Phoenixin-14 as a novel direct regulator of porcine luteal cell functions†.

Phoenixin is a neuropeptide with a well-established role in the central regulation of reproductive processes; however, knowledge regarding its role in the ovary is limited. One of the main active phoenixin isoforms is phoenixin-14, which acts through G protein-coupled receptor 173. Our research hypothesis was that phoenixin-14 is expressed in porcine corpus luteum and exerts luteotropic action by affecting the endocrine function of luteal cells through G protein-coupled receptor 173 and protein kinase signaling. Luteal cells were cultured to investigate the effect of phoenixin-14 (1-1000 nM) on endocrine function. We showed that phoenixin-14 and G protein-coupled receptor 173 are produced locally in porcine corpus luteum and their levels change during the estrous cycle. We detected phoenixin-14 immunostaining in the cytoplasm and G protein-coupled receptor 173 in the cell membrane. Plasma phoenixin levels were highest during the early luteal phase. Interestingly, insulin, luteinizing hormone, progesterone, and prostaglandins decreased phoenixin-14 levels in luteal cells. Phoenixin-14 increased progesterone, estradiol, and prostaglandin E2 secretion, but decreased prostaglandin F2α, upregulated the expression of steroidogenic enzymes, and downregulated receptors for luteinizing hormone and prostaglandin. Also, phoenixin-14 increased the expression of G protein-coupled receptor 173 and the phosphorylation of extracellular signal-regulated kinase 1/2, protein kinase B, inhibited the phosphorylation of protein kinase A, and had mixed effect on AMP-activated protein kinase alpha and protein kinase C. G protein-coupled receptor 173 and extracellular signal-regulated kinase 1/2 mediated the effect of phoenixin-14 on endocrine function of luteal cells. Our results suggest that phoenixin is produced by porcine luteal cells and can be a new regulator of their function.

Phoenixin-14 maintains the contractile type of vascular smooth muscle cells in cerebral aneurysms rats.

The rupture of intracranial aneurysm (IA) is the primary reason contributing to the occurrence of life-threatening subarachnoid hemorrhages. The oxidative stress-induced phenotypic transformation from the contractile phenotype to the synthetic phenotype of vascular smooth muscle cells (VSMCs) plays a pivotal role in IA formation and rupture. Our study aimed to figure out the role of phoenixin-14 in VSMC phenotypic switching during the pathogenesis of IA by using both cellular and animal models. Primary rat VSMCs were isolated from the Willis circle of male Sprague-Dawley rats. VSMCs were stimulated by hydrogen peroxide (H2O2) to establish a cell oxidative damage model. After pretreatment with phoenixin-14 and exposure to H2O2, VSMC viability, migration, and invasion were examined through cell counting kit-8 (CCK-8), wound healing, and Transwell assays. Intracellular reactive oxygen species (ROS) production in VSMCs was evaluated by using 2',7'-Dichlorofluorescin diacetate (DCFH-DA) fluorescence probes and flow cytometry. Rat IA models were established by ligation of the left common carotid arteries and posterior branches of both renal arteries. The histopathological changes of rat intracranial blood vessels were observed through hematoxylin and eosin staining. The levels of contractile phenotype markers (alpha-smooth muscle actin [α-SMA] and smooth muscle 22 alpha [SM22α]) in VSMCs and rat arterial rings were determined through real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Our results showed that H2O2 stimulated the production of intracellular ROS and induced oxidative stress in VSMCs, while phoenixin-14 pretreatment attenuated intracellular ROS levels in H2O2-exposed VSMCs. H2O2 exposure promoted VSMC migration and invasion, which, however, was reversed by phoenixin-14 pretreatment. Besides, phoenixin-14 administration inhibited IA formation and rupture in rat models. The decrease in α-SMA and SM22α levels in H2O2-exposed VSMCs and IA rat models was antagonized by phoenixin-14. Collectively, phoenixin-14 ameliorates the progression of IA through preventing the loss of the contractile phenotype of VSMCs.

Premetazoan Origin of Neuropeptide Signaling.

Neuropeptides are a diverse class of signaling molecules in metazoans. They occur in all animals with a nervous system and also in neuron-less placozoans. However, their origin has remained unclear because no neuropeptide shows deep homology across lineages, and none have been found in sponges. Here, we identify two neuropeptide precursors, phoenixin (PNX) and nesfatin, with broad evolutionary conservation. By database searches, sequence alignments, and gene-structure comparisons, we show that both precursors are present in bilaterians, cnidarians, ctenophores, and sponges. We also found PNX and a secreted nesfatin precursor homolog in the choanoflagellate Salpingoeca rosetta. PNX, in particular, is highly conserved, including its cleavage sites, suggesting that prohormone processing occurs also in choanoflagellates. In addition, based on phyletic patterns and negative pharmacological assays, we question the originally proposed GPR-173 (SREB3) as a PNX receptor. Our findings revealed that secreted neuropeptide homologs derived from longer precursors have premetazoan origins and thus evolved before neurons.

The role of phoenixin in the proliferation and migration of ectopic epithelial cells in vitro.

AIM: Endometriosis is one of the most common gynecologic diseases in women of reproductive age. The pathophysiology of endometriosis is still not fully understood. Phoenixin (PNX-14) is a newly discovered neuropeptide that regulates the hypothalamo-pituitary-gonadal (HPG) axis and reproductive functions. Recently, we reported that PNX-14, its precursor protein and receptor were expressed in human endometrium. Moreover, PNX-14 serum levels in endometriosis were reduced. This study aimed to evaluate the in vitro biological functions of physiological PNX-14 concentrations on the ectopic endometrium Z12 cells. METHODS: The proliferation and migration of Z12 cells were assessed using the xCELLigence® RTCA DP system following 72 h of stimulation with 0.05 and 0.2 nM of PNX-14. GPR173 and small integral membrane protein 20 (SMIM20) gene expression was evaluated using quantitative polymerase chain reaction (qPCR) and the protein levels of GPR173 were analyzed using Western blot analysis. RESULTS: PNX-14 at the concentration observed in the serum of patients with endometriosis (0.05 nM) reduced GPR173 and increased SMIM20 expression, while protein levels of GPR173 remained unchanged. Cell proliferation was increased by the 0.02 nM PNX-14- the concentration found in healthy subjects. The 0.2 nM of PNX-14 decreased SMIM20 expression with no change to GPR173 expression and reduced ectopic epithelial cell proliferation during the first 5 h after stimulation. However, at 72 h, the proliferation increased. CONCLUSIONS: This study shows that PNX-14 at endometriosis specific concentration desensitized ectopic epithelium to PNX-14, and increased the expression of SMIM20 to restore the physiological levels of PNX-14.

PNX14 but not PNX20 as a novel regulator of preadipocyte differentiation via activating Epac-ERK signaling pathway in Gallus gallus.

Small integral membrane protein 20 (SMIM20) could generate two main peptides, PNX14 and PNX20, which participate in multiple biological roles such as reproduction, inflammation and energy metabolism in mammals. However, little is known about their physiological functions in non-mammalian vertebrates. Using chicken (c-) as an animal model, we found cSMIM20 was moderately expressed in adipose tissues, and its expression was gradually increased during the differentiation of chicken preadipocytes, suggesting that it may play an important role in chicken adipogenesis. Further research showed cPNX14 could facilitate the differentiation of chicken preadipocytes into mature adipocytes by enhancing expression of adipogenic genes including PPARγ, CEBPα and FABP4, and promoting the formation of lipid droplets. This pro-adipogenic effect of cPNX14 was completely attenuated by Epac-specific and ERK inhibitor. Interestingly, cPNX20 failed to regulate the adipogenic genes and lipid droplet content. Collectively, our findings reveal that cPNX14 but not cPNX20 can serve as a novel adipogenesis mediator by activating the Epac-ERK signaling pathway in chickens.

Characterizing the SREB G protein-coupled receptor family in fish: Brain gene expression and genomic differences in upstream transcription factor binding sites.

The SREB (Super-conserved Receptors Expressed in Brain) family of orphan G protein-coupled receptors is highly conserved in vertebrates and consists of three members: SREB1 (orphan designation GPR27), SREB2 (GPR85), and SREB3 (GPR173). SREBs are associated with processes ranging from neuronal plasticity to reproductive control. Relatively little is known about similarities across the entire family, or how mammalian gene expression patterns compare to non-mammalian vertebrates. In fish, this system may be particularly complex, as some species have gained a fourth member (SREB3B) while others have lost genes. To better understand the system, the present study aimed to: 1) use qPCR to characterize sreb and related gene expression patterns in the brains of three fish species with different systems, and 2) identify possible differences in transcriptional regulation among the receptors, using upstream transcription factor binding sites across 70 ray-finned fish genomes. Overall, regional patterns of sreb expression were abundant in forebrain-related areas. However, some species-specific patterns were detected, such as abundant expression of receptors in zebrafish (Danio rerio) hypothalamic-containing sections, and divergence between sreb3a and sreb3b in pufferfish (Dichotomyctere nigroviridis). In addition, a gene possibly related to the system (dkk3a) was spatially correlated with the receptors in all three species. Genomic regions upstream of sreb2 and sreb3b, but largely not sreb1 or sreb3a, contained many highly conserved transcription factor binding sites. These results provide novel information about expression differences and transcriptional regulation across fish that may inform future research to better understand these receptors.

Distribution and Chemistry of Phoenixin-14, a Newly Discovered Sensory Transmission Molecule in Porcine Afferent Neurons.

Phoenixin-14 (PNX), initially discovered in the rat hypothalamus, was also detected in dorsal root ganglion (DRG) cells, where its involvement in the regulation of pain and/or itch sensation was suggested. However, there is a lack of data not only on its distribution in DRGs along individual segments of the spinal cord, but also on the pattern(s) of its co-occurrence with other sensory neurotransmitters. To fill the above-mentioned gap and expand our knowledge about the occurrence of PNX in mammalian species other than rodents, this study examined (i) the pattern(s) of PNX occurrence in DRG neurons of subsequent neuromeres along the porcine spinal cord, (ii) their intraganglionic distribution and (iii) the pattern(s) of PNX co-occurrence with other biologically active agents. PNX was found in approximately 20% of all nerve cells of each DRG examined; the largest subpopulation of PNX-positive (PNX+) cells were small-diameter neurons, accounting for 74% of all PNX-positive neurons found. PNX+ neurons also co-contained calcitonin gene-related peptide (CGRP; 96.1%), substance P (SP; 88.5%), nitric oxide synthase (nNOS; 52.1%), galanin (GAL; 20.7%), calretinin (CRT; 10%), pituitary adenylate cyclase-activating polypeptide (PACAP; 7.4%), cocaine and amphetamine related transcript (CART; 5.1%) or somatostatin (SOM; 4.7%). Although the exact function of PNX in DRGs is not yet known, the high degree of co-localization of this peptide with the main nociceptive transmitters SP and CGRP may suggests its function in modulation of pain transmission.

Linking mitochondrial dynamics and fertility: promoting fertility by phoenixin through modulation of ovarian expression of GnRH receptor and mitochondrial dynamics proteins DRP-1 and Mfn-2.

Obesity is linked to reproductive disorders. Novel neuropeptide phoenixin demonstrated many therapeutic actions. In this study, we aim to evaluate phoenixin's potential effect in obesity-induced infertility through modulating mitochondrial dynamics. Ninety adult female rats were divided to 4 groups: (I), fed with normal pellet diet; (II), given phoenixin; (III), fed with high-fat diet. Rats that developed obesity and infertility were divided to 2 groups: (III-A), received no further treatment; (III-B), given phoenixin. Our results showed that phoenixin treatment in obese infertile rats significantly decreased serum levels of insulin and testosterone and ovarian levels of dynamin-related protein1(Drp1),reactive oxygen species ROS, TNF-α, MDA, and caspase-3. Phoenixin treatment also significantly increased serum estrogen progesterone, LH, and FSH together with ovarian levels of GnRH receptor (GnRHR), mitofusin2(Mfn2), mitochondrial transmembrane potential (ΔΨm), and electron transport chain (ETC) complex-I significantly when compared with obese group. Ovarian histopathological changes were similarly improved by phoenixin. Our data demonstrate phoenixin's role in improving obesity-induced infertility.

Phoenixin-14 alleviates inflammatory smooth muscle cell-induced endothelial cell dysfunction in vitro.

BACKGROUND: Intracranial aneurysm (IA) is cerebrovascular disorder which refers to local vessel wall damage to intracranial arteries, forming abnormal bulge. Both endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are closely associated with IA formation and rupture. Inflammatory SMCs (iSMCs) were reported to induce EC dysfunction and result in IA progression. Phoenixin-14 (PNX-14) is a recently discovered brain peptide with pleiotropic roles, which participates in reproduction, cardio protection, lipid deposition and blood glucose metabolism. PNX-14 was previously reported to protect brain endothelial cells against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced cell injury. Therefore, our study was designed to investigate the influence of PNX-14 on iSMCs-induced endothelial dysfunction. METHODS: Inflammation in SMCs was induced by cyclic mechanical stretch. Human umbilical vein endothelial cells (HUVECs) were exposed to SMC- or iSMC-conditioned medium and then treated with 100 nM PNX-14 for 24 h. The levels of proinflammatory cytokines (IL-1ß, IL-6 and TNF-α) in cell supernatants were analyzed by ELISA. Cell viability, apoptosis, angiogenesis and migration were subjected to CCK-8 assay, flow cytometry analysis, tube formation assay and Transwell migration assay. The protein levels of proinflammatory cytokines and apoptosis markers (Bcl-2 and Bax) were evaluated by western blotting. RESULTS: Cyclic mechanical stretch upregulated IL-1ß, IL-6 and TNF-α levels in SMCs. Treatment with SMC- or iSMC-conditioned medium HUVECs inhibited cell viability, angiogenesis and migration and induced apoptosis in HUVECs. iSMC-conditioned medium has more significant effects on cell functions. However, the influence of SMC- or iSMC-conditioned medium treatment on HUVEC biological functions were reversed by PNX-14 treatment. PNX-14 exerts no significant influence on the biological functions of HUVECs treated with SMC medium. CONCLUSION: PNX-14 alleviates iSMCs-induced endothelial cell dysfunction in vitro.

Mediatory role of the central NPY, melanocortine and corticotrophin systems on phoenixin-14 induced hyperphagia in neonatal chicken.

Animal research indicates the neuropeptide Y (NPY), corticotrophin and melanocortin systems have a mediatory role in reward, however, how these substances interact with phenytoin-14 (PNX-14) induced food intake in birds remains to be identified. Accordingly, in this research eight tests were carried out to investigate the potential interactions of the NPY, melanocortin, as well as corticotrophin systems with PNX-14 on food consumption in neonatal chickens. In the first experiment, chickens were intracerebroventricular (ICV) injected with phosphate-buffered saline (PBS) and PNX-14 (0.8, 0.16, and 3.2 nmol). In second experiment, PBS, the antagonist of CRF1/CRF2 receptors (astressin-B, 30 µg) and PNX-14 + astressin-B were injected. In the rest of the experiments chicken received astressin2-B (CRF2 receptor antagonist; 30 µg), SHU9119 (MCR3/MCR4 receptor antagonist, 0.5nomol), MCL0020 (MCR4 receptor agonist, 0.5 nmol), B5063 (NPY1 receptor antagonist, 1.25 µg), SF22 (NPY2 receptor antagonist, 1.25 µg) and SML0891 (NPY5 receptor antagonist, 1.25 µg) rather than astressin-B. Then, cumulative intake of food was recorded for 2 h. Based on the findings, PNX-14 (0.16 and 3.2 nmol) led to increment in food consumption compared with the control (P < 0.05). Co-administration of the PNX-14 and astressin-B promoted PNX-14-induced hyperphagia (P < 0.05). Co-injection of the PNX-14 + astressin2-B potentiated hyperphagia PNX-14 (P < 0.05). Co-injection of PNX-14 + B5063 inhibited the effects of the PNX-14 (P < 0.05). The co-administration of the PNX-14 and SML0891 potentiated hypophagic effects of the PNX-14 (P < 0.05). The results showed that PNX-14-induced hyperphagia mediates via NPY1, NPY5, and CRF1/CRF2 receptors in neonatal chickens.

Phoenixin-20 ameliorates brain infarction by promoting microglia M2 polarization in an ischemic stroke model.

Ischemic stroke is one of the most common causes of death worldwide. The transformation of microglia from the classic M1 to the alternative M2 state has been shown to have both deleterious and immunosuppressive roles in neuroinflammation. Microglial polarization toward the M2 phase is currently proposed to be a beneficial phenotype in brain ischemic injury. Phoenixin-20 is a newly identified pleiotropic neuropeptide expressed abundantly in different brain regions. In this study, we found that administration of Phoenixin-20 in ischemic stroke middle cerebral artery occlusion (MCAO) mice significantly reduced the brain infarction area but improved the neurological deficit score. Gene expression analysis showed Phoenixin-20 treatment inhibited pro-inflammatory M1 phase microglial markers: a cluster of differentiation molecule 11b (CD11b), cluster of differentiation molecule 86 (CD86), inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and increased anti-inflammatory M2 phase markers (found in Inflammatory Zone 1 (FIZZ1), Arginase 1 (Arg-1), Chitinase 3-like 3 (YM1), and interleukin-10 (IL-10)) in the infarcted brain. We further investigated the molecular mechanism of Phoenixin-20 in cultured microglia. We found that treatment with it induced signature genes expression in microglial M2 state, including Fizz1, Arg-1, YM1, and IL-10, indicating the promotion of microglial polarization toward the M2 state. Furthermore, we found that treatment with the M2 phase cytokine interleukin 4 (IL-4) induced the expression of microglial G Protein-Coupled Receptor (GPR173), which is the receptor of Phoenixin-20. Silencing of the microglial signal transducer and activator of transcription 6 (STAT6) partially blocked the effect of IL-4 on GPR173, suggesting that STAT6 is the upstream regulator of GPR173. Finally, we showed that the silencing of GPR173 completely abolished the effect of Phoenixin-20 in microglia, indicating the dependency of its regulatory role on GPR173. Collectively, our study demonstrates that Phoenixin-20 has a protective role in the acute stroke model. Our cell-based study demonstrates Phoenixin-20 promotes microglia toward M2 transformation, which could be the mechanism of its neuroprotection.

Phoenixin-14 reduces the frequency of interictal-like events in mice brain slices.

Phoenixin-14 (PNX-14) has a wide bioactivity in the central nervous system. Its role in the hypothalamus has been investigated, and it has been reported that it is involved in the regulation of excitability in hypothalamic neurons. However, its role in the regulation of excitability in entorhinal cortex and the hippocampus is unknown. In this study, we investigated whether i. PNX-14 induces any synchronous discharges or epileptiform activity and ii. PNX-14 has any effect on already initiated epileptiform discharges. We used 350 µm thick acute horizontal hippocampal-entorhinal cortex slices obtained from 30- to 35-day-old mice. Extracellular field potential recordings were evaluated in the entorhinal cortex and hippocampus CA1 region. Bath application of PNX-14 did not initiate any epileptiform activity or abnormal discharges. 4-Aminopyridine was applied to induce epileptiform activity in the slices. We found that 200 nM PNX-14 reduced the frequency of interictal-like events in both the entorhinal cortex and hippocampus CA1 region which was induced by 4-aminopyridine. Furthermore, PNX-14 led to a similar suppression in the total power of local field potentials of 1-120 Hz. The frequency or the duration of the ictal events was not affected. These results exhibited for the first time that PNX-14 has a modulatory effect on synchronized neuronal discharges which should be considered in future therapeutic approaches.

Longitudinal study on novel neuropeptides phoenixin, spexin and kisspeptin in adolescent inpatients with anorexia nervosa - association with psychiatric symptoms.

OBJECTIVES: It is hypothesized that novel neuropeptides such as phoenixin (PNX), spexin (SPX), and kisspeptin (KISS) are involved in the pathogenesis of eating disorders. The study presented here analyzed neuropeptide concentrations during the course of anorexia nervosa (AN) and aimed to correlate those values with anthropometric and psychometric measurements. METHODS: A longitudinal study was carried outin 30 AN adolescent patients and 15 age-matched healthy female controls. Selected neuroprotein serum levels were analyzed in malnourished patients (accAN) and following partial weight recovery (norAN), and these values were compared with the control group. RESULTS: In accAN patients, decreased serum PNX levels were detected while SPX serum concentrations were lower in the accAN and norAN patients. No differences were observed in KISS concentrations in all studied groups. CONCLUSIONS: In malnourished adolescent inpatients with AN, serum PNX and SPX level were decreased. The partial weight recovery normalized PNX concentrations but failed to normalize SPX levels. Therefore these two neuropeptides might be crucial for the etiology and course of the AN. The KISS levels did not change in the course of AN. The PNX levels were associated with some symptoms of eating disorders which may indicate its potential contribution in the regulation of emotions and behaviors in AN.

Role of the Novel Peptide Phoenixin in Stress Response and Possible Interactions with Nesfatin-1.

The novel peptide phoenixin was shown to be involved in several physiological processes ranging from reproduction to food intake. Interest in this protein has steadily increased over the last few years and its known implications have become much broader, playing a role in glucose homeostasis, anxiety, nociception, and pruritus. Phoenixin is expressed in a multitude of organs such as the small intestine, pancreas, and in the hypothalamus, as well as several other brain nuclei influencing numerous physiological functions. Its highly conserved amino-acid sequence amongst species leads to the assumption, that phoenixin might be involved in essential physiological functions. Its co-expression and opposing functionality to the extensively studied peptide nesfatin-1 has given rise to the idea of a possible counterbalancing role. Several recent publications focused on phoenixin's role in stress reactions, namely restraint stress and lipopolysaccharide-induced inflammation response, in which also nesfatin-1 is known to be altered. This review provides an overview on the phoenixins and nesfatin-1 properties and putative effects, and especially highlights the recent developments on their role and interaction in the response to response.

Levels of the neuropeptide phoenixin-14 and its receptor GRP173 in the hypothalamus, ovary and periovarian adipose tissue in rat model of polycystic ovary syndrome.

Phoenixin (PNX) is a newly discovered peptide produced by proteolytic cleavage of a small integral membrane protein 20 (Smim20), which acts as an important regulator of energy homeostasis and reproduction. Since dysfunction of reproduction is characteristic in polycystic ovarian syndrome (PCOS), the role of PNX in pathogenesis of PCOS needs further investigation. The objective of this study was to determine expression of Smim20, PNX-14 and its receptor GRP173 in the hypothalamus, ovary and periovarian adipose tissue (PAT) of letrozole induced PCOS rats. Phosphorylation of extracellular signal-regulated kinase (ERK1/2), protein kinases A (PKA) and B (Akt) were also estimated. We observed that PCOS rats had high weight gain and a number of ovarian cyst, high levels of testosterone, luteinizing hormone and PNX-14, while low estradiol. Smim20 mRNA expression was higher in the ovary and PAT, while PNX-14 peptide production was higher only in the ovary of PCOS rat. Moreover, in PCOS rats Gpr173 level was lower in PAT but at the protein level increased only in the ovary. Depending on the tissues, kinases phosphorylation were significantly differ in PCOS rats. Our results showed higher levels of PNX-14 in PCOS rats and indicated some novel findings regarding the mechanisms of PCOS pathophysiology.

A novel regulator of thirst behavior: phoenixin.

There are examples of physiological conditions under which thirst is inappropriately exaggerated, and the mechanisms for these paradoxical ingestive behaviors remain unknown. We are interested in thirst mechanisms across the female life cycle and have identified a novel mechanism through which ingestive behavior may be activated. We discovered a previously unrecognized endogenous hypothalamic peptide, phoenixin (PNX), identified physiologically relevant actions of the peptide in brain and pituitary gland to control reproductive hormone secretion in female rodents, and in the process identified the previously orphaned G protein-coupled receptor Gpr173 to be a potential receptor for the peptide. Labeled PNX binding distribution in brain parallels areas known to be important in ingestive behaviors as well in areas where gonadal steroids feedback to control estrous cyclicity (Stein LM, Tullock CW, Mathews SK, Garcia-Galiano D, Elias CF, Samson WK, Yosten GLC, Am J Physiol Regul Integr Comp Physiol 311: R489-R496, 2016). We have demonstrated upregulation of Gpr173 during puberty, fluctuations across the estrous cycle, and, importantly, upregulation during the last third of gestation. It is during this hypervolemic, hyponatremic state that both vasopressin secretion and thirst are inappropriately elevated in humans. Here, we show that central administration of PNX stimulated water drinking in both males and females under ad libitum conditions, increased water drinking after overnight fluid deprivation, and increased both water and 1.5% NaCl ingestion under fed and hydrated conditions. Importantly, losartan pretreatment blocked the effect of PNX on water drinking, and knockdown of Gpr173 by use of short interfering RNA constructs significantly attenuated water drinking in response to overnight fluid deprivation. These actions, together with the stimulatory action of PNX on vasopressin secretion, suggest that this recently discovered neuropeptide may impact the recruitment of critically important neural circuits through which ingestive behaviors and endocrine mechanisms that maintain fluid and electrolyte homeostasis are regulated.

Phoenixin-20 suppresses food intake, modulates glucoregulatory enzymes, and enhances glycolysis in zebrafish.

Phoenixin is a 20-amino acid peptide (PNX-20) cleaved from the small integral membrane protein 20 (SMIM20), with multiple biological roles in mammals. However, its role in nonmammalian vertebrates is poorly understood. This research aimed to determine whether PNX-20 influences feeding and metabolism in zebrafish. The mRNAs encoding SMIM20 and its putative receptor, super conserved receptor expressed in brain 3 (SREB3), are present in both central and peripheral tissues of zebrafish. Immunohistochemical analysis confirmed the presence of PNX-like immunoreactivity in the gut and in zebrafish liver (ZFL) cell line. We also found that short-term fasting (7 days) significantly decreased smim20 mRNA expression in the brain, gut, liver, gonads, and muscle, which suggests a role for PNX-20 in food intake regulation. Indeed, single intraperitoneal injection of 1,000 ng/g body wt PNX-20 reduced feeding in both male and female zebrafish, likely in part by enhancing hypothalamic cart and reducing hypothalamic/gut preproghrelin mRNAs. Furthermore, the present results demonstrated that PNX-20 modulates the expression of genes involved in glucose transport and metabolism in ZFL cells. In general terms, such PNX-induced modulation of gene expression was characterized by the upregulation of glycolytic genes and the downregulation of gluconeogenic genes. A kinetic study of the ATP production rate from both glycolytic and mitochondrial pathways demonstrated that PNX-20-treated ZFL cells exhibited significantly higher ATP production rate associated with glycolysis than control cells. This confirms a positive role for PNX-20 on glycolysis. Together, these results indicate that PNX-20 is an anorexigen with important metabolic roles in zebrafish.

Phoenixin-14 protects human brain vascular endothelial cells against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced inflammation and permeability.

Stroke is one of the world's most deadly pathologies, and the rate of stroke recurrence is high. However, due to the complex nature of ischemia and reperfusion injury, there is presently no reliable treatment. The main factors driving brain damage from ischemic stroke are neuronal cell death resulting from oxidative stress, inflammation, and failure of the blood brain barrier. While under normal conditions, the blood brain barrier acts as a selectively permeable membrane allowing solutes and other substances to pass into the tissues of the central nervous system, ischemia and reperfusion alter the expression of tight junction proteins such as occludin, which leads to unmitigated perfusion and loss of homeostasis. Phoenixin-14 is a 14-amino acid neuropeptide that has been shown to play a role in regulating reproduction, blood sugar metabolism, pain, anxiety, and more recently, certain aspects of ischemic cardiac injury. In the present study, we found that phoenixin-14 confers protective effects against oxygen-glucose deprivation/reoxygenation (OGD/R) injury in bEnd.3 brain endothelial cells. Phoenixin-14 attenuated oxidative stress via downregulation of ROS and NOX1 and inhibited HMGB1 expression. Additionally, phoenixin-14 increased the expression of eNOS and NO, which play a protective role. Phoenixin-14 reduced endothelial monolayer permeability by increasing the expression of occludin. Finally, we found that the effects of phoenixin-14 on the expression of eNOS and occludin are dependent on the KLF2 transcriptional pathway, as evidenced by the results of our KLF2 knockdown experiment. Thus, phoenixin-14 may serve as a novel therapeutic agent for ischemic stroke.

Phoenixin 14 inhibits ischemia/reperfusion-induced cytotoxicity in microglia.

The process of ischemia/reperfusion (IR) in ischemic stroke often leads to significant cell death and permanent neuronal damage. Safe and effective treatments are urgently needed to mitigate the damage caused by IR injury. The naturally occurring pleiotropic peptide phoenixin 14 (PNX-14) has recently come to light as a potential treatment for IR injury. In the present study, we examined the effects of PNX-14 on several key processes involved in ischemic injury, such as pro-inflammatory cytokine expression, oxidative stress, and the related cascade mediated through the toll-like receptor 4 (TLR4) pathway, using BV2 microglia exposed to oxygen-glucose deprivation and reoxygenation (OGD/R). Our results demonstrate an acute ability of PNX-14 to regulate the expression levels of proinflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6). PNX-14 also prevented oxidative stress by reducing the generation of reactive oxygen species (ROS) and increasing the level of the antioxidant glutathione (GSH). Importantly, PNX-14 inhibited high-mobility group box 1 (HMGB1)/TLR4/myeloid differentiation primary response 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway, by inhibiting the activation of TLR4 and preventing the nuclear translocation of p65 protein. We further confirmed the cerebroprotective effects of PNX-14 in an MCAO rat model, which resulted in reduced infarct volume and decreased microglia activation. Together, the results of this study implicate a possible protective role of PNX-14 against various aspects of IR injury in vitro.

The protective effects of phoenixin-14 against lipopolysaccharide-induced inflammation and inflammasome activation in astrocytes.

INTRODUCTION: Neuroinflammation is a key aspect of various injuries and diseases of the central nervous system and brain, including stroke, Alzheimer's, Parkinson's, multiple sclerosis, etc. Phoenixin-14 is a naturally occurring pleiotropic peptide involved in reproduction, anxiety, pain, and other functions. MATERIALS AND METHODS: Primary astrocytes were isolated from new-born pups of c57bl/6 mice. The gene expression of GPR173, CHOP, and GADD34 was measured by real-time PCR. Protein expression was assessed by western blot analysis. Secretions of IL-1ß and IL-18 were determined by ELISA. RESULTS: Phoenixin-14 (PNX-14) is a ligand for the G protein-coupled receptor GPR173, which we demonstrate to be expressed in astrocytes and suppressed by exposure to lipopolysaccharide (LPS). Endoplasmic reticulum (ER) stress resulting from injury or disease leads to the unfolded protein response, which is mediated by the activation of transcription factors including eIF-2α, ATF4, and CHOP, and regulated by GADD34. ER stress also leads to a robust neuroinflammatory response, which is mediated by HMGB1-induced activation of the NLRP3 inflammasome and subsequent production of IL-1ß and IL-18. In the present study, we demonstrate that PNX-14 could attenuate LPS-induced ER stress response and NLRP3 inflammasome activation in mouse cerebral astrocytes. Our findings show that PNX-14 could suppress the production of ROS as well as the decrease in SOD induced by LPS. PNX-14 also inhibited HMGB1-mediated NLRP3 inflammasome activation and production of IL-1ß and IL-18. Through a GPR173 siRNA knockdown experiment, we further demonstrate that GPR173 knockdown abolished the effects of PNX-14 on LPS-induced NLRP3 expression and IL-18 production. CONCLUSION: These findings suggest that PNX-14 may have potential in the treatment of neuroinflammation.