Transcriptomic analysis of mechanism underlying the effect of induced molting on semen quality and reproductive performance in aged Houdan roosters.

The reproductive performance of breeder roosters has significant economic importance in the poultry industry. Breeder roosters have severely reduced semen quality with age and will be at risk of culling in the following years. In order to extend the use of breeder roosters, we drew on the induced molting model of hens and selected 35 Houdan roosters aged 50 wk for induced molting. By comparing the body weight, testicular weight, semen quality, and reproductive performance before and after induced molting, we found that induced molting could restore the body weight and testicular weight to the levels before molting (P > 0.05). At the same time, it significantly improved sperm motility (P < 0.05) and also improved reproductive performance such as fertilization rate and hatching rate. To further reveal the mechanism underlying the effects of induced molting on semen quality and reproductive performance in aged Houdan roosters, we collected testes from 3 periods: 1 d before fasting (F0), 15 d after fasting (F15), and 32 d after recovery feeding (R32) for transcriptome sequencing analysis. A total of 5,671 genes were detected in F0, F15, and R32, and trend analysis of the 5,671 differential genes showed 2 significant trends (profile 5 and profile 2). KEGG enrichment analysis of the genes in the 2 profiles, revealed significantly enriched pathway regulation of actin cytoskeleton. In the regulation of actin cytoskeleton pathway, we found a protein kinase gene (SRC) and a senescence gene (ROCK2). SRC was highly expressed at F15, leading to the phosphorylation of key substrates, which in turn disrupted the Sertoli cell spermatid connection and the spermiogenesis process, resulting in no mature spermatozoa produced from F15, SRC expression was inhibited at R32, the expression level was reduced, and mature spermatozoa reappeared. The senescence gene ROCK2 was highly expressed at F15 compared to F0 and R32, which may have been responsible for inducing senescence atrophy in the testes.

Hippocampal proteins discovery of Panax quinquefolius and Acorus gramineus ameliorating cognitive impairment in diabetic rats.

RATIONALE: Early diagnosis of diabetic cognitive impairment (DCI) and investigation of effective medicines are significant to prevent or delay the occurrence of irreversible dementia. OBJECTIVES: In this study, proteomics was applied to investigate the changes of hippocampal proteins after administration of Panax quinquefolius-Acorus gramineus (PQ-AG) to DCI rats, with a view to discover the differentially expressed proteins of PQ-AG action and elucidated the potential biological relationships. METHODS: The model and PQ-AG group rats were injected intraperitoneally with streptozotocin, and the PQ-AG group rats were continuously administered with PQ-AG. Social interaction and Morris water maze were performed to evaluate the behavior of rats on the 17th week after the model was established, and DCI rats were screened out from the model group by a screening approach. The hippocampal protein differences were investigated with proteomics in DCI and PQ-AG-treated rats. RESULTS: The learning and memory abilities and contact duration of DCI rats were improved after 16 weeks of PQ-AG administration. Altogether, 9 and 17 differentially expressed proteins were observed in control versus DCI rats and in DCI versus PQ-AG-treated rats, respectively. Three proteins were confirmed with western blotting analyses. These proteins were mainly involved in the pathways of JAK-STAT, apoptosis, PI3K/AKT, fork-head box protein O3, fructose, and mannose metabolism. CONCLUSIONS: This suggested that PQ-AG ameliorated cognitive impairment of diabetic rats by influencing the above pathways and providing an experimental basis for the mechanism of DCI and PQ-AG.

Rhamnogalacturonan-I enriched pectin from steamed ginseng ameliorates lipid metabolism in type 2 diabetic rats via gut microbiota and AMPK pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng C. A. Meyer (Ginseng) has traditionally been used to treat diabetes. Polysaccharide is the main active component of ginseng, and has been proved to have hypoglycaemic and hypolipidaemic effects, but its mechanism remains unclear. AIM OF THE STUDY: This study aimed to evaluate the effect and the potential mechanism of rhamnogalacturonan-I enriched pectin (GPS-1) from steamed ginseng on lipid metabolism in type 2 diabetes mellitus (T2DM) rats. MATERIALS AND METHODS: GPS-1 was prepared by water extraction, ion-exchange and gel chromatography. High-glucose/high-fat diet combined with streptozotocin was used to establish T2DM rat models, and lipid levels in serum and liver were tested. 16S rRNA sequencing and gas chromatography-mass spectrometry were used to detect the changes of gut microbiota and metabolites. The protein and mRNA levels of lipid synthesis-related genes were detected by Western blot and quantitative real-time polymerase chain reaction. RESULTS: The polyphagia, polydipsia, weight loss, hyperglycaemia, hyperlipidaemia and hepatic lipid accumulation in T2DM rats were alleviated after GPS-1 intervention. GPS-1 modulated the gut microbiota composition of T2DM rats, increased the levels of short-chain fatty acids, and promoted the secretion of glucagon-like peptide-1 and peptide tyrosine tyrosine. Further, GPS-1 activated AMP-activated protein kinases, phosphorylated acetyl-CoA carboxylase, reduced the expression of sterol regulatory element-binding protein-1c and fatty acid synthases in T2DM rats. CONCLUSIONS: The regulation effects of GPS-1 on lipid metabolism in T2DM rats are related to the regulation of gut microbiota and activation of AMP-activated protein kinase pathway.

Anti-Aging Effect of Momordica charantia L. on d-Galactose-Induced Subacute Aging in Mice by Activating PI3K/AKT Signaling Pathway.

Anti-aging is a challenging and necessary research topic. Momordica charantia L. is a common edible medicinal plant that has various pharmacological activities and is often employed in daily health care. However, its anti-aging effect on mice and the underlying mechanism thereof remain unclear. Our current study mainly focused on the effect of Momordica charantia L. on d-galactose-induced subacute aging in mice and explored the underlying mechanism. UHPLC-Q-Exactive Orbitrap MS was applied to qualitatively analyze the chemical components of Momordica charantia L. ethanol extract (MCE). A subacute aging mice model induced by d-galactose (d-gal) was established to investigate the anti-aging effect and potential mechanism of MCE. The learning and memory ability of aging mice was evaluated using behavioral tests. The biochemical parameters, including antioxidant enzyme activity and the accumulation of lipid peroxides in serum, were measured to explore the effect of MCE on the redox imbalance caused by aging. Pathological changes in the hippocampus were observed using hematoxylin and eosin (H&E) staining, and the levels of aging-related proteins in the PI3K/AKT signaling pathway were assessed using Western blotting. The experimental results demonstrated that a total of 14 triterpenoids were simultaneously identified in MCE. The behavioral assessments results showed that MCE can improve the learning and memory ability of subacute mice. The biochemical parameters determination results showed that MCE can improve the activity of antioxidant enzymes and decrease the accumulation of lipid peroxides in aging mice significantly. Furthermore, aging and injury in the hippocampus were ameliorated. Mechanistically, the results showed a significant upregulation in the protein expression of P-PI3K/PI3K and P-AKT/AKT (p < 0.01), as well as a significant reduction in cleaved caspase-3/caspase-3, Bax and P-mTOR/mTOR (p < 0.01). Our results confirm that MCE could restore the antioxidant status and improve cognitive impairment in aging mice, inhibit d-gal-induced apoptosis by regulating the PI3K/AKT signaling pathway, and rescue the impaired autophagy caused by mTOR overexpression, thereby exerting an anti-aging effect.

Metabolomics study of cerebrospinal fluid from diabetic rats with cognitive impairment simultaneously treated with Panax quinquefolius and Acorus gramineus.

A metabolomics approach was used to explore the effects of Panax quinquefolius (PQ) and Acorus gramineus (AG) on learning and memory in rats with diabetic-induced cognitive impairment. Thirty Wistar rats were divided into three groups, namely, the normal group, model group, and PQ-AG group (PQ-AG group, 1.80 g/kg/d). Diabetes was induced by intraperitoneal injection of streptozotocin (65 mg/kg). Cerebrospinal fluid (CSF) was collected via cisterna magna puncture, and the Morris water maze method was used to evaluate learning and memory in rats after 11 weeks of PQ-AG treatment. Metabolic profiling of CSF samples was performed by using UPLC-Q-TOF-MS. Compared with the normal group, the escape latency of the Morris water maze was significantly prolonged in model group rats after 12 weeks (p < 0.01). Compared with the model group, however, the escape latency was significantly shortened in PQ-AG group rats (p < 0.05). In multivariate statistical analysis, we identified 33 potential biomarkers, and six biomarkers were altered by PQ-AG. These biomarkers were involved in the metabolism of pyrimidine; nicotinate, and nicotinamide; glycine, serine, and threonine; and ascorbate and aldarate. Taken collectively, our results indicate that PQ-AG can attenuate diabetic-induced cognitive impairment by affecting a variety of metabolic pathways. Our results provide an experimental basis for studying the mechanism of action of PQ-AG.

The botanical origin and antioxidant, anti-BACE1 and antiproliferative properties of bee pollen from different regions of South Korea.

BACKGROUND: Bee pollen (BP) has been used as a traditional medicine and food diet additive due to its nutritional and biological properties. The potential biological properties of bee pollen vary greatly with the botanical and geographical origin of the pollen grains. This study was conducted to characterize the botanical origin and assess the antioxidant effects of ethanol extracts of 18 different bee pollen (EBP) samples from 16 locations in South Korea and their inhibitory activities on human ß-amyloid precursor cleavage enzyme (BACE1), acetylcholinesterase (AChE), human intestinal bacteria, and 5 cancer cell lines. METHODS: The botanical origin and classification of each BP sample was evaluated using palynological analysis by observing microscope slides. We measured the biological properties, including antioxidant capacity, inhibitory activities against human BACE1, and AChE, and antiproliferative activities toward five cancer cell lines, of the 18 EBPs. In addition, the growth inhibitory activities on four harmful intestinal bacteria, six lactic acid-producing bacteria, two nonpathogenic bacteria, and an acidulating bacterium were also assessed. RESULTS: Four samples (BP3, BP4, BP13 and BP15) were found to be monofloral and presented four dominant pollen types: Quercus palustris, Actinidia arguta, Robinia pseudoacacia, and Amygdalus persica. One sample (BP12) was found to be bifloral, and the remaining samples were considered to be heterofloral. Sixteen samples showed potent antioxidant activities with EC50 from 292.0 to 673.9 µg mL- 1. Fourteen samples presented potent inhibitory activity against human BACE1 with EC50 from 236.0 to 881.1 µg mL- 1. All samples showed antiproliferative activity toward the cancer cell lines PC-3, MCF-7, A549, NCI-H727 and AGS with IC50 from 2.7 to 14.4 mg mL- 1, 0.9 to 12.7 mg mL- 1, 5.0 to > 25 mg mL- 1, 2.7 to 17.7 mg mL- 1, and 2.4 to 8.7 mg mL- 1, respectively. In addition, total phenol and flavonoid contents had no direct correlation with antioxidant, anti-human BACE1, or antiproliferative activities. CONCLUSION: Fundamentally, Korean bee pollen-derived preparations could be considered a nutritional addition to food to prevent various diseases related to free radicals, neurodegenerative problems, and cancers. The botanical and geographical origins of pollen grains could help to establish quality control standards for bee pollen consumption and industrial production.

Comparative Pharmacokinetic Studies of Four Ginsenosides in Rat Plasma by UPLC-MS/MS after Oral Administration of Panax quinquefolius-Acorus gramineus and Panax quinquefolius Extracts.

Panax quinquefolius (PQ) and Acorus gramineus (AG) are drug target pairs in traditional Chinese medicine (TCM), which are used to treat age-related diseases. In the present study, we simultaneously determined the contents of four main bioactive ginsenosides (Rb1, Rb2, Rd, and Re) in rat plasma using an ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. Plasma specimens were purified by using the solid-phase extraction procedure, and separation was performed on Waters ACQUITY UPLC BEH C18 (100 mm × 2.1 mm, 1.7 µm) in multiple reaction monitoring (MRM) mode and negative electrospray ionization (ESI) mode. The established UPLC-MS/MS method showed good linear correlation (r ≥ 0.9978), stability (-11.93 to 12.11%), precision (RSD < 14.63%), and recovery (76.43%-95.20%). The lower limit of quantification was 3.6 ng/mL for Rb1, 1.6 ng/mL for Rb2, 1.2 ng/mL for Rd, and 2.5 ng/mL for Re. This validated method was successfully employed to investigate the pharmacokinetics of the four ginsenosides in rat plasma after oral administration of PQ-AG and PQ extracts. The results revealed the pharmacokinetic profiles of PQ-AG drug pair and clarified that AG played a critical role in stimulating the absorption of active ginsenosides in PQ. Collectively, our findings provided valid and reliable evidence for the rational use of PQ-AG in clinical practice.

The core structure characterization and of ginseng neutral polysaccharide with the immune-enhancing activity.

The study aims to clarify the structural domain required for the immune enhancement of ginseng neutral polysaccharide (GPN). GPN was first obtained through water extraction and ion-exchange chromatography from Panax ginseng. GPN was hydrolyzed by α-amylase for 24 h and fractionated through gel permeation chromatography to give two final fragments GPNE-I and GPNE-II, with molecular weight of 8.03 × 104 Da for GPNE-I and 3.15 × 104 Da for respectively. FT-IR, methylation and 1D/2D NMR analysis demonstrated that GPNE-I was a heteropolysaccharide consisting mainly of a glucan domain and type I and II arabinogalactans (AG-I and AG-II). GPNE II was a glucan consisting of (1 → 4)-α-d-Glcp backbone with a substitution at O-6 on every two residues. (1 → 3)-α-d-Glcp and (1 → 6)-α-d-Glcp were located at the branches. In the two fractions, both α- and ß-t-Glcp as reductive terminals and →4)-α-Glcp as a non-reducing end were detected. The branching degrees of GPNE-I and GPNE-II were 38.17% and 50.78%, respectively. Immunological experiments revealed that GPNE-I exhibited more effectively stimulated lymphocyte proliferation than GPN and GPNE-II, indicating the former showed potential for immunomodulators applications, indicating that GPNE-I might be the core active domain and necessary for GPN to promote lymphocyte proliferation.

Zm908p11, encoded by a short open reading frame (sORF) gene, functions in pollen tube growth as a profilin ligand in maize.

Double fertilization of flowering plants depends on the targeted transportation of sperm to the embryo sac by the pollen tube. Currently, little is known about the underlying molecular mechanisms that regulate pollen germination and pollen tube growth in maize (Zea mays). Here, a maize pollen-predominant gene Zm908, with several putative short open reading frames (sORFs), was isolated and characterized. The longest ORF of Zm908 encodes a small protein of 97 amino acids. This was designated as Zm908p11 and is distributed throughout the maize pollen tube. Western blot detected the small peptide in mature pollen. Quantitative reverse transcription-PCR and northern blot analysis revealed that Zm908p11 was expressed predominantly in mature pollen grains. Ectopic overexpression of full-length Zm908 and Zm908p11 in tobacco resulted in defective pollen, while transgenic tobacco plants with a site-specific mutation or a frameshift mutation of Zm908p11 showed normal pollen development. Overexpression of Zm908p11 in maize decreased pollen germination efficiency. Maize pollen cDNA library screening and protein-protein interaction assays demonstrated that Zm908p11 interacts with maize profilin 1 (ZmPRO1). A microarray analysis identified 273 up-regulated and 203 down-regulated genes in the overexpressing transgenic Zm908p11 pollen. Taken together, these results indicate that Zm908 functions as Zm908p11, and binds to profilins as a novel ligand, with a required role during pollen tube growth in maize. Accordingly, a model is proposed for the role of Zm908p11 during pollen tube growth in maize.

A low molecular weight proteome comparison of fertile and male sterile 8 anthers of Zea mays.

During maize anther development, somatic locular cells differentiate to support meiosis in the pollen mother cells. Meiosis is an important event during anther growth and is essential for plant fertility as pollen contains the haploid sperm. A subset of maize male sterile mutants exhibit meiotic failure, including ms8 (male sterile 8) in which meiocytes arrest as dyads and the locular somatic cells exhibit multiple defects. Systematic proteomic profiles were analysed in biological triplicates plus technical triplicates comparing ms8 anthers with fertile sibling samples at both the premeiotic and meiotic stages; proteins from 3.5 to 20 kDa were fractionated by 1-D PAGE, cleaved with Lys-C and then sequenced using a LTQ Orbitrap Velos MS paradigm. Three hundred and 59 proteins were identified with two or more assigned peptides in which each of those peptides were counted at least two or more times (0.4% peptide false discovery rate (FDR) and 0.2% protein FDR); 2761 proteins were identified with one or more assigned peptides (0.4% peptide FDR and 7.6% protein FDR). Stage-specific protein expression provides candidate stage markers for early anther development, and proteins specifically expressed in fertile compared to sterile anthers provide important clues about the regulation of meiosis. 49% of the proteins detected by this study are new to an independent whole anther proteome, and many small proteins missed by automated maize genome annotation were validated; these outcomes indicate the value of focusing on low molecular weight proteins. The roles of distinctive expressed proteins and methods for mass spectrometry of low molecular weight proteins are discussed.

Maize csmd1 exhibits pre-meiotic somatic and post-meiotic microspore and somatic defects but sustains anther growth.

Maize male reproductive development is complex and lengthy, and anther formation and pollen maturation are precisely and spatiotemporally regulated. Here, we document that callose, somatic, and microspore defect 1 (csmd1), a new male-sterile mutant, has both pre-meiotic somatic and post-meiotic gametophyte and somatic defects. Chromosome behavior and cell developmental events were monitored by nuclear staining viewed by bright field microscopy; cell dimensions were charted by Volocity analysis of confocal microscopy images. Aniline blue staining and quantitative assays were performed to record callose deposition, and expression of three callose synthase genes was measured by qRT-PCR. Despite numerous defects and unlike other maize male-sterile mutants that show growth arrest coincident with locular defects, csmd1 anther elongation is nearly normal. Pre-meiotically and during prophase I, there is excess callose surrounding the meiocytes. Post-meiotically csmd1 epidermal cells have impaired elongation but excess longitudinal divisions, and uninucleate microspores cease growth; the microspore nucleoli degrade followed by cytoplasmic vacuolization and haploid cell collapse. The single vascular bundle within csmd1 anthers senesces precociously, coordinate with microspore death. Although csmd1 anther locules contain only epidermal and endothecial cells at maturity, locules are oval rather than collapsed, indicating that these two cell types suffice to maintain an open channel within each locule. Our data indicate that csmd1 encodes a crucial factor important for normal anther development in both somatic and haploid cells, that excess callose deposition does not cause meiotic arrest, and that developing pollen is not required for continued maize anther growth.