3D hUC-MSC spheroids exhibit superior resistance to autophagy and apoptosis of granulosa cells in POF rat model.

In brief: This study reveals that orthotopic transplantation of 3D hUC-MSC spheroids is more effective than monolayer-cultured hUC-MSCs in improving POF and distinctly reducing oxidative stress through the paracrine effect, thereby preventing apoptosis and autophagy of GCs. Abstract: Premature ovarian failure (POF) is a common reproductive disease in women younger than 40 years old, and studies have demonstrated that the application of human umbilical cord mesenchymal stem cells (hUC-MSCs) is a promising therapy strategy for POF. Given the previously established therapeutic advantages of 3D MSC spheroids, and to evaluate their effectiveness, both 3D hUC-MSC spheroids and monolayer-cultured hUC-MSCs were employed to treat a cyclophosphamide-induced POF rat model through orthotopic transplantation. The effects of these two forms on POF were subsequently assessed by examining apoptosis, autophagy, and oxidative damage in ovarian granulosa cells (GCs). The results indicated that hUC-MSC spheroids exhibited superior treatment effects on resisting autophagy, apoptosis, and oxidative damage in GCs compared to monolayer-cultured hUC-MSCs. To further elucidate the impact of hUC-MSC spheroids in vitro, a H2O2-induced KGN cells model was established and co-cultured with both forms of hUC-MSCs. As expected, the hUC-MSC spheroids also exhibited superior effects in resisting apoptosis and autophagy caused by oxidative damage. Therefore, this study demonstrates that 3D hUC-MSC spheroids have potential advantages in POF therapy; however, the detailed mechanisms need to be further investigated. Furthermore, this study will provide a reference for the clinical treatment strategy of POF.

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.

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.

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.

Effects of postnatal exposure to tetrabromobisphenol A on testis development in mice and early key events.

Whether or not tetrabromobisphenol A (TBBPA) has reproductive developmental toxicity remains controversial. Here, we evaluated the effects of postnatal TBBPA exposure of dams (before weaning) and pups through drinking water (15, 150, 1500 ng/mL) on testis development in mice. On postnatal day (PND) 56, we found that TBBPA exerted little effects on testis weight, anogenital distance, sperm parameters, and the serum testosterone level, but resulted in dose-dependent reductions in the seminiferous tubule area coupled with decreased Sertoli cells and spermatogonia and the number of stage VII-VIII seminiferous tubules, and cytoskeleton damage in Sertoli cells, along with down-regulated expression of marker genes for Sertoli cells, spermatogonia and spermatocyte. Further study revealed that the reduced tubule area coupled decreased Sertoli cell and germ cell numbers and marker gene expression also occurred in TBBPA-treated testes on PND 7, along with reduced cell proliferation and disordered arrangement of Sertoli cell nuclei. On PND 15, most of these testicular alterations were still observed in TBBPA-treated males, and cytoskeleton damage in Sertoli cells became observable. All observations convincingly demonstrate that postnatal exposure to TBBPA disturbed testis development in early life and ultimately caused adverse outcomes in adult testes, and that cell proliferation inhibition, the reduction in the seminiferous tubule area coupled decreased Sertoli cell and germ cell numbers and marker gene expression, and cytoskeleton damage in Sertoli cells, are early events contributing to adverse outcomes in adult testes. Our study improves the understanding of reproductive developmental toxicity of TBBPA, highlighting its risk for human health.

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.

Di-(2-ethylhexyl) phthalate exposure induces female reproductive toxicity and alters the intestinal microbiota community structure and fecal metabolite profile in mice.

Di-(2-ethylhexyl) phthalate (DEHP) is one of the most commonly used plasticizers, and it is widely applied in various plastic products. DEHP is an endocrine-disrupting chemical (EDC) that has been shown to disrupt the function of reproductive system in females. Although many studies have shown that DEHP potentially causes female reproductive toxicity, including depletion of the primordial follicle and decreased sex hormone production, the specific mechanisms by which DEHP affects female reproduction remain unknown. In recent years, research focused on the intestinal flora has provided an idea to eliminate our confusion, and gut bacterial dysbiosis may contribute to female reproductive toxicity. In the present study, the feces of DEHP-exposed mice were collected and analyzed using 16S rRNA amplicon sequencing and untargeted global metabolite profiling of metabolomics. DEHP obviously causes reproductive toxicity, including the ovarian organ coefficient, estradiol level, histological features of the ovary and estrus. Furthermore, DEHP exposure alters the structure of the intestinal microbiota community and fecal metabolite profile in mice, suggesting that the reproductive toxicity may be caused by gut bacterial dysbiosis and altered metabolites, such as changes in the levels of short-chain fatty acid (SCFA). Additionally, it is well known that changes in gut microbiota and fecal metabolites cause inflammation and tissue oxidative stress, expectedly, we found oxidative stress in the ovary and systemic inflammation in DEHP exposed mice. Thus, based on our findings, DEHP exposure may cause gut bacterial dysbiosis and altered metabolite profiles, particularly SCFA profiles, leading to oxidative stress in the ovary and systemic inflammation to ultimately induce female reproductive toxicity.

Effects of cryopreservation and long-term culture on biological characteristics and proteomic profiles of human umbilical cord-derived mesenchymal stem cells.

BACKGROUND: Human umbilical cord-derived MSCs (hUC-MSCs) have been identified as promising seeding cells in tissue engineering and clinical applications of regenerative medicine due to their advantages of simple acquisition procedure and the capability to come from a young tissue donor over the other MSCs sources. In clinical applications, large scale production is required and optimal cryopreservation and culture conditions are essential to autologous and allogeneic transplantation in the future. However, the influence of cryopreserved post-thaw and long-term culture on hUC-MSCs remains unknown, especially in terms of specific protein expression. Therefore, biological characteristics and proteomic profiles of hUC-MSCs after cryopreserving and long-term culturing were investigated. METHODS: Firstly, hUC-MSCs were isolated from human umbilical cord tissues and identified through morphology, surface markers and tri-lineage differentiation potential at passage 3, and then the biological characteristics and proteomic profiles were detected and compared after cryopreserving and long-term culturing at passage 4 and continuously cultured to passage 10 with detection occurring here as well. The proteomic profiles were tested by using the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique and differential protein were confirmed by mass spectrometry. RESULTS: The results showed no significant differences in phenotypes including morphology, surface marker and tri-lineage differentiation potential but have obvious changes in translation level, which is involved in metabolism, cell cycle and other pathways. CONCLUSION: This suggests that protein expression may be used as an indicator of hUC-MSCs security testing before applying in clinical settings, and it is also expected to provide the foundation or standardization guide of hUC-MSCs applications in regenerative medicine.

Risk and mechanism of glucose metabolism disorder in the offspring conceived by female fertility maintenance technology.

Although female fertility maintenance technology (FFMT) provides an effective option for preserving fertility in patients with cancer suffering from fertility loss due to cancer treatment, previous studies have shown that the technique has certain potential risks and requires an assessment of the health status of the offspring since FFMT may lead to glucose metabolism disorder in offspring mice. The present animal study examined the glucose metabolism of adult mice offspring born from ovarian tissue cryopreservation and orthotopic allotransplantation. The mice were divided into three groups: normal, fresh ovary transplantation, and cryopreserved ovary transplantation. We recorded fasting blood glucose, glucose tolerance, and fasting serum insulin level for six months. Liver DNA, RNA, and proteins were extracted to detect the interaction between DNA methylation and Grb10 expression and insulin signaling pathway factors such as P-IGF1R, P-IRS2, P-AKT, and Grb10. Female recipient mice that received FFMT could successfully give birth after mating. The average litter size and total litter size of the cryopreserved and fresh groups showed marked differences compared with the normal group. Compared with the normal group, the fasting blood glucose and fasting serum insulin levels were higher in the cryopreserved and fresh groups. The mRNA and protein expressions of Grb10 were higher in the fresh and cryopreserved groups. Compared with the normal group, the DNA methylation status of four of the 11 sites of the Grb10 promoter was lower in the cryopreserved group. Grb10 overexpression inhibited the downstream phosphorylation protein factor expression (p-IGF-1R, p-IRS2, and p-Akt) of the IGF-1R signaling pathway. Female fertility maintenance technology (FFMT), including ovarian tissue cryopreservation (OTC), and orthotopic allotransplantation techniques might lead to glucose metabolism disorders in offspring mice.

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.

Integrated transcriptomic and metabolomic analysis reveals the underlying mechanisms for male reproductive toxicity of polystyrene nanoplastics in mouse spermatocyte-derived GC-2spd(ts) cells.

BACKGROUND: Polystyrene nanoplastics (PS-NPs), are ubiquitous pollution sources in human environments, posing significant biosafety and health risks. While recent studies, including our own, have illustrated that PS-NPs can breach the blood-testis barrier and impact germ cells, there remains a gap in understanding their effects on specific spermatogenic cells such as spermatocytes. METHODS AND RESULTS: Herein, we employed an integrated approach encompassing phenotype, metabolomics, and transcriptomics analyses to assess the molecular impact of PS-NPs on mouse spermatocyte-derived GC-2spd(ts) cells. Optimal exposure conditions were determined as 24 h with 50 nm PS-NPs at 12.5 µg/mL and 90 nm PS-NPs at 50 µg/mL for subsequent multi-omics analysis. Our findings revealed that PS-NPs significantly influenced proliferation and viability, causing alterations in transcriptome and metabolome profiles. Transcriptomics analysis of GC-2spd(ts) cells exposed to PS-NPs indicated the pivotal involvement of cell proliferation and cycle, autophagy, ferroptosis, and redox reaction pathways in PS-NP-induced effects on the proliferation and viability of GC-2spd(ts) cells. Furthermore, metabolomics analysis identified major changes in amino acid metabolism, cyanoamino acid metabolism, and purine and pyrimidine metabolism following PS-NP exposure. CONCLUSION: Our integrated approach, combining metabolomics and transcriptomics profiles with phenotype data, enhances our understanding of the adverse effects of PS-NPs on germ cells.

Therapeutic efficacy and in vivo distribution of human umbilical cord-derived mesenchymal stem cell spheroids transplanted via B-Ultrasound-guided percutaneous portal vein puncture in rhesus monkey models of liver fibrosis.

BACKGROUND: Liver fibrosis can progress to end-stage cirrhosis and liver cancer. Mesenchymal stem cells (MSCs) were considered the most promising therapeutic strategy, but most of the MSCs injected intravenously traditionally are trapped in the lungs, rapidly reducing their survival ability. MSC spheroids cultured in 3D have shown higher tolerance to fluid shear stress and better survival than dissociated MSCs. Simulating the route of orthotopic liver transplantation, transplanting MSC spheroids into the liver via hepatic portal vein may impact superior therapeutic effects. METHODS: In the present study, human umbilical cord-derived MSC spheroids (hUC-MSCsp) were transplanted into rhesus monkey models of liver fibrosis via B-ultrasound-guided percutaneous portal vein puncture with minimized body invasion. The therapeutic effect is evaluated through hematology, ultrasound, and pathology. To study the effect of hUC-MSCsp on gene expression in rhesus monkeys with liver injury, transcriptome sequencing analysis was performed on the livers of rhesus monkeys. The distribution of transplanted hUC-MSCsp was traced with RNA scope technology. RESULTS: We found that hUC-MSCsp significantly restored liver function, including ALT, AST, ALB, GLOB and bilirubin. hUC-MSCsp also significantly reduced liver collagen deposition and inflammatory infiltration, and promote dismission of liver ascites. Subsequently, the therapeutic effects were further validated in TGF-ß1/Smad pathway by global transcription profile. The distribution of transplanted hUC-MSCsp were also tracked, and we found that hUC-MSCsp distributed in the liver in a sphere status at 1 h after transplantation. After 16 days, the hUC-MSCsp were dispersed into dissociated cells that were predominantly distributed in the spleen, and a significant number of dissociated cells were still present in the liver. CONCLUSIONS: This study reveals the distributions of transplanted hUC-MSCsp after liver portal vein transplantation, and provides a novel approach and new insights into the molecular events of potential molecular events underlying the treatment of liver fibrosis with hUC-MSCsp.

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.

Toxicity to the Male Reproductive System after Exposure to Polystyrene Nanoplastics: A Macrogenomic and Metabolomic Analysis.

Nanoplastics (NPs) cause serious contamination of drinking water and potential damage to human health. This study aimed to investigate the effects of NPs with different particle sizes and concentrations on the reproductive function of male mice. In this study, free drinking water exposure was used to expose male BALB/C mice to PS-NPs (20 nm, 200 nm, and 1000 nm) at 0.1 mg/L, 1 mg/L, and 5 mg/L for 4 months. The male reproductive function of the mice was assessed after NPs exposure, and fecal and blood samples were collected for macrogenomics and metabolomics. The results showed that PS-NPs resulted in mice with reduced testicular organ coefficients, decreased sperm quality, altered testicular tissue structure, disturbed sex hormone levels, and abnormal levels of inflammatory factors and oxidative stress. Furthermore, this study found that NP exposure affected the alteration of gut communities and metabolic pathways related to male reproduction, such as Clostridium and glutathione metabolism. Importantly, we found an effect of NP particle size on reproductive function. In the future, more attention should be paid to the smaller particle sizes of NPs.

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.

Identification of biomarkers and the mechanisms of multiple trauma complicated with sepsis using metabolomics.

Sepsis after trauma increases the risk of mortality rate for patients in intensive care unit (ICUs). Currently, it is difficult to predict outcomes in individual patients with sepsis due to the complexity of causative pathogens and the lack of specific treatment. This study aimed to identify metabolomic biomarkers in patients with multiple trauma and those with multiple trauma accompanied with sepsis. Therefore, the metabolic profiles of healthy persons designated as normal controls (NC), multiple trauma patients (MT), and multiple trauma complicated with sepsis (MTS) (30 cases in each group) were analyzed with ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS)-based untargeted plasma metabolomics using collected plasma samples. The differential metabolites were enriched in amino acid metabolism, lipid metabolism, glycometabolism and nucleotide metabolism. Then, nine potential biomarkers, namely, acrylic acid, 5-amino-3-oxohexanoate, 3b-hydroxy-5-cholenoic acid, cytidine, succinic acid semialdehyde, PE [P-18:1(9Z)/16:1(9Z)], sphinganine, uracil, and uridine, were found to be correlated with clinical variables and validated using receiver operating characteristic (ROC) curves. Finally, the three potential biomarkers succinic acid semialdehyde, uracil and uridine were validated and can be applied in the clinical diagnosis of multiple traumas complicated with sepsis.

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.

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.

Exploring the Rescue Strategy for Cardiac Arrest in Makeshift (FangCang) Hospital Workers during the Pneumonia Outbreak Associated with COVID-19.

BACKGROUND: Beginning in Dec 2019, a novel coronavirus, designated SARS-CoV-2, has caused an international outbreak of respiratory illness termed COVID-19. The workers in the FangCang hospital have to work for more than 8 h and the work is high strength. Furthermore, to protect health and prevent serious cross-infection, they need to wear isolation equipment when working in FangCang hospital. These characteristics increase the risk of cardiac arrest (CA), which seriously endangers the lives of workers. METHODS: We participated in the rescue of the patient and workers at first-line in FangCang hospital, and summarized the rescue strategies for workers rescuing. RESULTS: Workers with CA were rescued in time according our guideline and showed zero dead in FangCang hospital. CONCLUSION: This study establishes the strategy for the CA of workers including the establishment of an in-FangCang resuscitation team, the establishment of a dedicated rescue room, and the formulation of rescue measures and procedures for CA of workers in the FangCang hospital. Therefore, we aimed to provide a strategy for the rescue of workers with CA in the FangCang hospital and share the success in rescuing with the world.

In vitro differentiation of rhesus macaque bone marrow- and adipose tissue-derived MSCs into hepatocyte-like cells.

Orthotopic liver or hepatocyte transplantation is effective for the treatment of acute liver injury and end-stage chronic liver disease. However, both of these therapies are hampered by the extreme shortage of organ donors. The clinical application of cell therapy through the substitution of hepatocytes with mesenchymal stem cells (MSCs) that have been differentiated into hepatocyte-like cells (HLCs) for liver disease treatment is expected to overcome this shortage. Bone marrow and adipose tissue are two major sources of MSCs [bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived MSCs (AT-MSCs), respectively]. However, knowledge about the variability in the differentiation potential between BM-MSCs and AT-MSCs is lacking. In the present study, the hepatogenic differentiation potential of rhesus macaque BM-MSCs and AT-MSCs was compared with the evaluation of morphology, immunophenotyping profiles, differentiation potential, glycogen deposition, urea secretion and hepatocyte-specific gene expression. The results indicated that BM-MSCs and AT-MSCs shared similar characteristics in terms of primary morphology, surface markers and trilineage differentiation potential (adipogenesis, osteogenesis and chondrogenesis). Subsequently, the hepatogenic differentiation potential of BM-MSCs and AT-MSCs was evaluated by morphology, glycogen accumulation, urea synthesis and expression of hepatocyte marker genes. The results indicated that rhesus BM-MSCs and AT-MSCs had hepatogenic differentiation ability. To the best of our knowledge, this is the first report to detect the hepatogenic differentiation potential of rhesus macaque BM-MSCs and AT-MSCs. The present study provides the basis for the selection of seed cells that can trans-differentiate into HLCs for cytotherapy of acute or chronic liver injuries in either clinical or veterinary practice.