Pathway analysis of spermidine anti-oxidative stress and inducing autophagy in granulosa cells of Sichuan white geese.

Spermidine, a natural polyamine, has been proven antioxidant function, but its pathway and mechanism of action remain unclear. Based on the oxidative stress model by 3-nitropropionic acid (3-NPA), the study explored the pathways by spermidine to rescue oxidative stress via autophagic process in goose granulosa cells by RNA-seq and RNA interference. In transcriptional regulation, in addition to KEGG pathways related to cell proliferation and differentiation, lots of KEGG pathways associated with inflammation, metabolism, and signaling were also significantly enriched in 3-NPA vs. 3-NPA + spermidine treatments. Six key genes (JUN, CD44, KITLG, RND2, BMP4 and KALRN) involved in spermidine-mediated anti-oxidative stress were screened. Furthermore, the experimental results showed that spermidine (80 µmol/L) significantly increased autophagic gene expression in goose granulosa cells, while EP300-siRNA or MAP1S-siRNA also significantly increased autophagic process. The autophagic gene expressions were no difference between EP300-siRNA and EP300-siRNA + spermidine treatments, although spermidine significantly increased autophagic process of granulosa cells compared to MAP1S-siRNA alone. In addition, inhibition of mTOR pathway significantly increased autophagic gene expression, which was further enhanced by spermidine in combined with mTOR inhibitor. These results suggest that spermidine can alleviate oxidative stress by inducing autophagy regulated by EP300, MAP1S and mTOR as well as regulating other independent gene expressions in goose granulosa cells.

Polyamines in Ovarian Aging and Disease.

Ovarian aging and disease-related decline in fertility are challenging medical and economic issues with an increasing prevalence. Polyamines are a class of polycationic alkylamines widely distributed in mammals. They are small molecules essential for cell growth and development. Polyamines alleviate ovarian aging through various biological processes, including reproductive hormone synthesis, cell metabolism, programmed cell death, etc. However, an abnormal increase in polyamine levels can lead to ovarian damage and promote the development of ovarian disease. Therefore, polyamines have long been considered potential therapeutic targets for aging and disease, but their regulatory roles in the ovary deserve further investigation. This review discusses the mechanisms by which polyamines ameliorate human ovarian aging and disease through different biological processes, such as autophagy and oxidative stress, to develop safe and effective polyamine targeted therapy strategies for ovarian aging and the diseases.

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.

Effects of exogenous spermidine on autophagy and antioxidant capacity in ovaries and granulosa cells of Sichuan white geese.

Autophagy can inhibit ovarian senescence induced by oxidative stress and regulate follicle development and atresia, but its mechanism is still unclear. Exogenous spermidine can induce autophagy and scavenge reactive oxygen species (ROS). In this experiment, oxidative stress in Sichuan white geese ovaries and follicular granulosa cells (GCs) was caused by 3-nitropropionic acid (3-NPA) and spermidine was added to explore the effect of exogenous spermidine inducing autophagy and inhibiting oxidative stress in vivo and in vitro. Research results showed that putrescine, spermidine and spermine contents in goose ovaries in the group treated with spermidine combined with 3-NPA were 2.70, 1.94, and 1.70 times higher than those in the group treated with 3-NPA, respectively (P < 0.05). The contents of spermidine and spermine in GCs were 1.37 and 0.89 times higher in the spermidine in combination with the 3-NPA group than in the 3-NPA group, respectively (P < 0.05). LC3 and p62 were mainly expressed in the follicular granulosa layer. The LC3-II/I ratio and p62 level in GCs in the spermidine combined with 3-NPA treatment group were 1.37 and 0.77 times higher than that of the 3-NPA treatment group, respectively (P < 0.05). 3-NPA treatment significantly increased ROS level and the apoptosis rate in GCs, while the combined treatment of spermidine and 3-NPA reversed this change (P < 0.05). In conclusion, spermidine alleviated the oxidative damage induced by 3-NPA by improving the antioxidant capacity of ovaries and follicular GCs of Sichuan white geese and may be alleviated by inducing autophagy in GCs.

This study investigated the effects of exogenous spermidine on oxidative stress induced by 3-nitropropionic acid (3-NPA) in ovaries and granulosa cells of Sichuan white geese. In ovarian tissue, spermidine can reduce malondialdehyde accumulation induced by 3-NPA by increasing antioxidant enzyme activity, thus alleviating the oxidative damage induced by 3-NPA. In addition, spermidine can also improve the morphological structure of follicles and alleviate the structural damage caused by 3-NPA. Our results showed that autophagy-associated proteins are mainly concentrated in the granulosa layer of follicles and spermidine can alter their expression. Subsequently, we found that spermidine could induce autophagy and reduce the accumulation of reactive oxygen species and apoptosis rate induced by 3-NPA in granulosa cells. Therefore, we speculate that spermidine can alleviate oxidative stress induced by 3-NPA by inducing autophagy in granulosa cells. In conclusion, spermidine can relieve oxidative stress induced by 3-NPA by increasing the activity of antioxidant enzymes, and may also relieve oxidative stress by inducing autophagy.

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.

Spermidine alleviating oxidative stress and apoptosis by inducing autophagy of granulosa cells in Sichuan white geese.

Spermidine have been reported a role in antioxidative, antiaging, and antiinflammatory. Oxidative stress causes granulosa cell (GC) apoptosis, follicular atresia, and impairs poultry reproductive functions. Studies have found that autophagy is the protective mechanism against antioxidant stress and apoptosis in cells. However, the relationship between spermidine-induced autophagy, oxidative stress, and apoptosis in goose GCs remains unclear. In this study, we investigated the autophagy mechanism to mediate spermidine effects on the alleviation of oxidative stress and apoptosis in goose GCs. Follicular GCs were treated with spermidine combination with 3-Nitropropanoic acid (3-NPA), rapamycin (RAPA), and chloroquine (CQ) or with hydrogen peroxide, RAPA, and CQ. Spermidine upregulated the ratio of LC3-II/I, inhibited the accumulation of p62 protein, and induced autophagy. 3-NPA treatment significantly increased ROS production, MDA content, SOD activity, cleaved CASPASE-3 protein expression, and decreased BCL-2 protein expression in follicular GCs. Spermidine inhibited oxidative stress and apoptosis induced by 3-NPA. In addition, hydrogen peroxide-induced oxidative stress was inhibited by spermidine. However, the inhibitory effect of spermidine was eliminated under chloroquine. Our results demonstrated that spermidine relieved oxidative stress and apoptosis of GCs by inducing autophagy, indicating that spermidine has a great potential to maintain proteostasis and sustain granulosa cell viability in geese.

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.

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.

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.

Spermidine improves the antioxidant capacity and morphology of intestinal tissues and regulates intestinal microorganisms in Sichuan white geese.

Introduction: Intestinal health is very important to the health of livestock and poultry, and is even a major determining factor in the performance of livestock and poultry production. Spermidine is a type of polyamine that is commonly found in a variety of foods, and can resist oxidative stress, promote cell proliferation and regulate intestinal flora. Methods: In this study, we explored the effects of spermidine on intestinal health under physiological states or oxidative stress conditions by irrigation with spermidine and intraperitoneal injection of 3-Nitropropionic acid (3-NPA) in Sichuan white goose. Results and discussion: Our results showed that spermidine could increase the ratio of intestinal villus to crypt and improve intestinal morphology. In addition, spermidine can also reduce malondialdehyde (MDA) accumulation caused by 3-NPA by increasing superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) enzyme activity, thus alleviating intestinal damage. Furthermore, spermidine can regulate intestinal digestive enzyme activities and affect intestinal digestion and absorption ability. Spermidine can also promote an increase in intestinal microbial diversity and abundance and alleviate the change of microflora structure caused by 3-NPA. In conclusion, spermidine promotes the production of beneficial intestinal metabolites such as Wikstromol, Alpha-bisabolol and AS 1-5, thus improving the level of intestinal health. Taken together, these results indicate that spermidine can improve intestinal health by improving intestinal morphology, increasing antioxidant capacity and regulating intestinal flora structure.

Hydrogen bonding sewing interface.

The strong force that originates from breaking covalent bonds can be easily quantified through various testing platforms, while weak interfacial sliding resistance (ISR), originating from hydrogen bonding or van der Waals (vdW) forces, is very challenging to measure. Facilitated by an in-house nanomechanical testing system, we are able to precisely quantify and clearly distinguish the interfacial interactions between individual carbon fibers and several substrates governed by either hydrogen bonding or vdW forces. The specific ISR of the interface dominated by vdW forces is 3.55 ± 0.50 µN mm-1 and it surprisingly increases to 157.86 ± 44.18 µN mm-1 if the interface is bridged by hydrogen bonding. The ad hoc studies demonstrate that hydrogen bonding rather than vdW forces has great potential in sewing the interface if both surfaces are supportive of the formation of hydrogen bonds. The findings will enlighten the engineering of interfacial interactions and further mediate the entire mechanical performance of structures.