhibited in the 200-MLP pre-treatment group (p < 0.05). The expressions of endoplasmic reticulum stress-related genes (forkhead box O1α (foxO1α), binding immunoglobulin protein (bip), activating transcription factor 6 (atf6), and C/EBP-homologous protein (chop)), Ca2+ transport-related genes (voltage-dependent anion-selective channel 1 (vdac1), mitofusin 2 (mfn2), and mitochondrial Ca2+ uniporter (mcu)), and interleukin 6 (il-6) and bcl2-associated x (bax) were significantly lower in the 200-MLP pre-treatment group than in the HP group (p < 0.05), which rebounded to normal levels in the NC group (p > 0.05). These results indicated that MLP pre-treatment attenuated H2O2-induced PBL oxidative damage in the M. amblycephala by inhibiting endoplasmic reticulum stress and maintaining mitochondrial function. These findings also support the possibility that MLPs can be exploited as a natural dietary supplement for M. amblycephala, as they protect against oxidative damage.

Regulation of sirt1 and foxO1 in glucose metabolism of Megalobrama amblycephala.

Both silent information regulator 2 homolog 1 (sirt1) and forkhead box transcription factor 1 (foxO1) are crucial transcription factors involved in glucolipid metabolism and energy regulation. The presnt study aimed to understand their regulatory roles in glucose metabolism. Molecular cloning and sequencing of sirt1 gene of Megalobrama amblycephala (masirt1) was conducted and cellular localization of both the factors were analysed. Their effects and action patterns in the glucose metabolism of Megalobrama amblycephala (M. amblycephala) were investigated through acute and long-term glucose tolerance assays. The results revealed that the full-length masirt1 cDNA sequence was 2350 bp and closely related to Sinocyclocheilus rhinocerous. Sirt1 and foxO1 were found to be mutually dependent and localized in the nucleus. Acute glucose tolerance tests revealed that the expression levels of both factors in the liver of M. amblycephala showed an initial increase followed by a decrease. Plasma glucose levels in M. amblycephala significantly increased at 2 and 12 h (P < 0.05). In a long-term breeding experiment with high-sugar feeding, the expressions of the sirt1 and foxO1 genes in the kidney and intestine of M. amblycephala exhibited synergistic changes. The 51WS groups had significantly higher levels of sirt1 and foxO1 gene expression in the kidney and intestine compared to the 0WS and 17WS groups (P < 0.05). Overall, masirt1 is evolutionarily highly conserved, and the interaction site of sirt1 and foxO1 is located in the nucleus. In long-term hyperglycemic regulation, sirt1 and foxO1 exhibit synergistic regulatory effects in the kidney and intestine of M. amblycephala. This study provides insights into how sirt1 and foxO1 regulate glucose metabolism in M. amblycephala.

Mulberry leaf meal: A potential feed supplement for juvenile Megalobrama amblycephala "Huahai No. 1".

This study was performed to evaluate the potential application of mulberry leaf meal (ML) and fermented mulberry leaf meal (FML) as feed supplements in aquatic animals for developing varieties of practical and economical feed ingredients. Juveniles Megalobrama amblycephala were fed a basal diet (35.7% crude protein, 10.4% crude lipid; control group) supplemented with 2.22% and 4.44% mulberry leaf meals (ML2, ML4) and fermented mulberry leaf meals (FML2, FML4) for 8 weeks. Generally, the two-way ANOVA showed the supplementation level exhibited a prominent effect on the growth performance and physiological status of fish. Furthermore, the two-way ANOVA showed the supplementary fermented mulberry leaf meal increased plasma complement 4 (C4) content (P < 0.05). The weight gain rate (WGR, 145.87%) and the specific growth rate (SGR, 1.63%) were significantly increased in FML2 group compared with the control group (P < 0.05). The muscle crude lipid content and hepatosomatic index (HSI) were higher in FML2 group than that in ML2 group (P < 0.05). The hepatic GSH content in ML4 group and CAT, T-SOD activities in FML4 group were significantly increased compared with the control group (P < 0.05). The hepatic MDA content in FML4 group was significantly decreased compared with the FML2 group (P < 0.05). Total cholesterol (TC) contents showed a significant decrease in ML4 and FML4 groups compared with the control group (P < 0.05). Regarding the gene expression, sirtiun 1 (Sirt1) gene expression was elevated in FML2 group compared with the ML2 group (P < 0.05). Compare to the control group, FML2 diet significantly increased the expression of i-kappa-B alpha (IKBα) gene in liver, and decreased the expression of forkhead box O1 α (FoxO1α), toll-like receptors 4 (TLR4) and nuclear factor-kappa B (NF-κB) genes (P < 0.05). In conclusion, 2.22% FML promoted the growth performance of M. amblycephala and enhanced the anti-inflammatory responses by inhibiting TLR4/NF-κB signaling pathway. On the other hand, 4.44% FML reduced plasma lipid content (hypolipedemic effect) and improved the hepatic antioxidant capacity of M. amblycephala.

Molecular characterization and expression of sirtuin 2, sirtuin 3, and sirtuin 5 in the Wuchang bream (Megalobrama amblycephala) in response to acute temperature and ammonia nitrogen stress.

This study sought to characterize sirtuin 2 (sirt2), sirtuin 3 (sirt3), and sirtuin 5 (sirt5) in Megalobrama amblycephala (M. amblycephala) by cloning the open reading frame (ORF) of sirt2, sirt3, and sirt5. The full-lengths of the resulting M. amblycephala sirt2, sirt3, and sirt5 cDNA sequences were 1845, 1534, and 1920 bp, respectively, with 92%, 98%, and 91% similarities to Danio rerio sequences. Based on our bioinformatic analyses and predictions, the sirt2 and sirt3 genes of M. amblycephala were classified within the Sir2 I family, whereas sirt5 belonged to the Sir2 III family. Furthermore, sirt2, sirt3, and sirt5 were widely distributed in different M. amblycephala tissues. Particularly, sirt2 and sirt5 were highly expressed in gills, intestines, and liver (P < 0.05), whereas sirt3 was highly expressed in gills, kidney, liver, and spleen (P < 0.05). A 2 × 2 factorial experiment was also conducted to analyze sirt2, sirt3, and sirt5 expression patterns in response to acute temperature (25 and 32 °C) and ammonia nitrogen (0 and 20 mg/L) stress. Notably, these two stressors were found to interactively affect sirt2, sirt3, and sirt5 expression patterns in M. amblycephala liver. At the higher water temperature (32 °C) and ammonia nitrogen concentration (20 mg/L) tested herein, sirt2, sirt3, and sirt5 had similar expression levels and exhibited a down-regulation trend at 6 and 48 h post-stress but became up-regulated thereafter to counteract the stressors at 96 h post-stress.