Nrf2-mediated ferroptosis of spermatogenic cells involved in male reproductive toxicity induced by polystyrene nanoplastics in mice. / Nrf2ä»‹å¯¼çš„ç”Ÿç²¾ç»†èƒžé“æ­»äº¡å‚ä¸Žäº†èšè‹¯ä¹™çƒ¯çº³ç±³å¡‘æ–™å¯¼è‡´çš„å°é¼ é›„æ€§ç”Ÿæ®–æ¯’æ€§.

Microplastics (MPs) and nanoplastics (NPs) have become hazardous materials due to the massive amount of plastic waste and disposable masks, but their specific health effects remain uncertain. In this study, fluorescence-labeled polystyrene NPs (PS-NPs) were injected into the circulatory systems of mice to determine the distribution and potential toxic effects of NPs in vivo. Interestingly, whole-body imaging found that PS-NPs accumulated in the testes of mice. Therefore, the toxic effects of PS-NPs on the reproduction systems and the spermatocytes cell line of male mice, and their mechanisms, were investigated. After oral exposure to PS-NPs, their spermatogenesis was affected and the spermatogenic cells were damaged. The spermatocyte cell line GC-2 was exposed to PS-NPs and analyzed using RNA sequencing (RNA-seq) to determine the toxic mechanisms; a ferroptosis pathway was found after PS-NP exposure. The phenomena and indicators of ferroptosis were then determined and verified by ferroptosis inhibitor ferrostatin-1 (Fer-1), and it was also found that nuclear factor erythroid 2-related factor 2 (Nrf2) played an important role in spermatogenic cell ferroptosis induced by PS-NPs. Finally, it was confirmed in vivo that this mechanism of Nrf2 played a protective role in PS-NPs-induced male reproductive toxicity. This study demonstrated that PS-NPs induce male reproductive dysfunction in mice by causing spermatogenic cell ferroptosis dependent on Nrf2.

A novel perspective on di-hexyl phthalate (2-ethylhexyl)-induced reproductive toxicity in females: Lipopolysaccharide synergizes with mono-2-ethylhexyl ester to cause inflammatory apoptosis rather than autophagy in ovarian granulosa cells.

Di-hexyl phthalate (2-ethylhexyl) (DEHP) has been confirmed to cause female reproductive toxicity in humans and model animals by affecting the survival of ovarian granulosa cells (GCs), but the interrelationships between DEHP's on autophagy, apoptosis, and inflammation in GCs are not clear. Our previous study demonstrated that DEHP exposure resulted in the disturbance of intestinal flora associated with serum LPS release, which in turn led to impaired ovarian function. LPS has also been shown to determine cell fate by modulating cellular autophagy, apoptosis, and inflammation. Therefore, this study investigated the role and link between LPS and autophagy, apoptosis, and inflammation of GCs in DEHP-induced ovarian injury. Here, we constructed an in vivo injury model by continuous gavage of 0-1500 mg/kg of DEHP in female mice for 30 days and an in vitro injury model by treatment of human ovarian granulosa cells (KGN) cells with mono-2- ethylhexyl ester (MEHP, an active metabolite of DEHP in vivo). In addition, the expression of relevant pathway molecules was detected by immunohistochemistry, immunofluorescence, qRT-PCR, and Western blotting after the addition of the autophagy inhibitor 3-methyladenine (3-MA), the apoptosis inhibitor Z-VAD- FMK and the NF-κB inhibitor BAY11-7082. The current study found that autophagy and apoptosis were significantly activated in GCs of DEHP-induced atretic follicles in vivo and found that MEHP-induced KGN cells autophagy and apoptosis were independent and potentially cytotoxic of each other in vitro. Further studies confirmed that DEHP exposure resulted in LPS release from the intestinal tract and entering the ovary, thereby participating in DEHP-induced inflammation of GCs. In addition, we found that exogenous LPS synergized with MEHP could activate the NF-κB signaling pathway to induce inflammation and apoptosis of GCs in a relatively prolonged exposure condition. Meanwhile, inhibition of inflammatory activation could rescue apoptosis and estrogen secretion function of GCs induced by MEHP combined with LPS. These results indicated that the increased LPS influenced by DEHP might cooperate with MEHP to induce inflammatory apoptosis of GCs, an important cause of ovarian injury in mice.

Human umbilical cord mesenchymal stem cells alleviate chemotherapy-induced premature ovarian insufficiency mouse model by suppressing ferritinophagy-mediated ferroptosis in granulosa cells.

Primary ovarian insufficiency (POI) in younger women (under 40) manifests as irregular periods, high follicle-stimulating hormone (FSH), and low estradiol (E2), often triggered by chemotherapy. Though mesenchymal stem cell (MSC) therapy shows promise in treating POI, its exact mechanism remains unclear. This study reveals that human umbilical cord-derived MSCs (hUC-MSCs) can protect ovarian granulosa cells (GCs) from cyclophosphamide (CTX)-induced ferroptosis, a form of cell death driven by iron accumulation. CTX, commonly used to induce POI animal model, triggered ferroptosis in GCs, while hUC-MSCs treatment mitigated this effect, both in vivo and in vitro. Further investigations using ferroptosis and autophagy inhibitors suggest that hUC-MSCs act by suppressing ferroptosis in GCs. Interestingly, hUC-MSCs activate a protective antioxidant pathway in GCs via NRF2, a stress-response regulator. Overall, our findings suggest that hUC-MSCs improve ovarian function in CTX-induced POI by reducing ferroptosis in GCs. This study not only clarifies the mechanism behind the benefits of hUC-MSCs but also strengthens the case for their clinical use in treating POI. Additionally, it opens up a new avenue for protecting ovaries from chemotherapy-induced damage by regulating ferroptosis.

Effects of trigonelline, diosgenin, and Cistanche deserticola polysaccharide on the culture of female germline stem cells in vitro.

Female germline stem cells (FGSCs) are renewable sources of oocytes that play an indispensable role in re-establishing mammal fertility. Here, we have established FGSCs from neonatal mice, which exhibit characteristics of germline stem cells. We show that compared with monomeric trigonelline and diosgenin, macromolecular compounds Cistanche deserticola polysaccharides (CDPs) in Chinese herbal medicine can enhance the ability of FGSCs to differentiate into oocytes at appropriate concentrations while maintaining self-renewal in vitro. In contrast, trigonelline and diosgenin inhibited the expression of germ cell-specific genes while reducing cell proliferation activity. In summary, CDPs could induce the differentiation and self-renewal of FGSCs in vitro. The comparison of the effects of the active components of different types of Chinese medicine will provide a reference for the development of clinical drugs in the future, and help to elucidate the development process of FGSCs.

Activation of the p62-Keap1-Nrf2 pathway protects against oxidative stress and excessive autophagy in ovarian granulosa cells to attenuate DEHP-induced ovarian impairment in mice.

Di-(2-ethylhexyl) phthalate (DEHP) is widely used in various plastics but has been demonstrated to cause female reproductive toxicity. However, the exact mechanism underlying the ovarian damage induced by DEHP remains unclear. In this study, DEHP was administered orally to 5-week-old female mice for 30 days at doses of 0, 250, 500, and 1000 mg/kg/day. The findings demonstrated that DEHP exposure disrupted ovarian function and follicular development as well as induced oxidative stress and autophagy in ovarian granulosa cells (GCs). Further, 200 µM mono-(2-ethylhexyl) phthalate (MEHP), the primary metabolite of DEHP in vivo, induced autophagy in both human ovarian granulosa cells line (KGN) and mouse primary GCs within 24 h in vitro. However, it did not affect the p62-dependent autophagy flux. Furthermore, MEHP-induced autophagy was inhibited by the autophagy inhibitor 3-MA and exacerbated by the autophagy activator rapamycin, indicating that MEHP induces excessive autophagy in GCs. Subsequently, we found that MEHP-induced autophagic cell death was primarily attributed to oxidative damage from elevated intracellular ROS levels. Meanwhile, MEHP exposure induced nuclear translocation of erythroid-derived factor 2-related factor (Nrf2), a key regulator of antioxidant activity resulting in activating antioxidant effects. Interestingly, we also found that MEHP-induced increase in p62 competitively binds Keap1, thereby facilitating nuclear translocation of Nrf2 and establishing a positive feedback loop in antioxidant regulation. Therefore, this study demonstrated that inhibition of Nrf2 could aggravate oxidative damage and enhance excessive autophagy caused by MEHP, while activation of Nrf2 could reverse the trend. These findings have also been reinforced in studies of cultured ovaries in vitro. Our study suggests that the p62-Keap1-Nrf2 pathway may serve as a potential protective mechanism against DEHP-induced oxidative stress and excessive autophagy in mouse GCs.

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) alleviate excessive autophagy of ovarian granular cells through VEGFA/PI3K/AKT/mTOR pathway in premature ovarian failure rat model.

BACKGROUND: Premature ovarian failure (POF) is one of the leading causes of female infertility and is accompanied by abnormal endocrine, seriously affecting female quality of life. Previous studies have demonstrated that mesenchymal stem cells (MSCs) transplantation is a promising therapeutic strategy for POF. However, the mechanism remains obscure. This study aims to investigate the therapeutic effect of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on ovarian function in the POF rat model and explore the underlying mechanisms. METHODS: The ovarian function was evaluated by ovarian morphology, histology, estrous cycle, hormone levels (AMH, E2, FSH, and LH), and fertility ability to investigate the effect of hUC-MSCs on the POF rats model. The cytokines levels were assayed in serum using protein array to explore the mechanisms of hUC-MSCs therapy for POF. The excessive autophagy levels were evaluated using a co-culture system of 3D MSCs spheroids with human ovarian granulosa cell line (KGN) or primary ovarian granulosa cells (GCs) to understand the paracrine effect of hUC-MSCs on GCs. The related proteins expression of autophagy and PI3K/AKT/mTOR pathway was detected using Western Blotting and/or in various inhibitors supplement to further demonstrate that vascular endothelial growth factor A (VEGFA) secreted by hUC-MSCs can alleviate excessive autophagy of ovarian GCs via PI3K/AKT/mTOR signaling pathway. The ovarian culture model in vitro was applied to confirm the mechanism. RESULTS: The ovarian function of POF and the excessive autophagy of ovarian GCs were restored after hUC-MSCs transplantation. The protein array result demonstrated that VEGF and PI3K/AKT might improve ovarian function. in vitro experiments demonstrated that VEGFA secreted by hUC-MSCs could decrease oxidative stress and inhibit excessive autophagy of ovarian GCs via PI3K/AKT/mTOR pathway. The ovarian culture model results confirmed this mechanism in vitro. CONCLUSION: The hUC-MSCs can alleviate excessive autophagy of ovarian GCs via paracrine VEGFA and regulate the PI3K/AKT/mTOR signaling pathway, thereby improving the ovarian function of POF.

Roles of follicle stimulating hormone and sphingosine 1-phosphate co-administered in the process in mouse ovarian vitrification and transplantation.

Some major challenges of ovarian tissue vitrification and transplantation include follicle apoptosis induced by cryopreservation and ischemia-reperfusion injury, as well as ovarian follicle loss during post-transplantation. This research aimed to investigate the protective effects and underlying mechanisms of follicle-stimulating hormone (FSH) and Sphingosine-1-phosphate (S1P) on vitrified and post-transplantation ovaries. Ovaries from 21-day-old mice were cryopreservation by vitrification with 0.3 IU/mL FSH, 2 µM S1P, and 0.3 IU/mL FSH + 2 µM S1P, respectively, for follicle counting and detection of apoptosis-related indicators. The results demonstrated that FSH and S1P co-intervention during the vitrification process could preserve the primordial follicle pool and inhibit follicular atresia by suppressing cell apoptosis. The thawed ovaries were transplanted under the renal capsule of 6-8 week-old ovariectomized mice and removed 24 h or 7 days after transplantation. The results indicated that FSH and S1P co-intervention can inhibit apoptosis and autophagy in ovaries at 24 h after transplantation, and promote follicle survival by up-regulating Cx37 and Cx43 expression, enhanced angiogenesis in transplanted ovaries by promoting VEGF expression, as well as increased the E2 levels to restore ovarian endocrine function at 7 days after transplantation. The hypoxia and ischemia cell model was established by CoCl2 treatment for hypoxia in human granulosa-like tumor cell line (KGN), as well as serum-free culture system was used for ischemia. The results confirmed that ischemia-hypoxia-induced apoptosis in ovarian granulosa cells was reduced by FSH and S1P co-intervention, and granulosa cell autophagy was inhibited by up-regulating the AKT/mTOR signaling pathway. In summary, co-administration of FSH and S1P can maintain ovarian survival during ovarian vitrification and increase follicle survival and angiogenesis after transplantation.

Integrated fecal microbiome and metabolome analysis explore the link between polystyrene nanoplastics exposure and male reproductive toxicity in mice.

Microplastics (MPs) and nanoplastics (NPs) are novel environmental pollutants that are ubiquitous in the environment and everyday life. NPs can easily enter the tissues and have more significant potential health risks due to their smaller diameter. Previous studies have shown that NPs can induce male reproductive toxicity, but the detailed mechanisms remain uncertain. In this study, intragastric administration treated mice with polystyrene NPs (PS-NPs, 50, and 90 nm) at 3 and 15 mg/mL/day doses for 30 days. Then, the fresh fecal samples were collected from those mice that the exposure doses of 50 nm PS-NPs at 3 mg/mL/day and 90 nm at 15 mg/mL/day for subsequent investigations of 16S rRNA and metabolomics according to significant toxicological effects (Sperm number, viability, abnormality, and testosterone level). The conjoint analysis findings indicated that PS-NPs disrupted the homeostasis of the gut microbiota, metabolism, and male reproduction, suggesting that abnormal gut microbiota-metabolite pathways may be important in PS-NPs-induced male reproductive toxicity. Meanwhile, the common differential metabolites such as 4-deoxy-Erythronic acid, 8-iso-15-keto-PGE2, apo-10'-violaxanthin, beta-D-glucosamine, isokobusone, oleamide, oxoadipic acid, sphingosine induced by 50 and 90 nm PS-NPs might be used as biomarkers to explore PS-NPs-induced male reproductive toxicity. In addition, this study systematically demonstrated that nano-scale PS-NPs induced male reproductive toxicity via the crosstalk of gut microbiota and metabolites. It also provided valuable insights into the toxicity of PS-NPs, which was conducive to reproductive health risk assessment for public health prevention and treatment.

The Activation of Reticulophagy by ER Stress through the ATF4-MAP1LC3A-CCPG1 Pathway in Ovarian Granulosa Cells Is Linked to Apoptosis and Necroptosis.

Female infertility is caused by premature ovarian failure (POF), which is triggered by the endoplasmic reticulum (ER) stress-mediated apoptosis of granulosa cells. The ER unfolded protein response (UPRer) is initiated to promote cell survival by alleviating excessive ER stress, but cellular apoptosis is induced by persistent or strong ER stress. Recent studies have reported that reticulophagy is initiated by ER stress. Whether reticulophagy is activated in the ER stress-mediated apoptosis of granulosa cells and which pathway is initiated to activate reticulophagy during the apoptosis of granulosa cells are unknown. Therefore, the role of reticulophagy in granulosa cell death and the relationship between ER stress and reticulophagy were investigated in this work. Our results suggest that the ER stress inducer tunicamycin causes POF in mice, which is attributed to the apoptosis of granulosa cells and is accompanied by the activation of UPRer and reticulophagy. Furthermore, granulosa cells were treated with tunicamycin, and granulosa cell apoptosis was triggered and increased the expression of UPRer and reticulophagy molecules. The expression of ATF4 was then downregulated by RNAi, which decreased the levels of autophagy and the reticulophagy receptor CCGP1. Furthermore, ATF4 targets MAP1LC3A, as revealed by the ChIP sequencing results, and co-IP results demonstrated that MAP1LC3A interacts with CCPG1. Therefore, reticulophagy was activated by ER stress through the ATF4-MAP1LC3A-CCPG1 pathway to mitigate ER stress. Additionally, the role of reticulophagy in granulosa cells was investigated by the knockdown of CCPG1 with RNAi. Interestingly, only a small number of granulosa cells died by apoptosis, whereas the death of most granulosa cells occurred by necroptosis triggered by STAT1 and STAT3 to impair ER proteostasis and the ER protein quality control system UPRer. Taken together, the results indicate that the necroptosis of granulosa cells is triggered by up- and downregulating the reticulophagy receptor CCPG1 through STAT1/STAT3-(p)RIPK1-(p)RIPK3-(p)MLKL and that reticulophagy is activated by ER stress through the ATF4-MAP1LC3A-CCPG1 pathway.

Oocyte Arrested at Metaphase II Stage were Derived from Human Pluripotent Stem Cells in vitro.

Initiation of meiosis is the most difficult aspect of inducing competent oocytes differentiation from human stem cells in vitro. Human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) were cultured with follicle fluid, cytokines and small molecule to induced oocyte-like cells (OLCs) formation through a three-step induction procedure. Expression of surface markers and differentiation potential of germ cells were analyzed in vitro by flow cytometry, gene expression, immunocytochemistry, western blotting and RNA Sequencing. To induce the differentiation of hiPSCs into OLCs, cells were firstly cultured with a primordial germ cell medium for 10 days. The cells exhibited similar morphological features to primordial germ cells (PGCs), high expressing of germ cell markers and primordial follicle development associated genes. The induced PGCs were then cultured with the primordial follicle-like cell medium for 5 days to form the induced follicle-like structures (iFLs), which retained both primordial oocytes-like cells and granulosa-like cells. In the third step, the detached iFLs were harvested and transferred to the OLC-medium for additional 10 days. The cultured cells developed cumulus-oocyte-complexes (COCs) structures and OLCs with different sizes (50-150 µm diameter) and a zona pellucida. The in vitro matured OLCs had polar bodies and were arrested at metaphase II (MII) stage. Some OLCs were self-activated and spontaneously developed into multiple-cell structures similar to preimplantation embryos, indicating that OLCs were parthenogenetically activated though in vitro fertilization potential of OLCs are yet to be proved. in vitro maturation of OLCs derived from hiPSCs provides a new means to study human germ cell formation and oogenesis.

Optimized study of an in vitro 3D culture of preantral follicles in mice.

BACKGROUND: In vitro culture of preantral follicles is a promising technology for fertility preservation. OBJECTIVES: This study aims to investigate an optimized three-dimensional (3D) fetal bovine serum (FBS)-free preantral follicle culture system having a simple and easy operation. METHODS: The isolated follicles from mouse ovaries were randomly divided in an ultra-low attachment 96-well plates supplement with FBS or bovine serum albumin (BSA) culture or encapsulated with an alginate supplement with FBS or BSA culture. Meanwhile, estradiol (E2) concentration was assessed through enzyme-linked immunosorbent assay of culture supernatants. The diameter of follicular growth was measured, and the lumen of the follicle was photographed. Spindle microtubules of oocytes were detected via immunofluorescence. The ability of oocytes to fertilize was assessed using in vitro fertilization. RESULTS: The diameters were larger for the growing secondary follicles cultured in ultra-low attachment 96-well plates than in the alginate gel on days 6, 8, and 10 (p < 0.05). Meanwhile, the E2 concentration in the BSA-supplemented medium was significantly higher in the alginate gel than in the other three groups on days 6 and 8 (p < 0.05), and the oocytes in the FBS-free system could complete meiosis and fertilization in vitro. CONCLUSIONS: The present study furnishes insights into the mature oocytes obtained from the 3D culture of the preantral follicle by using ultra-low attachment 96-well plate with an FBS-free system in vitro and supports the clinical practices to achieve competent, mature oocytes for in vitro fertilization.

Mechanism of Mitochondrial Homeostasis Controlling Ovarian Physiology.

Ovarian cells, including oocytes, granulosa/cumulus cells, theca cells, and stromal cells, contain abundant mitochondria, which play indispensable roles in the processes of ovarian follicle development. Ovarian function is closely controlled by mitochondrial proteostasis and mitostasis. While mitochondrial proteostasis and mitostasis are disturbed by several factors, leading to dysfunction of ovarian function and initiating the mitochondrial unfolded protein response (UPRmt) and mitophagy to maintain or recover ovarian function and mitochondrial function, clear interactions between the 2 pathways in the ovary have not been fully elucidated. Here, we comprehensively summarize the molecular networks or regulatory mechanisms behind further mitochondrial research in the ovary. This review provides novel insights into the interactions between the UPRmt and mitophagy in ovarian functions.

MIIP functions as a novel ligand for ITGB3 to inhibit angiogenesis and tumorigenesis of triple-negative breast cancer.

Migration and invasion inhibitory protein (MIIP) has been identified as a tumor suppressor in various cancer types. Although MIIP is reported to exert tumor suppressive functions by repressing proliferation and metastasis of cancer cells, the detailed mechanism is poorly understood. In the present study, we found MIIP is a favorable indicator of prognosis in triple-negative breast cancer. MIIP could inhibit tumor angiogenesis, proliferation, and metastasis of triple-negative breast cancer cells in vivo and in vitro. Mechanistically, MIIP directly interacted with ITGB3 and suppressed its downstream signaling. As a result, ß-catenin was reduced due to elevated ubiquitin-mediated degradation, leading to downregulated VEGFA production and epithelial mesenchymal transition. More importantly, we found RGD motif is essential for MIIP binding with ITGB3 and executing efficient tumor-suppressing effect. Our findings unravel a novel mechanism by which MIIP suppresses tumorigenesis in triple-negative breast cancer, and MIIP is thus a promising molecular biomarker or therapeutic target for the disease.

Melatonin prevents cyclophosphamide-induced primordial follicle loss by inhibiting ovarian granulosa cell apoptosis and maintaining AMH expression.

Cyclophosphaty -45mide (Cyc) chemotherapy in young female cancer patients is associated with an increased risk of premature ovarian insufficiency (POI). This study was designed to investigate the protective role of melatonin (Mel) as an adjuvant against Cyc-induced POI. Female mice received a single intraperitoneal (i.p.) dose of Cyc (75 mg/kg). Mel protection was achieved in mice after i.p. injection of melatonin (50 mg/kg) every 24 h for four consecutive days prior to chemotherapy initiation and for 14 additional days. Ovarian reserve testing, hormonal assays for follicle-stimulating hormone, luteinizing hormone, and anti-Müllerian hormone (AMH), assessment of the oxidative stress status, and measurement of the relative expression of genes in PTEN/AKT/FOXO3a and mitochondrial apoptosis pathways were performed. The results showed that treatment with 50 mg/kg Mel significantly prevented Cyc-induced over-activation of primordial follicles by maintaining the plasma level of AMH and subsequently preventing litter size reduction in mice treated with Cyc chemotherapy. Importantly, Mel treatment significantly prevented ovarian granulosa cell loss by inhibiting the mitochondrial apoptotic pathway. Identifying the protective actions of Mel against Cyc-induced primordial follicle loss has important implications for fertility maintenance in young cancer patients undergoing chemotherapy.

Zoledronic acid generates a spatiotemporal effect to attenuate osteoarthritis by inhibiting potential Wnt5a-associated abnormal subchondral bone resorption.

This study aimed to determine the effects of zoledronic acid (ZOL) on OA in rats and explored the molecular mechanism of osteoclast activation in early OA. A knee OA rat model was designed by surgically destabilizing the medial meniscus (DMM). Seventy-two male rats were randomly assigned to Sham+phosphate-buffered saline (PBS), DMM+PBS, and DMM+ZOL groups; rats were administered with 100 µg/Kg ZOL or PBS, twice weekly for 4 weeks. After 2, 4, 8, and 12 weeks of OA induction, the thickness of the hyaline and calcified cartilage layers was calculated using hematoxylin and eosin staining, degenerated cartilage stained with Safranin O-fast green staining was evaluated and scored, tartrate-resistant acid phosphatase (TRAP)-stained osteoclasts were counted, changes in subchondral bone using micro-computed tomography were analyzed, and PINP and CTX-I levels were detected using enzyme-linked immunosorbent assay. Using these results, 18 male rats were randomly assigned to three groups. Four weeks after surgery, Wnt5a, RANKL, CXCL12, and NFATc1 protein levels were measured in subchondral bone using western blotting, and mRNA levels of genes related to osteoclastogenesis in subchondral bone were measured using quantitative polymerase chain reaction. Bone marrow-derived macrophages were isolated as osteoclast precursors, and cell differentiation, migration, and adhesion were assessed by TRAP staining and Transwell assays, revealing that DMM induced knee OA in rats. Progressive cartilage loss was observed 12 weeks after OA induction. Subchondral bone remodeling was dominated by bone resorption during early OA (within 4 weeks), whereas bone formation was increased 8 weeks later. ZOL suppressed bone resorption by inhibiting Wnt5a signaling in early OA, improved the imbalance of subchondral bone remodeling, reduced cartilage degeneration, and delayed OA progression. Additionally, ZOL delayed OA progression and reduced cartilage degeneration via a spatiotemporal effect in DMM-induced OA. Osteoclast activity in early OA might be associated with Wnt5a signaling, indicating a possible novel strategy for OA treatment.

Cistanche deserticola polysaccharides extracted from Cistanche deserticola Y.C. Ma promote the differentiation of mouse female germline stem cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese herbal medicine Cistanche deserticola Y.C. Ma has been recorded and treatment for infertility and impotence since ancient times, which is widely distributed in northwest China, and is mainly composed of phenylethanol glycosides, iridoids, lignans, polysaccharides, alkaloids, etc. C. deserticola polysaccharides (CDPs) is one of its main active ingredients, studies of its effect on germline stem cells are limited so far. AIM OF THE STUDY: The aim of this study was to clarify that CDPs promoted the differentiation of FGSCs in vitro, and to initially clarify its possible cell signaling pathways. MATERIAL AND METHODS: The cells were randomly divided into two groups. Normal FGSCs culture medium and the optimal concentration of CDPs (0.5 µg/mL) were added for culture, which was the selected treatment concentration that could promote cell differentiation on the basis of maintaining cell viability. After treatment for different time periods (12 h, 24 h, 36 h, 48 h), the cell proliferation and differentiation were evaluated by CCK-8, real-time PCR (qPCR), cell immunofluorescence and Western blot. Subsequently, RNA-Seq and data analysis were used to preliminarily analyze and verify the different genes and possible signal pathways. RESULTS: Under the treatment of CDPs, cell viability was relatively better, and the expression of meiotic markers stimulated by retinoic acid gene 8 protein (Stra8) and synaptonemal complex protein 3 (Sycp3) significantly increased. In addition, their cell morphology was more similar to oocytes. Comparison of gene expression in FGSCs identified key differential expression genes (DEGs) by RNA-Seq that consisted of 549 upregulated and 465 downregulated genes. The DEGs enriched in the functional categories of germline cell development and relevant signaling pathways, which jointly regulate self-renewal and differentiation of FGSCs. The transforming growth factor ß (TGF-ß) signaling pathway and bone morphogenetic protein (BMP) signaling pathway might be activated to synergistically influence cell differentiation during the CDPs treatment of FGSCs. CONCLUSION: These findings indicated that CDPs could promote the differentiation of FGSCs in vitro and could be regulated by different DEGs and signal transduction. Preliminary mechanism studies have shown that CDPs can exert their biological activities by regulating the TGF-ß and BMP signaling pathways.

A novel anticancer property of Lycium barbarum polysaccharide in triggering ferroptosis of breast cancer cells.

Breast cancer is one of the most malignant tumors and is associated with high mortality rates among women. Lycium barbarum polysaccharide (LBP) is an extract from the fruits of the traditional Chinese herb, L. barbarum. LBP is a promising anticancer drug, due to its high activity and low toxicity. Although it has anticancer properties, its mechanisms of action have not been fully established. Ferroptosis, which is a novel anticancer strategy, is a cell death mechanism that relies on iron-dependent lipid reactive oxygen species (ROS) accumulation. In this study, human breast cancer cells (Michigan Cancer Foundation-7 (MCF-7) and MD Anderson-Metastatic Breast-231 (MDA-MB-231)) were treated with LBP. LBP inhibited their viability and proliferation in association with high levels of ferroptosis. Therefore, we aimed to ascertain whether LBP reduced cell viability through ferroptosis. We found that the structure and function of mitochondria, lipid peroxidation, and expression of solute carrier family 7 member 11 (SLC7A11, also known as xCT, the light-chain subunit of cystine/glutamate antiporter system Xc-) and glutathione peroxidase 4 (GPX4) were altered by LBP. Moreover, the ferroptosis inhibitor, Ferrostatin-1 (Fer-1), rescued LBP-induced ferroptosis-associated events including reduced cell viability and glutathione (GSH) production, accumulation of intracellular free divalent iron ions and malondialdehyde (MDA), and down-regulation of the expression of xCT and GPX4. Erastin (xCT inhibitor) and RSL3 (GPX4 inhibitor) inhibited the expression of xCT and GPX4, respectively, which was lower after the co-treatment of LBP with Erastin and RSL3. These results suggest that LBP effectively prevents breast cancer cell proliferation and promotes ferroptosis via the xCT/GPX4 pathway. Therefore, LBP exhibits novel anticancer properties by triggering ferroptosis, and may be a potential therapeutic option for breast cancer.

Metformin ameliorates polycystic ovary syndrome in a rat model by decreasing excessive autophagy in ovarian granulosa cells via the PI3K/AKT/mTOR pathway.

Polycystic ovary syndrome (PCOS) is a common gynecological disease accompanied by a variety of clinical features, including anovulation, hyperandrogenism, and ovarian abnormalities, resulting in infertility. PCOS affects approximately 6%-15% of all reproductive-age women worldwide. Metformin, a popular drug used to treat PCOS in patients, has beneficial effects in reducing hyperandrogenism and inducing ovulation; however, the mechanisms by which metformin ameliorates PCOS are not clear. Hence, we aimed to explore the mechanisms of metformin in treating PCOS. In the present study, we first treated a letrozole-induced PCOS rat model with metformin, detected the pathological recovery of PCOS, and then assessed the effects of metformin on H2O2-induced autophagy in ovarian granulosa cells (GCs) by detecting the level of oxidative stress and the expression of autophagy-associated proteins and key proteins in the PI3K/AKT/mTOR pathway. We demonstrated that metformin ameliorated PCOS in a rat model by downregulating autophagy in GCs, and metformin decreased the levels of oxidative stress and autophagy in H2O2-induced GCs and affected the PI3K/AKT/mTOR signaling pathway. Taken together, our results indicate that metformin ameliorates PCOS in a rat model by decreasing excessive autophagy in GCs via the PI3K/AKT/mTOR pathway, and this study provides evidence for targeted reduction of excessive autophagy of ovarian granulosa cells and improvement of PCOS.

A proteomic analysis of Bcl-2 regulation of cell cycle arrest: insight into the mechanisms.

B cell lymphoma 2 (Bcl-2) is an important antiapoptotic gene that plays a dual role in the maintenance of the dynamic balance between the survival and death of cancer cells. In our previous study, Bcl-2 was shown to delay the G0/G1 to S phase entry by regulating the mitochondrial metabolic pathways to produce lower levels of adenosine triphosphate (ATP) and reactive oxygen species (ROS). However, the detailed molecular mechanisms or pathways by which Bcl-2 regulates the cell cycle remain unknown. Here, we compared the effects of Bcl-2 overexpression with an empty vector control in the NIH3T3 cell line synchronized by serum starvation, and evaluated the effects using proteomic analysis. The effect of Bcl-2 on cell cycle regulation was detected by monitoring Bcl-2 and p27 expression. The result of subsequent proteomic analysis of Bcl-2 overexpressing cells identified 169 upregulated and 120 downregulated proteins with a 1.5-fold change. These differentially expressed proteins were enriched in a number of signaling pathways predominantly involving the ribosome and oxidative phosphorylation, according to the data of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. These results indicated that Bcl-2 potentially acts at the translation level to influence proteins or enzymes of the respiratory chain or in the ribosome, and thereby regulates the cell cycle. Additionally, differentially expressed proteins involved in oxidative phosphorylation were determined to account for most of the effects of Bcl-2 on the cell cycle mediated by the mitochondrial pathway investigated in our previous study. These results can provide assistance for additional in-depth studies on the regulation of the cell cycle by Bcl-2. The results of the proteomic analysis determined the mechanism of Bcl-2-dependent delay of the cell cycle progression. In summary, the results of this study provide a novel mechanistic basis for identifying the key proteins or pathways for designing and developing precisely targeted cancer drugs.

Oxymatrine inhibits neuroinflammation byRegulating M1/M2 polarization in N9 microglia through the TLR4/NF-κB pathway.

Microglia are the primary immune cells involved in the immune response, inflammation, and injury repair in the central nervous system. Under different stimuli, the dual polarization of classically-activated M1 microglia and anti-inflammatory selectively-activated M2 microglia is observed. Oxymatrine (OMT) exerts various anti-inflammatory and neuroprotective effects, but the mechanism underlying its action remains unclear. In the present study, we investigated the effects of OMT on the polarization of M1/M2 microglia in a lipopolysaccharide (LPS)-induced inflammation model in order to elucidate the potential molecular mechanism of action of OMT in vitro. We first used a Cell Counting Kit-8 (CCK-8) to evaluate the effects of different concentrations OMT on the viability of N9 microglia to determine the appropriate concentration for follow-up experiments. Next, Griess reagent and enzyme-linked immunosorbent assay (ELISA) kits were used to detect the expression of the inflammation-related factors nitric oxide (NO), tumour necrosis factor-alpha (TNF-α), and interleukin (IL)-6, -1ß, and -10. To evaluate the protective effects of OMT, the ultrastructure of the cells was observed using electron microscopy. Immunofluorescence, flow cytometry, and western blotting were performed to evaluate the effects of OMT on the following markers of M1 and M2 microglia: CD16/32, CD206, Arginase-10 (Arg-1), and inducible nitric oxide synthase (iNOS). Lastly, western blotting and quantitative polymerase chain reaction (qPCR) were used to detect factors associated with the Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) signalling pathway in order to explore the potential mechanism by which OMT regulates microglial polarization. The viability of N9 cells did not decrease when treated with a concentration of 1000 µg/mL OMT. Electron microscopy revealed that a concentration of 100 µg/mL OMT exerted a protective effect on N9 cells stimulated by LPS. The results of the present study indicated that OMT inhibited the over-activation of microglia, increased the levels of the M2 marker IL-10, decreased the levels of the M1 markers NO, TNF-α, IL-6, and IL-1ß, promoted the polarization of N9 microglia to the M2 phenotype, and regulated M1/M2 polarization in the microglia by inhibiting TLR4/NF-κB signalling, which effectively attenuated the LPS-induced inflammatory response.