GRIM-19 deficiency promotes macrophage polarization to the M1 phenotype partly through glycolysis in unexplained recurrent spontaneous abortion†.

The occurrence of unexplained recurrent spontaneous abortion (URSA) is closely related to immune system disorders, however, the underlying mechanisms remain unclear. The purpose of this study was to investigate the expression of GRIM-19 in URSA and the possible pathogenesis of URSA according to macrophage polarization. Here, we showed that GRIM-19 was downregulated in the uterine decidual macrophages of patients with URSA and that GRIM-19 downregulation was accompanied by increased M1 macrophage polarization. Furthermore, the expression levels of glycolytic enzymes were substantially enhanced in the uterine decidual macrophages of URSA patients, and glycolysis in THP-1-derived macrophages was further enhanced by the downregulation of GRIM-19. Additionally, the increase of M1 macrophages resulting from the loss of GRIM-19 was significantly reversed in cells treated with 2-deoxy-D-glucose (2-DG, an inhibitor of glycolysis). To provide more direct evidence, GRIM-19 deficiency was shown to promote macrophage polarization to the M1 phenotype in GRIM-19+/- mouse uteri. Overall, our study provides evidence that GRIM-19 deficiency may play a role in regulating macrophage polarization in URSA, and that glycolysis may participate in this process.

N-acetylcysteine alleviated tris(2-chloroisopropyl) phosphate-induced sperm motility decline and functional dysfunction in mice through reversing oxidative stress and DNA damage.

The decline in male fertility caused by environmental pollutants has attracted worldwide attention nowadays. Tris(2-chloroisopropyl) phosphate (TCPP) is a chlorine-containing organophosphorus flame retardant applied in many consumer products and has multiple side effects on health. However, whether TCPP impairs spermatogenesis remains unclear. In this study, we found that TCPP reduced the sperm motility and blastocyst formation, inhibited proliferation and induced apoptosis in mice testes and spermatocyte cell line GC-2. Moreover, TCPP induced imbalance of oxidant and anti-oxidant, DNA damage and mitochondrial dysfunction, thus induced abnormal spermatogenesis. In this process, p53 signaling pathway was activated and N-acetylcysteine treatment partially alleviated the side effects of TCPP, including decrease of sperm motility, activation of p53 signaling pathway and DNA damage. Finally, our study verified that TCPP elevated reactive oxygen species (ROS), decreased mitochondrial membrane potential and induced apoptosis in human semen samples. Overall, ROS mediated TCPP-induced germ cell proliferation inhibition and apoptosis, which finally led to the decline of sperm motility.

Cresyl Diphenyl Phosphate exposure induces reproductive functional defects in men and male mice.

Cresyl Diphenyl Phosphate (CDP), as a novel organophosphate esters (OPEs), achieves widely used and exposed in multiple industries. However, its male reproductive toxicity and underlying mechanism remains unclear. In vivo, male mice were gavaged with CDP (0, 4, 20, or 100 mg/kg/d) for 8 weeks. And we treated TM3, TM4 and GC-2 cells with 0, 10, 25, and 50 µM CDP for 24 h to detect its reproductive toxicity effect in vitro. In our study, we revealed that CDP inhibited proliferation and induced apoptosis in mice testis and GC-2 cells, thereby leading to the decreased sperm quality. In mechanism, CDP trigger the oxidative stress and ROS production, thus partially causing DNA damage and cell apoptosis. Moreover, CDP exposure causes injury to Ledyig cells and Sertoli cells, thus disturbing the testicular microenvironment and inhibiting spermatogonia proliferation. In conclusion, this research reveals multiple adverse impacts of CDP on the male reproductive system and calls for further study of the toxicological effects of CDP on human health.

HIF-1α is positively associated with endometrial receptivity by regulating PKM2.

PURPOSE: Numerous advancements have been introduced into the field of assisted reproductive technology (ART) in the past four decades. Nonetheless, implantation failure is still a key limiting step for a successful pregnancy. Building of endometrial receptivity (ER) is essential for successful implantation. However, the fundamental biological processes and mechanisms of ER remain elusive. Our study investigates the function of hypoxia inducible factor-1α (HIF-1α) during ER establishment and shed lights on the novel molecular mechanism by which HIF-1α regulates ER-related gene expression network. METHODS: Levels of HIF-1α, homeobox A10 (HOXA10), insulin-like growth factor-binding protein 1 (IGFBP1), pyruvate kinase M2 (PKM2), and lactate dehydrogenase A (LDHA) in endometrial tissues were measured via real-time PCR, immunoblotting and immunohistochemistry. The correlation between HIF-1α and HOXA10, IGFBP1, PKM2, LDHA were analyzed separately. Ishikawa cells were treated with vector HIF-1α, HIF-1α-siRNA, and PKM2-siRNA. After transfection, the levels of HOXA10, IGFBP1, LDHA, and PKM2 were measured via real-time PCR and immunoblotting, and the lactate concentrations and cell migration of Ishikawa cells were measured. RESULTS: Levels of HIF-1α, IGFBP1, HOXA10, LDHA, and PKM2 were significantly decreased in recurrent implantation failure (RIF) patients and levels of HOXA10, IGFBP1, PKM2, and LDHA were correlated with HIF-1α in endometrium. Then in a cellular model established by HIF-1α vector and HIF-1α-siRNA, the expression of HOXA10, IGFBP1, LDHA, PKM2, and lactate concentrations were dramatically upregulated and downregulated. And the expression of HOXA10, and IGFBP1 were dramatically decreased by PKM2-siRNA. CONCLUSIONS: HIF-1α plays a crucial role in the building of ER through regulating glycolysis.

True intratracheal oxygen concentration delivered by SentriO Oxy™ masks under various respiratory conditions: a bench study.

SentriO Oxy™ is a newly available, Food and Drug Administration-approved oxygenation mask system that provides high oxygenation, even on low-flow (5-10 L/min) oxygen. This study aimed to accurately measure the intratracheal fraction of inspired oxygen (FiO2) using SentriO Oxy™ masks under relatively low oxygen flow rates. A manikin-ventilator-test lung simulation system was used. We measured FiO2 at the level of the carina, 5 minutes after applying 45 different respiratory parameter combinations using SentriO Oxy™ masks. Tidal volume (TV) was set to 300, 500, and 700 mL; respiratory rate (RR) was set to 8, 12, 16, 20, and 24 breaths per minute; and oxygen flow rate was set to 6, 8, and 10 L/min. Our hypothesis was that FiO2 would be proportional to the difference between oxygen flow rate and minute ventilation. FiO2 measured by smaller TV, lower RR, or higher oxygen flows revealed a significantly higher value, confirming our hypothesis. In addition, using linear regression analysis, we found that TV, RR, and oxygen flow were all significant factors influencing the measured FiO2. Our experiment proposed two prediction equations considering the oxygen flow rate, TV, and RR. The results of our study may provide information and prediction of FiO2 for clinicians to use SentriO Oxy™ masks during sedative anesthetic procedures under low oxygen flow rates.

Decreased expression of GRIM-19 induces autophagy through the AMPK/ULK1 signaling pathway during adenomyosis†.

The processes underlying adenomyosis are similar to those of tumor metastasis, and it is defined as progressive invasion by the endometrium and the subsequent creation of ectopic lesions. GRIM-19 regulates cell death via the mitochondrial respiratory chain. Stress following oxygen deprivation can induce tumor cell autophagy, leading to cell invasion and migration. Here, we revealed that GRIM-19 negatively regulates autophagy, and, at least in adenomyosis, decreased expression of GRIM-19 is accompanied by an increased level of autophagy and 5'-adenosine monophosphate-activated protein kinase-Unc-51 like autophagy activating kinase 1 (AMPK-ULK1) activation. Upregulation of GRIM-19 expression in human primary endometrial cells and ISHIKAWA cells inhibits autophagy via the AMPK-ULK1 pathway and helps control cell invasion and migration. In addition, we also identified increased expression of AMPK and ULK1, and higher levels of autophagy in the uterine tissues of GRIM-19+/- mice. Importantly, the function of the GRIM-19-AMPK-ULK1 axis in regulating autophagy in adenomyosis is similar to that of tumor tissues, which may help elucidate the regulation of adenomyosis tumor-like behavior, and is expected to help identify novel targets for the diagnosis and treatment of adenomyosis.

The effect and mechanism of Grim 19 on mouse sperm quality and testosterone synthesis.

Abnormal sperm parameters such as oligospermia, asthenospermia, and teratozoospermia result in male factor infertility. Previous studies have shown that mitochondria play an important role in human spermatozoa motility. But the related pathogenesis is far from elucidated. The aim of this study was to investigate the association between gene associated with retinoid-interferon-induced mortality 19 (GRIM19) and asthenospermia. In this study, Grim19 knockout model (Grim19+/- mouse) was created through genome engineering. We showed that compared with WT mice, the sperm count and motility of Grim19+/- mice were significantly reduced. Grim19 may contribute to sperm count and vitality by influencing the mitochondrial membrane potential, intracellular reactive oxygen species production, and increasing cell apoptosis. The spermatogenic cells of all levels in the lumen of the seminiferous tubules were sparsely arranged, and the intercellular space became larger in the testis tissue of Grim19+/- mice. The serum testosterone concentration is significantly reduced in Grim19+/- mice. The expression of steroid synthesis-related proteins STAR, CYP11A1, and HSD3B was decreased in Grim19+/- mice. To further confirm whether changes in testosterone biosynthesis were due to Grim19 downregulation, we validated this result using Leydig cells and TM3 cells. We also found that Notch signaling pathway was involved in Grim19-mediated testosterone synthesis to some extent. In conclusion, we revealed a mechanism underlying Grim19 mediated spermatozoa motility and suggested that Grim19 affected the synthesis of testosterone and steroid hormones in male mouse partly through regulating Notch signal pathways.

Augmented CCL5/CCR5 signaling in brown adipose tissue inhibits adaptive thermogenesis and worsens insulin resistance in obesity.

Chemokine (C-C motif) ligand 5 (CCL5) and CCR5, one of its receptors have been reported to be highly expressed in white adipose tissue (WAT) and are associated with the progression of inflammation and the development of insulin resistance in obese humans and mice. However, the role of CCL5/CCR5 signaling in obesity-associated dysregulation of energy metabolism remains unclear. Here, we demonstrate that global CCL5/CCR5 double knockout (DKO) mice have higher cold stress-induced energy expenditure and thermogenic function in brown adipose tissue (BAT) than wildtype (WT) mice. DKO mice have higher cold stress-induced energy expenditure and thermogenic function in BAT than WT mice. KEGG pathway analysis indicated that deletion of CCL5/CCR5 further facilitated the cold-induced expression of genes related to oxidative phosphorylation (OxPhos) and lipid metabolic pathways. In primary brown adipocytes of DKO mice, the augmentation of CL-316243-stimulated thermogenic and lipolysis responses was reversed by co-treatment with AMPKα1 and α2 short interfering RNA (siRNA). Overexpression of BAT CCL5/CCR5 genes by local lentivirus injection in WT mice suppressed cold stress-induced lipolytic processes and thermogenic activities. In contrast, knockdown of BAT CCL5/CCR5 signaling further up-regulated AMPK phosphorylation as well as thermogenic and lipolysis responses to chronic adrenergic stimuli and subsequently decreased level of body weight gain. Chronic knockdown of BAT CCL5/CCR5 signaling improved high-fat diet (HFD)-induced insulin resistance in WT mice. It is suggested that obesity-induced augmentation of adipose tissue (AT) CCL5/CCR5 signaling could, at least in part, suppress energy expenditure and adaptive thermogenesis by inhibiting AMPK-mediated lipolysis and oxidative metabolism in thermogenic AT to exacerbate the development of obesity and insulin resistance.

Linalyl Acetate Ameliorates Mechanical Hyperalgesia Through Suppressing Inflammation by TSLP/IL-33 Signaling.

Neuropathic pain is a debilitating chronic disorder, significantly causing personal and social burdens, in which activated neuroinflammation is one major contributor. Thymic stromal lymphopoietin (TSLP) and interleukin (IL)-33 is important for chronic inflammation. Linalyl acetate (LA) is main component of lavender oil with an anti-inflammatory property through TSLP signaling. The aim of the study is to investigate how LA regulates mechanical hyperalgesia after sciatic nerve injury (SNI). Adult Sprague-Dawley male rats were separated into 3 groups: control group, SNI group and SNI with LA group. LA was administrated intraperitoneally one day before SNI. Pain behavior test was evaluated through calibration forceps testing. Ipsilateral sciatic nerves (SNs), dorsal root ganglions (DRGs) and spinal cord were collected for immunofluorescence staining and Western blotting analyses. SNI rats were more sensitive to hyperalgesia response to mechanical stimulus since operation, which was accompanied by spinal cord glial cells reactions and DRG neuro-glial interaction. LA could relieve the pain sensation, proinflammatory cytokines and decrease the expression of TSLP/TSLPR complex. Also, LA could reduce inflammation through reducing IL-33 signaling. This study is the first to indicate that LA can modulate pain through TSLP/TSLPR and IL-33 signaling after nerve injury.

GRIM19 downregulation-induced pyroptosis of macrophages through NLRP3 pathway in adenomyosis.

RESEARCH QUESTION: Does the absence of GRIM19 affect pyroptosis of macrophages? Is the release of IL-1ß caused by pyroptosis a relevant factor in the regulation of adenomyosis progression? DESIGN: Endometrial tissues were collected from patients with (n = 12) and without (n = 12) adenomyosis. GRIM19 expression of adenomyosis tissues was analysed by western blot and real-time polymerase chain reaction (RT-PCR). In GRIM19 knockdown macrophages, pyroptosis-related factors expressions were also measured by western blot and RT-PCR. The human endometrial stromal cells (HESC) were co-cultured with GRIM19-depleted macrophages and IL-1ß neutralizing antibody to detect the effects of pyroptosis of macrophages on apoptosis, proliferation and migration of HESC. RESULTS: The expression of GRIM19 was significantly lower in adenomyosis (P = 0.0002). In THP-1-derived macrophages, the expression of NLRP3 (P < 0.0001), ASC (P = 0.0176), caspase-1 (P = 0.0368), GSDMD (P = 0.0453) and IL-1ß (P = 0.0208) are increased after downregulation of GRIM19. GRIM19 knockdown induced the release of IL-1ß (P = 0.0195) in THP-1-derived macrophages. The apoptosis of HESC co-cultured with GRIM19 knockdown macrophages was significantly inhibited (P < 0.0001), the proliferation (P = 0.0254) and migration (P < 0.0001) were markedly promoted. Existence of IL-1ß neutralizing antibody in supernatants recovered the effects (P < 0.0001) of GRIM19 knockdown macrophages on HESC. CONCLUSIONS: GRIM19 downregulation induces pyroptosis of macrophages through NLRP3 pathway, increases the secretion of IL-1ß and promotes adenomyosis progression.

Granulocytic MDSC with Deficient CCR5 Alleviates Lipogenesis and Inflammation in Nonalcoholic Fatty Liver Disease.

C-C chemokine receptor type 5 (CCR5) positively contributes to the pathogenesis of nonalcoholic fatty liver disease (NAFLD), a common metabolic liver disease associated with chronic inflammation. CCR5 signaling also facilitates the immunosuppressive activity of a group of immature myeloid cells known as granulocytic myeloid-derived suppressor cells (g-MDSCs). While both hepatocyte and g-MDSC express CCR5, how CCR5 coordinates these two distinct cell types in the hepatic microenvironment remains largely unknown. Here, we used in vivo and ex vivo approaches to define the molecular details of how CCR5 mediates the crosstalk between hepatocytes and g-MDSCs in a mouse model of NAFLD. Global CCR5-deficient mice exhibited more severe steatosis, increased hepatic gene expression of lipogenesis, and exacerbated liver damage in diet-induced obesity. Either NAFLD or CCR5-deficiency per se is causative for the increase of g-MDSCs. Purified g-MDSCs have a higher survival rate in the fatty liver microenvironment, and blockade of CCR5 significantly decreases g-MDSCs' expression of anti-inflammatory factors. On the other hand, the null of CCR5 signaling increases hepatocytes' expression of lipogenic genes in the NAFLD microenvironment. Most importantly, inhibiting g-MDSCs' CCR5 signaling in the fatty liver microenvironment dramatically reduces STAT3 signaling, lipogenic, and pro-inflammatory gene expression in primary hepatocytes. Adoptive cell transfer experiments further demonstrate that CCR5-deficient g-MDSCs mitigate hepatic lipogenic gene expression without facilitating pro-inflammatory cytokine production and liver damage in NAFLD mice. These results suggest that targeting g-MDSCs' CCR5 signaling might serve as a potential therapeutic strategy for NAFLD.

Activation of oxytocin receptor in the trigeminal ganglion attenuates orofacial ectopic pain attributed to inferior alveolar nerve injury.

This study explores the effects of oxytocin receptor (OXTR) in the trigeminal ganglion (TG) on orofacial neuropathic pain. We demonstrate that OXTR activation in the TG relieves the orofacial ectopic pain as well as inhibits the upregulated expression of calcitonin gene-related peptide (CGRP), IL-1ß, and TNFα in the TG and spinal trigeminal nucleus caudalis (SpVc) of rats with inferior alveolar nerve transection. OXTR, a G protein-coupled receptor, has been demonstrated to play a significant role in analgesia after activation by its canonical agonist oxytocin (OXT) in the dorsal root ganglion. However, the role of OXTR in the trigeminal nervous system on the orofacial neuropathic pain is still little known. In the present study, we aimed to investigate the regulation effect and mechanism of OXTR in the TG) and SpVc) on orofacial ectopic pain induced by trigeminal nerve injury. The inferior alveolar nerve (IAN) was transected to establish a ectopic pain model. A behavioral test with electronic von Frey filament demonstrated IAN transection (IANX) evoked mechanical hypersensitivity in the whisker pad from day 1 to at least day 14 after surgery. In addition, administration of OXT (50 and 100 µM) into the TG attenuated the mechanical hypersensitivity induced by IANX, which was reversed by pretreatment with L-368,899 (a selective antagonist of OXTR) into the TG. In addition, immunofluorescence showed the expression of OXTR in neurons in the TG and SpVc. Furthermore, Western blot analysis indicated that the upregulated expression of OXTR, CGRP, IL-1ß, and TNFα in the TG and SpVc after IANX was inhibited by the administration of OXT into the TG. And the inhibition effect of OXT on the expression of CGRP, IL-1ß, and TNFα was abolished by preapplication of OXTR antagonist L-368,899 into the TG.NEW & NOTEWORTHY This study explores the effects of oxytocin receptor (OXTR) in the trigeminal ganglion (TG) on orofacial neuropathic pain. We demonstrate that OXTR activation in the TG relieves the orofacial ectopic pain as well as inhibits the upregulated expression of calcitonin gene-related peptide, IL-1ß, and TNF-α in the TG and spinal trigeminal nucleus caudalis of rats with inferior alveolar nerve transection.

GRIM19 is involved in WT1 expression and epithelial-to-mesenchymal transition in adenomyotic lesions.

The epithelial-to-mesenchymal transition may play a role in adenomyosis. GRIM19 expression is downregulated in adenomyotic lesions, and the effects of this downregulation in adenomyosis remain relatively unclear. In this study, we aimed to explore whether aberrant GRIM19 expression is associated with the epithelial-to-mesenchymal transition in adenomyosis and found that the expression of both GRIM19 and WT1 was low, and epithelial-to-mesenchymal transition, which included significant changes in CDH1, CDH2 and KRT8 expression, occurred in adenomyotic lesions, as confirmed by Western blotting and quantitative real-time PCR. We provided novel insights into WT1 expression in adenomyosis, revealing that WT1 expression was increased in the endometrial glands of adenomyotic lesions by immunohistochemistry. In vitro, knockdown of GRIM19 expression by small interfering RNA (siRNA) promoted the proliferation, migration and invasion of Ishikawa cells, as measured by Cell Counting Kit-8, wound healing assay and Transwell assays. Western blotting and quantitative real-time PCR confirmed that WT1 expression increased and epithelial-to-mesenchymal transition was induced, including the upregulation of CDH2 and downregulation of CDH1 and KRT8after transfecting the GRIM19 siRNA to Ishikawa cells. Furthermore, WT1 expression was upregulated and epithelial-to-mesenchymal transition was observed, including downregulation of CDH1 and KRT8in GRIM19 gene-knockdown mice. Upregulation of Wt1 expression in the endometrial glands of Grim19 knockdown mice was also verified by immunohistochemistry. Taken together, these results reveal that low expression of GRIM19 in adenomyosis may upregulate WT1 expression and induce epithelial-to-mesenchymal transition in the endometria, providing new insights into the pathogenesis of adenomyosis.

Increased Expression of Thymic Stromal Lymphopoietin in Chronic Constriction Injury of Rat Nerve.

Thymic stromal lymphopoietin (TSLP) is a well-known cytokine for T helper 2 inflammatory responses. A nerve injury activates the neuroinflammation cascade and neuron-glia interaction in dorsal root ganglions (DRG)s, leading to neuropathic pain. Therefore, this study was to investigate the role of TSLP after nerve injury. Male Sprague-Dawley rats were divided as an experimental group with chronic constriction injury (CCI) to the sciatic nerve and a control group. The mechanical pain threshold response was determined by calibration forceps. After assessment of mechanical allodynia, the ipsilateral spinal cord, DRG, sciatic nerve and skin were harvested. Immunofluorescence staining was performed to identify cell types with various markers. Western blot analyses were performed to evaluate protein expressions. Mechanical allodynia developed after CCI and persisted for the next 14 days. Astrocyte reactions occurred and continued until day 14, too. After CCI, DRG and the sciatic nerve also had significantly increased expressions of TSLP/TSLP-R/STAT5. The TSLPR was localized to sensory neuronal endings innervating the skin. This study is the first to demonstrate that the TSLP complex and the STAT5 pathway in nerve are potential therapeutic targets because of their roles in pain regulation after nerve injury.

Activation of the RAS/B-RAF-MEK-ERK pathway in satellite glial cells contributes to substance p-mediated orofacial pain.

The cross talk between trigeminal ganglion (TG) neurons and satellite glial cells (SGCs) is crucial for the regulation of inflammatory orofacial pain. Substance P (SP) plays an important role by activating neurokinin (NK)-I receptors in this cross talk. The activation of extracellular signal-regulated kinase (ERK) 1/2, protein kinase A (PKA) and protein kinase C (PKC) in neurons and SGCs of peripheral ganglions by peripheral inflammation is associated with inflammatory hypersensitivity. This study tested the hypothesis that SP evoked SP-NK-I receptor positive feedback via the Renin-Angiotensin System/B-Protein Kinase A-Rapidly Accelerates Fibrosarcoma-MEK-Extracellular Signal-Regulated Kinase (RAS/PKA-RAF-MEK-ERK) pathway, which is involved in pain hypersensitivity. Inflammatory models were induced in vivo by injecting Complete Freund's adjuvant (CFA) into the whisker pad of rats. SP was administrated to SGCs in vitro for investigating, whether SP regulates the expression of NK-I receptor in the SGC nucleus. The effects of RAS-RAF-MEK, PKA and PKC pathways in this process were measured by co-incubating SGCs with respective Raf, PKA, PKC and MEK inhibitors in vitro and by pre-injecting these inhibitors into the TG in vivo. SP significantly upregulated NK-I receptor, p-ERK1/2, Ras, B-Raf, PKA and PKC in SGCs under inflammatory conditions. In addition, L703,606 (NK-I receptor antagonist), U0126 (MEK inhibitor), Sorafenib (Raf inhibitor) and H892HCL (PKA inhibitor) but not chelerythrine chloride (PKC inhibitor) significantly decreased NK-I mRNA and protein levels induced by SP. The allodynia-related behavior evoked by CFA was inhibited by pre-injection of L703,606, U0126, Sorafenib and H892HCL into the TG. Overall, SP upregulates NK-I receptor in TG SGCs via PKA/RAS-RAF-MEK-ERK pathway activation, contributing to a positive feedback of SP-NK-I receptor in inflammatory orofacial pain.

Signal transducer and activator of transcription 5a (STAT5a) represses mitochondrial gene expression through direct binding to mitochondrial DNA.

Signal transducer and activator of transcription (STAT) proteins are latent cytoplasmic transcription factors essential for cytokine signaling. Our previous study showed that interleukin-3 (IL-3) induced STAT5 translocation to mitochondria and binding to mitochondrial DNA (mtDNA) in vitro. In this report, we further demonstrated in vivo binding of endogenous STAT5a to mtDNA transcriptional control region and reduced gene expression from all three mtDNA promoters after IL-3 stimulation. To specifically define the function of mitochondrial STAT5a, we generated mitochondrial-targeting wild-type and mutant STAT5a proteins. Compared with non-targeting STAT5a, mitochondrial-targeting wild-type STAT5a significantly reduced mitochondrial gene expression in transfected HEK293 cells. The level of attenuation was amplified in cells expressing constitutively active STAT5a, but abrogated in cells expressing DNA-binding-defective STAT5a. STAT5a-mediated repression of mtDNA expression also positively correlated with STAT5a binding to the E2 subunit of pyruvate dehydrogenase complex (PDC-E2), both a gate-keeping metabolic enzyme and a component of mtDNA nucleoid in mitochondrial matrix. Metabolic shift away from mitochondrial respiration is known in many cytokine-stimulated cells and cancer cells. STAT5a-mediated repression of mitochondrial gene expression and its interaction with PDC-E2 may provide important insights into its underlying mechanisms.

Interaction of M2 macrophages and endometrial cells induces downregulation of GRIM-19 in endometria of adenomyosis.

RESEARCH QUESTION: Does the aggregation of M2 macrophages affect the expression of gene associated with retinoid-interferon-induced mortality 19 (GRIM-19) in adenomyosis? DESIGN: Endometrial tissues were collected from patients with (n = 15) and without (n = 15) adenomyosis. Tissues were analysed for GRIM-19 and toll-like receptor 4 (TLR4) expression by immunohistochemistry and western blotting. Apoptosis was analysed by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay. Human endometrial stromal cells (HESC) were transfected with GRIM-19 small interfering RNA (SiRNA) to knockdown GRIM-19 expression. The HESC were co-cultured with M2 macrophages to detect the influence of M2 macrophages in HESC cells. Analyses included GRIM-19, caspase-3 and TLR4 expression by western blotting, and GRIM-19 and TLR4 by quantitative real-time polymerase chain reaction. Apoptosis was measured by flow cytometry and TUNEL assay. Cell proliferation (Cell Counting Kit-8 assay) and migration assays were carried out. RESULTS: The expression of GRIM-19 was significantly lower in adenomyosis lesions compared with controls (P < 0.001). Deficiency of GRIM-19 induced by siRNA decreased apoptosis and increased proliferation and migration in HESC. A significant decrease in GRIM-19 expression occurred in HESC after co-culture with M2 macrophages (P = 0.018). After co-culture with M2 macrophage, apoptosis decreased and proliferation and cell invasion in HESC increased. Protein (P = 0.006) and mRNA (P = 0.013) expression of TLR4 in HESC also reduced after this co-culture. Up-regulation of GRIM-19 occurred in HESC treated with the activator TLR4 (P = 0.016). Up-regulation of GRIM-19 was significantly reversed in cells treated with the TLR4 inhibitor (P = 0.011). CONCLUSION: M2 macrophages may be involved in regulating the expression of GRIM-19 partly through the TLR4 signalling axis in adenomyosis.

[Early Mitotic Inhibitor 1 Regulates DNA Re-replication Mediated by Human Papillomavirus Subtype 16 E7 in Response to DNA Damage].

Objective To explore the molecular mechanism of human papillomavirus subtype 16(HPV-16)E7 oncogene-induced DNA re-replication in response to DNA damage. Methods Flow cytometry was performed to examine the cell cycle changes in RPE1 E7 cells stably expressing HPV-16 E7 and its control cell RPE1 Vector after DNA damage.Immunoblotting assay was used to evaluate the early mitotic inhibitor 1(Emi1)expression in RPE1 E7 and RPE1 Vector cells with or without DNA damage.The changes of the proportion of polyploidy was detected by flow cytometry in DNA-damaged RPE1 E7 cells interfered by Emi1 small interfering RNA. Results Compared with the control cells,the proportion of polyploids in RPE1 E7 cells was significantly increased in response to DNA damage(t=6.397,P=0.0031).Emi1 protein expression was significantly increased in DNA damaged RPE1 E7 cells(t=8.241,P=0.0012).The polyploid ratio of RPE1 E7 cells was significantly reduced after Emi1 was interfered by two independent small interfering RNAs(t=2.916,P=0.0434;t=3.452,P=0.0260). Conclusion In response to DNA damage,Emi1 promoted DNA re-replication caused by HPV-16 E7.

Expression of GRIM-19 in unexplained recurrent spontaneous abortion and possible pathogenesis.

STUDY QUESTION: Is aberrant expression of gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) associated with unexplained recurrent spontaneous abortion (URSA)? SUMMARY ANSWER: GRIM-19 deficiency may regulate regulatory T cell/T helper 17 cell (Treg/Th17) balance partly through reactive oxygen species (ROS)-mammalian target of rapamycin (mTOR) signaling axis in URSA. WHAT IS KNOWN ALREADY: Immunological disorders may cause impaired maternal immune tolerance to the fetus and result in fetal rejection. The differentiation of Treg and Th17 cells is controlled by phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling pathway. GRIM-19 participates in the immune response, but its role in URSA is largely unknown. STUDY DESIGN, SIZE, DURATION: The current study included 28 URSA patients and 30 non-pregnant healthy women. PARTICIPANTS/MATERIALS, SETTING, METHODS: The proportion of Treg and Th17 cells in peripheral blood of URSA patients and control subjects were assessed with flow cytometry. The expression of GRIM-19 in peripheral blood lymphocytes (PBLs) was measured with quantitative real-time PCR and western blot analysis. Furthermore, the ROS level in the PBLs of URSA patients and control subjects were assessed by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Then, Akt/mTOR expression in the PBLs was measured. Downregulation of GRIM-19 in Jurkat cells was performed by specific siRNA. Then, intracellular ROS production and the expression of p-mTOR, which is known to enhance Th17 differentiation and decrease Treg cell differentiation, were detected. Finally, N-acetylcysteine (NAC) was used to decrease the intracellular ROS level, and the expression of p-mTOR was measured. MAIN RESULTS AND THE ROLE OF CHANCE: The proportion of Treg cells was reduced in URSA patients, whereas the proportion of Th17 cells was increased. The expression of GRIM-19 was significantly lower in PBLs of URSA patients. Furthermore, there is a considerable increase in intracellular ROS production and a high level of p-Akt and p-mTOR expression in the PBLs of URSA patients compared with the control subjects. In parallel to this, downregulation of GRIM-19 in the Jurkat cells by siRNA results in an increased ROS production and an increased expression of p-mTOR. Importantly, the upregulation of p-mTOR resulting from GRIM-19 loss was significantly reversed in the cells treatment with ROS inhibitor N-acetyl-l-cysteine (NAC), indicating that ROS was indeed required for GRIM-19 depletion induced p-mTOR expression. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: A large number of researches have confirmed that the differentiation of Treg and Th17 cells is controlled by PI3K/Akt/mTOR signaling pathway. We have not shown the regulatory role of ROS and PI3K/Akt/mTOR in Treg and Th17 differentiation in this study. WIDER IMPLICATIONS OF THE FINDINGS: Our study has demonstrated that GRIM-19 deficiency may play a role in regulating Treg/Th17 balance partly through ROS-mTOR signaling axis in URSA. The present study offers a new perspective to the roles of GRIM-19 in immunoregulation. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (Grant numbers 81571511, 81701528, 81370711 and 30901603), the Shandong Provincial Natural Science Foundation (Grant numbers ZR2017PH052 and ZR2013HM090) and the Science Foundation of Qilu Hospital of Shandong University, Fundamental Research Funds of Shandong University (Grant numbers 2015QLQN50 and 2015QLMS24). The authors declare that there is no conflict of interest that could prejudice the impartiality of the present research.

Expression of NDUFA13 in asthenozoospermia and possible pathogenesis.

Asthenozoospermia is a common cause of male infertility, which is characterized by reduced forward motility of spermatozoa. The cause and pathogenesis of asthenozoospermia are not fully understood. The purpose of this study was to investigate the expression of nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) 1 alpha subcomplex, 13 (NDUFA13) in the spermatozoa of men with asthenozoospermia and its possible pathogenesis. Protein content of NDUFA13 in spermatozoa was measured by Western blot analysis. The results showed that NDUFA13 expression in spermatozoa was significantly lower in men with asthenozoospermic than in men with normozoospermia (P < 0.01). Immunofluorescence experiments showed that NDUFA13 was expressed predominantly in the sperm mid-piece. A lower mitochondrial membrane potential, a higher intracellular reactive oxygen species (ROS) level and more apoptotic cells were also detected in men with asthenozoospermia. NDUFA13-specific small interfering RNA was used in the mouse spermatocyte GC2-spd cell line to down-regulate the expression of NDUFA13. The knockdown of NDUFA13 in the GC2-spd cells caused a collapse of mitochondrial membrane potential, an increase in ROS level and more apoptotic cells. Our study showed that NDUFA13 deficiency may be associated with asthenozoospermia through the disturbance of spermatozoa mitochondrial membrane potential and by increasing apoptosis and intracellular ROS.