Efficacy of Endoscopic Tissue Adhesive in Patients with Gastrointestinal Tumor Bleeding.

BACKGROUND: Gastrointestinal tumors bleeding remains a significantly clinical challenge due to its resistance to conventional endoscopic hemostasis methods. While the efficacy of endoscopic tissue adhesives (ETA) in variceal bleeding has been established, its role in gastrointestinal tumor bleeding (GITB) remains ambiguous. AIMS: This study aims to assess the feasibility and effectiveness of ETA in the treatment of GITB. METHODS: The study enrolled 30 patients with GITB who underwent hemostasis through Histoacryl® tissue glue injection. Hemostasis success rates, ETA-related adverse events, and re-bleeding rates were evaluated. RESULTS: ETA application achieved successful hemostasis at all tumor bleeding sites, with immediate hemostasis observed in all 30 (100.0%) patients. Among the initially hemostasis cases, 5 patients (17.0%) experienced re-bleeding within 30 days, and the 60 day re-bleeding rate was 20.0% (6/30). Expect for one case of vascular embolism, no adverse events related with ETA application were reported. The 6 month survival was 93%. CONCLUSION: ETA demonstrated excellent immediate hemostasis success rate in GITB cases and showed promising outcomes in prevention re-bleeding.

[Gly14]-humanin exerts a protective effect against D-galactose-induced primary ovarian insufficiency in mice.

RESEARCH QUESTION: Is there a protective effect of the humanin derivative [Gly14]-humanin (HNG) on a D-gal-induced mouse model of primary ovarian insufficiency (POI), and what is the underlying mechanism? DESIGN: D-gal (200 mg/kg/day) was injected subcutaneously for 6 weeks to induce the mouse POI model. Mice treated with HNG were injected intraperitoneally with different concentrations for 6 weeks. Ovarian morphology, function, levels of sex hormones and states of oxidative stress in the ovary and body were evaluated. RESULTS: Compared with the D-gal group, 10 mg/kg HNG improved the abnormal ovarian morphology and oestrous cycle (P = 0.0036), increased the number of ovarian follicles (P = 0.0016) and litters (P = 0.0127), and increased the levels of oestrogen (P = 0.0043) and AMH (P = 0.0147). Antioxidant indicators in the ovaries and serum of mice, including total antioxidant capacity (P = 0.0004 and P = 0.0032, respectively), catalase (P = 0.0173 and P = 0.0103, respectively) and glutathione (both P < 0.0001) were significantly increased. The oxidation indicator malondialdehyde decreased significantly (all P < 0.01). Apoptosis of ovarian granulosa cells was significantly reduced (P = 0.0140) as was the expression of senescence-related proteins p53, p21 and p16 (all P < 0.01). The level of autophagy in ovarian tissue of mice treated with high increased (significantly increased LC3 protein [P < 0.0001] and significantly reduced p62 protein [P = 0.0007]). CONCLUSIONS: HNG inhibited D-gal-induced oxidative stress, apoptosis and ovarian damage, promoting ovarian autophagy. HNG may be a potential prophylactic agent against POI.

Fluorene-9-bisphenol exposure damages the testis in mice through a novel mechanism of ferroptosis.

Fluorene-9-bisphenol (BHPF) is an emerging global endocrine-disrupting chemical found in numerous household products as a substitute of bisphenol A. Many studies have reported various toxicities associated with BHPF. However, the effect of BHPF on male reproduction, particularly on the structural integrity of the blood testis barrier (BTB) in mice, has not yet been extensively studied. Ferroptosis, a newly identified form of cell death, occurs in the testicular tissue following exposure to BPA, affecting male fertility. We investigated whether ferroptosis plays a role in BHPF-induced testicular damage. The findings indicated that BHPF exposure led decreases in serum testosterone (T) concentration and sperm concentration and motility in mice. Furthermore, BHPF disrupted the BTB by interfering with key BTB-related proteins, including Cx43, ß-catenin, and ZO-1. Moreover, BHPF induced ferroptosis through the induction of lipid peroxidation, iron overload, oxidative stress, and mitochondrial dysfunction in the testicular tissue. Inhibition of ferroptosis using Fer-1 mitigated the BHPF-induced damage to the BTB and ferroptosis in TM4 cells. Overall, our findings indicated the detrimental effects of BHPF on male reproductive function in mice, suggesting ferroptosis as a mechanism underlying testicular damage.

Knockdown of SETD5 inhibited glycolysis and tumor growth in gastric cancer cells by down-regulating Akt signaling pathway.

Gastric cancer (GC) is the 5th most common cancer and the 3rd leading cause of cancer-related death worldwide. It is of great significance to study the underlying molecular mechanism of GC, and targeting glycolysis is a good strategy to treat GC. SET domain containing 5 (SETD5) contains a catalytic methyltransferase SET domain, which is known as a lysine methyltransferase that affects the progression of multiple cancers. However, its possible role in GC was still unclear. Here, we revealed that SETD5 was highly expressed in GC and was associated with a poor prognosis. Further through the in vitro experiments, we revealed that the downregulation of SETD5 inhibited the proliferation and migration of GC cells. Knockdown of SETD5 inhibited glucose consumption and glycolysis. Further studies have shown that SETD5 knockdown restrained the Akt signaling pathway. Therefore, we thought that SETD5 could act as a GC target.

The humanin analogue (HNG) alleviates intrauterine adhesions by inhibiting endometrial epithelial cells ferroptosis: a rat model-based study.

STUDY QUESTION: Does a humanin analogue (HNG) have a therapeutic effect on intrauterine adhesions (IUAs) caused by uterine cavity surgery in a rat model? SUMMARY ANSWER: HNG supplementation attenuated the development of endometrial fibrosis and IUAs, improved fertility, and contributed to the regulation of endometrial fibrosis by inhibiting endometrial ferroptosis in rats with IUAs. WHAT IS KNOWN ALREADY: IUAs, which are characterized by endometrial fibrosis, are a common cause of female infertility. Humanin (rattin in rats) is a mitochondrial-derived peptide that is widely expressed in multiple tissues. S14G-humanin (HNG) is an HNG that has been reported to have a protective effect against myocardial fibrosis. STUDY DESIGN, SIZE, DURATION: Endometrial tissues from three patients with IUAs and three controls were tested for humanin expression. Two animal models were used to evaluate the modelling effect of IUAs and the preventive effect of HNG against IUAs. In the first model, 40 rats were equally randomized to control and Day 7, 14, and 21 groups to establish the IUA model. In the second model, 66 rats were equally randomized to the control, IUA, and IUA + humanin analogue (HNG) groups. Erastin was used to induce ferroptosis in the Ishikawa cell line. PARTICIPANTS/MATERIALS, SETTING, METHODS: The endometrium was scraped with a surgical spatula, combined with lipopolysaccharide treatment, to establish the rat model of IUAs. Rats were intraperitoneally injected with 5 mg/kg/day HNG for 21 consecutive days beginning from the day of operation to evaluate the therapeutic effect on IUAs. Haematoxylin-eosin and Masson's trichrome staining were used to assess endometrial morphology and evaluate fibrosis. Ferroptosis-related markers, namely nuclear factor E2-related factor 2 (Nrf2), acyl-CoA synthetase long-chain family member 4 (ACSL4), haeme oxygenase-1 (HO-1), solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), and ferritin, were measured by immunohistochemistry and western blotting to determine whether ferroptosis was involved in the development of IUAs and to assess the attenuative effect of HNG on ferroptosis. Additionally, the female rats were mated with male rats with normal fertility to assess fertility. MAIN RESULTS AND THE ROLE OF CHANCE: Humanin was widely expressed in endometrial cells, including epithelial and stromal cells, in both humans and rats. Humanin expression levels were downregulated in the endometria of patients and rats with IUAs relative to the endometria of controls. Endometrial thickness and the number of glands were significantly decreased on Day 7, 14, and 21 after endometrial scraping when compared with the controls (all P < 0.05), whereas the fibrotic area was significantly increased (P < 0.05). Among the tested ferroptosis markers, the expression levels of Nrf2, SLC7A11, and GPX4 were significantly downregulated and those of ACSL4, HO-1, and ferritin were significantly upregulated after endometrial scraping relative to their expression levels in controls (all P < 0.05). The mating rates in the control, IUA, and IUA + HNG groups were 100% (10/10), 40% (4/10), and 80% (8/10), respectively. The number of embryos in rats with IUAs (mean ± SD: 1.6 ± 2.1) was significantly less than the number in the controls (11.8 ± 1.5). HNG supplementation significantly attenuated this decrease in the number of implanted embryos (6.3 ± 4.5) (P < 0.01). Further results showed that HNG significantly attenuated the altered expression levels of proteins involved in ferroptosis in the endometria of rats with IUAs. Moreover, in vitro experiments showed that HNG significantly attenuated the erastin-induced decrease in the viability of the Ishikawa cell line and also attenuated the increase in reactive oxygen species production and the downregulation of GPX4. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: The findings of this study showed that HNG inhibited ferroptosis and reduced fibrosis in a rat model of IUAs. However, we could not establish a causal relationship between ferroptosis and the development of IUAs. WIDER IMPLICATIONS OF THE FINDINGS: HNG may be effective at alleviating fibrosis during the development of IUAs, and the inhibition of ferroptosis is a promising new strategy for IUA therapy. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation of China (No. 82171647); the '1000 Talent Plan' of Yunnan Province (No. RLQN20200001); and the Basic Research Project of the Yunnan Province-Outstanding Youth Foundation (No. 202101AW070018). The authors declare no competing financial interests.

Bioinformatics analysis of the common targets of miR-223-3p, miR-122-5p, and miR-93-5p in polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is one of the most common gynecological endocrine disorders. MicroRNAs (miRNAs) play extensive roles in the pathogenesis of PCOS and can serve as potential diagnostic markers. However, most studies focused on the regulatory mechanisms of individual miRNAs, and the combined regulatory effects of multiple miRNAs remain unclear. The aim of this study was to identify the common targets of miR-223-3p, miR-122-5p, and miR-93-5p; and assess the transcript levels of some of these targets in PCOS rat ovaries. Transcriptome profiles of granulosa cells from PCOS patients were obtained from the Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs). A total of 1,144 DEGs were screened, 204 of which were upregulated and 940 were downregulated. According to the miRWalk algorithm, 4,284 genes were targeted by all three miRNAs at the same time, and intersection with DEGs was used to obtain candidate target genes. A total of 265 candidate target genes were screened, and the detected target genes were subjected to Gene ontology (GO) and KEGG pathway enrichment, followed by PPI network analysis. Then, qRT-PCR was used to determine the levels of 12 genes in PCOS rat ovaries. The expressions of 10 of these genes were found to be consistent with our bioinformatics results. In conclusion, JMJD1C, PLCG2, SMAD3, FOSL2, TGFB1, TRIB1, GAS7, TRIM25, NFYA, and CALCRL may participate in the development of PCOS. Our findings contribute to the identification of biomarkers that may promote the effective prevention and treatment of PCOS in the future.

Toxicity of polystyrene nanoparticles for mouse ovary and cultured human granulosa cells.

The issue of global environmental contamination of microplastics has recently been receiving widespread attention. However, the effects of polystyrene nanoparticles (Nano-PS) on the female reproductive system remain unclear. We investigated the toxicity and explored the potential underlying mechanisms of Nano-PS in both mouse ovarian tissue in vivo and human ovarian granulosa cell lines in vitro. In vivo experiments: Mice were fed different concentrations of Nano-PS for 8 weeks. In vitro experiments: COV434 cells were treated with increasing concentrations of Nano-PS. In the present study, ovarian reserve was found to decrease significantly, while oxidative stress and apoptosis levels increased. Nano-PS increased the proportion of metestrum and diestrus periods, and decreased the proportion of estrous period. The implantation rates and the number of pups per litter decreased. In COV434 cells, Nano-PS reduced cell viability and mitochondrial membrane potential, increased the expression of apoptotic and oxidative stress markers and led to subsequent cell cycle arrest. Specifically, Nano-PS exert their toxic effects on mouse ovarian tissue and COV434 cells by inducing oxidative stress. A potential strategy to overcome this could be to activate the nuclear factor-E2-related factor 2 (Nrf2) signaling pathway to mitigate Nano-PS-induced oxidative stress.

Endoplasmic reticulum stress promotes endometrial fibrosis through the TGF-ß/SMAD pathway.

In brief: Intrauterine adhesion (IUA) is one of the main causes of female infertility. This study reveals that endoplasmic reticulum stress activation upregulates the TGF-ß/SMAD pathway to induce epithelial-mesenchymal transition and promote endometrial fibrosis in an IUA model. Abstract: IUA is a common gynecological disease and is a leading cause of female infertility. Mechanical or infectious damage to the endometrial basal layer can lead to endometrial fibrosis, which is the most common cause of IUA. Endoplasmic reticulum stress (ERS), the transforming growth factor beta signaling pathway (TGF-ß/SMAD) and epithelial-mesenchymal transition (EMT) are important factors promoting endometrial fibrosis. The purpose of this study was to determine the up- and downstream regulatory relationships of the above three in the process of endometrial fibrosis. The rat IUA model was induced by double injury method and prophylactic injection of the ERS inhibitor 4-phenylbutyric acid (4-PBA) was given in vivo. The ERS activator tunicamycin and the TGF-ß/SMAD pathway inhibitor A 83-01 were used in human endometrial epithelial cells (HEECs) in vitro. Masson's trichrome, Sirius red staining, immunohistochemistry, immunofluorescence and Western blot analyses were used to determine ERS, TGF-ß/SMAD pathway, EMT and fibrosis markers in the uterine tissue and HEECs of the different treatment groups. In animal experiments, ERS and the TGF-ß/SMAD pathway had been activated and EMT occurred in an in vivo model of IUA but was suppressed in animals treated with prophylactic 4-PBA. In in vitro experiments, tunicamycin-treated HEECs had increased the activation of ERS, the abundance of TGF-ß/SMAD pathway and fibrosis markers while EMT occurred, but the TGF-ß/SMAD pathway and EMT were significantly inhibited in the tunicamycin+A 83-01 group. Our data suggest that increased ERS can induce EMT and promote endometrial fibrosis through the TGF-ß/SMAD pathway.

High MEIS3 Expression Indicates a Poor Prognosis for Patients with Stage II/III Colorectal Cancer.

BACKGROUND: The Wnt/ß-catenin signaling pathway plays crucial roles in tumor budding and the epithelial-mesenchymal transition (EMT). Myeloid ecotropic viral insertion site 3 (MEIS3)-a direct target of Wnt/ß-catenin-promotes vagal neural crest cell migration into the gut tissue during development; however, its role in cancer progression remains unclear. In this study, the role of MEIS3 in colorectal cancer (CRC) progression was investigated. METHODS: We analyzed the association between MEIS3 protein expression and the clinical stages of CRC patients, and the effect on tumor cell migration and invasion by wound healing and transwell assays. Finally, we analyzed the association between MEIS3 expression and the disease-free survival (DFS) and overall survival of CRC patients through Kaplan-Meier analysis. RESULTS: We found that MEIS3 expression was strongly associated with CRC progression and could be employed to assess DFS in postoperative patients. MEIS3-positive cells were mainly distributed in the growth front and tumor-stroma interface of the CRC tissues, which contain abundant EMT-active and tumor budding cells dominating cancer metastasis. Moreover, MEIS3 promoted CRC cell migration and invasion by regulating effectors including laminin subunit beta 1, matrix metalloprotein 2, and vimentin. MEIS3 protein expression increased with CRC progression according to the clinical stage, which could be used as a biomarker to stratify CRC patients. The 5-year DFS of MEIS3-high patients was poorer than that of MEIS3-low patients (40.6% vs. 61.7%; p < 0.0001). Moreover, the 5-year DFS of stage II patients with MEIS3-high expression (53.4%) was comparable to that of stage III patients with MEIS3-low expression (49.5%), while the 5-year DFS of MEIS3-high patients in stage III (30.9%) was comparable to that of stage IV patients (29.6%). CONCLUSIONS: This study showed that MEIS3 can promote cancer cell metastasis and thus may be a promising biomarker for higher rates of recurrence in postoperative patients with stage II/III CRC.

N6-methyladenosine RNA demethylase ALKBH5 is testis-specifically downregulated in hybrid male sterile dzo and is a target gene of bta-miR-200a.

N6-methyladenosine (m6A) is the most common RNA methylation modification in mammals, which is controlled in the male germline to ensure coordinated gene expression in the entire process of spermatogenesis. Dzo is the male offspring of a cross between the domestic cattle (Bos taurus) and yak (Bos grunniens), and is sterile. This study aimed to investigate whether m6A-associated genes are linked with dzo male sterility. The mRNA expression pattern of m6A-associated genes and spermatogenesis-related genes modified by m6A was characterized in cattle, yak, and dzo. Compared with fertile cattle and yak, m6A erasing (ALKBH5 and FTO), writing (METTL14, WTAP, and ZC3H13), and reading (YTHDC2, YTHDF1, and YTHDF2) were testis-specifically downregulated in infertile dzo. The expression of m6A target genes in spermatogonial self-renewal and proliferation (BCL6B, FOXO1, TAF4B, and FGFR1) and differentiation genes (DNMT3B and SOHLH2) were dereguleted in dzo testis. Immunofluorescent staining showed that intense ALKBH5 immunoreactivity was present in spermatogonia, primary spermatocytes, and round spermatids of cattle and yak testis. However, the number of ALKBH5 immunoreactive-positive cells were significantly reduced in dzo testis, especially in primary spermatocytes and round spermatids. Whole genome bisulfite-seq data showed that the promoter regions of FTO and YTHDC2 genes were hypermethylated in dzo testis. Moreover, bta-miR-200a was significantly downregulated in dzo testis, and it targeted the m6A-associated genes such as ALKBH5, FTO, WTAP, and YTHDF2. In conclusion, mRNA of ALKBH5 was testis-specifically downregulated in dzo, which may be because fewer specific spermatogenic cells express this gene. The role of m6A-associated genes in dzo male sterility and the interaction of DNA methylation and miRNA with m6A-associated protein expression need to be further explored.

EndMT: New findings on the origin of myofibroblasts in endometrial fibrosis of intrauterine adhesions.

BACKGROUND: Intrauterine adhesion (IUA) is one of the leading causes of infertility and the main clinical challenge is the high recurrence rate. The key to solving this dilemma lies in elucidating the mechanisms of endometrial fibrosis. The aim of our team is to study the mechanism underlying intrauterine adhesion fibrosis and the origin of fibroblasts in the repair of endometrial fibrosis. METHODS: Our experimental study involving an animal model of intrauterine adhesion and detection of fibrosis-related molecules. The levels of molecular factors related to the endothelial-to-mesenchymal transition (EndMT) were examined in a rat model of intrauterine adhesion using immunofluorescence, immunohistochemistry, qPCR and Western blot analyses. Main outcome measures are levels of the endothelial marker CD31 and the mesenchymal markers alpha-smooth muscle actin (α-SMA) and vimentin. RESULTS: Immunofluorescence co-localization of CD31 and a-SMA showed that 14 days after moulding, double positive cells for CD31 and a-SMA could be clearly observed in the endometrium. Decreased CD31 levels and increased α-SMA and vimentin levels indicate that EndMT is involved in intrauterine adhesion fibrosis. CONCLUSIONS: Endothelial cells promote the emergence of fibroblasts via the EndMT during the endometrial fibrosis of intrauterine adhesions.

Cold-inducible RNA-binding protein regulates cyclin B1 against spermatogenesis arrest caused by heat stress.

BACKGROUND: Spermatogenesis arrest and spermatogenic cell apoptosis occur in the testes of heat-stressed mice. Although heat stress-induced spermatogenic cell apoptosis is due to the decreased expression of cold-inducible RNA-binding protein (CIRBP), it remains unclear whether spermatogenesis arrest is also affected by CIRBP. Additionally, the specific mechanism by which CIRBP regulates spermatogenic cell apoptosis or inhibits spermatogenesis remains to be elucidated. OBJECTIVES: To investigate the mechanism by which CIRBP contributes to heat stress-induced testicular spermatogenesis arrest. MATERIALS AND METHODS: Target mRNAs downstream of CIRBP in testicular tissue of BALB/c mice, exposed or not to heat stress, were sequenced. Sequencing data were subjected to bioinformatics analysis to identify key mRNAs and pathways associated with heat stress-induced spermatogenic damage. The link between CIRBP and its target mRNA Ccnb1 (cyclin B1) was verified by western blotting, flow cytometry, and RNA pulldown assays, and the ability of CIRBP to inhibit germ cell cycle arrest by regulating cyclin B1 expression was investigated in a mouse spermatocyte cell line (GC-2spd). RESULTS: Changes in mRNA expression downstream of CIRBP were mainly associated with the cell cycle and RNA binding, transport and splicing. Cyclin B1 was found to regulate the G2/M transition during the first meiotic division of spermatogenic cells. Further, CIRBP was shown to bind directly to the 3'-untranslated region of Ccnb1 mRNA and was associated with cyclin B1-induced inhibition of spermatogenesis arrest. DISCUSSION AND CONCLUSION: In conclusion, our results provide strong evidence that CIRBP may exert its key function in heat stress-induced testicular spermatogenic cell injury partly by regulating the expression of Ccnb1, the product of which inhibits spermatogenesis arrest.

Construction of Potential Gene Expression and Regulation Networks in Prostate Cancer Using Bioinformatics Tools.

OBJECTIVE: To identify the key genes involved in prostate cancer and their regulatory network. METHODS: The dataset of mRNA/miRNA transcriptome sequencing was downloaded from The Cancer Genome Atlas/the Gene Expression Omnibus database for analysis. The "edgeR" package in the R environment was used to normalize and analyze differentially expressed genes (DEGs) and miRNAs (DEmiRNAs). First, the PANTHER online tool was used to analyze the function enrichment of DEGs. Next, a protein-protein interaction (PPI) network was constructed using STRING and Cytoscape tools. Finally, miRNA-gene regulatory networks were constructed using the miRTarBase. RESULTS: We identified 4339 important DEGs, of which 2145 were upregulated (Up-DEGs) and 2194 were downregulated (Down-DEGs). Functional enrichment analysis showed that the Up-DEGs were related to the immune system and the cell cycle in prostate cancer, whereas the Down-DEGs were related to the nucleic acid metabolic process and metabolism pathways. Twelve core protein clusters were found in the PPI network. Further, the constructed miRNA-gene interaction network showed that 11 downregulated miRNAs regulated 16 Up-DEGs and 22 upregulated miRNAs regulated 22 Down-DEGs. CONCLUSION: We identified 4339 genes and 70 miRNAs that may be involved in immune response, cell cycle, and other key pathways of the prostate cancer regulatory network. Genes such as BUB1B, ANX1A1, F5, HTR4, and MUC4 can be used as biomarkers to assist in the diagnosis and prognosis of prostate cancer.

Differential expression of microRNAs in human endometrium after implantation of an intrauterine contraceptive device containing copper.

Intrauterine devices containing copper placement will release a large amount of Cu2+ into the uterine fluid, leading to local endometrial damage and inflammation, which is considered to be one of the causes of abnormal uterine bleeding. Studies have shown that the metabolism and function of metal ions are related to the regulation of microRNA. The aims of this study were to investigate changes in endometrial microRNA levels after implantation of an intrauterine device containing copper and to preliminarily explore the signalling pathways involved in abnormal uterine bleeding. The subjects were fertile women, aged 25-35, without major obstetrics and gynaecology diseases. Human endometrial tissues were collected before implantation or removal of the intrauterine device containing copper. High-throughput microRNA sequencing was performed on human endometrial tissues, and real-time quantitative PCR, western blotting and immunohistochemistry were used to detect the expression of relevant genes. MicroRNA sequencing results showed that 72 miRNAs were differentially expressed in the endometrial tissue after the insertion of the intrauterine device containing copper. Implantation of an intrauterine device containing copper implantation can up-regulate the expression of miR-144-3p in endometrial tissue, and therefore, decreases the mRNA and protein expression levels of genes related to endometrial injury and tissue repair, including the MT/NF-κB/MMP damage pathway and the THBS-1/TGF-ß/SMAD3 repair pathway. In this study, the molecular mechanisms of abnormal uterine bleeding due to an intrauterine device containing copper were preliminarily investigated. The information will be beneficial for the clinical treatment of abnormal uterine bleeding caused by intrauterine device.

Copper Nanoparticles Induce Oxidative Stress via the Heme Oxygenase 1 Signaling Pathway in vitro Studies.

PURPOSE: The toxicity of copper nanoparticle (CuNP) exposure in the ovaries has attracted attention recently, but the precise molecular mechanism involved requires further investigation. We investigated the cytotoxicity of CuNPs in ovarian granulosa cells and the protective effect of heme oxygenase 1 (HO-1) against CuNP-induced damage. METHODS: Human ovarian granulosa cells (COV434) were treated with CuNPs, and cytotoxicity was evaluated using Cell Counting Kit-8 and flow cytometry assays. Oxidative stress was identified using biochemical markers of oxidation and anti-oxidation. The protein levels of mitogen-activated protein kinase 14 (MAPK14), phospho-MAPK14, nuclear factor erythroid 2-related factor 2 (Nrf2), and HO-1 were measured by immunoblotting. Subsequently, for oxidative stress parameter detection, the cells were pre-treated with hemin to induce HO-1 expression prior to CuNP treatment. RESULTS: Exposure to CuNPs decreased cell viability and the mitochondrial membrane potential, increased the apoptosis rate, and induced oxidative stress. Furthermore, hemin pretreatment induced HO-1 expression in cells, which partially reduced the accumulation of reactive oxygen species induced by CuNPs and increased the levels of antioxidant enzymes. CONCLUSION: CuNPs exert cytotoxic effects on human ovarian granulosa cells by inducing oxidative stress, and may induce HO-1 expression via the MAPK14-Nrf2 signaling pathway. Moreover, HO-1 protects against oxidative stress induced by CuNPs.

Humanin Alleviates Insulin Resistance in Polycystic Ovary Syndrome: A Human and Rat Model-Based Study.

Polycystic ovary syndrome (PCOS), the most common endocrine disorder in women of reproductive age, is characterized by hyperandrogenism and insulin resistance (IR); however, the pathogenesis of local ovarian IR in PCOS remains largely unclear. Humanin, a mitochondria-derived peptide, has been reported to be associated with IR. Our previous study confirmed that humanin is expressed in multiple cell types in the ovary and is present in follicular fluid. However, it remains unknown whether humanin participates in the pathogenesis of local ovarian IR or whether humanin supplementation can improve IR in PCOS patients. In this study, we compared humanin concentrations in follicular fluid from PCOS patients with and without IR. We further investigated the effect of humanin analogue (HNG) supplementation on IR in a rat model of dehydroepiandrosterone-induced PCOS. Humanin concentrations in the follicular fluid were found to be significantly lower in PCOS patients with IR than in those without IR. HNG supplementation attenuated both the increases in the levels of fasting plasma glucose and fasting insulin in rats with PCOS and the decreases in phosphorylation of IRS1, PI3K, AKT, and GLUT4 proteins in the granulosa cells of these rats. Combined supplementation with HNG and insulin significantly improved glucose consumption in normal and humanin-siRNA-transfected COV434 cells. In conclusion, downregulated humanin in the ovaries may be involved in the pathogenesis of IR in PCOS, and exogenous supplementation with HNG improved local ovarian IR through modulation of the IRS1/PI3K/Akt signaling pathway in a rat model. This finding supports the potential future use of HNG as a therapeutic drug for PCOS.

Humanin regulates oxidative stress in the ovaries of polycystic ovary syndrome patients via the Keap1/Nrf2 pathway.

Polycystic ovary syndrome (PCOS) is the most common endocrinological pathology among women of reproductive age, whereas the pathogenesis is still not fully understood. Systemic and ovarian oxidative stress (OS) imbalance is a pivotal feature of PCOS. Humanin, a mitochondria-derived peptide, has been reported to function as an antioxidant in cardiomyocytes, pancreatic beta cells and other cells, but how this function is regulated remains unclear. In this study, we investigated whether humanin expression differs in the granulosa cells (GCs) of PCOS patients versus controls, and whether humanin alleviates OS in PCOS ovaries. Sixteen PCOS patients and 28 age- and BMI-matched controls undergoing IVF were recruited, and their serum, follicular fluid and GCs were collected for humanin analysis. Dehydroepiandrosterone-induced rat PCOS models, and vitamin K3-induced OS COV434 cell lines were applied to investigate the mechanism. Humanin expression was significantly down-regulated in the ovaries of PCOS patients relative to those of non-PCOS patients. Exogenous humanin supplementation significantly attenuated body weight gain, ovarian morphological abnormalities, endocrinological disorders and ovarian and systemic OS in PCOS rat models. Our study further demonstrated that this attenuation effect was involved in the modulation of the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor-erythroid 2-related factor 2 (Nrf2) signalling pathway. In summary, this study reported for the first time that decreased expression of humanin in the GCs was associated with oxidative imbalance in PCOS. Humanin alleviates OS in ovarian GCs of PCOS patients via modulation of the Keap1/Nrf2 signalling pathway.

The role of miR-128-3p through MAPK14 activation in the apoptosis of GC2 spermatocyte cell line following heat stress.

BACKGROUND: MicroRNAs play a crucial role in the regulation of spermatogenesis. For example, miR-128-3p expression is known to decrease significantly after testicular hyperthermia, but the regulatory effect of this change on the spermatogenesis damage caused by heat stress remains unclear. OBJECTIVES: This study aimed to verify whether the target gene of miR-128-3p is MAPK14, which affects spermatogenic cell proliferation and apoptosis under testicular hyperthermia. MATERIALS AND METHODS: Mouse testis and GC2 spermatocyte cell line heat stress models were established. miR-128-3p expression before and after heat stress was analyzed by reverse transcription polymerase chain reaction. MAPK14 and p-MAPK14 expression was detected by Western blot, and cell apoptosis was analyzed by Annexin V-FITC/PI. Subsequently, miR-128-3p inhibitors and mimics were used to interfere with spermatocytes before and after heat stress, respectively, for correlation detection. RESULTS: Compared with the control group, the heat stress group showed decreased miR-128-3p expression, increased p-MAPK14 expression, and decreased cell proliferation activity. In the GC2-spd cell line in vitro, miR-128-3p inhibitors were found to upregulate p-MAPK14 expression, reduce cell proliferation activity, and increase apoptosis, consistent with the results obtained in the heat treatment alone. Furthermore, miR-128-3p mimics transfected in the GC2 cells after heat stress reduced p-MAPK14 expression, alleviated the decrease in cell proliferation, and decreased the apoptosis level. CONCLUSIONS: The downregulation of miR-128-3p expression plays an important role in spermatogenesis damages after testicular hyperthermia, which is probably attributable to the activation of the MAPK signaling pathway. Downregulated miR-128-3p expression induces the apoptosis and inhibits the proliferation of spermatogenic cells by promoting MAPK14 phosphorylation.