Spermidine suppresses oxidative stress and ferroptosis by Nrf2/HO-1/GPX4 and Akt/FHC/ACSL4 pathway to alleviate ovarian damage.

AIMS: Oxidative stress is considered to be one of the culprits of ovarian dysfunction. Spermidine (SPD) is a natural aliphatic polyamine that is widely present in living organisms and has been shown to exert preventive effects on various ageing-related diseases. This study seeks to investigate the potential preventive and protective effects of SPD on ovarian oxidative damage. MAIN METHODS: Ovarian oxidative stress model in C57BL/6 mice was established by 3-nitropropionic acid. Female mice were administrated 10 mg/kg or 15 mg/kg SPD. The estrous cycle, serum hormone levels and mating test were measured to evaluate ovarian function. Follicle counts and AMH levels to assess ovarian reserve. Masson's trichrome to assess ovarian fibrosis. TUNEL analysis to evaluate follicular granulosa cells (GCs) apoptosis. Oxidative stress and autophagy indicators (Nrf2, HO-1, GPX4, LC3B, P62) were measured in vivo and in vitro. RNA-sequencing was performed on SPD-treated GC to study the effects of SPD on Akt and FHC/ACSL4 signaling. KEY FINDINGS: SPD supplementation improved ovarian endocrine function and reproductive capacity in oxidative stress mice. SPD regularized the estrous cycle and alleviated oxidative stress. Furthermore, SPD increased the ovarian reserve, reducing GC apoptosis by activating the Nrf2/HO-1/GPX4 pathway. RNA-sequencing showed that SPD induced 230 genes changes in porcine GC, which were mainly involved in oocyte meiosis, arginine biosynthesis and glutathione metabolism pathways. SPD attenuated H2O2-induced ferroptosis by regulating Akt/FHC/ACSL4 signaling. SIGNIFICANCE: SPD alleviates oxidative stress and ferroptosis by regulating the Nrf2/HO-1/GPX4 and Akt/FHC/ACSL4 pathway, which may be a novel potential strategy to protect ovarian oxidative damage.

Correlation analysis of polyamine metabolism and reproductive hormone levels in goose ovarian follicles.

To investigate the relationship between polyamine metabolism and reproductive hormones in ovarian follicles of Sichuan white geese, follicle polyamine content and reproductive hormone levels and gene expressions related to polyamine metabolism, steroidogenesis and steroid hormone receptors were detected by HPLC, ELISA and RT-qPCR. The results showed that the overall trend of spermidine and spermine levels increased first and then decreased as increasing follicle size, with the highest level in F3 and F5 follicles (P < 0.05). Putrescine and 17ß-estradiol (E2) levels in hierarchical follicles were significantly lower than those in prehierarchical follicles (P < 0.05). Progesterone (P4) first increased and then decreased, with the highest level in the F5 follicle (P < 0.05). The expression levels of estrogen receptor 1 (ER1) showed an overall increase as increasing follicle size (except in F3 follicles), while estrogen receptor 2 (ER2) in hierarchical follicles was significantly lower than that in the prehierarchical follicles (P < 0.05). In addition, the overall expression level of progesterone receptor (PR) decreased, with no significant differences among F1, F2 and F3 follicles (P > 0.05). Yolk putrescine contents were positively correlated with yolk E2 concentrations and PR expression levels (P < 0.05), A significant positive correlation of spermidine levels with yolk P4 concentrations and PR expressions was also observed, as well as the spermine levels with yolk P4 concentrations (P < 0.05). In summary, polyamines were involved in the regulation of follicular development in geese, and this regulation played a role in affecting steroidogenesis and the expression of genes related to hormone receptors.

Ferritin heavy chain participated in ameliorating 3-nitropropionic acid-induced oxidative stress and apoptosis of goose follicular granulosa cells.

Oxidative stress is the major culprits responsible for ovarian dysfunction by damaging granulosa cells (GCs). Ferritin heavy chain (FHC) may participate in the regulation of ovarian function by mediating GCs apoptosis. However, the specific regulatory function of FHC in follicular GCs remains unclear. Here, 3-nitropropionic acid (3-NPA) was utilized to establish an oxidative stress model of follicular GCs of Sichuan white geese. To explore the regulatory effects of FHC on oxidative stress and apoptosis of primary GCs in geese by interfering or overexpressing FHC gene. After transfection of siRNA-FHC to GCs for 60 h, the expressions of FHC gene and protein decreased significantly (P < 0.05). After FHC overexpression for 72 h, the expressions of FHC mRNA and protein upregulated considerably (P < 0.05). The activity of GCs was impaired after interfering with FHC and 3-NPA coincubated (P < 0.05). When overexpression of FHC combined with 3-NPA treatment, the activity of GCs was remarkably enhanced (P < 0.05). After interference FHC and 3-NPA treatment, NF-κB and NRF2 gene expression decreased (P < 0.05), the intracellular reactive oxygen species (ROS) level increased greatly (P < 0.05), BCL-2 expression reduced, BAX/BCL-2 ratio intensified (P < 0.05), the mitochondrial membrane potential decreased notably (P < 0.05), and the apoptosis rate of GCs aggravated (P < 0.05). While overexpression of FHC combined with 3-NPA treatment could promote BCL-2 protein expression and reduce BAX/BCL-2 ratio, indicating that FHC regulated the mitochondrial membrane potential and apoptosis of GCs by mediating the expression of BCL-2. Taken together, our research manifested that FHC alleviated the inhibitory effect of 3-NPA on the activity of GCs. FHC knockdown could suppress the expression of NRF2 and NF-κB genes, reduce BCL-2 expression and augment BAX/BCL-2 ratio, contributing to the accumulation of ROS and jeopardizing mitochondrial membrane potential, as well as exacerbating GCs apoptosis.

Exploration of the Antioxidant Effect of Spermidine on the Ovary and Screening and Identification of Differentially Expressed Proteins.

Spermidine is a naturally occurring polyamine compound that has many biological functions, such as inducing autophagy and anti-inflammatory and anti-aging effects. Spermidine can affect follicular development and thus protect ovarian function. In this study, ICR mice were fed exogenous spermidine drinking water for three months to explore the regulation of ovarian function by spermidine. The results showed that the number of atretic follicles in the ovaries of spermidine-treated mice was significantly lower than that in the control group. Antioxidant enzyme activities (SOD, CAT, T-AOC) significantly increased, and MDA levels significantly decreased. The expression of autophagy protein (Beclin 1 and microtubule-associated protein 1 light chain 3 LC3 II/I) significantly increased, and the expression of the polyubiquitin-binding protein p62/SQSTM 1 significantly decreased. Moreover, we found 424 differentially expressed proteins (DEPs) were upregulated, and 257 were downregulated using proteomic sequencing. Gene Ontology and KEGG analyses showed that these DEPs were mainly involved in lipid metabolism, oxidative metabolism and hormone production pathways. In conclusion, spermidine protects ovarian function by reducing the number of atresia follicles and regulating the level of autophagy protein, antioxidant enzyme activity, and polyamine metabolism in mice.

Effects of Spermidine on Mouse Gut Morphology, Metabolites, and Microbial Diversity.

Spermidine is a class of biologically active organic small molecules that play an important role in maintaining intestinal homeostasis. The specific objective of this study was to explore the effects of spermidine on intestinal morphology, metabolites, and microbial diversity in mice. We showed that 0.3 mmol/L of spermidine significantly promoted the growth of ileal villi (p < 0.05), and 3.0 mmol/L of spermidine significantly increased the body weight of mice and promoted the growth of jejunum villi (p < 0.05). The 16S rDNA sequencing results indicated that 3.0 mmol/L of spermidine affected the balance of the intestinal flora by increasing the abundance of intestinal Lactic acid bacteria and reducing the abundance of harmful bacteria (Turicibacter and Alistipes). Additionally, spermidine affects the levels of microbial metabolites such as succinic acid and Pantetheine. In summary, spermidine affects intestinal morphology and regulates intestinal flora and metabolites, and this study has provided a new understanding of spermidine's effects on the intestinal tract.

Effects of ODC on polyamine metabolism, hormone levels, cell proliferation and apoptosis in goose ovarian granulosa cells.

Ornithine decarboxylase (ODC) plays an indispensable role in the process of polyamine biosynthesis. Polyamines are a pivotal part of living cells and have diverse roles in the regulation of cell proliferation and apoptosis, aging and reproduction. However, to date, there have been no reports about ODC regulating follicular development in goose ovaries. Here, we constructed ODC siRNA and overexpression plasmids and transfected them into goose primary granulosa cells (GCs) to elucidate the effects of ODC interference and overexpression on the polyamine metabolism, hormone levels, cell apoptosis and proliferation of granulosa cells. After interfering with ODC in GCs, the mRNA and protein levels of ODC and the content of putrescine were greatly decreased (P < 0.05). When ODC was overexpressed, ODC mRNA and protein levels and putrescine content were greatly increased (P < 0.05). The polyamine-metabolizing enzyme genes ornithine decarboxylase antizyme 1 (OAZ1) and spermidine / spermine-N1-acetyltransferase (SSAT) were significantly increased, and spermidine synthase (SPDS) was significantly decreased when ODC was downregulated (P < 0.05). OAZ1, SPDS and SSAT were significantly increased when ODC was upregulated (P < 0.05). In addition, after interference with ODC, progesterone (P4) levels in the culture medium of GCs increased greatly (P < 0.05), while the overexpression of ODC caused the P4 level to decrease significantly (P < 0.05). After ODC downregulation, granulosa cell activity was significantly reduced, the apoptosis rate was significantly increased, and the BCL-2 / BAX ratio was downregulated (P < 0.05). Under ODC overexpression, the activity of GCs was notably increased, the apoptosis rate was significantly reduced, and the BCL-2 / BAX protein ratio was upregulated (P < 0.05). Our study successfully induced ODC interference and overexpression in goose ovarian GCs, and ODC regulated mainly putrescine content in GCs with a slight influence on spermidine and spermine. Moreover, ODC participated in the adjustment of P4 levels in the culture medium of GCs, promoted granulosa cell proliferation and inhibited granulosa cell apoptosis.

[Effects of α-enolase gene interference expression on proliferation and apoptosis of follicular granulosa cells from Zi geese].

Objective: To study the effects of α-enolase (ENO1) gene interference expression on proliferation, and cell cycle of follicular granulosa cells from Zi geese. Methods: F1 follicular granulosa cells were primary cultured (mixed culture), which were divided into four groups: ENO1 interference expression group (RNAi), unrelated sequence group (NC), culture group (Control), transfection reagent group (Lip). The apoptosis rate and cell cycle phase of the interference group and the control group were detected by the flow cytometry. Results: ENO1 gene interference expression slowed the proliferation of granulosa cells, increased the apoptosis, and increased the proportion of granulosa cells in G2/M phase. Conclusion: ENO1 gene interference expression could cause G2/M phase arrest in primary cultured goose follicular granulosa cells, induce cell apoptosis and inhibit cell proliferation.

Effects of α-enolase Gene Silencing on Reproductive-related Hormone Receptor Expression and Steroid Hormone Synthesis of Primary Granulosa Cells from Goose F1 Follicles.

INTRODUCTION: Enolases are enzymes in the glycolytic pathway, which catalyse the reversible conversion of D-2-phosphoglycerate into phosphoenol pyruvate in the second half of the pathway. In this research, the effects of α-enolase (ENO1) on steroid reproductive-related hormone receptor expression and on hormone synthesis of primary granulosa cells from goose F1 follicles were studied. MATERIAL AND METHODS: Primary granulosa cells from the F1 follicles of eight healthy 8-month-old Zi geese were separated and cultured. An ENO1 interference expression vector was designed, constructed and transfected into primary cultured granulosa cells. The mRNA expression levels of follicle-stimulating hormone receptor (FSHR), luteinising hormone receptor (LHR), oestrogen receptor α (ER α), oestrogen receptor ß (ER ß), growth hormone receptor (GHR) and insulin-like growth factor binding protein-1 (IGFBP-1) in the cells were evaluated as were the secretion levels of oestradiol, activin, progesterone, testosterone, inhibin and follistatin in cell supernatant. RESULTS: α-enolase gene silencing reduced the expression of FSHR, LHR, ERα, ERß, GHR, and IGFBP-1 mRNA, potentiated the secretion of oestrogen, progesterone, testosterone, and follistatin of granulosa cells, and hampered the production of activin and inhibin. CONCLUSION: ENO1 can regulate the reactivity of granulosa cells to reproductive hormones and regulate cell growth and development by adjusting their hormone secretion and reproductive hormone receptor expression. The study provided a better understanding of the functional action of ENO1 in the processes of goose ovary development and egg laying.

Latent Factor Decoding of Multi-Channel EEG for Emotion Recognition Through Autoencoder-Like Neural Networks.

Robust cross-subject emotion recognition based on multichannel EEG has always been hard work. In this work, we hypothesize that there exist default brain variables across subjects in emotional processes. Hence, the states of the latent variables that relate to emotional processing must contribute to building robust recognition models. Specifically, we propose to utilize an unsupervised deep generative model (e.g., variational autoencoder) to determine the latent factors from the multichannel EEG. Through a sequence modeling method, we examine the emotion recognition performance based on the learnt latent factors. The performance of the proposed methodology is verified on two public datasets (DEAP and SEED) and compared with traditional matrix factorization-based (ICA) and autoencoder-based approaches. Experimental results demonstrate that autoencoder-like neural networks are suitable for unsupervised EEG modeling, and our proposed emotion recognition framework achieves an inspiring performance. As far as we know, it is the first work that introduces variational autoencoder into multichannel EEG decoding for emotion recognition. We think the approach proposed in this work is not only feasible in emotion recognition but also promising in diagnosing depression, Alzheimer's disease, mild cognitive impairment, etc., whose specific latent processes may be altered or aberrant compared with the normal healthy control.

Identification, functional prediction, and key lncRNA verification of cold stress-related lncRNAs in rats liver.

Cold stimulation reduces the quality of animal products and increases animal mortality, causing huge losses to the livestock industry in cold regions. Long non-coding RNAs (lncRNAs) take part in many biological processes through transcriptional regulation, intracellular material transport, and chromosome remodeling. Although cold stress-related lncRNAs have been reported in plants, no research is available on the characteristic and functional analysis of lncRNAs after cold stress in rats. Here, we built a cold stress animal model firstly. Six SPF male Wistar rats were randomly divided to the acute cold stress group (4 °C, 12 h) and the normal group (24 °C, 12 h). lncRNA libraries were constructed by high-throughput sequencing (HTS) using rat livers. 2,120 new lncRNAs and 273 differentially expressed (DE) lncRNAs were identified in low temperature environments. The target genes of DElncRNA were predicted by cis and trans, and then functional and pathway analysis were performed to them. GO and KEGG analysis revealed that lncRNA targets were mainly participated in the regulation of nucleic acid binding, cold stimulation reaction, metabolic process, immune system processes, PI3K-Akt signaling pathway and pathways in cancer. Next, a interaction network between lncRNA and its targets was constructed. To further reveal the mechanism of cold stress, DElncRNA and DEmRNA were extracted to reconstruct a co-expression sub-network. We found the key lncRNA MSTRG.80946.2 in sub-network. Functional analysis of key lncRNA targets showed that targets were significantly enriched in fatty acid metabolism, the PI3K-Akt signaling pathway and pathways in cancer under cold stress. qRT-PCR confirmed the sequencing results. Finally, hub lncRNA MSTRG.80946.2 was characterized, and verified its relationship with related mRNAs by antisense oligonucleotide (ASO) interference and qRT-PCR. Results confirmed the accuracy of our analysis. To sum up, our work was the first to perform detailed characterization and functional analysis of cold stress-related lncRNAs in rats liver. lncRNAs played crucial roles in energy metabolism, growth and development, immunity and reproductive performance in cold stressed rats. The MSTRG.80946.2 was verified by network and experiments to be a key functional lncRNA under cold stress, regulating ACP1, TSPY1 and Tsn.

Metabonomics analysis of Zi goose follicular granulosa cells using ENO1 gene expression interference.

The Zi goose is native to North-east China and is noted for its high egg production. Alpha enolase (ENO1) is a glycolytic enzyme which functions as a plasminogen receptor in follicular granulosa cells (FGCs), with several studies showing that FGCs can support follicular development. By transfecting the ENO1 interfering plasmid (shRNA) into FGCs, ENO1 expression in these cells was downregulated, suggesting the successful knock-down of ENO1 in these cells. In this knock-down model, we detected 13 metabolites from FGCs using LC/MS. When compared with the non-coding shRNA (NC) group, the lower level metabolites were (R)-(+)-citronellic acid, altretamine, 3-hydroxycaproic acid, heptadecanoic acid, cholecalciferol vitamin D3, indole, benzoic acid, capric acid, caffeic acid, azelaic acid, 3,4-dihydroxyhydrocinnamic acid and cholic acid, while oleic acid was detected at high levels. To further examine the results of metabolomics, six key metabolites were verified by gas chromatography-mass spectrometry (GC-MS). We found that vitamin D3, indole, benzoic acid, capric acid and cholic acid were significantly downregulated in the shRNA group, while oleic acid was significantly upregulated. This observation was consistent with the metabolomics data. Through these studies, we found that decreased ENO1 levels altered certain metabolite levels in FGCs.

MicroRNA-mediated susceptible poplar gene expression regulation associated with the infection of virulent Melampsora larici-populina.

BACKGROUND: Rust caused by Melampsora larici-populina is one of the most damaging diseases of poplars. Rust is considered to be a model pathogen for genetic studies because both pathogen and host genomes are available. The poplar 'Robusta', whose general rust resistance is defeated by virulent rust E4, provides suitable host material for studies of the gene regulation involved in rust resistance/susceptibility. In this study, we investigated the microRNA-mediated susceptible poplar gene expression regulation associated with the infection of virulent rust. We were particularly interested in delineating the host-pathogen interactions with a specific focus on microRNAs (miRNAs). RESULTS: To study the susceptibility of poplar to M. larici-populina, small RNA (sRNA) libraries, a degradome cDNA library and digital gene expression libraries were constructed for rust-inoculated and rust-free susceptible poplar 'Robusta' leaves through high-throughput sequencing. Altogether, 12,722 regulating interactions were identified. The results delineated the framework of post-transcriptional regulation of gene expression in the susceptible poplar, which was infected by the virulent rust. The results indicated that pathogen-associated molecular patterns (PAMPs) and PAMP-triggered immunity were induced by the infection of virulent rust E4 and that miRNAs still functioned at this stage. After this stage, miRNA-regulated R genes, such as TIR-NBS-LRR and CC-NBS-LRR, were not fully functional. Additionally, the rust-responsive miRNAs did not regulate the signaling component genes related to the salicylic acid pathway or the hypersensitive response. CONCLUSIONS: We found that the defense-related post-transcriptional regulation of the susceptible poplar 'Robusta' functions normally only at the stage of PAMPs and PAMP-triggered immunity (PTI). More importantly, the miRNA-mediated post-transcriptional regulation of defense signal pathway genes were inactivated by the infection of virulent rust at the stage of effector-triggered susceptibility and during the following stages of salicylic acid and hypersensitive responses. This inactivation was the major characteristic of 'Robusta' susceptibility.