The protective effects of orexin-A in alleviating cell senescence against interleukin-1ß (IL-1ß) in chondrocytes.

Osteoarthritis (OA) is one of the most important causes of global disability, and dysfunction of chondrocytes is an important risk factor. The treatment of OA is still a challenge. Orexin-A is a hypothalamic peptide, and its effects in OA are unknown. In this study, we found that exposure to interleukin-1ß (IL-1ß) reduced the expression of orexin-2R, the receptor of orexin-A in TC-28a2 chondrocytes. Importantly, the senescence-associated ß-galactosidase (SA-ß-gal) staining assay demonstrated that orexin-A treatment ameliorates IL-1ß-induced cellular senescence. Importantly, the presence of IL-1ß significantly reduced the telomerase activity of TC-28a2 chondrocytes, which was rescued by orexin-A. We also found that orexin-A prevented IL-1ß-induced increase in the levels of Acetyl-p53 and the expression of p21. It is shown that orexin-A mitigates IL-1ß-induced reduction of sirtuin 3 (SIRT3). Silencing of SIRT3 abolished the protective effects of orexin-A against IL-1ß-induced cellular senescence. These results imply that orexin-A might serve as a promising therapeutic agent for OA.

CD200R activation on naïve T cells by B cells induces suppressive activity of T cells via IL-24.

CD200 is an anti-inflammatory protein that facilitates signal transduction through its receptor, CD200R, in cells, resulting in immune response suppression. This includes reducing M1-like macrophages, enhancing M2-like macrophages, inhibiting NK cell cytotoxicity, and downregulating CTL responses. Activation of CD200R has been found to modulate dendritic cells, leading to the induction or enhancement of Treg cells expressing Foxp3. However, the precise mechanisms behind this process are still unclear. Our previous study demonstrated that B cells in Peyer's patches can induce Treg cells, so-called Treg-of-B (P) cells, through STAT6 phosphorylation. This study aimed to investigate the role of CD200 in Treg-of-B (P) cell generation. To clarify the mechanisms, we used wild-type, STAT6 deficient, and IL-24 deficient T cells to generate Treg-of-B (P) cells, and antagonist antibodies (anti-CD200 and anti-IL-20RB), an agonist anti-CD200R antibody, CD39 inhibitors (ARL67156 and POM-1), a STAT6 inhibitor (AS1517499), and soluble IL-20RB were also applied. Our findings revealed that Peyer's patch B cells expressed CD200 to activate the CD200R on T cells and initiate the process of Treg-of-B (P) cells generation. CD200 and CD200R interaction triggers the phosphorylation of STAT6, which regulated the expression of CD200R, CD39, and IL-24 in T cells. CD39 regulated the expression of IL-24, which sustained the expression of CD223 and IL-10 and maintained the cell viability. In summary, the generation of Treg-of-B (P) cells by Peyer's patch B cells was through the CD200R-STAT6-CD39-IL-24 axis pathway.

Molecular incompatibility between pig CD200 and human CD200 receptor in in vitro xenogeneic immune responses.

Overexpression of human CD200 (hCD200) in porcine endothelial cells (PECs) has been reported to suppress xenogeneic immune responses of human macrophages against porcine endothelial cells. The current study aimed to address whether the above-mentioned beneficial effect of hCD200 is mediated by overcoming the molecular incompatibility between porcine CD200 (pCD200) and hCD200 receptor or simply by increasing the expression levels of CD200 without any molecular incompatibility across the two species. We overexpressed hCD200 or pCD200 using lentiviral vectors with V5 marker in porcine endothelial cells and compared their suppressive activity against U937-derived human macrophage-like cells (hMCs) and primary macrophages. In xenogeneic coculture of porcine endothelial cells and human macrophage-like cells or macrophages, hCD200-porcine endothelial cells suppressed phagocytosis and cytotoxicity of human macrophages to a greater extent than pCD200-porcine endothelial cells. Secretion of tumor necrosis factor-α, interleukin-1ß, and monocyte chemoattractant protein-1 from human macrophages and expression of M1 phenotypes (inducible nitric oxide synthase, dectin-1, and CD86) were also suppressed by hCD200 to a greater extent than pCD200. Furthermore, in signal transduction downstream of CD200 receptor, hCD200 induced Dok2 phosphorylation and suppressed IκB phosphorylation to a greater extent than pCD200. The above data supported the possibility of a significant molecular incompatibility between pCD200 and human CD200 receptor, suggesting that the beneficial effects of hCD200 overexpression in porcine endothelial cells could be mediated by overcoming the molecular incompatibility across the species barrier rather than by simple overexpression effects of CD200.

CD200R promotes high glucose-induced oxidative stress and damage in human retinal pigment epithelial cells by activating the mTOR signaling pathway.

Diabetic retinopathy (DR) is established as the primary cause of visual impairment and preventable blindness, posing significant social and economic burdens on healthcare systems worldwide. Oxidative stress has been identified as a major contributor to DR, yet the precise role of the transmembrane glycoprotein CD200R in this context remains elusive. We studied human retinal pigment epithelia ARPE-19 cells to investigate the role of CD200R in high-glucose (HG) induced oxidative stress. Under HG conditions, we found a significant increase in CD200R expression in a time-dependent pattern. Conversely, knockdown of CD200R effectively alleviated oxidative stress and restored cell viability in HG-treated ARPE-19 cells, a phenomenon corroborated by the addition of a reactive oxygen species (ROS) scavenger. Exploration of the AKT/mTOR signaling pathway confirmed its mediating role regarding CD200R knockdown suppression of the expression of key proteins induced by HG conditions. Additionally, we found that the inhibition of mTOR signaling with Rapamycin effectively countered HG-induced oxidative stress in ARPE-19 cells, suggesting a promising therapeutic target against oxidative stress in the context of DR. This study establishes the crucial role of CD200R in HG-induced oxidative stress and identifies potential therapeutic avenues for the treatment of DR.

The differential effects of blocking retinal orexin receptors on the expression of retinal c-fos and hypothalamic Vip, PACAP, Bmal1, and c-fos in Male Wistar Rats.

Orexin A and B (OXA and OXB) and their receptors are expressed in the majority of retinal neurons in humans, rats, and mice. Orexins modulate signal transmission between the different layers of the retina. The suprachiasmatic nucleus (SCN) and the retina are central and peripheral components of the body's biological clocks; respectively. The SCN receives photic information from the retina through the retinohypothalamic tract (RHT) to synchronize bodily functions with environmental changes. In present study, we aimed to investigate the impact of inhibiting retinal orexin receptors on the expression of retinal Bmal1 and c-fos, as well as hypothalamic c-fos, Bmal1, Vip, and PACAP at four different time-points (Zeitgeber time; ZT 3, 6, 11, and ZT-0). The intravitreal injection (IVI) of OX1R antagonist (SB-334867) and OX2R antagonist (JNJ-10397049) significantly up-regulated c-fos expression in the retina. Additionally, compared to the control group, the combined injection of SB-334867 and JNJ-10397049 showed a greater increase in retinal expression of this gene. Moreover, the expression of hypothalamic Vip and PACAP was significantly up-regulated in both the SB-334867 and JNJ-10397049 groups. In contrast, the expression of Bmal1 was down-regulated. Furthermore, the expression of hypothalamic c-fos was down-regulated in all groups treated with SB-334867 and JNJ-10397049. Additionally, the study demonstrated that blocking these receptors in the retina resulted in alterations in circadian rhythm parameters such as mesor, amplitude, and acrophase. Finally, it affected the phase of gene expression rhythms in both the retina and hypothalamus, as identified through cosinor analysis and the zero-amplitude test. This study represents the initial exploration of how retinal orexin receptors influence expression of rhythmic genes in the retina and hypothalamus. These findings could provide new insights into how the retina regulates the circadian rhythm in both regions and illuminate the role of the orexinergic system expression within the retina.

Myeloid cell expression of CD200R is modulated in active TB disease and regulates Mycobacterium tuberculosis infection in a biomimetic model.

A robust immune response is required for resistance to pulmonary tuberculosis (TB), the primary disease caused by Mycobacterium tuberculosis (Mtb). However, pharmaceutical inhibition of T cell immune checkpoint molecules can result in the rapid development of active disease in latently infected individuals, indicating the importance of T cell immune regulation. In this study, we investigated the potential role of CD200R during Mtb infection, a key immune checkpoint for myeloid cells. Expression of CD200R was consistently downregulated on CD14+ monocytes in the blood of subjects with active TB compared to healthy controls, suggesting potential modulation of this important anti-inflammatory pathway. In homogenized TB-diseased lung tissue, CD200R expression was highly variable on monocytes and CD11b+HLA-DR+ macrophages but tended to be lowest in the most diseased lung tissue sections. This observation was confirmed by fluorescent microscopy, which showed the expression of CD200R on CD68+ macrophages surrounding TB lung granuloma and found expression levels tended to be lower in macrophages closest to the granuloma core and inversely correlated with lesion size. Antibody blockade of CD200R in a biomimetic 3D granuloma-like tissue culture system led to significantly increased Mtb growth. In addition, Mtb infection in this system reduced gene expression of CD200R. These findings indicate that regulation of myeloid cells via CD200R is likely to play an important part in the immune response to TB and may represent a potential target for novel therapeutic intervention.

Imaging flow cytometry of tumoroids: A new method for studying GPCR expression.

Fluorescence confocal microscopy is commonly used to analyze the regulation membrane proteins expression such as G protein-coupled receptors (GPCRs). With this approach, the internal movement of GPCRs within the cell can be observed with a high degree of resolution. However, these microscopy techniques led to complex and time-consuming analysis and did not allow a large population of events to be sampled. A recent approach termed imaging flow cytometry (IFC), which combines flow cytometry and fluorescence microscopy, had two main advantages to study the regulation of GPCRs expression such as orexins receptors (OXRs): the ability (1) to analyze large numbers of cells and; (2) to visualize cell integrity and fluorescent markers localization. Here, we compare these two technologies using the orexin A (OxA) ligand coupled to rhodamine (OxA-rho) to investigate anti-tumoral OX1R expression in human digestive cancers. IFC has been adapted for cancer epithelial adherent cells and also to 3D cell culture tumoroids which partially mimic tumoral structures. In the absence of specific antibody, expression of OX1R is examined in the presence of OxA-rho. 2D-culture of colon cancer cells HT-29 exhibits a maximum level of OX1R internalization induced by OxA with 19% ± 3% colocalizing to early endosomes. In 3D-culture of HT-29 cells, internalization of OX1R/OxA-rho reached its maximum at 60 min, with 30.7% ± 6.4% of OX1R colocalizing with early endosomes. This is the first application of IFC to the analysis of the expression of a native GPCR, OX1R, in both 2D and 3D cultures of adherent cancer cells.

The integrative role of orexin-1 and orexin-2 receptors within the hippocampal dentate gyrus in the modulation of the stress-induced antinociception in the formalin pain test in the rat.

The stressful experiences, by triggering a cascade of hormonal and neural changes, can produce antinociception commonly referred to as stress-induced antinociception (SIA). Orexin neuropeptides have an essential role in stress responses and pain modulation. The dentate gyrus receives orexinergic projections and has been shown to be involved in pain processing. The current study investigated the possible role of orexin-1 and orexin-2 receptors (OX1r and OX2r, respectively) within the dentate gyrus in SIA in a rat model of formalin-induced pain behavior in one hind paw. Male Wistar rats weighing 230-250 g underwent stereotaxic surgery and a cannula was implanted in their brains, above the dentate gyrus region. Either SB334867 or TCS OX2 29 (OX1r and OX2r antagonists, respectively) was microinjected into the dentate gyrus region at a range of doses at 1, 3, 10, and 30 nmol (control group received DMSO 12% as vehicle), 5 min before the forced swim stress (FSS) exposure. The formalin test was performed to assess pain-related behaviors. The results indicated that FSS exposure relieves pain-related behavior in the early and late phases of the formalin test. Blockade of intra-dentate gyrus OX1 or OX2 receptors reduced the antinociceptive responses induced by FSS in the formalin test, with more impact during the late phase. Our findings support the potential role of intra-dentate gyrus orexin receptors as target sites of orexin neurons in painful and stressful situations. Therefore, understanding the exact mechanisms of SIA and the role of the orexinergic system in this phenomenon can lead to identifying the strategies to guide future research and offer a new approach to discovering new pain therapeutic agents.

Orexins and Prostate Cancer: State of the Art and Potential Experimental and Therapeutic Perspectives.

Prostate cancer (PCa) is the second most common cancer in humans. Peptides have recently been used as targeted therapeutics in cancers, due to their extensive multi-functional applications. Two hypothalamic peptides, orexins A (OXA) and B (OXB) and their specific receptors, orexin receptor 1 (OX1R) and 2 (OX2R), orchestrate several biological processes in the central nervous system and peripheral organs. However, in addition to their role in physiological responses, orexins are involved in numerous inflammatory and/or neoplastic pathologies. The presence and expression of orexins in different cancer models, including prostate cancer, and their role in inducing pro- or anti-apoptotic responses in tumor cell lines, suggest that the orexinergic system might have potential therapeutic action or function as a diagnostic marker in PCa. In addition to the traditional animal models for studying human PCa, the canine model might also serve as an additional tool, due to its clinical similarities with human prostate cancer.

Anandamide improves food intake and orexinergic neuronal activity in the chronic sleep deprivation induction model in rats by modulating the expression of the CB1 receptor in the lateral hypothalamus.

Sleep deprivation alters orexinergic neuronal activity in the lateral hypothalamus (LH), which is the main regulator of sleep-wake, arousal, appetite, and energy regulation processes. Cannabinoid receptor (CBR) expression in this area is involved in modulating the function of orexin neurons. In this study, we investigated the effects of endocannabinoid anandamide (AEA) administration on improving food intake and appetite by modulating the activity of orexin neurons and CB1R expression after chronic sleep deprivation. Adult male Wistar rats (200-250 g) were randomly divided into three groups: control + vehicle (Control), chronic sleep deprivation + vehicle (SD), and chronic sleep deprivation +20 mg/kg AEA (SD + A). For SD induction, the rats were kept in a sleep deprivation device for 18 h (7 a.m. to 1 a.m.) daily for 21 days. Weight gain, food intake, the electrical power of orexin neurons, CB1R mRNA expression in hypothalamus, CB1R protein expression in the LH, TNF-α, IL-6, IL-4 levels and antioxidant activity in hypothalamus were measured after SD induction. Our results showed that AEA administration significantly improved food intake (p < 0.01), Electrical activity of orexin neurons (p < 0.05), CB1R expression in the hypothalamus (p < 0.05), and IL-4 levels (p < 0.05). AEA also reduced mRNA expression of OX1R and OX2R (p < 0.01 and p < 0.05 respectively), also IL-6 and TNF-α (p < 0.01) and MDA level (p < 0.05) in hypothalamic tissue. As a consequence, AEA modulates orexinergic system function and improves food intake by regulating the expression of the CB1 receptor in the LH in sleep deprived rats.

Restraint stress potentiates sensitivity to the antinociceptive effect of morphine through orexin receptors in the ventral tegmental area.

Orexin signaling in the ventral tegmental area (VTA) plays a critical role in stress and addictive behaviors. On the other hand, exposure to stress potentiates behavioral sensitization to drugs of abuse such as morphine. This study aimed to elucidate the role of orexin receptors within the VTA in restraint stress (RS)-induced morphine sensitization. Adult male albino Wistar rats underwent stereotaxic surgery, and two stainless steel guide cannulae were bilaterally implanted into the VTA. Different doses of SB334867 or TCS OX2 29 as orexin-1 (OX1) and orexin-2 (OX2) receptor antagonists were microinjected into the VTA five min before exposure to RS, respectively. A duration of three hours was considered for applying the RS, and 10 min after RS exposure, animals received a subcutaneous injection of an ineffective dose of morphine (1 mg/kg) for three consecutive days followed by a five-day drug/stress-free period. On the ninth day, the tail-flick test evaluated the sensitivity to the antinociceptive effects of morphine. The results demonstrated that the sole application of RS or morphine (1 mg/kg) could not induce morphine sensitization; however, concurrent application of RS and morphine could induce morphine sensitization. Besides, intra-VTA administration of OX1 R or OX2 R antagonists before paired administration of morphine and RS blocked morphine sensitization. The role of OX1 R and OX2 R in the induction of stress-induced morphine sensitization was almost identical. This study provides new insight into the role of orexin signaling in the VTA in the potentiation of morphine sensitization induced by RS and morphine co-administration.

Orexin system in buffalo ovarian follicles and effect of orexin on oestradiol production.

Orexin is a ligand for orexin receptors OX1R and OX2R; it is a neuropeptide with pleiotropic functions, including regulation of reproduction. The current study was carried out to investigate the mRNA expression of the prepro-orexin gene (PPO) and orexin receptors (OX1R and OX2R) in ovarian follicles during different stages of their development in the ovary of water buffalo (Bubalus bubalis) and to determine the role of orexin on oestradiol production. Ovarian follicles were classified into four groups based on size and oestradiol (E2 ) level in the follicular fluid (FF) as follows: (i) small or F1, (ii) medium or F2, (iii) large or F3, and (iv) dominant/pre-ovulatory follicle or F4. In follicles, the mRNA expression of PPO and OX1R was greater in F3 and F4 follicles in granulosa cells (GC) and theca interna (TI) cells. OX2R expression did not vary among the different follicular stages in GC. Orexin-A and orexin receptors were localized in the cytoplasm of GC and TI, and intensity was higher in F3 and F4 follicles. In addition, we cultured GC and treated them at 0.1, 1.0, and 10 ng/mL orexin-A alone or in the presence of FSH (30 ng/mL) or IGF-I (10 ng/mL) for 48 h. There was a significant (p < .05) increase in oestradiol (E2 ) secretion and cytochrome P0450 family 19 subfamily A member 1 (CYP19A1) expression from GC at 1.0 and 10.0 ng/mL orexin-A in the presence of 30 ng/mL follicle-stimulating hormone (FSH) or 10 ng/mL insulin-like growth factor-I (IGF-I). In conclusion, the present study provided evidence that the orexin system is expressed in buffalo ovarian follicles, and orexin-A in the presence of FSH and IGF-I has a stimulatory effect on oestradiol secretion from the GC of water buffalo.

The stress-induced antinociceptive responses to the persistent inflammatory pain involve the orexin receptors in the nucleus accumbens.

Stress suppresses the sense of pain, a physiological phenomenon known as stress-induced analgesia (SIA). Brain orexin peptides regulate many physiological functions, including wakefulness and nociception. The contribution of the orexinergic system within the nucleus accumbens (NAc) in the modulation of antinociception induced by forced swim stress (FSS) remains unclear. The present study addressed the role of intra-accumbal orexin receptors in the antinociceptive responses induced by FSS during the persistent inflammatory pain model in the rat. Stereotaxic surgery was performed unilaterally on 106 adult male Wistar rats weighing 250-305 g. Different doses (1, 3, 10, and 30 nmol/ 0.5 µl DMSO) of orexin-1 receptor (OX1r) antagonist (SB334867) or OX2 receptor antagonist (TCS OX2 29) were administered into the NAc five minutes before exposure to FSS for a 6-min period. The formalin test was carried out using formalin injection (50 µl; 2.5%) into the rat's hind paw plantar surface, which induces biphasic pain-related responses. The first phase begins immediately after formalin infusion and takes 3-5 min. Subsequently, the late phase begins 15-20 min after formalin injection and takes 20-40 min. The findings demonstrated that intra-accumbal microinjection of SB334867 or TCS OX2 29 attenuated the FSS-induced antinociception in both phases of the formalin test, with the TCS OX2 29 showing higher potency. Moreover, the effect of TCS OX2 29 was more significant during the early phase of the formalin test. The results suggest that OX1 and OX2 receptors in the NAc might modulate the antinociceptive responses induced by the FSS.

Inhibition of the retinal orexin receptors affects the hypothalamic-pituitary-gonadal axis through retinal pituitary adenylate cyclase activating polypeptide (PACAP) in male Wistar rats.

Orexins A and B (OXA and OXB) and their receptors are expressed in the retina of both human and rodents and play a vital role in regulating signal transmission circuits in the retina. There is an anatomical-physiological relationship between the retinal ganglion cells and suprachiasmatic nucleus (SCN) through glutamate as a neurotransmitter and retinal pituitary adenylate cyclase-activating polypeptide (PACAP) as a co-transmitter. SCN is the main brain center for regulating the circadian rhythm, which governs the reproductive axis. The impact of retinal orexin receptors on the hypothalamic-pituitary-gonadal axis has not been investigated. Retinal OX1R or/and OX2R in adult male rats by 3 µl of SB-334867 (1 µg) or/and 3 µl of JNJ-10397049 (2 µg) were antagonized via intravitreal injection (IVI). Four time-periods were considered (3, 6, 12, and 24 h) for the controls without any treatment, SB-334867, JNJ-10397049, and SB-334867 + JNJ-10397049 groups. Antagonizing retinal OX1R or/and OX2R resulted in a significant elevation of retinal PACAP expression compared to control animals. In addition, expression of GnRH increased non-significantly in the hypothalamus over the 6 h of the study, and the serum concentration of LH decreased significantly in the SB-334867 group after 3 h of injection. Furthermore, testosterone serum levels declined significantly, especially within 3 h of injection; serum levels of progesterone were also exposed to a significant rise at least within 3 h of injection. However, the retinal PACAP expression changes were mediated by OX1R more effectively than by OX2R. In this study, we report the retinal orexins and their receptors as light-independent factors by which the retina affects the hypothalamic-pituitary-gonadal axis.

The hypothalamic RFamide, QRFP, increases feeding and locomotor activity: The role of Gpr103 and orexin receptors.

Here we show that central administration of pyroglutamylated arginine-phenylamine-amide peptide (QRFP/26RFa) increases both food intake and locomotor activity, without any significant effect on energy expenditure, thermogenesis or reward. Germline knock out of either of the mouse QRFP receptor orthologs, Gpr103a and Gpr103b, did not produce a metabolic phenotype. However, both receptors are required for the effect of centrally administered QRFP to increase feeding and locomotor activity. As central injection of QRFP activated orexin/hypocretin neurons in the lateral hypothalamus, we compared the action of QRFP and orexin on behaviour. Both peptides increased arousal and locomotor activity. However, while orexin increased consummatory behaviour, QRFP also affected other appetitive behaviours. Furthermore, the feeding but not the locomotor response to QRFP, was blocked by co-administration of an orexin receptor 1 antagonist. These results suggest that QRFP agonism induces both appetitive and consummatory behaviour, but only the latter is dependent on orexin/hypocretin receptor signalling.

Orexin receptors in the CA1 region of hippocampus modulate the stress-induced antinociceptive responses in an animal model of persistent inflammatory pain.

Stress activates multiple neural pathways and neurotransmitters that often suppress pain perception, the phenomenon called stress-induced analgesia (SIA). Orexin neurons from the lateral hypothalamus project to entire brain structures such as the hippocampus. The present study examined this hypothesis that orexinergic receptors in the CA1 region of the hippocampus may play a modulatory role in the development of SIA in formalin test as an animal model of persistent inflammatory pain. One hundred-two adult male Wistar rats were administered with intra-CA1 orexin-1 receptor (OX1r) antagonist, SB334867, at the doses of 3, 10, 30, and 100 nmol or TCS OX2 29 as orexin-2 receptor (OX2r) antagonist at the doses of 1, 3, 10, and 30 nmol. Five min later, rats were exposed to forced swim stress (FSS) for a 6-min period. Then, pain-related behaviors induced by formalin injection were measured at the 5-min blocks during a 60-min period of formalin test. The current study indicated that solely stress exposure elicits antinociception in the early and late phases of the formalin test. The FSS-induced analgesia was prevented by intra-CA1 administration of SB334867 or TCS OX2 29 during either phase of the formalin test. Moreover, the contribution of the OX2r in the mediation of analgesic effect of stress was more prominent than that of the OX1r during both phases of the formalin test. It is suggested that OX1r and OX2r in the CA1 region of the hippocampus are involved in stress-induced analgesia in the animal model of persistent inflammatory pain.

MicroRNA-129-5p-regulated microglial expression of the surface receptor CD200R1 controls neuroinflammation.

CD200R1 is an inhibitory surface receptor expressed in microglia and blood macrophages. Microglial CD200R1 is known to control neuroinflammation by keeping the microglia in resting state, and therefore, tight regulation of its expression is important. CCAAT/enhancer-binding protein ß (CEBPß) is the known regulator of CD200R1 transcription. In the present study, our specific intention was to find a possible posttranscriptional regulatory mechanism of CD200R1 expression. Here we investigated a novel regulatory mechanism of CD200R1 expression following exposure to an environmental stressor, arsenic, combining in silico analysis, in vitro, and in vivo experiments, as well as validation in human samples. The in silico analysis and in vitro studies with primary neonatal microglia and BV2 microglia revealed that arsenic demethylates the promoter of a microRNA, miR-129-5p, thereby increasing its expression, which subsequently represses CD200R1 by binding to its 3'-untranslated region and shuttling the CD200R1 mRNA to the cytoplasmic-processing body in mouse microglia. The role of miR-129-5p was further validated in BALB/c mouse by stereotaxically injecting anti-miR-129. We found that anti-miR-129 reversed the expression of CD200R1, as well as levels of inflammatory molecules IL-6 and TNF-α. Experiments with a CD200R1 siRNA-induced loss-of-function mouse model confirmed an miR-129-5p→CD200R1→IL-6/TNF-α signaling axis. These main findings were replicated in a human cell line and validated in human samples. Taken together, our study revealed miR-129-5p as a novel posttranscriptional regulator of CD200R1 expression with potential implications in neuroinflammation and related complications.

Orexin-1 receptor signaling in ventral tegmental area mediates cue-driven demand for cocaine.

Drug-associated sensory cues increase motivation for drug and the orexin system is importantly involved in this stimulus-enhanced motivation. Ventral tegmental area (VTA) is a major target by which orexin signaling modulates reward behaviors, but it is unknown whether this circuit is necessary for cue-driven motivation for cocaine. Here, we investigated the role of VTA orexin signaling in cue-driven motivation for cocaine using a behavioral economics (BE) paradigm. We found that infusion of the orexin-1 receptor (Ox1R) antagonist SB-334867 (SB) into VTA prior to BE testing reduced motivation when animals were trained to self-administer cocaine with discrete cues and tested on BE with those cues. SB had no effect when animals were trained to self-administer cocaine without cues or tested on BE without cues, indicating that learning to associate cues with drug delivery during self-administration training was necessary for cues to recruit orexin signaling in VTA. These effects were specific to VTA, as injections of SB immediately dorsal had no effect. Moreover, intra-VTA SB did not have an impact on locomotor activity, or low- or high-effort consumption of sucrose. Finally, we microinjected a novel retrograde adeno-associated virus (AAVretro) containing an orexin-specific short hairpin RNA (OxshRNA) into VTA to knock down orexin in the hypothalamus-VTA circuit. These injections significantly reduced orexin expression in lateral hypothalamus (LH) and decreased cue-driven motivation. These studies demonstrate a role for orexin signaling in VTA, specifically when cues predict drug reward.

Orexin-A Regulates Follicular Growth, Proliferation, Cell Cycle and Apoptosis in Mouse Primary Granulosa Cells via the AKT/ERK Signaling Pathway.

Granulosa cells (GCs) are essential for follicular growth, development, and atresia. The orexin-A (OXA) neuropeptide is widely involved in the regulation of various biological functions. OXA selectively binds to orexin receptor type 1 (OX1R) and mediates all its biological actions via OX1R. This study aimed to explore the expression of OXA and OX1R and their regulatory role in GCs proliferation, cell cycle progression, apoptosis, oocyte maturation, and underlying molecular mechanisms of these processes and elucidate its novel signaling pathway. Western blotting and RT-qPCR showed that OXA and OX1R were expressed during different developmental stages of GCs, and siRNA transfection successfully inhibited the expression of OX1R at the translational and transcriptional levels. Flow cytometry revealed that OX1R knockdown upregulated GCs apoptosis and triggered S-phase arrest in cell cycle progression. RT-qPCR and Western blotting showed significantly reduced expression of Bcl-2 and elevated expression of Bax, caspase-3, TNF-α, and P21 in OX1R-silenced GCs. Furthermore, the CCK-8 assay showed that knockdown of OX1R suppressed GCs proliferation by downregulating the expression of PCNA, a proliferation marker gene, at the translational and transcriptional levels. Western blotting revealed that knockdown of OX1R resulted in a considerable decrease of the phosphorylation level of the AKT and ERK1/2 proteins, indicating that the AKT/ERK1/2 pathway is involved in regulating GCs proliferation and apoptosis. In addition, OX1R silencing enhanced the mRNA expression of GDF9 and suppressed the mRNA expression of BMP15 in mouse GCs. Collectively, these results reveal a novel regulatory role of OXA in the development of GCs and folliculogenesis by regulating proliferation, apoptosis, and cell cycle progression. Therefore, OXA can be a promising therapeutic agent for female infertility.

Orexin-A alleviates astrocytic apoptosis and inflammation via inhibiting OX1R-mediated NF-κB and MAPK signaling pathways in cerebral ischemia/reperfusion injury.

Orexin-A (OXA) is a neuropeptide with neuroprotective effect by reducing cerebral ischemia/reperfusion injury (CIRI). Inflammation and apoptosis mediated by astrocyte activation are the key pathological mechanisms for CIRI. We thus attempted to confirm neuroprotective effects of OXA on astrocytic inflammation and apoptosis in CIRI and clarify the relative mechanisms. A middle cerebral artery occlusion and reperfusion (MCAO/R) rat model and U251 glioma cells model subjected to oxygen glucose deprivation and reperfusion (OGD/R) were established, with or without OXA treatment. Neurological deficit score was determined, and cerebral infarct volume was evaluated by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Western Blot was used to detect the expressions of NF-κB p65, p-p65, p-ERK, p-p38, GFAP, OX1R, IL-1ß, TNF-α, IL-6, iNOS, Bcl-2, Bax, CytC, cleaved caspase-9 and cleaved caspase-3 in vivo and in vitro. Pro-inflammatory cytokines in cell supernatant IL-1ß, TNF-α and IL-6 were determined by ELISA. Hoechst 33342 staining was used to detect the apoptosis of astrocyte. Immunofluorescent staining was performed to assess the nuclear translocation of p65 and the expression of GFAP. The results showed that OXA significantly improved neurological deficit score and decreased the volume of infarct area in brain. OXA decreased inflammatory mediators, inhibited astrocyte activation and nuclear translocation of NF-κB and phosphorylation of NF-κB, MAPK/ERK and MAPK/p38. Besides, OXA suppressed apoptosis via upregulating the ratio of Bcl-2/Bax and downregulating cytochrome C, cleaved-caspase-9 and cleaved caspase-3. Overall, it was concluded that OXA exerts neuroprotective effect during CIRI through attenuating astrocytes apoptosis, astrocytes activation and pro-inflammatory cytokines production, by Inhibiting OX1R-mediated NF-κB, MAPK/ERK and MAPK/p38 signaling pathways. The progress in our study is helpful to elucidate the molecular mechanisms of OXA neuroprotection, which could lead to the development of new treatment strategies for ischemic stroke.