Huaier Polysaccharide Alleviates Dextran Sulphate Sodium Salt-Induced Colitis by Inhibiting Inflammation and Oxidative Stress, Maintaining the Intestinal Barrier, and Modulating Gut Microbiota.

The incidence of ulcerative colitis (UC) is increasing annually, and UC has a serious impact on patients' lives. Polysaccharides have gained attention as potential drug candidates for treating ulcerative colitis (UC) in recent years. Huaier (Trametes robiniophila Murr) is a fungus that has been used clinically for more than 1000 years, and its bioactive polysaccharide components have been reported to possess immunomodulatory effects, antitumour potential, and renoprotective effects. In this study, we aimed to examine the protective effects and mechanisms of Huaier polysaccharide (HP) against UC. Based on the H2O2-induced oxidative stress model in HT-29 cells and the dextran sulphate sodium salt (DSS)-induced UC model, we demonstrated that Huaier polysaccharides significantly alleviated DSS-induced colitis (weight loss, elevated disease activity index (DAI) scores, and colonic shortening). In addition, HP inhibited oxidative stress and inflammation and alleviated DSS-induced intestinal barrier damage. It also significantly promoted the expression of the mucin Muc2. Furthermore, HP reduced the abundance of harmful bacteria Escherichia-Shigella and promoted the abundance of beneficial bacteria Muribaculaceae_unclassified, Anaerotruncus, and Ruminococcaceae_unclassified to regulate the intestinal flora disturbance caused by DSS. Nontargeted metabolomics revealed that HP intervention would modulate metabolism by promoting levels of 3-hydroxybutyric acid, phosphatidylcholine (PC), and phosphatidylethanolamine (PE). These results demonstrated that HP had the ability to mitigate DSS-induced UC by suppressing oxidative stress and inflammation, maintaining the intestinal barrier, and modulating the intestinal flora. These findings will expand our knowledge of how HP functions and offer a theoretical foundation for using HP as a potential prebiotic to prevent UC.

GnRH-driven FTO-mediated RNA m6A modification promotes gonadotropin synthesis and secretion.

BACKGROUND: Gonadotropin precisely controls mammalian reproductive activities. Systematic analysis of the mechanisms by which epigenetic modifications regulate the synthesis and secretion of gonadotropin can be useful for more precise regulation of the animal reproductive process. Previous studies have identified many differential m6A modifications in the GnRH-treated adenohypophysis. However, the molecular mechanism by which m6A modification regulates gonadotropin synthesis and secretion remains unclear. RESULTS: Herein, it was found that GnRH can promote gonadotropin synthesis and secretion by promoting the expression of FTO. Highly expressed FTO binds to Foxp2 mRNA in the nucleus, exerting a demethylation function and reducing m6A modification. After Foxp2 mRNA exits the nucleus, the lack of m6A modification prevents YTHDF3 from binding to it, resulting in increased stability and upregulation of Foxp2 mRNA expression, which activates the cAMP/PKA signaling pathway to promote gonadotropin synthesis and secretion. CONCLUSIONS: Overall, the study reveals the molecular mechanism of GnRH regulating the gonadotropin synthesis and secretion through FTO-mediated m6A modification. The results of this study allow systematic interpretation of the regulatory mechanism of gonadotropin synthesis and secretion in the pituitary at the epigenetic level and provide a theoretical basis for the application of reproductive hormones in the regulation of animal artificial reproduction.

Turtle peptide and its derivative peptide ameliorated DSS-induced ulcerative colitis by inhibiting inflammation and modulating the composition of the gut microbiota.

Ulcerative colitis (UC) is a recurrent intestinal disease with an increasing incidence worldwide that seriously affects the life of patients. Turtle peptide (TP) is a bioactive peptide extracted from turtles that has anti-inflammatory, antioxidant and anti-aging properties. However, studies investigating the effect of TP on the progression of UC are lacking. The aim of this study was to investigate effects and underlying mechanisms of TP and its derivative peptide GPAGPIGPV (GP-9) in alleviating UC in mice. The results showed that 500 mg/kg TP treatment significantly ameliorated colitis symptoms and oxidative stress in UC mice. TP alleviated intestinal barrier damage in UC mice by promoting mucosal repair and increasing the expression of tight junction proteins (ZO1, occludin and claudin-1). TP also modulated the composition of the gut microbiota by increasing the abundance of the beneficial bacteria Anaerotignum, Prevotellaceae_UCG-001, Alistipes, and Lachno-spiraceae_NK4A136_group and decreasing the abundance of the harmful bacteria Prevotella_9 and Parasutterella. Furthermore, we characterized the peptide composition of TP and found that GP-9 ameliorated the symptoms of dextran sodium sulfate (DSS)-induced colitis in mice by inhibiting the TLR4/NF-κB signaling pathway. In conclusion, TP and its derivative peptides ameliorated DSS-induced ulcerative colitis by inhibiting the expression of inflammatory factors and modulating the composition of the intestinal microbiota; this study provides a theoretical basis for the application of TP and its derivative peptides for their anti-inflammatory activity.

Circ-ERC2 Is Involved in Melatonin Synthesis by Regulating the miR-125a-5p/MAT2A Axis.

The circadian rhythm of melatonin secretion in the pineal gland is highly conserved in vertebrates. Melatonin levels are always elevated at night. Acetylserotonin O-methyltransferase (ASMT) is the last enzyme in the regulation of melatonin biosynthesis (N-acetyl-5-hydroxytryptamine-melatonin). S-adenosylmethionine (SAM) is an important methyl donor in mammals and can be used as a substrate for the synthesis of melatonin. Methionine adenosyltransferase (MAT) catalyzes the synthesis of SAM from methionine and ATP and has a circadian rhythm. CircRNA is an emerging type of endogenous noncoding RNA with a closed loop. Whether circRNAs in the pineal gland can participate in the regulation of melatonin synthesis by binding miRNAs to target mat2a as part of the circadian rhythm is still unclear. In this study, we predicted the targeting relationship of differentially expressed circRNAs, miRNAs and mRNAs based on the results of rat pineal RNA sequencing. Mat2a siRNA transfection confirmed that mat2a is involved in the synthesis of melatonin. Circ-ERC2 and miR-125a-5p were screened out by software prediction, dual-luciferase reporter experiments, cell transfection, etc. Finally, we constructed a rat superior cervical ganglionectomy model (SCGx), and the results showed that circ-ERC2 could participate in the synthesis of melatonin through the miR-125a-5p/MAT2A axis. The results of the study revealed that circ-ERC2 can act as a molecular sponge of miR-125a-5p to regulate the synthesis of melatonin in the pineal gland by targeting mat2a. This experiment provides a basis for research on the circadian rhythm of noncoding RNA on pineal melatonin secretion.

TRH Regulates the Synthesis and Secretion of Prolactin in Rats with Adenohypophysis through the Differential Expression of miR-126a-5p.

Prolactin (PRL) is an important hormone that is secreted by the pituitary gland and plays an important role in the growth, development and reproduction of organisms. Thyrotropin-releasing hormone (TRH) is a common prolactin-releasing factor that regulates the synthesis and secretion of prolactin. In recent studies, microRNAs (miRNAs) have been found to play a key role in the regulation of pituitary hormones. However, there is a lack of systematic studies on the regulatory role that TRH plays on the pituitary transcriptome, and the role of miRNAs in the regulation of PRL synthesis and secretion by TRH lacks experimental evidence. In this study, we first investigated the changes in PRL synthesis and secretion in the rat pituitary gland after TRH administration. The results of transcriptomic analysis after TRH treatment showed that 102 genes, including those that encode Nppc, Fgf1, PRL, Cd63, Npw, and Il23a, were upregulated, and 488 genes, including those that encode Lats1, Cacna2d1, Top2a, and Tfap2a, were downregulated. These genes are all involved in the regulation of prolactin expression. The gene expression of miR-126a-5p, which regulates the level of PRL in the pituitary gland, was screened by analysis prediction software and by a dual luciferase reporter system. The data presented in this study demonstrate that TRH can regulate prolactin synthesis and secretion through miR-126a-5p, thereby improving our understanding of the molecular mechanism of TRH-mediated PRL secretion and providing a theoretical basis for the role of miRNAs in regulating the secretion of pituitary hormones.

Comprehensive analysis of differences in N6-methyladenosine RNA methylomes in the rat adenohypophysis after GnRH treatment.

N6-methyladenosine is considered to be the most common and abundant internal chemical modification among the more than 150 identified chemical RNA modifications. It is involved in most biological processes and actively participates in the regulation of animal reproduction. However, the potential function of m6 A in the pituitaries of mammals is not yet clear. It is also unknown whether m6 A is involved in the secretion and regulation of FSH by GnRH, which in turn affects mammalian reproduction. In this study, rats were treated with gonadorelin to simulate physiological GnRH-mediated regulation of FSH synthesis and secretion, and m6 A-seq was used to analyze the differential m6 A modification of the rat pituitary after gonadorelin treatment. A whole-transcriptome map of m6 A in the rat pituitary gland before and after gonadorelin treatment was successfully created. A total of 6413 differential peaks were identified, of which 3764 m6 A peaks were upregulated and 2649 m6 A peaks were downregulated. Among the 709 differentially expressed genes, 250 genes were discovered with differential methylation modifications. Intriguingly, the altered m6 A peaks within mRNAs were enriched in steroid biosynthetic processes and responses to cAMP. The results of the study will lay a foundation for further exploration of the potential role of m6 A modification in the regulation of reproductive hormone secretion and provide a theoretical basis for the application of GnRH analogs in mammalian artificial reproduction.

CircRNA-WNK2 Acts as a ceRNA for miR-328a-3p to Promote AANAT Expression in the Male Rat Pineal Gland.

Noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and circular RNAs (circRNAs), which are expressed with a daily rhythm in the rat pineal gland, are associated with the regulation of melatonin secretion and other biological functions. However, the mechanisms of these molecules in the rat pineal gland are not yet fully understood. In this study, we found that circR-WNK2 was highly expressed at night, which may be involved in the regulation of melatonin secretion through the competitive endogenous RNA (ceRNA) mechanism. By dual luciferase reporter, RNA pull-down, and fluorescence in situ hybridization (FISH) assays, we found that miR-328a-3p can target circR-WNK2 and the Aa-nat mRNA 3'UTR. Transfection experiments indicated that circR-WNK2 could competitively bind to miR-328a-3p, reduce miR-328a-3p expression, and promote Aa-nat gene expression and melatonin secretion. And by constructing a superior cervical ganglionectomy (SCGx) rat model, we found that ncRNAs expression in the pineal gland was regulated by signals from the suprachiasmatic nucleus. This finding supports the hypothesis that these noncoding RNAs may interact to shape the circadian rhythm through transcriptional processing in melatonin synthesis.

Regulation of FSH Synthesis by Differentially Expressed miR-488 in Anterior Adenohypophyseal Cells.

Gonadotropin-releasing hormone (GnRH), which is synthesized and released by the hypothalamus, promotes the synthesis and secretion of follicle-stimulating hormone (FSH), thereby regulating the growth and reproduction of animals. GnRH analogues have been widely used in livestock production. MiRNAs, which are endogenous non-coding RNAs, have been found to play important roles in hormone regulation and other physiological processes in recent years. However, the roles of miRNAs in GnRH-mediated regulation of FSH secretion have rarely been studied. Herein, we treated bovine anterior adenohypophyseal cells with an exogenous GnRH analogue and found that miR-488 was differentially expressed. Through a combination of TargetScan prediction and dual luciferase reporter analysis, miR-488 was confirmed to be able to target the FSHB gene. Based on this finding, we verified the expression of Fshß and Lhß mRNA in the rat adenohypophysis before and after exogenous GnRH treatment in vivo and in vitro. Experiments on rat anterior adenohypophyseal cells showed that overexpression of miR-488 significantly inhibited Fshß expression and FSH synthesis, while knockdown of miR-488 had the opposite effects. Our results demonstrate that GnRH relies on miR-488 to regulate FSH synthesis, providing additional useful evidence for the significance of miRNAs in the regulation of animal reproduction.

Identification of Circular RNAs in the Anterior Pituitary in Rats Treated with GnRH.

The pituitary gland, an important endocrine organ, can secrete a variety of reproductive hormones under the action of hypothalamus-secreted gonadotropin-releasing hormone (GnRH) and plays important roles in animal reproduction. Circular RNAs (circRNAs) are a class of RNA molecules with stable covalently closed circular structures. CircRNAs are equipped with miRNA response elements (MREs), which can regulate the expression of target genes by competitively binding miRNAs. However, whether the expression levels of circRNAs in the pituitary gland change under the action of GnRH and whether such changes can further affect the secretion of reproductive hormones are still unclear. In this study, we performed RNA sequencing (RNA-Seq) of GnRH-treated rats to identify differentially expressed circRNAs. The results revealed 1433 related circRNAs, 14 of which were differentially expressed. In addition, we randomly selected five differentially expressed circRNAs and tested their relative expression levels by RT-qPCR, the results of which were consistent with the RNA sequencing results. Finally, we predicted targeted relationships between the differentially expressed circRNAs and FSHb-LHb-associated miRNAs. In all, a total of 14 circRNAs were identified that may act on the secretion and regulation of reproductive hormones in GnRH-treated rats. Our expression profiles of circRNAs in the anterior pituitaries of rats treated with GnRH can provide insights into the roles of circRNAs in mammalian development and reproduction.

Identifying daily changes in circRNAs and circRNA-associated-ceRNA networks in the rat pineal gland.

Circular RNAs (circRNAs) are a new class of covalently closed circular RNA molecules that are involved in many biological processes. However, information about circRNAs in the pineal gland, particularly that of rats, is limited. To establish resources for the study of the rat pineal gland, we performed transcriptome analysis of the pineal glands during the day and night. In this study, 1413 circRNAs and 1989 miRNAs were identified in the pineal gland of rats during the night and day using the Illumina platform. Forty differentially expressed circRNAs and 93 differentially expressed miRNAs were obtained, among which 20 circRNAs and 37 miRNAs were significantly upregulated during the day and 20 circRNAs and 56 miRNAs were significantly upregulated during the night. As circRNAs have been reported to work as miRNA sponges, we predicted 15940 interactions among 40 circRNAs, 93 miRNAs and 400 mRNAs with differential diurnal expression using miRanda and TargetScan to build a ceRNA regulatory network in the rat pineal gland. The diurnal expression profile of circRNAs in the rat pineal gland may provide additional information about the role of circRNAs in regulating changes in melatonin circadian rhythms. The analyzed data reported in this study will be an important resource for future studies to elucidate the altered physiology of circRNAs in diurnal rhythms.

Roles of differential expression of miR-543-5p in GH regulation in rat anterior pituitary cells and GH3 cells.

Growth hormone (GH) is an important hormone released by the pituitary gland that plays a key role in the growth and development of organisms. In our study, TargetScan analysis and the dual luciferase reporter assays were used to predict and screen for miRNAs that might act on the rat Gh1 gene, and we identified miR-543-5p. Then, the GH3 cell line and the primary rat pituitary cells were transfected with miRNA mimic, inhibitor, and siRNA. We detected the Gh1 gene expression and the GH secretion by real-time PCR and ELISAs, respectively, to verify the regulatory effect of miR-543-5p on GH secretion. The results showed that miR-543-5p can inhibit Gh1 mRNA expression and reduce GH secretion. MiR-543-5p inhibitor upregulated Gh1 mRNA expression and increased GH secretion compared with the negative control. In summary, miR-543-5p downregulates Gh1 expression, resulting in a decrease in GH synthesis and secretion, which demonstrates the important role of miRNAs in regulating GH and animal growth and development.

Pituitary tissue-specific miR-7a-5p regulates FSH expression in rat anterior adenohypophyseal cells.

The follicle-stimulating hormone (FSH), which is synthesized and secreted by the anterior pituitary gland, plays an important role in regulating reproductive processes. In this study, using the TargetScan program, we predicted that microRNAs (miRNAs) regulate FSH secretion. Dual-luciferase reporter assays were performed and identified miR-7a-5p. MiR-7a-5p has been reported to regulate diverse cellular functions. However, it is unclear whether miR-7a-5p binds to mRNAs and regulates reproductive functions. Therefore, we constructed a suspension of rat anterior pituitary cells and cultured them under adaptive conditions, transfected miR-7a-5p mimics or inhibitor into the cell suspension and detected expression of the FSHb gene. The results demonstrated that miR-7a-5p downregulated FSHb expression levels, while treatment with miR-7a-5p inhibitors upregulated FSHb expression levels relative to those of negative control groups, as shown by quantitative PCR analysis. The results were confirmed with a subsequent experiment showing that FSH secretion was reduced after treatment with mimics and increased in the inhibitor groups, as shown by enzyme-linked immunosorbent assay. Our results indicated that miR-7a-5p downregulates FSHb expression levels, resulting in decreased FSH synthesis and secretion, which demonstrates the important role of miRNAs in the regulation of FSH and animal reproduction.

Prelamin A overexpression promotes detrusor calcification/aging in urinary incontinence via prelamin A accumulation.

Urinary incontinence (UI) is known as a distressing condition particularly among older adults, and negatively associated with health-related quality of life in both males and females. Prelamin A accumulation has been found in all progeroid laminopathies and is obviously linked to cell and organism aging. Therefore, this study was expected to investigate the effect of prelamin A on detrusor on UI. Prelamin A expression in clinical and animal samples was detected. To investigate the degree of prelamin A accumulation and detrusor calcification/aging, the detrusor cells were subcultured separately into low and high passage. The low-passage subculture cells were treated with transfection of overexpressed prelamin A plasmid, and transfection of overexpressed prelamin A plasmid and application of farnesyl transferase inhibitor (FTIs) H-9279, respectively. Zmpste24, Icmt and lamin A/C expression were detected to explore how prelamin A affected detrusor calcification/aging. Prelamin A was overexpressed in aged detrusor cells, indicating prelamin A expression was positively related to the age of subjects. The degree of prelamin A accumulation and detrusor calcification/aging was higher in aged rats and high passage subculture cells. Zmpste24, Icmt and lamin A/C were poorly expressed in cells transfected with overexpressed prelamin A, as well as cell proliferation activity decreased and calcium deposition and apoptotic rate increased. Furthermore, we also found that the effect of overexpressed prelamin A was lost when cells were treated with H-9279. These findings provide evidence that prelamin A overexpression impairs degradation of its farnesylated form, thus causing prelamin A accumulation which induces detrusor calcification/aging in UI.