Orexin B protects dopaminergic neurons from 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity associated with reduced extracellular signal-regulated kinase phosphorylation.

BACKGROUND: The loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) is a major pathological hallmark of Parkinson's disease (PD). Orexin B (OXB) has been reported to promote the growth of DA neurons. However, the roles of OXB in the degeneration of DA neurons still remained not fully clear. METHODS: An in vivo PD model was constructed by administrating 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice. Pole test was performed to investigate the motor function of mice and the number of DA neurons was detected by immunofluorescence (IF). A PD cell model was established by treating SH-SY5Y cells with 1-methyl-4-phenylpyridinium (MPP+). OXB was added to the culture medium 2 h after MPP + treatment. Microscopic analysis was carried out to investigate the function of OXB in the cell model of PD 24 h after MPP + challenge. RNA-Seq analysis of the PD cell model was performed to explore the possible mechanisms. Western blot was used to detect the phosphorylation levels of extracellular signal-regulated kinase (ERK). RESULTS: OXB significantly decreased the DA neurons death caused by MPTP, alleviated MPP+-induced neurotoxicity in SH-SY5Y cells, and robustly enhanced the weight and motor ability of PD mice. Besides, RNA-Seq analysis demonstrated that the mitogen-activated protein kinase (MAPK) pathway was involved in the pathology of PD. Furthermore, MPP + led to increased levels of phosphorylation of ERK (p-ERK), OXB treatment significantly decreased the levels of p-ERK in MPP+-treated SH-SY5Y cells. CONCLUSIONS: This study demonstrated that OXB exerts a neuroprotective role associated with reduced ERK phosphorylation in the PD model. This suggests that OXB may have therapeutic potential for treatment of PD.

Mutual Effects of Orexin and Bone Morphogenetic Proteins on Catecholamine Regulation Using Adrenomedullary Cells.

Orexins are neuronal peptides that play a prominent role in sleep behavior and feeding behavior in the central nervous system, though their receptors also exist in peripheral organs, including the adrenal gland. In this study, the effects of orexins on catecholamine synthesis in the rat adrenomedullary cell line PC12 were investigated by focusing on their interaction with the adrenomedullary bone morphogenetic protein (BMP)-4. Orexin A treatment reduced the mRNA levels of key enzymes for catecholamine synthesis, including tyrosine hydroxylase (Th), 3,4-dihydroxyphenylalanie decarboxylase (Ddc) and dopamine ß-hydroxylase (Dbh), in a concentration-dependent manner. On the other hand, treatment with BMP-4 suppressed the expression of Th and Ddc but enhanced that of Dbh with or without co-treatment with orexin A. Of note, orexin A augmented BMP-receptor signaling detected by the phosphorylation of Smad1/5/9 through the suppression of inhibitory Smad6/7 and the upregulation of BMP type-II receptor (BMPRII). Furthermore, treatment with BMP-4 upregulated the mRNA levels of OX1R in PC12 cells. Collectively, the results indicate that orexin and BMP-4 suppress adrenomedullary catecholamine synthesis by mutually upregulating the pathway of each other in adrenomedullary cells.

Selenium alleviates modafinil-induced neurobehavioral toxicity in rat via PI3K/Akt/mTOR/GSK3B signaling pathway and suppression of oxidative stress and apoptosis: in vivo and in silico study.

Nonmedical use of modafinil (MOD) led to increased rates of overdose toxicity, road accidents, addiction, withdrawal, suicide, and mental illnesses. The current study aims to determine the probable MOD brain toxicity and elucidate the possible role of selenium (Se) in ameliorating the neurotoxicity in rat models. Fifty-four male Albino rats were randomly assigned into nine groups. The groups were G1 (control negative), G2 (Se0.1), G3 (Se0.2), G4 (MOD300), G5 (MOD600), G6 (Se0.1 + MOD300), G7 (Se0.2 + MOD300), G8 (Se0.1 + MOD600), and G9 (Se0.2 + MOD600). After finishing the experiment, blood and brain tissue were harvested for biochemical and histological investigation. Neurobehavior parameters were assessed. Tissue neurotransmitter levels and oxidative stress markers were assessed. Gene expression of PI3K/Akt/mTOR-GSK3B, orexin, and orexin receptor2 was measured by qRT-PCR. Histological and immunohistochemistry assessments, as well as molecular docking, were carried out. MOD-induced neurobehavioral toxicity exhibited by behavioral and cognitive function impairments, which are associated with decreased antioxidant activities, increased MDA levels, and decreases in neurotransmitter levels. Brain levels of mRNA expression of PI3K, Akt, and mTOR were decreased, while GS3K, orexin, and orexin receptors were significantly elevated. These disturbances were confirmed by histopathological brain changes with increased silver and Bax immunostaining and decreased crystal violet levels. MOD induced neurotoxic effects in a dose-dependent manner. Compared with the MOD groups, SE coadministration significantly attenuates MOD-induced toxic changes. Docking study shows the protective role of Se as an apoptosis inhibitor and inflammation inhibitor. In conclusion, Se could be used as a biologically effective antioxidant compound to protect from MOD neurobehavioral toxicity in Wistar rats by reversing behavioral alterations, inflammation, apoptosis, and oxidative injury.

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.

C-terminal peptide of preproorexin enhances brain-derived neurotrophic factor expression in rat cerebrocortical cells and recognition memory in mice.

During the production of orexin A and B from preproorexin, a common precursor protein, in hypothalamic orexin neurons, C-terminal peptide (herein called preproorexin C-peptide) is concomitantly produced via post-translational processing. The predicted three-dimensional structure of preproorexin C-peptide is similar among mammalian species, suggestive of a conserved function in the mammalian brain. However, C-peptide has long been regarded as a non-functional peptide. We herein examined the effects of rat and/or mouse preproorexin C-peptide on gene expression and cell viability in cultured rat cerebrocortical cells and on memory behavior in C57BL/6J mice. Rat and mouse C-peptides both increased brain-derived neurotrophic factor (Bdnf) mRNA levels. Moreover, C-peptide enhanced high K+-, glutamate-, and BDNF-induced increases in Bdnf mRNA levels without affecting forskolin-induced Bdnf expression. H-89, a protein kinase A inhibitor, blocked C-peptide-induced Bdnf expression, whereas rolipram, a phosphodiesterase inhibitor, enhanced this effect. Intracellular cyclic AMP concentrations were elevated by C-peptide. These results demonstrate that preproorexin C-peptide promoted Bdnf mRNA expression by a cyclic AMP-dependent mechanism. Eleven amino acids at the N terminus of rat preproorexin C-peptide exerted similar effects on Bdnf expression as full-length preproorexin C-peptide. Preproorexin C-peptide also exerted protective effects against CoCl2-induced neuronal cell death. An intracerebroventricular injection of mouse preproorexin C-peptide induced c-fos and Bdnf expression in the cerebral cortex and hippocampus and enhanced novel object recognition memory in mice. Collectively, the present results show that preproorexin C-peptide is a functional substance, at least in some pharmacological and neuronal settings.

Similar functional roles of the Orexin-1 and Orexin-2 receptors within the dentate gyrus area of the hippocampus in the stress-induced antinociceptive responses in the acute pain model in the rat.

Studies establish that the brain's Orexin system is involved in pain modulation. Orexin-1 and orexin-2 receptors (OX1 and OX2r, respectively) are essential in responsiveness to stressful stimuli. Some evidence indicates that the hippocampus's dentate gyrus (DG) potentially modulates pain and stress. The present study examined the involvement of OX1 and OX2 receptors within the DG in response to acute pain after exposure to forced swim stress (FSS). Five to seven days post-stereotaxic surgery, the baseline tail-flick latency (TFL) was taken from the animal, then rats unilaterally received through an implanted cannula either different doses of OX1r antagonist (SB334867; 1, 3, 10, and 30 nmol), OX2r antagonist (TCS OX2 29; 1, 3, 10 and 30 nmol), or vehicle (0.5 µl solution of 12% DMSO). After 5 min, rats were exposed to the FSS for six minutes. Subsequently, the tail-flick test was conducted, and the TFLs were measured at the 60-min time set intervals. Results indicated that FSS produces antinociceptive responses in the tail-flick test. Two-way ANOVA analysis showed that Microinjection of OX1r and OX2r antagonists into the DG region of the brain reduced FSS-induced analgesia in the tail-flick test. The decrement effects of these two antagonists were almost the same. Additionally, results showed that the role of both receptors was the same in modulating stress-induced analgesia (SIA). These findings show that the orexin system in the hippocampal DG region might be partially involved in the SIA in acute pain.

Efficiency of Orexin-A for Inflammatory Flare and Mucosal Healing in Experimental Colitis: Comparison with the Anti-TNF Alpha Infliximab.

Inflammatory bowel diseases are chronic inflammation of the intestinal mucosa characterized by relapsing-remitting cycle periods of variable duration. Infliximab (IFX) was the first monoclonal antibody used for the treatment of Crohn's disease and ulcerative colitis (UC). High variability between treated patients and loss of IFX efficiency over time support the further development of drug therapy. An innovative approach has been suggested based on the presence of orexin receptor (OX1R) in the inflamed human epithelium of UC patients. In that context, the aim of this study was to compare, in a mouse model of chemically induced colitis, the efficacy of IFX compared to the hypothalamic peptide orexin-A (OxA). C57BL/6 mice received 3.5% dextran sodium sulfate (DSS) in drinking water for 5 days. Since the inflammatory flare was maximal at day 7, IFX or OxA was administered based on a curative perspective at that time for 4 days using intraperitoneal injection. Treatment with OxA promoted mucosal healing and decreased colonic myeloperoxidase activity, circulating concentrations of lipopolysaccharide-binding protein, IL-6 and tumor necrosis factor alpha (TNFα) and decreased expression of genes encoding cytokines in colonic tissues with better efficacy than IFX allowing for more rapid re-epithelization. This study demonstrates the comparable anti-inflammatory properties of OxA and IFX and shows that OxA is efficient in promoting mucosal healing, suggesting that OxA treatment is a promising new biotherapy.

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.

Hypocretin-1 suppresses malignant progression of glioblastoma cells through Notch1 signaling pathway.

Hypocretin-1 is a multifunctional neuropeptide that has been identified as a potential antitumor agent for its role in inhibiting tumor growth, including in colon cancer, neuroendocrine tumor, and prostate cancer. However, the role and mechanism of hypocretin-1 in the occurrence and development of malignant glioma have not been well studied. Therefore, we investigated the effect of hypocretin-1 on glioblastoma proliferation, apoptosis, migration and invasion and its mechanism. We found that the hypocretin-1 receptor was expressed in both glioma cell lines and glioma tissues. Hypocretin-1 treatment can inhibit glioblastoma cell proliferation, migration and invasion, and induce cell apoptosis. Meanwhile, hypocretin-1 treatment significantly reduces tumor growth rate and tumor weight. In addition, mechanistic studies have found that hypocretin-1 exerts antitumor effects by inhibiting NOTCH signaling pathway. Overexpression of NICD significantly reversed the antitumor effect of hypocretin on glioblastoma. Taken together, these findings suggest that hypocretin-1 inhibits glioblastoma proliferation, migration and invasion and induces apoptosis in vitro and in vivo through NOTCH signaling pathway.

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.

The Effectiveness of Nigella sativa and Ginger as Appetite Suppressants: An Experimental Study on Healthy Wistar Rats.

Background: Obesity is a global pandemic that is associated with high morbidity and mortality. Natural herbs are commonly used for weight reduction and appetite suppression. Therefore, we aim to investigate the role and mechanism of Nigella sativa (NS) and ginger on weight reduction and appetite regulation. Methods: This experimental study was performed at Imam Abdulrahman Bin Faisal University. Twenty-five female rats were distributed into 5 groups: NS (oral 1000mg/kg), Ginger (500 mg/kg), NS-ginger (both interventions), a positive control (intraperitoneal 50 µg/kg Liraglutide), and a negative control. Each intervention was given for 9 weeks. Food intake and body weight were assessed weekly. Serum lipid profile and peptides involved in appetite control (cholecystokinin (CCK), glucagon-like peptide 1(GLP-1), gastric inhibitory polypeptide (GIP), ghrelin, peptide YY, and orexin) were assayed at the end of the experiment. Results: None of the interventions showed a statistically significant difference regarding food consumption or weight gain (p > 0.05). However, the three interventions significantly reduced total cholesterol (TC), NS and NS-ginger significantly increased HDL, NS increased ghrelin and ginger increased orexin. Conclusion: The present dose and duration of NS, ginger, or in combination did not demonstrate a significant change in body weight or food consumption in comparison to the negative or positive controls. However, NS or ginger has improved the lipid profile by reducing TC and increasing HDL. In addition, NS or ginger can influence some of the peptides involved in appetite regulation such as the increase in ghrelin induced by NS and the reduction of orexin induced by ginger. We believe that these latter effects are novel and might indicate a promising effect of these natural products on appetite regulation.

Nicotine suppresses central post-stroke pain via facilitation of descending noradrenergic neuron through activation of orexinergic neuron.

Central post-stroke pain (CPSP) is a type of central neuropathic pain, whose underlying mechanisms remain unknown. We previously reported that bilateral carotid artery occlusion (BCAO)-induced CPSP model mice showed mechanical hypersensitivity and decreased mRNA levels of preproorexin, an orexin precursor, in the hypothalamus. Recently, nicotine was shown to regulate the neuronal activity of orexin in the lateral hypothalamus (LH) and suppress inflammatory and neuropathic pain. In this study, we evaluated whether nicotine could suppress BCAO-induced mechanical allodynia through the activation of orexinergic neurons. Mice were subjected to BCAO for 30 min. Mechanical hypersensitivity was assessed by the von Frey test. BCAO mice showed hypersensitivity to mechanical stimuli three days after BCAO surgery. The intracerebroventricular injection of nicotine suppressed BCAO-induced mechanical hypersensitivity in a dose-dependent manner. These effects were inhibited by α7 or α4ß2-nicotinic receptor antagonists. After nicotine injection, the level of c-fos, a neuronal activity marker, increased in the LH and locus coeruleus (LC) of Sham and BCAO mice. Increased number of c-Fos-positive cells partly colocalized with orexin A-positive cells in the LH, as well as tyrosine hydroxylase-positive cells in the LC. Orexinergic neurons project to the LC area. Nicotine-induced antinociception tended to cancel by the pretreatment of SB334867, an orexin receptor1 antagonist into the LC. Intra-LH microinjection of nicotine attenuated BCAO-induced mechanical hypersensitivity. Nicotine-induced antinociception was inhibited by intrathecal pre-treatment with yohimbine, an α2 adrenergic receptor antagonist. These results indicated that nicotine may suppress BCAO-induced mechanical hypersensitivity through the activation of the descending pain control system via orexin neurons.

Effect of winter feeding frequency on growth performance, biochemical blood parameters, oxidative stress, and appetite-related genes in Takifugu rubripes.

Tiger pufferfish (Takifugu rubripes) is one of Asia's most economically valuable aquaculture species. However, winter production of this species in North China is limited by low water temperature and unavailability of high-quality feed, resulting in high mortality and low profitability. Therefore, the aim of this study was to evaluate the effect of feeding frequency (F1: one daily meal; F2: two daily meals; F3: four daily meals; F4: continuous diurnal feeding using a belt feeder) on the growth performance, plasma biochemistry, digestive and antioxidant enzyme activities, and expression of appetite-related genes in T. rubripes (initial weight: 266.80 ± 12.32 g) cultured during winter (18.0 ± 1.0 °C) for 60 days. The results showed that fish in the F3 group had the highest final weight, weight gain rate, specific growth rate, survival rate, and best feed conversion ratio. Additionally, daily feed intake increased significantly with increasing feeding frequency. The protein efficiency and lipid efficiency ratios of fish in the F3 group were significantly higher than those of fish in the other groups. Furthermore, total cholesterol, triglycerides, and glucose levels increased with increasing feeding frequency, peaking in the F2 group and decreasing under higher feeding frequencies. The antioxidant (superoxide dismutase, catalase, glutathione, and glutathione peroxidase) and digestive (trypsin, amylase, and lipase) enzyme activities of fish in the F1 group were significantly higher than those of fish in the F3 and F4 groups. Additionally, there was a decrease in orexin expression with increasing feeding frequency. In contrast, the expression levels of tachykinin, cholecystokinin, and leptin increased with increasing feeding frequency, peaking in the F4 group. Overall, the findings of this study indicated that a feeding frequency of four meals per day was optimal for improved growth performance of pufferfish juveniles cultured during winter.

Oxytocin acts centrally in the brain to improve leaky gut through the vagus nerve and a cannabinoid signaling in rats.

Brain oxytocin plays a role in gastrointestinal functions. Among them, oxytocin acts centrally to modulate gastrointestinal motility and visceral sensation. Intestinal barrier function, one of important gut functions, is also regulated by the central nervous system. Little is, however, known about a role of central oxytocin in the regulation of intestinal barrier function. The present study was performed to clarify whether brain oxytocin is also involved in regulation of intestinal barrier function and its mechanism. Colonic permeability was estimated in vivo by quantifying the absorbed Evans blue in colonic tissue in rats. Intracisternal injection of oxytocin dose-dependently abolished increased colonic permeability in response to lipopolysaccharide while intraperitoneal injection of oxytocin at the same dose failed to block it. Either atropine or surgical vagotomy blocked the central oxytocin-induced improvement of colonic hyperpermeability. Cannabinoid 1 receptor antagonist but not adenosine or opioid receptor antagonist prevented the central oxytocin-induced blockade of colonic hyperpermeability. In addition, intracisternal injection of oxytocin receptor antagonist blocked the ghrelin- or orexin-induced improvement of intestinal barrier function. These results suggest that oxytocin acts centrally in the brain to reduce colonic hyperpermeability. The vagal cholinergic pathway or cannabinoid 1 receptor signaling plays a vital role in the process. The oxytocin-induced improvement of colonic hyperpermeability mediates the central ghrelin- or orexin-induced improvement of intestinal barrier function. We would therefore suggest that activation of central oxytocin signaling may be useful for leaky gut-related diseases such as irritable bowel syndrome and autism.

Roles of Neuropeptides in Sleep-Wake Regulation.

Sleep and wakefulness are basic behavioral states that require coordination between several brain regions, and they involve multiple neurochemical systems, including neuropeptides. Neuropeptides are a group of peptides produced by neurons and neuroendocrine cells of the central nervous system. Like traditional neurotransmitters, neuropeptides can bind to specific surface receptors and subsequently regulate neuronal activities. For example, orexin is a crucial component for the maintenance of wakefulness and the suppression of rapid eye movement (REM) sleep. In addition to orexin, melanin-concentrating hormone, and galanin may promote REM sleep. These results suggest that neuropeptides play an important role in sleep-wake regulation. These neuropeptides can be divided into three categories according to their effects on sleep-wake behaviors in rodents and humans. (i) Galanin, melanin-concentrating hormone, and vasoactive intestinal polypeptide are sleep-promoting peptides. It is also noticeable that vasoactive intestinal polypeptide particularly increases REM sleep. (ii) Orexin and neuropeptide S have been shown to induce wakefulness. (iii) Neuropeptide Y and substance P may have a bidirectional function as they can produce both arousal and sleep-inducing effects. This review will introduce the distribution of various neuropeptides in the brain and summarize the roles of different neuropeptides in sleep-wake regulation. We aim to lay the foundation for future studies to uncover the mechanisms that underlie the initiation, maintenance, and end of sleep-wake states.

Effects of orexin A on PTGS2, PTGES, CBR1 and PGFS mRNA transcript abundances and prostaglandin E2 and F2α concentrations in culture medium of pig uterine explants collected during early gestation and the estrogenic cycle.

In this study, aims were to evaluate orexin A (OXA) effects on mRNA abundance of important enzymes involved in prostaglandin production, such as cyclooxygenase 2 (PTGS2), microsomal PGE2 synthase-1 (PTGES), PGF2α synthase (PGFS) and carbonyl reductase 1 (CBR1), as well as prostaglandin E2 (PGE2) and F2α (PGF2α) culture medium concentrations for endometrial and myometrial explants. Tissues were collected from gilts during specific phases of the estrogenic cycle or early gestational period. There were greater concentrations of PGE2 with OXA treatments of endometrial tissues collected on days 12-13 and 27-28, as well as PGF2α on days 10-11 of the gestational period. The PGF2α concentrations were less in tissues collected on days 27-28 of the gestational period. The OXA treatments resulted in lesser concentrations of PGE2 from myometrial tissues collected on days 10-11 and greater PGF2α on days 10-11 of the gestational period and 10-11 of the estrogenic cycle. Effects of OXA may occur due to actions at PTGS2, PTGES, PGFS and CBR1 genes because mRNA abundances for proteins encoded by these genes were affected by OXA. Results indicate there is an OXA effect on mRNA abundances and prostaglandin culture medium concentrations of uterine tissue collected at different stages of the gestational period or estrogenic cycle using different doses of OXA. It, therefore, is concluded OXA may affect de novo synthesis and secretion of PGE2 and PGF2α in the uterus of pigs.

Orexin A Suppresses the Expression of Exosomal PD-L1 in Colon Cancer and Promotes T Cell Activity by Inhibiting JAK2/STAT3 Signaling Pathway.

BACKGROUND: Colon cancer, ranked third in cancer related mortality, is the most common malignant cancer of digestive tract. Though immune checkpoint inhibitors show promising efficacy in colon cancer, a rather high unresponsive rate and recurrence rate requires further elucidation of the underlying regulatory mechanism of cancer-related immunity. AIMS: To study the regulatory function of Orexin A in the expression of exosomal PD-L1 and T cell activity. METHODS: Orthotopic colon cancer transplantation mice model were established to study the cancer growth and immune infiltration between Orexin A treated group and untreated group. In vitro studies using mouse CT-26 and human HCT-116 colon cancer cell model studied the effect of Orexin A on cellular and exosomal PD-L1 expression. Co-culturing Jurkat cells with exosomes delivered by cancer cells treated with Orexin A, PD-L1 knockdown and PBS studied different effects on T cell. Comparing Orexin A with WP1066, a JAK2/STAT3 inhibitor verified the mechanism of these changes. RESULTS: The growth rate of orthotopic transplanted colon cancer was slower in Orexin A treated group, with lower PD-L1 expression and higher immune infiltration. Orexin A could inhibit cellular and exosomal PD-L1 expression. The decreased expression of PD-L1 in exosomes could promote the activity of Jurkat cells secreting higher level of IFN-γ and IL-2. Orexin A showed a similar effect like WP1066 which proved JAK2/STAT3 signaling pathway was its downstream signaling pathway. CONCLUSIONS: Orexin A could suppress the expression of exosomal PD-L1 in colon cancer cells and promote T cells activity by inhibiting JAK2/STAT3 signaling pathway.

Zuogui Wan improves trabecular bone microarchitecture in ovariectomy-induced osteoporosis rats by regulating orexin-A and orexin receptor.

OBJECTIVE: To investigate the protective effects of Zuogui Wan (ZGW) on bone loss induced by ovariectomy (OVX) and its mechanism via orexin-A and orexin receptors in the osteoporosis rat model. METHODS: Fifty Sprague-Dawley female rats were randomly divided into sham-operated (sham) group and four OVX subgroups. Rats subjected to sham and OVX were treated with the vehicle (OVX, 1 mL/100 g weight, n = 10), 17ß-estradiol (E2, 50 µg*kg-1*d-1), and ZGW at the doses of 2.3 (ZGW-L) and 4.6 (ZGW-H) g/kg/day lyophilized powder daily for 3 months, respectively. The serum biochemical parameters of 17ß-estrogen (17ß-E2), tartrate-resistant acid phosphatase (TRACP-5b) and bone alkaline phosphatase (BALP) were measured by enzyme-linked immunosorbent assay. Hematoxylin-eosin staining was used to detect the changes in the morphological structure in bones. Microcomputed tomography was used to evaluate the bone mineral density and microarchitecture of the distal femur. The gene or protein expression of orexin-A, orexin receptor 1 (OX1R), orexin receptor 2 (OX2R), osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were assayed by either quantitative polymerase chain reaction or Western blot analysis. RESULTS: Compared with the OVX group, ZGW could reduce the serum level of TRACP-5b and increased the serum levels of BALP and17ß-E2 (P < 0.01). Meanwhile, ZGW could prevent bone loss and improved bone trabecular microarchitecture by increasing the trabeculae structure thickness and trabecular number, and arranging the trabeculae structure properly. Compared with the OVX group, it was upregulated for the orexin-A and OX2R mRNA or protein expression from the hypothalamus and tibiae, and OPG in the tibiae of ZGW groups (P < 0.01, < 0.05), while downregulated for the OX1R mRNA and protein expression in the tibiae and hypothalamus and RANKL from the tibiae (P < 0.01). CONCLUSION: ZGW exhibited a protective effect for PMOP that may be mediated via orexin-A and orexin receptors regulation.

The role of ventral tegmental area orexinergic afferents in depressive-like behavior in a chronic unpredictable mild stress (CUMS) mouse model.

Orexin has been implicated in comorbid diseases of depression, making it a promising target for anti-depression treatment. Although orexin neurons exhibit abnormal activity in depression, the neurocircuit mechanism of orexin remains unclear. As one of the important downstream factors of orexin neurons, the ventral tegmental area (VTA) is considered crucial to the mechanism of depression. However, the role of VTA orexinergic afferents in depression remains unclear. In this study, we applied a combination of opto/chemogenetic and neuropharmacology methods to investigate whether the VTA orexinergic afferents participate in the pathogenesis of depression in a chronic unpredictable mild stress (CUMS) mouse model. We found that c-Fos expression in these VTA-projecting orexin neurons specifically decreased in CUMS-treated mice. Optogenetic and chemogenetic activation of orexin terminals in the VTA significantly reversed depressive behavior. Microinjection of orexin-A, but not orexin-B, into the VTA significantly improved depressive-like behavior. Our study provided direct evidence that the VTA orexinergic afferents participate in the mechanism of depression, and the orexin-1 receptor plays a major role.

Activation of Orexinergic Neurons Inhibits the Anesthetic Effect of Desflurane on Consciousness State via Paraventricular Thalamic Nucleus in Rats.

BACKGROUND: Orexin, a neuropeptide derived from the perifornical area of the hypothalamus (PeFLH), promotes the recovery of propofol, isoflurane, and sevoflurane anesthesias, without influencing the induction time. However, whether the orexinergic system also plays a similar role in desflurane anesthesia, which is widely applied in clinical practice owing to its most rapid onset and offset time among all volatile anesthetics, has not yet been studied. In the present study, we explored the effect of the orexinergic system on the consciousness state induced by desflurane anesthesia. METHODS: The c-Fos staining was used to observe the activity changes of orexinergic neurons in the PeFLH and their efferent projection regions under desflurane anesthesia. Chemogenetic and optogenetic techniques were applied to compare the effect of PeFLH orexinergic neurons on the induction, emergence, and maintenance states between desflurane and isoflurane anesthesias. Orexinergic terminals in the paraventricular thalamic nucleus (PVT) were manipulated with pharmacologic, chemogenetic, and optogenetic techniques to assess the effect of orexinergic circuitry on desflurane anesthesia. RESULTS: Desflurane anesthesia inhibited the activity of orexinergic neurons in the PeFLH, as well as the neuronal activity in PVT, basal forebrain, dorsal raphe nucleus, and ventral tegmental area, as demonstrated by c-Fos staining. Activation of PeFLH orexinergic neurons prolonged the induction time and accelerated emergence from desflurane anesthesia but only influenced the emergence in isoflurane anesthesia, as demonstrated by chemogenetic and pharmacologic techniques. Meanwhile, optical activation of orexinergic neurons exhibited a long-lasting inhibitory effect on burst-suppression ratio (BSR) under desflurane anesthesia, and the effect may be contributed by the orexinergic PeFLH-PVT circuitry. The orexin-2 receptor (OX2R), but not orexin-1 receptor (OX1R), in the PVT, which had been inhibited most significantly by desflurane, mediated the proemergence effect of desflurane anesthesia. CONCLUSIONS: We discovered, for the first time, that orexinergic neurons in the PeFLH could not only influence the maintenance and emergence from isoflurane and desflurane anesthesias but also affect the induction under desflurane anesthesia. Furthermore, this specific effect is probably mediated by orexinergic PeFLH-PVT circuitry, especially OX2Rs in the PVT.