Prostaglandin E2 induces ovulation in prepubertal mice

The objective of this study was to determine the ability of prostaglandin E2 (PGE2) to induce ovulation and expression of PGE2 receptor (EP2 and EP4) and COX genes (COX-1 and COX-2) in the ovary and pituitary of prepubertal mice. The positive control consisted of the application of 5 μg of gonadotropin-releasing hormone (GnRH, n = 29); the negative control applied 0.5 mL of phosphate buffered saline (PBS, n=31); the treatment tested the application of 250 μg of PGE2 (n = 29), making a total of 89 prepubertal mice (BALB/c). Mice were euthanized 14 to 15 h after treatments to detect ovulation and tissue collection. A Chi-square test was used to compare the proportion of animals ovulating. Gene expressions and number of ovulation were analyzed by one-way ANOVA and Tukey’s test was used to compare means among groups. A greater proportion of mice (P < 0.001) ovulated after receiving GnRH (89.7%, 26/29) compared to PGE2 group (58.6%, 17/29). However, the proportion was higher compared to those treated with PBS (0%, 0/31). Ep2 gene expression in the pituitary was > two-fold higher (P < 0.05) in the PGE2 group compared to the PBS and GnRH groups. Further, PGE2 stimulated Cox1 (2.7 fold, P < 0.05) while GnRH stimulated Cox2 expression (6.5 fold, P < 0.05) in the pituitary when compared to the PBS group. In conclusion, our results support the hypothesis that PGE2 can induce ovulation in prepubertal mice with a concomitant increase in Ep2 and Cox1 gene expression in the pituitary gland.

Inhibition of Autotaxin and Lysophosphatidic Acid Receptor 5 Attenuates Neuroinflammation in LPS-Activated BV-2 Microglia and a Mouse Endotoxemia Model

Increasing evidence suggests that systemic inflammation triggers a neuroinflammatory response that involves sustained microglia activation. This response has deleterious consequences on memory and learning capability in experimental animal models and in patients. However, the mechanisms connecting systemic inflammation and microglia activation remain poorly understood. Here, we identify the autotaxin (ATX)/lysophosphatidic acid (LPA)/LPA-receptor axis as a potential pharmacological target to modulate the LPS-mediated neuroinflammatory response in vitro (the murine BV-2 microglia cell line) and in vivo (C57BL/6J mice receiving a single i.p. LPS injection). In LPS-stimulated (20 ng/mL) BV-2 cells, we observed increased phosphorylation of transcription factors (STAT1, p65, and c-Jun) that are known to induce a proinflammatory microglia phenotype. LPS upregulated ATX, TLR4, and COX2 expression, amplified NO production, increased neurotoxicity of microglia conditioned medium, and augmented cyto-/chemokine concentrations in the cellular supernatants. PF8380 (a type I ATX inhibitor, used at 10 and 1 µM) and AS2717638 (an LPA5 antagonist, used at 1 and 0.1 µM) attenuated these proinflammatory responses, at non-toxic concentrations, in BV-2 cells. In vivo, we demonstrate accumulation of PF8380 in the mouse brain and an accompanying decrease in LPA concentrations. In vivo, co-injection of LPS (5 mg/kg body weight) and PF8380 (30 mg/kg body weight), or LPS/AS2717638 (10 mg/kg body weight), significantly attenuated LPS-induced iNOS, TNFα, IL-1β, IL-6, and CXCL2 mRNA expression in the mouse brain. On the protein level, PF8380 and AS2717638 significantly reduced TLR4, Iba1, GFAP and COX2 expression, as compared to LPS-only injected animals. In terms of the communication between systemic inflammation and neuroinflammation, both inhibitors significantly attenuated LPS-mediated systemic TNFα and IL-6 synthesis, while IL-1β was only reduced by PF8380. Inhibition of ATX and LPA5 may thus provide an opportunity to protect the brain from the toxic effects that are provoked by systemic endotoxemia.

Ferritinophagy-Mediated Ferroptosis Involved in Paraquat-Induced Neurotoxicity of Dopaminergic Neurons: Implication for Neurotoxicity in PD

Parkinson’s disease (PD) is a progressive nervous system disorder. Until now, the molecular mechanism of its occurrence is not fully understood. Paraquat (PQ) was identified as a neurotoxicant and is linked to increased PD risk and PD-like neuropathology. Ferroptosis is recognized as a new form of regulated cell death. Here, we revealed a new underlying mechanism by which ferritinophagy-mediated ferroptosis is involved in PD induced by PQ. The effect of PQ on movement injury in mice was investigated by the bar fatigue and pole-climbing test. SH-SY5Y human neuroblastoma cells were used to evaluate the mechanism of ferroptosis. Our results showed that PQ induced movement injury by causing the decrease in tyrosine hydroxylase in mice. In vitro, PQ significantly caused the iron accumulation in cytoplasm and mitochondria through ferritinophagy pathway induced by NCOA4. Iron overload initiated lipid peroxidation through 12Lox, further inducing ferroptosis by producing lipid ROS. PQ downregulated SLC7A11 and GPX4 expression and upregulated Cox2 expression significantly, which were important markers in ferroptosis. Fer-1, an inhibitor of ferroptosis, could significantly ameliorate the ferroptosis induced by PQ. Meanwhile, Bcl2, Bax, and p-38 were involved in apoptosis induced by PQ. In conclusion, ferritinophagy-mediated ferroptosis pathway played an important role in PD occurrence. Bcl2/Bax and P-p38/p38 pathways mediated the cross-talk between ferroptosis and apoptosis induced by PQ. These data further demonstrated the complexity of PD occurrence. The inhibition of the ferroptosis and apoptosis together may be a new strategy for the prevention of neurotoxicity or PD in the future.

Cyclo-oxygenase 2, a putative mediator of vessel remodeling, is expressed in the brain AVM vessels and associates with inflammation

Background Brain arteriovenous malformations (bAVM) may rupture causing disability or death. BAVM vessels are characterized by abnormally high flow that in general triggers expansive vessel remodeling mediated by cyclo-oxygenase-2 (COX2), the target of non-steroidal anti-inflammatory drugs. We investigated whether COX2 is expressed in bAVMs and whether it associates with inflammation and haemorrhage in these lesions. Methods Tissue was obtained from surgery of 139 bAVMs and 21 normal Circle of Willis samples. The samples were studied with immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR). Clinical data was collected from patient records. Results COX2 expression was found in 78% (109/139) of the bAVMs and localized to the vessels’ lumen or medial layer in 70% (95/135) of the bAVMs. Receptors for prostaglandin E2, a COX2-derived mediator of vascular remodeling, were found in the endothelial and smooth muscle cells and perivascular inflammatory cells of bAVMs. COX2 was expressed by infiltrating inflammatory cells and correlated with the extent of inflammation (r = .231, p = .007, Spearman rank correlation). COX2 expression did not associate with haemorrhage. Conclusion COX2 is induced in bAVMs, and possibly participates in the regulation of vessel wall remodelling and ongoing inflammation. Role of COX2 signalling in the pathobiology and clinical course of bAVMs merits further studies.

Arg-Vasotocin Directly Activates Isotocin Receptors and Induces COX2 Expression in Ovoviviparous Guppies

Oxytocin (OT) is a crucial regulator of reproductive behaviors, including parturition in mammals. Arg-vasopressin (AVP) is a nonapeptide homologous to Arg-vasotocin (AVT) in teleosts that has comparable affinity for the OT receptor. In the present study, ovoviviparous guppies (Poecilia reticulata) were used to study the effect of AVT on delivery mediated by the activation of prostaglandin (PG) biosynthesis via isotocin (IT) receptors (ITRs). One copy each of it and avt and two copies of itrs were identified in guppies. The results of the affinity assay showed that various concentrations of AVT and IT (10−6, 10−7, and 10−8 mol/L) significantly activated itr1 (P &lt; 0.05). In vitro experiments revealed significant upregulation (P &lt; 0.05) of cyclooxygenase 2 (cox2), which is the rate-limiting enzyme involved in PG biosynthesis, and itr1 by AVT and IT. Furthermore, dual in situ hybridization detected positive signals for itr1 and cox2 at the same site, implying that ITR1 may regulate cox2 gene expression. Measurement of prostaglandin F2a (PGF2a) concentrations showed that AVT induced PGF2a synthesis (P &lt; 0.05) and that the effect of IT was not significant. Finally, intraperitoneal administration of PGF2a significantly induced premature parturition of guppies. This study is the first to identify and characterize AVT and ITRs in guppies. The findings suggest that AVT promotes PG biosynthesis via ITR and that PGF2a induces delivery behavior in ovoviviparous guppies.

Obtusifolin, an Anthraquinone Extracted from Senna obtusifolia (L.) H.S.Irwin & Barneby, Reduces Inflammation in a Mouse Osteoarthritis Model

Osteoarthritis (OA) is an age-related degenerative disease that causes cartilage dysfunction and inflammation. Obtusifolin, an anthraquinone extracted from Senna obtusifolia (L.) H.S.Irwin & Barneby seeds, has anti-inflammatory functions; it could be used as a drug component to relieve OA symptoms. In this study, we investigated the effects of obtusifolin on OA inflammation. In vitro, interleukin (IL)-1β (1 ng/mL)-treated mouse chondrocytes were co-treated with obtusifolin at different concentrations. The expression of matrix metalloproteinase (Mmp) 3, Mmp13, cyclooxygenase 2 (Cox2), and signaling proteins was measured by polymerase chain reaction and Western blotting; collagenase activity and the PGE2 level were also determined. In vivo, OA-induced C57BL/6 mice were administered obtusifolin, and their cartilage was stained with Safranin O to observe damage. Obtusifolin inhibited Mmp3, Mmp13, and Cox2 expression to levels similar to or more than those after treatment with celecoxib. Additionally, obtusifolin decreased collagenase activity and the PGE2 level. Furthermore, obtusifolin regulated OA via the NF-κB signaling pathway. In surgically induced OA mouse models, the cartilage destruction decreased when obtusifolin was administered orally. Taken together, our results show that obtusifolin effectively reduces cartilage damage via the regulation of MMPs and Cox2 expression. Hence, we suggest that obtusifolin could be a component of another OA symptom reliever.

Schisandrol A Suppresses Catabolic Factor Expression by Blocking NF-κB Signaling in Osteoarthritis

Schisandrol A possesses pharmacological properties and is used to treat various diseases; however, its effects on osteoarthritis (OA) progression remain unclear. Here, we investigated Schisandrol A as a potential therapeutic agent for OA. In vitro, Schisandrol A effects were confirmed based on the levels of expression of catabolic factors (MMPs, ADAMTS5, and Cox2) induced by IL-1β or Schisandrol A treatment in chondrocytes. In vivo, experimental OA in mice was induced using a destabilized medial meniscus (DMM) surgical model or oral gavage of Schisandrol A in a dose-dependent manner, and demonstrated using histological analysis. In vitro and in vivo analyses demonstrated that Schisandrol A inhibition attenuated osteoarthritic cartilage destruction via the regulation of Mmp3, Mmp13, Adamts5, and Cox2 expression. In the NF-κB signaling pathway, Schisandrol A suppressed the degradation of IκB and the phosphorylation of p65 induced by IL-1β. Overall, and Schisandrol A reduced the expression of catabolic factors by blocking NF-κB signaling and prevented cartilage destruction. Therefore, Schisandrol A attenuated OA progression, and can be used to develop novel OA drug therapies.

COX2 expression is associated with preoperative tumor volume but not with volumetric tumor growth in vestibular schwannoma

Objective Vestibular schwannomas (VS) are benign slow growing tumors arising from the vestibular nerve. The role of cyclooxygenase 2 (COX2) in tumor development of growth has been addressed in a few studies with contradictory results and suggestions. We recently analyzed the immunohistochemical expression of COX2 in 1044 VS samples and described an association of higher COX2 expression with proliferation but found no influence by regular intake of acetylsalicylic acid. We now collected volumetric radiographic data of the preoperative tumor volume and growth to further test the role of COX2 in VS growth. Methods Preoperative images of 898 primary sporadic vestibular schwannomas were assessed, and sufficient preoperative imaging was used for the volumetric measurement preoperative tumor volume (n = 747) and preoperative relative tumor growth (n = 171). Clinical parameters and results of the immunohistochemical expression of COX2 and MIB1 in resected tumor tissue samples were obtained from our prior study. ANOVA, CART-analysis and multivariate nominal logistic regression were used for statistical analysis. Results Larger preoperative tumor volumes were observed with tumors of younger patients (p = 0.0288) and with higher COX2 expression scores (p < 0.0001). Higher MIB1 expression was associated with smaller tumors (p = 0.0149) but with increased radiographic tumor growth (p = 0.0003). Patients of older age had tumors with slower growth rates (p = 0.0311). In the multivariate analysis only MIB1 expression was an independent significant factor regarding tumor growth (p = 0.0002). Conclusions Higher expression of COX2 in schwannoma is associated with an increased preoperative tumor volume but not with radiographic tumor growth over time.

Effect of Rubus idaeus Extracts in Murine Chondrocytes and Explants

Osteoarthritis is characterized by cartilage loss resulting from the activation of chondrocytes associated with a synovial inflammation. Activated chondrocytes promote an increased secretion of matrix proteases and proinflammatory cytokines leading to cartilage breakdown. Since natural products possess anti-inflammatory properties, we investigated the direct effect of Rubus idaeus extracts (RIE) in chondrocyte metabolism and cartilage loss. The effect of RIE in chondrocyte metabolism was analyzed in murine primary chondrocytes and cartilage explants. We also assessed the contribution of RIE in an inflammation environment by culturing mice primary chondrocytes with the supernatant of Raw 264.7 macrophage-like cells primed with RIE. In primary chondrocytes, RIE diminished chondrocyte hypertrophy (Col10), while increasing the expression of catabolic genes (Mmp-3, Mmp-13) and reducing anabolic genes (Col2a1, Acan). In cartilage explants, Rubus idaeus prevented the loss of proteoglycan (14.84 ± 3.07% loss of proteoglycans with IL1 alone vs. 3.03 ± 1.86% with IL1 and 100 µg/mL of RIE), as well as the NITEGE neoepitope expression. RIE alone reduced the expression of Il1 and Il6 in macrophages, without changes in Tnf and Cox2 expression. The secretome of macrophages pre-treated with RIE and transferred to chondrocytes decreases the gene and protein expression of Mmp-3 and Cox2. In conclusion, these data suggest that RIE may protect from chondrocyte catabolism and cartilage loss in inflammatory conditions. Further evaluations are need before considering RIE as a candidate for the treatment for osteoarthritis.