Dietary Eucommia ulmoides leaf extract improves laying performance by altering serum metabolic profiles and gut bacteria in aged laying hens.

The leaves of Eucommia ulmoides are rich in bioactive constituents that have potential gastrointestinal benefits for animals. In aged laying hens, intestinal health issues contribute to a significant decline in egg-laying capacity during intermediate and later stages. It remains unclear whether E. ulmoides leaf extract (ELE) can improve intestinal health and enhance egg production in elderly laying hens, and the underlying mechanisms are yet to be elucidated. Therefore, we conducted a study with 480 laying hens (65 weeks old) randomly allocated into four groups: a control group fed with the basal diet, and three treatment groups supplemented with 500, 1,000, and 2,000 mg/kg of ELE, respectively. The primary active constituents of ELE include flavonoids, polysaccharides, terpenoids, and phenolic acids. Dietary supplementation with ELE at 1,000 mg/kg (ELE1000) significantly improved laying performance and egg quality compared to the other groups. ELE1000 stimulated the maturation of intestinal epithelial cells, increased villus height, and reduced crypt depth. It also influenced the levels of proteins associated with tight junctions (claudin-1 and claudin-2) and intestinal inflammatory factors (IL-6, IL-1ß, and IL-2) in different intestinal sections. Integrative analysis of serum metabolomics and gut microbiota revealed that ELE1000 improved nutrient metabolism by modulating amino acid and ubiquinone biosynthesis and influenced the abundance of intestinal microbiota by enriching pivotal genera such as Bacteroides and Rikenellaceae_RC9_gut_group. We identified 15 metabolites significantly correlated with both gut microbiota and laying performance, e.g., DL-methionine sulfoxide, THJ2201 N-valerate metabolite, tetracarbonic acid, etc. In conclusion, ELE1000 improved laying performance in elderly laying hens by affecting intestinal morphology, barrier function, microbiota, and serum metabolite profiles. These findings suggest that ELE can be a beneficial feed additive for extending the peak producing period in aged laying hens.

In vitro antibacterial effects of Broussonetia papyrifera leaf extract and its anti-colitis in DSS-treated mice.

Recently, the hybrid Broussonetia papyrifera (BP) has been extensively cultivated and predominantly utilized in ruminants because of its high protein and bioactive compound content. In the present study, the effects of an ethanolic extract of BP leaves (BPE, 200 mg/kg) on mitigating 2% dextran sodium sulfate (DSS)-induced intestinal inflammation in mice were evaluated. BPE is rich in flavonoids, polyphenols, and polysaccharides, and displays potent antioxidant and antibacterial activities against pathogenic strains such as Clostridium perfringens, Salmonella Typhimurium, and Salmonella enterica subsp. enterica in vitro. In a mouse study, oral administration of DSS resulted in weight loss, incidence of diarrhea, enlargement of the liver and spleen, impaired colonic morphology, downregulation of both gene and protein expression related to intestinal antioxidant (Nrf2) and barrier function (ZO-1), decreased diversity of colonic microbiota, and 218 differentially altered colonic metabolites; however, co-treatment with BPE did not restore these modified aspects except for the liver index and colonic bacterial diversity. The singular treatment with BPE did not manifest evident side effects in normal mice but induced a mild occurrence of diarrhea and a notable alteration in the colonic metabolite profile. Moreover, a single BPE administration augmented the abundance of the commensal beneficial bacteria Faecalibaculum and Akkermansia genera. Overall, the extract of BP leaves did not demonstrate the anticipated effectiveness in alleviating DSS-induced intestinal inflammation.

Impact of fermented Broussonetia papyrifera on laying performance, egg quality, lipid metabolism, and follicular development of laying hens.

Hybrid Broussonetia papyrifera (BP) has been widely planted and commonly used as ruminant forage source after fermentation in China. Very less information is available to know the impact of fermented BP on laying hens, thus, we have investigated effects of dietary supplementation of Lactobacillus plantarum-fermented B. papyrifera (LfBP) on laying performance, egg quality, serum biochemical parameters, lipid metabolism, and follicular development of laying hens. A total of 288 HY-Line Brown hens (age, 23 wk) were randomly assigned into 3 treatment groups: control group (Con, a basal diet), LfBP1 and LfBP5 group (a basal diet supplemented with 1% or 5% LfBP). Each group has 8 replicates of twelve birds each. The results demonstrated that dietary supplementation of LfBP increased average daily feed intake (linear, P < 0.05), feed conversion ratio (linear, P < 0.05), and average egg weight (linear, P < 0.05) during the entire experimental period. In addition, dietary inclusion of LfBP enhanced the egg yolk color (linear, P < 0.01) but decreased the eggshell weight (quadratic, P < 0.05) and eggshell thickness (linear, P < 0.01). In serum, the LfBP supplementation linearly decreased the content of total triglyceride (linear, P < 0.01) but increased the content of high density lipoprotein-cholesterol (linear, P < 0.05). The gene expression related to hepatic lipid metabolism including acetyl-CoA carboxylase, fatty acid synthase, and peroxisome proliferator-activated receptor (PPARα) was down-regulated whereas liver X receptor was up-regulated in LfBP1 group. Moreover, LfBP1 supplementation remarkably reduced the F1 follicle number and ovarian gene expression of reproductive hormone receptors including estrogen receptor, follicle stimulating hormone receptor, luteinizing hormone receptor, progesterone receptor, prolactin receptor, and B cell lymphoma-2. In conclusion, dietary inclusion of LfBP could improve feed intake, egg yolk color, and lipid metabolism, but may cause a decline in eggshell quality with higher inclusion level, herein, 1% is suggested.

L-Arginine/nitric oxide regulates skeletal muscle development via muscle fibre-specific nitric oxide/mTOR pathway in chickens.

L-Arginine (L-Arg), the precursor of nitric oxide (NO), plays an important role in muscle function. Fast-twitch glycolytic fibres are more susceptible to age-related atrophy than slow-twitch oxidative fibres. The effect of L-Arg/NO on protein metabolism of fast- and slow-twitch muscle fibres was evaluated in chickens. In Exp. 1, 48 chicks at 1 day old were divided into 4 groups of 12 birds and subjected to 4 treatments: basal diet without supplementation or supplemented with 1% L-Arg, and water supplemented with or without L-nitro-arginine methyl ester (L-NAME, 18.5 mM). In Exp. 2, 48 chicks were divided into 4 groups of 12 birds fed with the basal diet and subjected to the following treatments: tap water (control), tap water supplemented with L-NAME (18.5 mM), or molsidomine (MS, 0.1 mM), or 18.5 mM L-NAME + 0.1 mM MS (NAMS). The regulatory effect of L-Arg/NO was further investigated in vitro with myoblasts obtained from chicken embryo pectoralis major (PM) and biceps femoris (BF). In vivo, dietary L-Arg supplementation increased breast (+14.94%, P < 0.05) and thigh muscle mass (+23.40%, P < 0.05); whereas, MS treatment had no detectable influence. However, L-NAME treatment blocked the beneficial influence of L-Arg on muscle development. L-Arg decreased (P < 0.05) protein synthesis rate, phosphorylated mTOR and ribosomal protein S6 kinase beta-1 (p70S6K) levels in breast muscle, which was recovered by L-NAME treatment. In vitro, L-Arg or sodium nitroprusside (SNP) reduced protein synthesis rate, suppressed phosphorylated mTOR/p70S6K and decreased atrogin-1 and muscle RING finger 1 (MuRF1) in myoblasts from PM muscle (P < 0.05). L-NAME abolished the inhibitory effect of L-Arg on protein synthesis and the mTOR/p70S6K pathway. However, myoblasts from BF muscle showed the weak influence. Moreover, blocking the mTOR/p70S6K pathway with rapamycin suppressed protein synthesis of the 2 types of myoblasts; whereas, the protein expression of atrogin-1 and MuRF1 levels were restricted only in myoblasts from PM muscle. In conclusion, L-Arg/NO/mTOR/p70S6K pathway enhances protein accumulation and muscle development in fast-twitch glycolytic muscle in chickens. L-Arg/NO regulates protein turnover in a muscle fibre specific way, which highlights the potential clinical application in fast-twitch glycolytic muscle fibres.

Nicotinamide mononucleotide supplementation protects the intestinal function in aging mice and D-galactose induced senescent cells.

The nicotinamide adenine dinucleotide (NAD+) level shows a temporal decrease during the aging process, which has been deemed as an aging hallmark. Nicotinamide mononucleotide (NMN), a key NAD+ precursor, shows the potential to retard the age-associated functional decline in organs. In the current study, to explore whether NMN has an impact on the intestine during the aging process, the effects of NMN supplementation on the intestinal morphology, microbiota, and NAD+ content, as well as its anti-inflammatory, anti-oxidative and barrier functions were investigated in aging mice and D-galactose (D-gal) induced senescent IPEC-J2 cells. The results showed that 4 months of NMN administration had little impact on the colonic microbiota and NAD+ content in aging mice, while it significantly increased the jejunal NAD+ content and improved the jejunal structure including increasing the villus length and shortening the crypt. Moreover, NMN supplementation significantly up-regulated the mRNA expression of SIRT3, SIRT6, nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), the catalytic subunit of glutamate-cysteine ligase (GCLC), superoxide dismutase 2 (SOD2), occludin, and claudin-1, but down-regulated the mRNA expression of tumor necrosis factor alpha (TNF-α). Specifically, in the D-gal induced senescent IPEC-J2 cells, 500 µM NMN restored the increased mRNA expression of interleukin 6 (IL6ST), IL-1A, nuclear factor (NF-κB1), and claudin-1 to normal levels to some extent. Furthermore, NMN treatment significantly affected the mRNA expression of antioxidant enzymes including NQO1, GCLC, SOD 2 and 3, and GSH-PX1, 3 and 4. In addition, 200 µM NMN enhanced the cell viability and total antioxidant capacity and lowered the reactive oxygen species level of senescent IPEC-J2 cells. Notably, NMN restored the down-regulated protein expression of occludin and claudin-1 induced by D-gal. The above data demonstrated the potential of NMN in ameliorating the structural and functional decline in the intestine during aging.

Dietary Enteromorpha polysaccharide-Zn supplementation regulates amino acid and fatty acid metabolism by improving the antioxidant activity in chicken.

BACKGROUND: Enteromorpha prolifera (E. prolifera) polysaccharide has become a promising feed additive with a variety of physiological activities, such as anti-oxidant, anti-cancer, anti-diabetic, immunomodulatory, hypolipidemic, and cation chelating ability. However, whether Enteromorpha polysaccharide-trace element complex supplementation regulates amino acid and fatty acid metabolism in chicken is largely unknown. This study was conducted to investigate the effects of E. prolifera polysaccharide (EP)-Zn supplementation on growth performance, amino acid, and fatty acid metabolism in chicken. METHODS: A total of 184 one-day-old Ross-308 broiler chickens were randomly divided into two treatment groups with 8 replicates, 12 chickens per replicate, and fed either the basal diet (control group) or basal diet plus E. prolifera polysaccharide-Zinc (400 mg EP-Zn/kg diet). RESULTS: Dietary EP-Zn supplementation significantly increased (P < 0.05) the body weight, average daily gain, muscle antioxidant activity, serum HDL level, and reduced serum TG and LDL concentration. In addition, dietary EP-Zn supplementation could modulate ileal amino acid digestibility and upregulate the mRNA expression of amino acid transporter genes in the jejunum, ileum, breast muscle, and liver tissues (P < 0.05). Compared with the control group, breast meat from chickens fed EP-Zn had higher (P < 0.05) Pro and Asp content, and lower (P < 0.05) Val, Phe, Gly, and Cys free amino acid content. Furthermore, EP-Zn supplementation upregulated (P < 0.05) the mRNA expressions of mTOR and anti-oxidant related genes, while down-regulated protein degradation related genes in the breast muscle. Breast meat from EP-Zn supplemented group had significantly lower (P < 0.05) proportions of Σn-3 PUFA, and a higher percentage of Σn-6 PUFA and the ratio of n-6/n-3 PUFA. Besides, EP-Zn supplementation regulated lipid metabolism by inhibiting the gene expression of key enzymes involved in the fatty acid synthesis and activating genes that participated in fatty acid oxidation in the liver tissue. CONCLUSIONS: It is concluded that EP-Zn complex supplementation regulates apparent ileal amino acid digestibility, enhances amino acid metabolism, and decreases oxidative stress-associated protein breakdown, thereby improving the growth performance. Furthermore, it promotes fatty acid oxidation and restrains fat synthesis through modulating lipid metabolism-related gene expression.

Optimization of ultrasound extraction of Tribulus terrestris L. leaves saponins and their HPLC-DAD-ESI-MSn profiling, anti-inflammatory activity and mechanism in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Tribulus terrestris L., as an annual herb plant from Zygophyllaceae, exhibits many biological activities, and its main chemical constituents are saponins. However, the extraction process, chemical compositions, anti-inflammatory effect and mechanism of total saponins from Tribulus terrestris L. leaves are still unclear. AIM OF THE STUDY: The present study extensively evaluated the extraction process, major components, anti-inflammatory action and mechanism of Tribulus terrestris L. leaves saponins. MATERIALS AND METHODS: The ultrasonic extraction and response surface methods were adopted for optimization of extraction technology of total saponins from Tribulus terrestris L. leaves, and its compositions were detected with LC-MSn method. The anti-inflammatory activity of total saponins was studied by lipopolysaccharide induced RAW 264.7 cells and acute lung injury mice models. RESULTS: The ultrasonic extraction parameters of saponins fraction, including ethanol concentration 30%, extraction time 55 min, ratio of solvent to material 35:1 ml/g and extraction temperature 46 °C, were screened by response surface method with the extracting rate 5.49%, and thirty compositions were detected with LC-MSn method. Moreover, saponins fraction can play a stronger anti-inflammatory effect by reducing the phagocytic activity and pulmonary edema, and protection of morphology of RAW 264.7 cells and lung tissues, and decreasing the content of NO and TNF-α. Moreover, it was revealed that total saponins extract can exert the anti-inflammatory action by the inhibition of the activation of the TLR4-TRAF6-NF-κB signalling pathway. CONCLUSION: These studies imply that Tribulus terrestris L. leaves saponins may be an important anti-inflammatory drug in clinic.

Rutin prevents inflammation induced by lipopolysaccharide in RAW 264.7 cells via conquering the TLR4-MyD88-TRAF6-NF-κB signalling pathway.

OBJECTIVES: Inflammation widely exists in many diseases and poses a great threat to human and animal health. Rutin, quercetin-3-rhamnosyl glucoside, has a variety of pharmacological effects, including anti-oxidant, anti-inflammatory, antibacterial, anticancer and radioresistance effects. The current study focused on evaluation of its anti-inflammatory activity and described the mechanism of rutin in lipopolysaccharide-induced RAW 264.7 cells. METHODS: The related gene and protein expression levels were investigated by quantification real-time PCR and western blotting, respectively. KEY FINDINGS: This study revealed that rutin can decrease inducible nitric oxide synthase (iNOS) gene and protein expression levels, effectively increase IκB gene expression, reduce toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), tumour necrosis factor receptor-associated factor 6 (TRAF6) and p65 gene expression and inhibit the phosphorylation of IκB and p65 and the proteins expression of TLR4, MyD88 and TRAF6. CONCLUSIONS: These results suggest that rutin might exert anti-inflammatory effect on LPS-stimulated RAW 264.7 cells and will be potentially useful as an adjuvant treatment for inflammatory diseases.

The regulatory effect of flavonoids extracted from Abutilon theophrasti leaves on gene expression in LPS-induced ALI mice via the NF-κB and MAPK signaling pathways.

Context: ALI is a common disease characterized by acute pulmonary inflammatory disorder. Abutilon theophrasti Medik. (Malvaceae), as a Chinese traditional medicine, is used for the treatment of inflammation. Its main constituents are flavonoid compounds. Objective: This study investigates the regulatory effect of a TFE from Abutilon theophrasti leaves on gene expression in LPS-induced ALI mice via the NF-κB and MAPK signaling pathways. Materials and methods: Kunming mice were intragastrically administered TFE (0.25, 0.5, 1.0 g/kg) for 5 days, and then ALI was induced via intranasal administration 40 µg of LPS in 10 µL PBS after intragastric administration on the 5th day, and PBS and DEX (2 mg/kg) were negative and positive control groups, respectively. Results: The relative expression of iNOS gene was 0.707, 0.507 and 0.483 for 0.25, 0.5 and 1.0 g/kg TFE, and COX-2 gene expression was also reduced after treatment by three concentrations of TFE with 0.768, 0.545, and 0.478. The mRNA expression levels of p65 were 0.61, 0.43 and 0.27 for 0.25, 0.5 and 1.0 g/kg TFE and IκB levels were increased in a dose-dependent manner with 3.99, 13.69 and 34.36. 0.5 and 1.0 g/kg TFE inhibited the expression of ERK1/2 with 0.59 and 0.38, p38MAPK with 0.62 and 0.54, and JNK with 0.37 and 0.29, and JNK mRNA expression was 0.60 for 0.25 g/kg TFE. Discussion and conclusion: These results indicate that the regulatory mechanisms of TFE on gene expression in LPS-induced ALI mice include inhibition of the NF-κB and MAPK signaling pathways.

L-Arginine Enhances Protein Synthesis by Phosphorylating mTOR (Thr 2446) in a Nitric Oxide-Dependent Manner in C2C12 Cells.

Muscle atrophy may arise from many factors such as inactivity, malnutrition, and inflammation. In the present study, we investigated the stimulatory effect of nitric oxide (NO) on muscle protein synthesis. Primarily, C2C12 cells were supplied with extra L-arginine (L-Arg) in the culture media. L-Arg supplementation increased the activity of inducible nitric oxide synthase (iNOS), the rate of protein synthesis, and the phosphorylation of mTOR (Thr 2446) and p70S6K (Thr 389). L-NAME, an NOS inhibitor, decreased NO concentrations within cells and abolished the stimulatory effect of L-Arg on protein synthesis and the phosphorylation of mTOR and p70S6K. In contrast, SNP (sodium nitroprusside), an NO donor, increased NO concentrations, enhanced protein synthesis, and upregulated mTOR and p70S6K phosphorylation, regardless of L-NAME treatment. Blocking mTOR with rapamycin abolished the stimulatory effect of both L-Arg and SNP on protein synthesis and p70S6K phosphorylation. These results indicate that L-Arg stimulates protein synthesis via the activation of the mTOR (Thr 2446)/p70S6K signaling pathway in an NO-dependent manner.