Exosome-mediated miR-200a delivery into TGF-ß-treated AGS cells abolished epithelial-mesenchymal transition with normalization of ZEB1, vimentin and Snail1 expression.

Exosomes are small extracellular vesicles that can be derived from human cells such as mesenchymal stem cells (MSCs). The size of exosomes is at nano-scale range and owing to their biocompatibility and other characteristics, they have been promising candidates for delivery of bioactive compounds and genetic materials in disease therapy, especially cancer therapy. Gastric cancer (GC) is a leading cause of death among patients and this malignant disease affects gastrointestinal tract that its invasiveness and abnormal migration mediate poor prognosis of patients. Metastasis is an increasing challenge in GC and microRNAs (miRNAs) are potential regulators of metastasis and related molecular pathways, especially epithelial-to-mesenchymal transition (EMT). In the present study, our aim was to explore role of exosomes in miR-200a delivery for suppressing EMT-mediated GC metastasis. Exosomes were isolated from MSCs via size exclusion chromatography. The synthetic miR-200a mimics were transfected into exosomes via electroporation. AGS cell line exposed to TGF-ß for EMT induction and then, these cells cultured with miR-200a-loaded exosomes. The transwell assays performed to evaluate GC migration and expression levels of ZEB1, Snail1 and vimentin measured. Exosomes demonstrated loading efficiency of 5.92 ± 4.6%. The TGF-ß treatment transformed AGS cells into fibroblast-like cells expressing two stemness markers, CD44 (45.28%) and CD133 (50.79%) and stimulated EMT. Exosomes induced a 14.89-fold increase in miR-200a expression in AGS cells. Mechanistically, miR-200a enhances E-cadherin levels (P < 0.01), while it decreases expression levels of ß-catenin (P < 0.05), vimentin (P < 0.01), ZEB1 (P < 0.0001) and Snail1 (P < 0.01), leading to EMT inhibition in GC cells. This pre-clinical experiment introduces a new strategy for miR-200a delivery that is of importance for preventing migration and invasion of GC cells.

Photobiomodulation therapy reverses spermatogenesis arrest in hyperthermia-induced azoospermia mouse model.

Testicular heat stress leads to impairment of spermatogenesis in mammals. Involved mechanism in this vulnerability to heat-induced injury remains unclear, and research is being conducted to find an approach to reverse spermatogenesis arrest caused by hyperthermia. Recently, different studies have utilized photobiomodulation therapy (PBMT) therapy for the improvement of sperm criteria and fertility. This study aimed at evaluating the effect of PBMT on the improvement of spermatogenesis in mouse models of hyperthermia-induced azoospermia. A total of 32 male NMRI mice were equally divided into four groups consisting of control, hyperthermia, hyperthermia + Laser 0.03 J/cm2, and hyperthermia + Laser 0.2 J/cm2. To induce scrotal hyperthermia, mice were anesthetized and placed in a hot water bath at 43 °C for 20 min for 5 weeks. Then, PBMT was operated for 21 days using 0.03 J/cm2 and 0.2 J/cm2 laser energy densities in the Laser 0.03 and Laser 0.2 groups, respectively. Results revealed that PBMT with lower intensity (0.03 J/cm2) increased succinate dehydrogenase (SDH) activity and glutathione (GSH)/oxidized glutathione (GSSG) ratio in hyperthermia-induced azoospermia mice. At the same time, low-level PBMT reduced reactive oxygen species (ROS), mitochondrial membrane potential, and lipid peroxidation levels in the azoospermia model. These alterations accompanied the restoration of spermatogenesis manifested by the elevated number of testicular cells, increased volume and length of seminiferous tubules, and production of mature spermatozoa. After conducting experiments and analyzing the results, it has been revealed that the use of PBMT at a dosage of 0.03 J/cm2 has shown remarkable healing effects in the heat-induced azoospermia mouse model.

Sertoli cell-conditioned medium restores spermatogenesis in azoospermic mouse testis.

The current study evaluates potential applications of Sertoli cell (SC)-conditioned medium (CM) and explores the effects of the conditioned medium on the spermatogenesis process in azoospermic mice. For this study, 40 adult mice (28-30 g) were divided into 4 experimental groups: (1) control, (2) DMSO 2% (10 µl), (3) busulfan (40 mg/kg single dose) and (4) busulfan/CM (10 µl). SCs were isolated from 4-week-old mouse testes. After using anesthetics, 10 µl of CM was injected over 3-5 min into each testis and subsequently, sperm samples were collected from the tail of the epididymis. Afterward, the animals were euthanized and testis samples were taken for histopathology experiments and RNA extraction in order to examine the expression of c-kit, STRA8 and PCNA genes. The data showed that CM notably increased the total sperm count and the number of testicular cells, such as spermatogonia, primary spermatocytes, round spermatids, SCs and Leydig cells compared with the control, DMSO and busulfan groups. Furthermore, the results showed that expression of c-kit and STRA8 was significantly decreased in the busulfan and busulfan/SC groups at 8 weeks after the last injection (p < 0.001) but no significant difference was found for PCNA compared with the control and DMSO groups (p < 0.05). These findings suggest that the Sertoli cell-conditioned medium may be beneficial as a practical approach for therapeutic strategies in reproductive and regenerative medicine.

The potential chemoprotective effects of melatonin against genotoxicity induced by diazinon in human peripheral blood lymphocytes.

The protection afforded by melatonin (MLT) against diazinon (DZN)-induced micronucleus formation, an index of DNA damage, in human blood lymphocytes was investigated. Whole blood samples were collected from five volunteers and were incubated with MLT at different concentrations (100, 200, 300, and 400 µM final concentration) for 1 h. The samples were then incubated with 750 µM DZN for 1 h. Subsequently, the lymphocytes were cultured with a mitogenic stimulant to evaluate micronucleus formation in cytokinesis-blocked binucleated cells. The incubation of lymphocytes with DZN induces additional genotoxicity. Pretreatment with MLT at these doses significantly reduced the micronucleus frequency in cultured lymphocytes (p < 0.05-p < 0.0001). The maximum decrease in the frequency of micronuclei was observed at 400 µM of MLT, which caused a reduction of 87%. MLT also exhibited an excellent and dose-dependent radical-scavenging activity against 1,1-diphenyl-2-picrylhydrazyl free radicals. Our study revealed that MLT has a potent antigenotoxic effect against DZN-induced DNA damage, which may be due to the scavenging of free radicals and increased antioxidant status. Because MLT is a natural compound and is considered safe, it can be used as a supplement to protect people exposed to chemical or environmental hazards.

Incidence of intracranial bleeding in mild traumatic brain injury patients taking oral anticoagulants: a systematic review and meta-analysis.

BACKGROUND: Traumatic brain injury (TBI) is one of the leading causes of disability and death worldwide. Most TBI cases occur in older people, because they are at a higher risk of accidental falling. As the population ages, the use of anticoagulants is increasing. Some serious complications of TBI, such as intracranial hemorrhage (ICH), may occur even in mild cases. According to the current guidelines regarding managing mild TBI patients, a CT head scan is recommended for all patients receiving anticoagulation. We aim to assess the incidence of ICH in patients with mild TBI taking oral anticoagulants. METHODS: Our systematic review and meta-analysis were performed using the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) checklist. The protocol was registered in PROSPERO (CRD42024503086). Twenty-eight studies evaluating patients with a mild TBI from ten countries with a total sample size of 11,172, 5671 on DOACs, and 5501 on VKAs were included in our meta-analysis. RESULTS: The random-effects overall incidence of ICH among oral anticoagulated patients with mild TBI was calculated to be 9.4% [95% CI 7.2-12.1%, I2 = 89%]. The rates of immediate ICH for patients taking DOACs and VKAs were 6.4% and 10.5%, respectively. The overall rate of immediate ICH in anticoagulated mild TBI patients was 8.5% [95% CI 6.6-10.9%], with a high heterogeneity between studies (I2 = 88%). Furthermore, the rates of delayed ICH in patients with mild TBI taking DOACs and VKAs were 1.6% and 1.9%, respectively. The overall incidence of delayed ICH among oral anticoagulated mild TBI patients was 1.7% [95% CI 1-2.8%, I2 = 79%]. The overall rate of ICH among mild TBI patients taking DOAC was calculated to be 7.3% [95% CI 5.2-10.3%], with significant heterogeneity between studies (I2 = 79%). However, the overall ICH rate is higher in patients who take only VKAs 11.3% [95% CI 8.6-14.7%, I2 = 83%]. Patients on DOACs were at lower risk of ICH after mild TBI compared to patients on VKAs (OR = 0.64, 95% CI 0.48-0.86, p < 0.01, I2 = 28%). CONCLUSION: Our meta-analysis confirms the need for performing brain CT scan in patients with mild TBI patients who receive oral anticoagulants before injury. Due to limited data, further multi-center, prospective studies are warranted to confirm the true incidence of traumatic ICH in patients on anticoagulants.

Effect of 1,25(OH)2-vitamin D3 on decidualization of human endometrial stromal cells.

Decidualization is the differentiation of endometrial stromal cells (eSC) to rounded, epithelioid-like cells during menstrual cycle and pregnancy. The impairment of this process leads to infertility and a variety of pregnancy disorders, including recurrent miscarriages and uteroplacental disorders. The aim of this study was to evaluate the effect of 1,25(OH)2-vitamin D3 (VD) on transformation of primary eSC into decidual cells. After isolation of eSC from biopsy samples of healthy fertile women and their characterization, the cells were cultured and propagated, and confluent cultures were decidualized for 12 days with progesterone (P4) and estradiol (E2) in presence or absence of VD. Prolactin (PRL) concentration was measured every 48 h in culture medium of eSCs, and ultrastructural changes were evaluated at the end of treatment. The results showed that PRL concentration in culture medium of eSCs was significantly increased in VD-treated decidual cells compared to control groups in a time-dependent manner. Ultrastructural analysis demonstrated that VD enhances many of the ultrastructural changes of decidualized cells including expansion of rough endoplasmic reticulum (rER), increased lipid droplets and high number of euchromatin round nuclei. These results suggest that VD may play an important role during early pregnancy by promoting cellular transformation associated with decidualization.

Conditioned medium derived from seminal extracellular vesicles-exposed endometrial stromal cells induces inflammatory cytokine secretion by macrophages.

OBJECTIVE: Seminal plasma (SP) contains large numbers of sub-cellular structures called extracellular vesicles (EV) which have been postulated to have immunological functions due to their bioactive contents including proteins and small non-coding RNAs. Although the response of endometrial cells to seminal EV (SEV) is recently being elucidated, the impact of these signaling vesicles on stroma-immune crosstalk is still unknown. Herein, we aimed to investigate the effect of conditioned medium (CM) derived from SEV-exposed endometrial stromal cells (eSC) on cytokine secretion by macrophages. STUDY DESIGN: SEV were isolated from SP samples of healthy donors and characterized by common methods needed for EV characterization, including size determination by dynamic light scattering (DLS), transmission electron microscopy (TEM), and western blot analysis of EV markers. Endometrial biopsies were obtained from healthy individuals and eSC were isolated and characterized. EV internalization assay was performed by labeling the SEV with PKH67 green fluorescent dye. Then, the eSC were exposed to SEV and the CM was collected. Finally, the CM from SEV-exposed eSC was added to the macrophage culture and the level of inflammatory (interleukin (IL)-1α and IL-6) and anti-inflammatory (IL-10) cytokines were measured in the culture supernatant of macrophages. RESULTS: The results demonstrated that the CM derived from SEV-exposed eSC induce IL-1α and IL-6 secretion by the macrophages, while the secretion of IL-10 was reduced. CONCLUSION: Our results support the idea that the stroma-immune interaction is affected by SEV. This effect may be a part of immunoregulatory function of SP inside upper female genital tract and have an obvious impact during peri-implantation period.

Pyrvinium pamoate induces in-vitro suppression of IL-6 and IL-8 produced by human endometriotic stromal cells.

Endometriosis, a chronic inflammatory disease, is identified by the presence of endometrial tissue outside the uterus. The prevalence of this disease among reproductive-age women is almost 10-15%. High levels of IL-6 and IL-8 have been found in the peritoneal fluid (PF) of women with endometriosis and are involved in its pathogenesis. Isolated stromal cells from 12 ectopic and eutopic endometrial biopsies of women with ovarian endometrioma and also 12 endometrial biopsies of nonendometriotic controls were treated with 1.1 µM pyrvinium pamoate, a Wnt/ß-catenin signaling pathway inhibitor, for 72 hrs. Before treatment, mRNA gene expression and secretion of IL-6 and IL-8 were significantly higher in ectopic (EESCs) than eutopic (EuESCs) and control (CESCs) endometrial stromal cells. After treatment, mRNA gene expression and also secretion of IL-6 and IL-8 were significantly reduced. Our Findings showed that pyrvinium pamoate suppresses the mRNA gene expression and secretion of IL-6 and IL-8 in human endometriotic stromal cells. Additional investigations on this compound are required before clinical application.

Seminal exosomes induce interleukin-6 and interleukin-8 secretion by human endometrial stromal cells.

OBJECTIVE: Exosomes are extracellular microvesicles that participate in intercellular communication. Seminal plasma (SP) contains very large amounts of exosomes which are deposited in female genital tract after insemination. Although the response of vaginal cells to seminal exosomes (SE) is recently being elucidated, the interaction of uterine cells with SE is still unknown. Here, we aimed to evaluate the effect of SE on cytokine secretion by human endometrial stromal cells (eSC). STUDY DESIGN: Exosomes were isolated from the semen samples of healthy men with proven fertility and characterized using common exosome characterization methods. Human eSC were isolated from endometrial biopsies obtained from healthy premenopausal women. For exosome internalization analysis, SE were labeled with PKH67 green fluorescent dye and incubated with the cells. For investigating the effect of SE on cytokine secretion of eSC, we measured levels of interleukin (IL)-6, IL-8, IL-10, IL-1α, and leukemia inhibitory factor (LIF) in the culture supernatants of control and experimental groups by enzyme-linked immunosorbent assay (ELISA) after 24 h of incubation. RESULTS: Our results demonstrated that SE are internalized by eSC and subsequently induce them to produce IL-6 and IL-8, the cytokines which are involved in the immunology of embryo implantation. CONCLUSION: The findings of the present study suggest that SE contribute to the immunoregulatory functions of SP in the uterus and may participate in embryo implantation process. Therefore dysfunction of intracellular machineries of SE biogenesis and secretion, inadequate production, defective transportation to the uterus and impaired communication with endometrium may play a distinct role in pathophysiology of embryo implantation failure.