PGE2 Release Research Articles

Effects of Curcumin on Biological Behavior and NF-κB/TNF-α Pathway in Mice with Metastatic Bone Pain of Breast Cancer Induced by Walker 256 Cells

Background. The active ingredient curcumin of traditional Chinese medicine was selected as the research object to investigate the possible mechanism of breast cancer metastatic bone pain in mouse walker 256 cells and the effect of curcumin on the NF-κB/TNF-α pathway in order to provide a new idea for clinical treatment of breast cancer metastatic bone pain. Methods. By establishing an animal model of breast cancer bone metastasis in walker 256 cells, the biological behavior of nude mice was observed on the 8th day after successful modeling. Meanwhile, the low dose group, middle dose group and high dose group of mice were given 15 mg/kg, 25 mg/kg, 50 mg/kg of curcumin solution intraperitoneally in 21 days, and the right cavity bone and spinal cord distended in mice (L4-L6) tissues were used to detect related factors, Immunohistochemical method was used to detect c-fos in spinal cord. Expression levels of RANK, NF-κB and TNF-α were detected by RT-PCR and Western blot. Meanwhile, serum levels of Cox2, il-6, leukotriene and PGE2 were detected. Results. Observing the biological behavior index of nude mice, we found that the mechanical pain and thermal pain threshold decreased (p Conclusions. On the 8th day after the success of the animal model of breast cancer bone metastasis in Walker 256 cells, abnormal biological behaviors such as heat pain, cold pain sensation and spontaneous hyperalgesia were observed. Further studies have found that the increased expression of rank on osteoclasts induced up-regulated expression of NF-κB and c-fos, induced expression of TNF-α gene, and could induce synthesis and release of leukotriene, PGE2 through direct activation of cyclooxygenase, inflammatory media IL-6 cascade reaction, resulting in pathological pain and hypersensitivity. Traditional Chinese medicine active ingredient curcumin could reduce RANK expression of osteoclast, inhibit cell NF-κB and spinal cord c-fos activity, reduce TNF-α expression, inhibit Cox2 activity, and reduce the synthesis and release of inflammatory factors leukotriene and PGE2, thus exerting its analgesic effect, which provides new ideas and methods for clinical treatment of metastatic bone pain in breast cancer.

The Effects of Gehuajiecheng Decoction on Expression of COX-2, Serum Level of PGE2, TNF-α and IL-6 in Alcoholic Hepatitis

Background: In this experimental study, we have researched Gehuajiecheng decoction mechanism of action in the treatment of alcoholic hepatitis so as to supply an effective drug in clinical practice. Methods: 40 male Wistar rats were randomly divided into 4 groups and treated with different drugs. The control group were given intragastric administration of corn oil (2g/kg/per day) and 50% glucose (20ml/kg/per day); the model group were given intragastric administration of 40% alcohol (8g/kg/per day) and corn oil (2g/kg/per day); Gehuajiecheng decoction group were given intragastric administration of Gehuajiecheng decoction (13g/kg/per day) and 40% alcohol (8g/kg/per day); the Celecoxib group were given intragastric administration of celecoxib (10mg/kg/per day) and 40% alcohol (8g/kg/per day). 30 days after treatment, the rats were anesthetized with sodium pentobarbital and the blood was collected. Serum levels of AST, ALT and GGT were measured by using chromatometry, PGE2, TNF-α and IL-6 levels were examined by using radioimmunology method. After taking blood samples, the rats were sacrificed, and the liver was determined by Western blotting analysis. Results: Compared to controls, model group showed significantly higher levels of serum AST, ALT, GGT, PGE2, TNF-α and IL-6, which can be corrected by administration of Gehuajiecheng decoction or celecoxib, since the group treated with Gehuajiecheng decoction or celecoxib showed significantly lower levels of serum AST, ALT, GGT, PGE2, TNF-α and IL-6 (p 0.05). Similarly, COX-2 expression in the model group was significantly higher than that of controls. However, its expression can be depleted by Gehuajiecheng decoction or celecoxib as COX-2 expression in the model groups (p<0.01). Conclusion: Gehuajiecheng decoction protects against alcohol-induced liver injuries via inhibiting expression of COX-2, decreasing release of PGE2, TNF-α and IL-6. It has the same effect as COX-2 selective inhibitor, therefore, we suggest that this decoction could be used as an effective method in the treatment of alcoholic hepatitis.

Sub-toxic levels of Co2+ are anti-inflammatory and protect cartilage from degradation caused by IL-1β

BackgroundCobalt ions from some orthopaedic implants induce a dose-dependent cytotoxic and pro-inflammatory response. Recent studies show that sub-toxic levels of cobalt influence actin organisation regulating fibroblasts and macrophages behaviour. However little is known about the influence of sub-toxic levels of cobalt on articular cartilage biology and biomechanics. Previously, we have reported that IL-1β signalling in chondrocytes, is regulated by primary cilia and associated intraflagellar transport. Since primary cilia expression is modulated by actin organisation, we set out to test the hypothesis that sub-toxic levels of cobalt regulate cilia expression and IL-1β signalling thereby influencing articular cartilage degradation. MethodsIsolated chondrocytes and bovine cartilage explants were subjected to Co2+ in the presence and absence of IL-1β. Primary cilia were monitored by confocal immunofluorescence. Nitric oxide and PGE2 release were used to monitor IL-1β signalling. Degradation of cartilage matrix was assessed by the release of sGAG and the biomechanical properties of the tissue in uniaxial unconfined compression. FindingsSub-toxic levels of Co2+ (50 μM) blocked IL-1β-induced primary cilia elongation in isolated chondrocytes. This was associated with disruption of pro-inflammatory signalling in both isolated chondrocytes and cartilage explants, and inhibition of cartilage matrix degradation and loss of biomechanical properties. InterpretationThis study reveals that low levels of cobalt ions are anti-inflammatory, preventing cartilage degradation in response to IL-1β. This mechanism is associated with regulation of primary cilia elongation. These observations provide new insight into the potential beneficial role of cobalt and may lead to novel mechanisms for controlling cartilage inflammation.

12-HETE is a regulator of PGE2 production via COX-2 expression induced by a snake venom group IIA phospholipase A2 in isolated peritoneal macrophages

The snake venom miotoxin (MT)-III is a group IIA secreted phospholipase A2 (sPLA2) with pro-inflammatory activities. Previous studies have demonstrated that MT-III has the ability to stimulate macrophages to release inflammatory lipid mediators derived from arachidonic acid metabolism. Among them, we highlight prostaglandin (PG)E2 produced by the cyclooxygenase (COX)-2 pathway, through activation of nuclear factor (NF)-κB. However, the mechanisms coordinating this process are not fully understood. This study investigates the regulatory mechanisms exerted by other groups of bioactive eicosanoids derived from 12-lipoxygenase (12-LO), in particular 12-hydroxyeicosatetraenoic (12-HETE), on group IIA sPLA2-induced (i) PGE2 release, (ii) COX-2 expression, and (iii) activation of signaling pathways p38 mitogen-activated protein kinases(p38MAPK), protein C kinase (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-κB. Stimulation of macrophages with group IIA sPLA2 resulted in release of 12-HETE without modification of 12-LO protein levels. Pre-treatment of these cells with baicalein, a 12-LO inhibitor, decreased the sPLA2-induced PGE2 production, significantly reduced COX-2 expression, and inhibited sPLA2-induced ERK; however, it did not affect p38MAPK or PKC phosphorylation. In turn, sPLA2-induced PGE2 release and COX-2 expression, but not NF-κB activation, was attenuated by pre-treating macrophages with PD98059 an inhibitor of ERK1/2. These results suggest that, in macrophages, group IIA sPLA2-induced PGE2 release and COX-2 protein expression are distinctly mediated through 12-HETE followed by ERK1/2 pathway activation, independently of NF-κB activation. These findings highlight an as yet undescribed mechanism by which 12-HETE regulates one of the distinct signaling pathways for snake venom group IIA sPLA2-induced PGE2 release and COX-2 expression in macrophages.

Amplification of the COX/TXS/TP receptor pathway enhances uridine diphosphate-induced contraction by advanced glycation end products in rat carotid arteries.

Advanced glycation end products (AGEs) play a pivotal role in vascular functions under various pathophysiological conditions. Although uridine diphosphate (UDP) is an important extracellular nucleotide, the relationship between AGEs and UDP regarding their effect on vascular functions remains unclear. Therefore, we investigated the effects of AGE-bovine serum albumin (AGE-BSA) on UDP-mediated responses in rat thoracic aorta and carotid arteries. In rat thoracic aorta, UDP-induced relaxation was observed and this relaxation was similar between control (1.0v/v% PBS) and AGE-BSA-treated (0.1mg/mL for 60min) groups. In contrast, contraction but not relaxation was obtained following UDP application to carotid arteries with and without endothelia; contraction was greater in the AGE-BSA-treated group than in the control group. The difference in UDP-induced contraction between the two groups was not abolished by the use of a nitric oxide synthase (NOS) inhibitor, whereas it was abolished by the use of cyclooxygenase (COX), thromboxane synthase (TXS), and thromboxane-prostanoid (TP) receptor antagonist. Further, the difference in UDP-induced contraction was not abolished by the use of a cPLA2 inhibitor, whereas it was abolished by the use of an iPLA2 inhibitor. UDP increased TXA2 release in both groups, and its level was similar in both groups. Moreover, the release of PGE2, PGF2α, and PGI2 was similar among the groups. Under NOS inhibition, TP receptor agonist-induced contraction increased in the AGE-BSA-treated group (vs. control group). In conclusion, the increase in UDP-induced carotid arterial contraction by AGE-BSA can be attributed to an increase in the COX/TXS/TP receptor pathway, particularly, TP receptor signaling.

Matteuinterins A–C, three new glycosides from the rhizomes of Matteuccia intermedia

Phytochemical investigation on the rhizomes of Matteuccia intermedia C.Chr. led to the isolation of three new compounds, named matteuinterins A–C (1–3), together with seven known compounds (4–10). Their structures were elucidated by extensive NMR analyses and chemical derivatization. Compounds 5–10 were evaluated for their anti-inflammatory activities on PGE2 release in LPS-stimulated RAW 264.7 murine macrophages. Compounds 5 and 10 exhibited inhibitory effect on PGE2 production in LPS-activated murine macrophages with IC50 values of 17.8 ± 1.5 and 30.3 ± 2.1 μM, respectively.

Novel insights into the mechanism of cyclophosphamide-induced bladder toxicity: chloroacetaldehyde's contribution to urothelial dysfunction in vitro.

The clinical use of cyclophosphamide and ifosfamide is limited by a resultant bladder toxicity which has been attributed to the metabolite acrolein. Another metabolite chloroacetaldehyde (CAA) associated with nephrotoxicity, has not been investigated for toxicity in the bladder and this study investigates the effects of acrolein and CAA on human urothelial cells in vitro. Human urothelial cells (RT4 and T24) were treated with acrolein or CAA and changes in cell viability, reactive oxygen species, caspase-3 activity and release of urothelial mediators ATP, acetylcholine, PGE2 were measured. The protective effects of N-acetyl cysteine (NAC) were also assessed. Both metabolites were toxic to human urothelial cells, however, CAA significantly decreased cell viability at a ten-fold lower concentration (10µM) than acrolein (100µM). This was associated with increased ROS production and caspase-3 activity. NAC protected cells from these changes. In RT4 cells 100µM acrolein caused a significant increase in basal and stretch-induced ATP, Ach and PGE2 release. In T24 cells chloroacetaldehyde (10µM) increased basal and stimulated ATP and PGE2 levels. Again, NAC protected against changes in urothelial mediator release following acrolein or CAA. This study is the first to report that CAA in addition to acrolein contributes to the urotoxicity of cyclophosphamide and ifosfamide. Both metabolites altered urothelial mediator levels which could contribute to the sensory and functional bladder changes experienced by patients after treatment with cyclophosphamide or ifosfamide. Alterations in urothelial cell viability and mediator release may be causally linked to oxidative stress, with NAC providing protection against these changes.

Prostaglandin E2 receptor EP3 subtype in the paraventricular hypothalamic nucleus mediates corticotropin-releasing factor-induced elevation of plasma noradrenaline levels in rats

Corticotropin-releasing factor (CRF) plays an important role in sympathetic regulation. Centrally administered CRF elevates plasma catecholamine levels, resulting in CRF-dependent hypertension and tachycardia. We previously reported that brain thromboxane A2 mediates CRF-induced elevation of plasma adrenaline levels, whereas prostanoids other than thromboxane A2 mediate elevations in plasma noradrenaline levels. However, the mechanism by which CRF induces elevations in plasma noradrenaline levels remains unknown. Previous studies have revealed that brain prostaglandin (PG) E2, but not other PGs, causes sympathetic activation. In this study, we examined the roles of brain PGE2 and its receptors in CRF-induced elevation of plasma noradrenaline levels in rats. Our results showed that intracerebroventricular pretreatment with an antagonist of the PGE2 receptor EP3 subtype, but not other subtypes, suppressed CRF-induced elevations in plasma noradrenaline levels. We also examined the role of PGE2 and EP3 receptors in the paraventricular hypothalamic nucleus (PVN), the major integrative center for sympathetic regulation, in CRF-induced elevation of plasma noradrenaline levels. Centrally administered CRF increased PGE2 levels in PVN microdialysates, and microinjection of an EP3 receptor agonist into the PVN elevated plasma noradrenaline levels. Bilateral blockade of EP3 receptors in the PVN suppressed the elevation of plasma noradrenaline levels evoked by intracerebroventricular administration and PVN-microinjection of CRF. Our results suggest that CRF stimulates PGE2 release into the PVN that activates EP3 receptors in the PVN, resulting in the elevation of plasma noradrenaline levels.

Evidence of necroptosis in osteoarthritic disease: investigation of blunt mechanical impact as possible trigger in regulated necrosis

Joint injuries are highly associated with cell death and development of posttraumatic osteoarthritis (PTOA). The present study focused on necroptosis as a possible modality of chondrocyte death after cartilage trauma and its relevance in OA disease in general. For this purpose, apoptosis- and necroptosis-associated markers were determined in highly degenerated (ICRS ≥ 3) as well as macroscopically intact cartilage tissue (ICRS ≤ 1) by means of real-time PCR and immunohistochemistry (IHC). Moreover, influence of blunt trauma and/or stimulation with cycloheximide (CHX), TNF-a, and caspase-inhibitor zVAD were investigated in cartilage explants (ICRS ≤ 1). Further characterization of necroptosis was performed in isolated chondrocytes. We found that gene expression levels of RIPK3 (4.2-fold, P < 0.0001) and MLKL (2.7-fold, P < 0.0001) were elevated in highly degenerated cartilage tissue, which was confirmed by IHC staining. After ex vivo trauma and/or CHX/TNF stimulation, addition of zVAD further enhanced expression of necroptosis-related markers as well as release of PGE2 and nitric oxide, which was in line with increased cell death and subsequent release of intracellular HMGB1 and dsDNA in CHX/TNF stimulated chondrocytes. However, trauma and/or chemically induced cell death and subsequent release of pro-inflammatory mediators could be largely attenuated by RIPK1-inhibitor necrostatin 1 or antioxidant N-acetylcysteine. Overall, the study provided clear evidence of necroptotic cell death in OA disease. Moreover, a possible link between cartilage injury and necroptotic processes was found, depending on oxidative stress and cytokine release. These results contribute to further understanding of cell death in PTOA and development of novel therapeutic approaches.

FTY720P Upregulates the Na+/K+ ATPase in HepG2 Cells by Activating S1PR3 and Inducing PGE2 Release.

Liver regeneration is induced by S1P and accompanied with an increase in hepatic Na+/K+ ATPase activity, suggesting a potential modulatory role of the sphingolipid on the ATPase activity. The ability of S1P to alter the ATPase activity was confirmed in a previous work which showed a time dependent effect, with an inhibition appearing at 15min and a stimulation at two hours. The aim of this work was to investigate if FTY720-P, an analogue of S1P used in the treatment of multiple sclerosis, exerts a similar effect at 2 hours. HepG2 cells were treated with FTY720-P for two hours and the activity of the Na+/K+ ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain. The involvement of NF-κB in the pathway was investigated by determining changes in the protein expression of IκB. FTY720-P induced a 2.5-fold increase in the activity of the Na+/K+ ATPase which was maintained in the presence of JTE-013, a specific blocker of S1PR2, but disappeared completely in presence of CAY 10444, a specific S1PR3 antagonist. The involvement of S1PR3 was supported by the stimulation observed with Cym5541, a S1PR3 agonist. FTY720-P increased the expression of COX2, and reduced that of IκB. Its effect was not manifested in presence of indomethacin, a COX inhibitor, or in presence of an NF-κB inhibitor. Exogenous PGE2 induced a significant stimulatory effect. Inhibiting PKC and ERK with respectively calphostin C and PD98059 abolished the effect of FTY720-P on the ATPase and on IκB, but not that of exogenous PGE2 indicating that the two kinases are upstream of NF-κB and PGE2. The PKC activator PMA increased the activity of the Na+/K+ ATPase as well as the expression of phopho-ERK, inferring that PKC is upstream of ERK. It was concluded that FTY720-P stimulates the Na+/K+ ATPase via PGE2 by activating sequentially S1PR3, PKC, ERK, NF-κB. The latter enhances COX-2 expression leading to PGE2 release.

Combinatorial Action of Triterpenoid, Flavonoid, and Alkaloid on Inflammation

In the present study, the synergistic effects of BASk, a combination of betulinic acid (B), apigenin (A), and skimmianine (Sk) in the ratio of 1:1:1, were studied to construct a novel drug mixture against inflammation via the TLR4-nuclear factor Kappa light chain enhancer of activated B cells (NFκB) signaling pathway. In silico drug likeness and docking studies recommended 3 bioactive compounds as suitable ligands for drug development. BASk inhibited TLR4 from its dimerization with MD2 and blocked the TLR4 signaling cascade. Reduced nuclear translocation of NFκB inhibited the release of pro-inflammatory mediators (IL-1β and TNF-α), COX-2 expression, and PGE2. Similarly, BASk exerted its protective role by reducing pro-inflammatory mediators and elevating anti-inflammatory cytokine, IL-10. This confirms the inhibiting potential of BASk in the activation of the TLR4-NFκB signaling cascade. Thus, BASk was superior in its anti-inflammatory effect on oxidized low density lipoprotein (ox-LDL) induced human peripheral blood mononuclear cells than its individual components synergistically. Since BASk inhibited COX-2 expression and further release of PGE2, it is a potent therapeutic agent with better efficacy against inflammation because COX-2 is the target site for treating inflammatory diseases. Thus, it can be clearly stated that this innovation will be a breakthrough in the treatment of inflammatory diseases.

Phenolic rich Cocos nucifera inflorescence extract ameliorates inflammatory responses in LPS-stimulated RAW264.7 macrophages and toxin-induced murine models

Anti-inflammatory and antinociceptive effects of the acetone extract of Cocos nucifera (CnAE), an important ingredient in several traditional drugs, have been studied using different in vitro and in vivo models. CnAE did not show any observable toxicity in RAW264.7 macrophages by MTT assay. The calorimetric analysis (total COX, 5-LOX, MPO, iNOS and NO), ELISA (IL-1β, IL-6, TNF-α and PGE2) and qRT-PCR (IL-1β, IL-6, TNF-α and NF-κB) were performed in LPS-induced RAW264.7 macrophages. Phosphorylation of NF-κBp65 and IκB was determined by western blotting. CnAE (100µg/mL) remarkably inhibited total COX (68.67%) and 5-LOX (63.67%) activities, and subsequent release of iNOS, NO and PGE2 (p ≤ 0.05) in RAW264.7 cells treated with LPS. ELISA showed CnAE markedly decreased the level of pro-inflammatory cytokines IL-1β (p ≤ 0.001), IL-6 (p ≤ 0.001) and TNF-α (p ≤ 0.001) in LPS treated RAW264.7 cells. CnAE (100µg/mL) also significantly down-regulated the mRNA expressions of pro-inflammatory cytokines (IL-1β, p ≤ 0.05; IL-6, p ≤ 0.01 and TNF-α, p ≤ 0.001) and NF-κB (p ≤ 0.001) against LPS-induction. Moreover, LPS-induced phosphorylation of IκB-α and NF-κB p65 was significantly inhibited by CnAE (100µg/mL). In vivo anti-inflammatory studies showed that CnAE (400mg/kg) significantly inhibited carrageenan-induced acute paw oedema (59.81%, p ≤ 0.001) and formalin-induced chronic paw oedema (52.90%, p ≤ 0.001) in mice. CnAE at a dose of 400mg/kg also showed a significant anti-nociceptive effect on acetic acid-induced writhing (48.21%, p ≤ 0.001) and Eddy's hot plate methods. These findings suggest that CnAE has significant anti-inflammatory and anti-nociceptive properties, mainly attributed to the inhibition of NF-κB/IκB signalling cascade.

Oxaliplatin induces prostaglandin E2 release in vascular endothelial cells.

Oxaliplatin (L-OHP) is known to induce adverse reactions at the injection site, including vascular pain, but the underlying mechanisms have not been clarified. Vascular pain during intravenous L-OHP administration can be inhibited by taking non-steroidal anti-inflammatory drugs (NSAIDs). In this study, we investigated the involvement of the arachidonic acid cascade and prostaglandin (PG) E2 and 15d-PGJ2 in vascular pain sensation during intravenous delivery of L-OHP. Cultured normal human umbilical cord vein endothelial cells (HUVECs) were treated with L-OHP or L-OHP + NSAID flurbiprofen for 2h and analyzed for the release of PGE2 and 15d-PGJ2 into culture supernatant by ELISA. The results showed that L-OHP significantly and dose-dependently increased PGE2 secretion by HUVECs; however, flurbiprofen effectively prevented PGE2 increase. On the other hand, cisplatin, another platinum anticancer drug, did not stimulate PGE2 production. Other PGs, including 15d-PGJ2, 6-keto PGF1α, PGF2α, and PGD2 were not increased by L-OHP or cisplatin. Protein expression analysis revealed that cyclooxygenase 1 and cytoplasmic PGE synthase involved in constitutive PG metabolism were expressed in HUVECs but not affected by L-OHP exposure. This study indicates that L-OHP treatment specifically upregulated PGE2 secretion by vascular endothelial cells, which may contribute to vascular pain, and that NSAIDs can be used to inhibit PGE2 release and attenuate L-OHP-induced hyperalgesia.

Synthesis and Biological Evaluation of New Benzylidenethiazolidine-2,4-dione Derivatives as 15-hydroxyprostaglandin Dehydrogenase Inhibitors to Control the Intracellular Levels of Prostaglandin E2 for Wound Healing

A novel series of benzylidenethiazolidine-2,4-dione derivatives was synthesized and investigated for 15-hydroxyprostaglandin dehydrogenase (15-PGDH)-scavenging activity, PGE2 release, and wound-healing activity. Among the tested derivatives, seven compounds (3, 9, 11, 12, 13, 14, and 25) resulted in a 50% inhibition of 15-PGDH at concentrations between 0.07 and 0.2 µM and increased PGE2 levels from 300 to over 600% in A549 cells treated with 5.0 and 10.0 µM of the compounds for 12 h. A scratch wound-healing assay using HaCaT cell line was conducted to verify the effects of 10 µM of these compounds on cell regeneration. The closure rate of the scratch wound healing showed that all compounds (3, 9, 11, 12, 13, 14, and 25) had greater wound regeneration effects than the cell growth factor, TGF-β1, which was used as the positive control. In particular, (Z)-N-benzyl-4-((2,4-dioxothiazolidin-5-ylidene)methyl)benzamide (compound 14) showed that the highest wound closure rate, which was 360%; this is about 3.6-fold higher than that of TGF-β1. Overall, these results show that compound 14 may be considered a promising candidate for the development of novel wound-healing agents.

Targeting mechanotransduction mechanisms and tissue weakening signals in the human amniotic membrane

Mechanical and inflammatory signals in the fetal membrane play an important role in extracellular matrix (ECM) remodelling in order to dictate the timing of birth. We developed a mechanical model that mimics repetitive stretching of the amniotic membrane (AM) isolated from regions over the placenta (PAM) or cervix (CAM) and examined the effect of cyclic tensile strain (CTS) on mediators involved in mechanotransduction (Cx43, AKT), tissue remodelling (GAGs, elastin, collagen) and inflammation (PGE2, MMPs). In CAM and PAM specimens, the application of CTS increased GAG synthesis, PGE2 release and MMP activity, with concomitant reduction in collagen and elastin content. Co-stimulation with CTS and pharmacological agents that inhibit either Cx43 or AKT, differentially influenced collagen, GAG and elastin in a tissue-dependent manner. SHG confocal imaging of collagen fibres revealed a reduction in SHG intensity after CTS, with regions of disorganisation dependent on tissue location. CTS increased Cx43 and AKT protein and gene expression and the response could be reversed with either CTS, the Cx43 antisense or AKT inhibitor. We demonstrate that targeting Cx43 and AKT prevents strain-induced ECM damage and promotes tissue remodelling mechanisms in the AM. We speculate that a combination of inflammatory and mechanical factors could perturb typical mechanotransduction processes mediated by Cx43 signalling. Cx43 could therefore be a potential therapeutic target to prevent inflammation and preterm premature rupture of the fetal membranes.

881 Hair growth and protection properties of an oligosaccharide and peptidic extract from Maca root

The hair follicle is a complex mini-organ that possesses the ability to self-renew through a specific growth cycle composed of 3 distinct phases: anagen (growth phase), catagen (regression phase) and telogen (hair elimination). During hair loss, in particular alopecia, the hair cycle is disrupted with a modification of the anagen/telogen ratio in favor of telogen phase. This results in an atrophy of the hair follicle thus producing shorter and thinner hair until it becomes microscopic. Maca (Lepidium meyenii) is a plant that grows at high altitudes in the Peruvian Andes. We have developed a patented active ingredient from Maca root, “Maca hydrolysate”. This extract, rich in oligosaccharides and peptides, is obtained from maca flour by a bioenzymatic process. Its biological efficacy has been evaluated in in vitro and ex vivo models on key factors involved in hair protection and hair growth. Maca extract was able to protect hair follicles from oxidative and inflammatory stress by decreasing the production of reactive oxygen species induced by lipid peroxide exposure in human follicle dermal papilla cells (HFDPC). The extract also decreased PGE2 release in HFDPC stressed by IL1-alpha, suggesting that Maca hydrolysate exerts anti-inflammatory effect. In a co-culture of human keratinocytes with HFDPC, the Maca extract significantly induced the production of FGF7 and beta–catenin, suggesting that it may induce the promotion of hair growth. Moreover, immunostainings of microdissected human hair follicles suggested that the extract could extend anagen phase and delay catagen. Accordingly, we have finally demonstrated that the extract significantly stimulated hair growth in ex vivo human hair follicles. Taken together, our data show that Maca hydrolysate extract is able to protect hair follicle and to promote hair growth, thereby slowing down hair loss. This new extract may be of particular interest in formulated products designed for the management of hair loss condition.

Paradoxical Effect of LTB4 on the Regulation of Stress-Induced Corticosterone Production.

Depression is a mental illness with a complex and multifactorial etiology, which has been associated with stress and inflammation. Infections, autoimmune diseases, envenomation, and trauma induce an inflammatory response that is characterized by increasing levels of circulating cytokines (e.g., IL-1β) and lipid mediators [e.g., PGE2 and leukotrienes B4 (LTB4)]. Recently, we showed that LTB4 production by the 5-lipoxygenase (5-LO) pathway regulates IL-1β and PGE2 release, reducing tissue damage in a model of sterile inflammation. Since IL-1β and PGE2 increase in serum of stressed patients and potentially trigger depression, we used an animal model of chronic unpredictable stress (CUS) to investigate the potential impact of LTB4 over depression-like symptoms. At basal conditions, 5-LO deficiency (Alox5−/−) reduces the preference for sucrose, while inducing a higher immobilization time on the tail suspension test when compared 129sv. Moreover, Alox5−/− mice present increased caspase-1 expression and elevated levels of IL-1β, IL-17 and PGE2 in the spleen, with increasing corticosterone levels in the frontal cortex but reducing systemic levels. Compared to 129sv mice, CUS induced higher levels of systemic, frontal cortex and hippocampal corticosterone, and also reduced sucrose preference, increased levels of splenic IL-1β, IL-17 and PGE2 and reduced levels of LTB4. Interestingly, CUS exposure did not alter the reduced sucrose preference shown by Alox5−/− mice but greatly enhanced splenic PGE2 production. Compared to Alox5−/− mice at basal conditions, CUS exposure also increased levels of systemic corticosterone, which remained lower than those of CUS-129sv animals. We also observed that treatment with LTB4 decreased caspase-1 expression and systemic levels of corticosterone in CUS-Alox5−/− mice but there was no significant impact on the reduced sucrose preference. Our results demonstrate that LTB4 controls the hypothalamic-pituitary-adrenal (HPA) axis by regulating levels of systemic corticosterone associated with the repression of caspase-1 expression and production of inflammatory mediators. One limitation of our study is that 129sv and Alox5−/− mice were not littermates, not sharing, therefore, the same intra-uterine and preweaning environment. Even so, taken together our results indicate that 5-LO activity is critical for the regulation of stress-induced symptoms, suggesting that the Alox5−/− mouse could be a natural model of corticosterone-independent reduced reward sensitivity.

In-vitro studies of the effects of interferons on endometrial metabolism in sheep

Primary cultures of ovine epithelial and stromal cells have been used to examine paracrine interactions between the endometrium and the preimplantation sheep blastocyst, and in particular the actions of the blastocyst alpha-interferon, ovine trophoblast protein-1 (oTP-1), on endometrial cell metabolism. The synthesis and secretion of several 'pregnancy-related' acidic proteins with molecular weights in the range 70,000-120,000 can be induced by addition of oTP-1 or human recombinant interferon (IFN) to cultured enriched epithelial endometrial cells. Consistent with the antiluteolytic role of oTP-1, dose-dependent attenuation of both PGE and PGF-2 alpha release has been demonstrated. Arachidonic acid added to the cells increased overall PG release but the inhibitory effects of interferons were still apparent. Immunocytochemical analysis of PG synthase demonstrated marked cyclic variation of its localization within the endometrium, but no differences in distribution or intensity of staining were apparent in endometrium of early pregnancy (Day 15) compared with that of the cycle (Day 15). It appears that conceptus-induced changes in PG release do not occur via changes in concentration or localization of PG synthase but rather by modifying its activity. Highly purified epithelial cells cultured on matrigel-coated millicell inserts retain important morphological features seen in vivo. Under such conditions, PGF-2 alpha and PGE release into the basal compartment was greater than that into the apical compartment. Stromal fibroblasts cultured under similar conditions secreted less PGF-2 alpha but more PGE than epithelial cells. Whilst the limitations of in-vitro studies are acknowledged, these findings are compatible with and markedly extend what is known of the action of embryonic interferons in vivo in the establishment of pregnancy in sheep.

Chemical Composition, Antioxidant and Anti-inflammatory Activity Evaluation of the Lebanese Propolis Extract.

Propolis is a resinous substance produced by bees and known to possess antioxidant, antimicrobial, antiproliferative and anti-inflammatory activities. This study is aimed at evaluating the in vivo and in vitro anti-inflammatory potential of the Crude Ethanolic Extract (CE) of Lebanese propolis and its Ethyl Acetate Fraction (EAF). Chemical content of propolis was characterized using high-performance liquid chromatography and LC-MS/MS. COX-2 and iNOS protein expression, nitric oxide (NO) and prostaglandin (PGE2) release in LPS-activated RAW monocytes were achieved respectively by western blot and spectrophotometry. Antioxidant activity was evaluated by DPPH free radical scavenging assay. Measurement of paw thickness in carrageenan-induced paw edema in mice and pathologic assessment of inflammation in paw sections were used to judge the anti-inflammatory properties of propolis. Pathology analysis revealed in the treated group significant reduction of immune cell infiltration and edema. Both extract and ethyl acetate fraction showed significant anti-inflammatory and antioxidant effects in LPS-treated RAW cells characterized by the inhibition of COX-2 and iNOS protein expression, as well as PGE2 and NO release. Chemical analysis of the crude extract and its ethyl acetate fraction identified 28 different compounds of which two phenolic acids and nine other flavonoids were also quantified. Ferulic acid, caffeic acid, chrysin, galangin, quercetin, and pinocembrin were among the most representative compounds. Lebanese propolis is rich in a various amount of flavonoids which showed promising antiinflammatory and antioxidant properties. Additionally, chemical analysis showed unique chemical compositions with the potential of identifying ingredients with interesting anti-inflammatory activities.

Activation of Toll-like receptor 2 induces B1 and B2 kinin receptors in human gingival fibroblasts and in mouse gingiva

The regulation of the kallikrein-kinin system is an important mechanism controlling vasodilation and promoting inflammation. We aimed to investigate the role of Toll-like receptor 2 (TLR2) in regulating kinin B1 and B2 receptor expression in human gingival fibroblasts and in mouse gingiva. Both P. gingivalis LPS and the synthetic TLR2 agonist Pam2CSK4 increased kinin receptor transcripts. Silencing of TLR2, but not of TLR4, inhibited the induction of kinin receptor transcripts by both P. gingivalis LPS and Pam2CSK4. Human gingival fibroblasts (HGF) exposed to Pam2CSK4 increased binding sites for bradykinin (BK, B2 receptor agonist) and des-Arg10-Lys-bradykinin (DALBK, B1 receptor agonist). Pre-treatment of HGF for 24 h with Pam2CSK4 resulted in increased PGE2 release in response to BK and DALBK. The increase of B1 and B2 receptor transcripts by P. gingivalis LPS was not blocked by IL-1β neutralizing antibody; TNF-α blocking antibody did not affect B1 receptor up-regulation, but partially blocked increase of B2 receptor mRNA. Injection of P. gingivalis LPS in mouse gingiva induced an increase of B1 and B2 receptor mRNA. These data show that activation of TLR2 in human gingival fibroblasts as well as in mouse gingival tissue leads to increase of B1 and B2 receptor mRNA and protein.