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.

Phoenixin-14 may ameliorate testicular damage caused by torsion-detorsion by reducing oxidative stress and inflammation in prepubertal rats.

The present study aimed to investigate the effects of Phoenixin-14 (PNX-14) on oxidative damage, inflammatory response, histopathological variations, and serum testosterone levels in testicular tissues. Forty-eight Wistar albino prepubertal male rats were divided into 4 groups (Sham, TTD, TT+PNX+TD, TTD+PNX) (n=12). The torsion period was 2 hours and the detorsion period was 24 hours in the testicular torsion/detorsion (TD) groups. A single PNX-14 (50 µg/kg) dose was injected into the rats in the TT+PNX TD group on the 90th minute of torsion, and it was injected into the rats in the TTD+PNX group at the beginning of detorsion. Oxidative damage in testicular tissues was determined based on superoxide dismutase (SOD), malondialdehyde (MDA), total antioxidant status (TAS) and total oxidant status (TOS), and inflammatory damage was determined based on TNF-α and IL-6 levels. Histopathological variations were investigated with the Periodic Acid Schiff (PAS) staining method in testicular tissues and analyzed based on Johnsen scores. Spermatogonia cells were examined immunohistochemically. Serum testosterone levels were determined with the enzyme-linked immunosorbent assay (ELISA). A significant increase in oxidative stress and inflammation parameters was determined in the TTD group when compared to the other groups (p<0.05). PNX-14 treatment led to a statistically significant decrease in these parameters and significantly repaired the TD damage in testicular tissue (p<0.05). Johnsen scoring revealed significant improvement in PNX-14 groups and an increase in spermatogonia count, supporting the biochemical findings (p<0.05). PNX-14 could be a potential therapeutic agent in testicular TD damage and further studies should be conducted to elucidate the present study findings.

The Levels of Phoenixin-14 and Phoenixin-20 in Patients with Type 2 Diabetes Mellitus.

BACKGROUND: New pathogenesis-related early detection markers are needed to prevent Type 2 Diabetes Mellitus (T2DM). OBJECTIVE: We aimed to determine phoenixin (PNX)-14 and PNX-20 levels in T2DM patients and investigate their relationship with diabetes. METHODS: 36 T2DM patients and 36 healthy controls were included in the study, and PNX-14 and PNX-20 levels in blood samples taken from the groups were measured by ELISA method. RESULTS: Patients' serum PNX-14 and PNX-20 levels were statistically significantly lower than in controls (p <0.001). A negative correlation was detected between PNX-14 and BMI, fasting blood sugar, HbA1c%, and HOMA-IR. A negative correlation was found between PNX-20 and BMI, fasting insulin and glucose, HbA1c%, and HO-MA-IR. A positive correlation was noticed between PNX-14 and PNX-20 levels. In ROC analyses, PNX-14 and PNX-20 performed almost equally in predicting T2DM. In predicting T2DM, the area under the ROC curve for PNX-14 was 0.874 (cutoff value 413.4 ng/L, sensitivity 89 %, specificity 72%), and for PNX-20 was 0.858 (cutoff value 228.7 ng/L, sensitivity 80 %, specificity 83 %). CONCLUSION: This study shows that serum PNX measurement may have a high level of evidence in predicting T2DM. PNX, related to pathogenesis, may be useful in diagnosing T2DM and other information to support clinical decision-making.

Expression and in vitro effect of phoenixin-14 on the porcine ovarian granulosa cells.

Phoenixin-14 (PNX-14) regulates energy metabolism via the G protein-coupled receptor 173 (GPR173); elevated plasma levels have been described in patients with polycystic ovary syndrome. The aims were to investigate the ovarian expression of PNX-14/GPR173 and the in vitro effect of PNX-14 on granulosa cells (Gc) function. Transcript and protein levels of PNX-14/GRP173 were analysed by real-time PCR, western blot and immunohistochemistry in the porcine ovarian follicles at days 2-3, 10-12 and 16-18 of the oestrous. For in vitro experiments, Gc were isolated from follicles at days 10-12 of the oestrous (4-6 mm) and PNX-14 at doses 1-1000 nM was added for 24-72 h to determine Gc proliferation. Cell cycle progression, E2 secretion, expression of proliferating cells nuclear antigen, cyclins, mitogen-activated kinase (MAP3/1; ERK1/2), protein kinase B (AKT) and signal transducer and activator of transcription 3 (STAT3) were studied. The involvement of these kinases in PNX-14 action on Gc proliferation was analysed using pharmacological inhibitors. Levels of GPR173 were increased in the ovarian follicles with oestrous progression, while only PNX-14 protein was the highest at days 10-12 of the oestrous. Immuno-signal of PNX-14 was detected in Gc and theca cells and oocyte, while GPR173 was mostly in theca. Interestingly, PNX-14 stimulated Gc proliferation, E2 secretion, cell cycle progression and cyclins expression and had a modulatory effect on MAP3/1, AKT and STAT3 activation. Our study suggests that PNX-14 could be an important factor for porcine reproduction by influencing ovarian follicle growth through direct action on Gc function.

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.

Increased Serum Levels of Phoenixin-14, Nesfatin-1 and Dopamine Are Associated with Positive Pregnancy Rate after Ovarian Stimulation.

BACKGROUND: We study the relationship between phoenixin (PNX-14), nesfatin-1 (NES-1), dopamine (DA) and oxytocin (OT) levels together with pregnancy rates in women after ovarian stimulation (OS). METHODS: In a prospective case-control study, 56 infertile women were enrolled from the Department of Gynecological Endocrinology University Hospital. Infertile women age < 40 years old, with polycystic ovary syndrome (PCOS), confirmed tubal patency and suitable sperm quality were included. Blood samples were drawn twice-before the initiation of OS and before the human chorionic gonadotropin (hCG) administration. Assessments of PNX-14, NES-1, DA and OT serum levels were performed. Pregnancy rates after OS were observed. RESULTS: Pregnant women showed higher baseline NES-1 and OT levels (+29.2% and +44%) but not PNX-14 and DA levels when compared to non-pregnant ones. In pregnant women, positive correlations between OT and prolactin, PRL (r = 0.47, p = 0.04), as well as between OT and NES-1 (r = 0.55, p = 0.02), were observed at baseline. At baseline, an OT level increase was associated with a positive pregnancy rate (per 100 pg/mL, OR = 1.39, 95% CI 1.04-1.74), while after OS, higher PNX-14 was a predictor of pregnancy (by 10 pg/mL, OR = 1.23, 95%CI 1.07-1.39). Post-stimulation PNX-14, NES-1 and DA concentrations were higher in pregnant women compared to non-pregnant ones (+17.4%, +26.1%, and +45.5%, respectively; all p < 0.05). In the pregnant group, OT levels were 2.7-times lower than in the remainder (p = 0.03). Moreover, in pregnant participants, a negative association between NES-1 and PNX (r = -0.53, p = 0.024) was observed. CONCLUSION: Elevated PNX-14, NES-1 and DA along with decreased OT levels were observed in women who achieved pregnancy.

Locked Out: Phoenixin-14 Does Not Cross a Stem-Cell-Derived Blood-Brain Barrier Model.

Phoenixin-14 is a recently discovered peptide regulating appetite. Interestingly, it is expressed in the gastrointestinal tract; however, its supposed receptor, GPR173, is predominantly found in hypothalamic areas. To date, it is unknown how peripherally secreted phoenixin-14 is able to reach its centrally located receptor. To investigate whether phoenixin is able to pass the blood-brain barrier, we used an in vitro mono-culture blood-brain barrier (BBB) model consisting of brain capillary-like endothelial cells derived from human induced-pluripotent stem cells (hiPSC-BCECs). The passage of 1 nMol and 10 nMol of phoenixin-14 via the mono-culture was measured after 30, 60, 90, 120, 150, 180, 210, and 240 min using a commercial ELISA kit. The permeability coefficients (PC) of 1 nMol and 10 nMol phoenixin-14 were 0.021 ± 0.003 and 0.044 ± 0.013 µm/min, respectively. In comparison with the PC of solutes known to cross the BBB in vivo, those of phoenixin-14 in both concentrations are very low. Here, we show that phoenixin-14 alone is not able to cross the BBB, suggesting that the effects of peripherally secreted phoenixin-14 depend on a co-transport mechanism at the BBB in vivo. The mechanisms responsible for phoenixin-14's orexigenic property along the gut-brain axis warrant further research.

Expression of phoenixin-14 and nesfatin-1 in the hypothalamo-pituitary-gonadal axis in the phases of the estrous cycle.

Phoenixin-14 (PNX-14) and nucleobindin 2 (NUCB2)/nesfatin-1 are regulatory neuropeptides expressed in the hypothalamus. These neuropeptides can be effective in hormonal regulation of the hypothalamo-pituitary-gonadal (HPG) axis and reproductive functions. In the present study, the distribution of PNX-14 and NUCB2/nesfatin-1 in the hypothalamus, pituitary, ovary, and uterus tissues during the phases of the estrous cycle in female rats was investigated. Eighteen Wistar Albino rats determined among animals showing regular estrous cycle by vaginal smear method were divided into three groups: proestrus (Group I), estrus (Group II) and diestrus (Group III). Serum gonadotropin-releasing hormone (GnRH), plasma PNX-14, and NUCB2/nesfatin-1 concentrations were the highest, moderate, and lowest in estrus, diestrus, and proestrus phases, respectively. PNX-14 immunoreactivity in the supraoptic and arcuate nuclei of the hypothalamus and NUCB2/nesfatin-1 immunoreactivity in the paraventricular nuclei were particularly evident in the estrus phase. These neuropeptide immunoreactivities were decreased in different cells of anterior pituitary during proestrus compared with those during estrus and diestrus. PNX-14 immunoreactivity in the ovary, especially during the estrus phase, was diffuse and intense in the granulosa and luteal cells and oocytes, and it was few and weak in theca cells. In addition, NUCB2/nesfatin-1 immunoreactivity was abundant and strong in granulosa and luteal cells, theca and interstitial cells, and oocytes during estrus. In the estrus phase, PNX-14 immunoreactivity was strong in the glandular epithelial cells and stromal cells of the endometrium, also NUCB2/nesfatin-1 immunoreactivity was strong in the epithelial and glandular epithelial cells. As a result, when the estrous cycle was evaluated, it was concluded that the changes in the distribution of PNX-14 and NUCB2/nesfatin-1 at all phases were related to GnRH and that these neuropeptides showed the highest immunoreactivity especially in the HPG axis and uterus tissues of estrus rats.

Phoenixin-14 Promotes the Recovery of Neurological Dysfunction After Spinal Cord Injury by Regulating Microglial Polarization via PTEN/Akt Signaling Pathway.

Spinal cord injury (SCI) is a destructive event in central nervous system (CNS) with the hallmark of deficits in neuronal function. Phoenixin-14 (PNX-14) is a reproductive peptide that also has neuroprotective effects. However, the role of PNX-14 in SCI has not yet been studied. In this study, we firstly investigated the effects of PNX-14 on the recovery of neurological dysfunction and microglial polarization in a SCI mice model. We demonstrated that PNX-14 improved the recovery of neurological dysfunction with increased Basso Mouse Scale (BMS) scores, reduced lesion area volume and Evans blue (EB) dye extravasation. PNX-14 alleviated neuronal apoptosis and neuroinflammation in mice underwent SCI. In vitro co-culture assay proved that PNX-14 protected neurons injury in response to LPS- activated BV-2 cells. PNX-14 suppressed the LPS- induced microglia M1 phenotype polarization with decreased expression of M1-associated markers (CD16 and iNOS) and increased expression of M2-associated markers (CD206 and Arg1). PNX-14 also suppressed LPS- caused decrease in anti-inflammatory cytokines TGF-ß, IL-10, and IL-13, as well increase in pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 in BV2 cells. PNX-14 treatment caused increased PTEN expression and decreased p-Akt expression in BV2 cells against LPS induction. While inhibition of PTEN by SF1670 reversed the effects of PNX-14 on LPS- induced phenotypic transition of BV2 cells. Taken together, we found that PNX-14 exerted protective effects on neurological dysfunction and inflammation in SCI mice through modulating microglial polarization via PTEN/Akt signaling pathway.

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.

Investigation of serum phoenixin levels in patients with hypertension

SUMMARY OBJECTIVE: Hypertension is a major modifiable risk factor for cardiovascular disease and premature death worldwide. Phoenixin is a newly identified neuropeptide with multiple bioactivity. However, there was no published data about phoenixin levels in hypertension. The aim of this study was to evaluate the relationship between phoenixin and hypertension. METHODS: This study was performed in 36 patients with hypertension and 36 healthy controls. Serum phoenixin-14 and phoenixin-20 levels were determined by Enzyme-Linked ImmunoSorbent Assay method. RESULTS: Serum phoenixin-14 and phoenixin-20 values were significantly lower in hypertension patients compared with the control group (p<0.001). The levels of phoenixin-14 were negatively correlated with weight (r=-0.376; p<0.005), body mass index (r=-0.407; p<0.001), systolic blood pressure (r=-0.586; p<0.001), and diastolic blood pressure (r=-0.319; p<0.01). There was a negative correlation between serum phoenixin-20 and weight (r=-0.378; p<0.005), body mass index (r=-0.383; p<0.005), systolic blood pressure (r=-0.551; p<0.001), and diastolic blood pressure (r=-0.306; p<0.01). We used receiver operating characteristic curve analyses to compare the diagnosis value of Phoenixin-14 and Phoenixin-20 levels in hypertensive patients. We found that Phoenixin-14 value is an area under the curve of 0.87 (cutoff value 404.7 ng/L, sensitivity 92%, specificity 72%) and Phoenixin-20 value is an area under the curve of 0.83 (cutoff value 209.9 ng/L, sensitivity 86%, specificity 75%). Phoenixin-14 did nearly show equally compared to phoenixin-20 in predicting hypertension. CONCLUSION: Serum phoenixin-14 and phoenixin-20 may be related to the pathogenesis of hypertension. Our findings indicated that serum phoenixin-14 and phoenixin-20 may serve as a novel biomarker for the diagnosis of hypertension.

Phoenixin-14 Ameliorates Cellular Senescence Against Morphine in M17 Neuronal Cells.

Drug dependence on morphine is commonly accompanied by neurodegenerative disorders. A previous study showed that prolonged exposure to morphine induces cellular senescence in neuronal cells by reducing telomere length. Phoenixin-14 is a newly discovered brain peptide with pleiotropic roles. However, it is unknown whether phoenixin-14 possesses a beneficial property against morphine-induced cellular senescence. Our results show that morphine reduced the expression of G protein-coupled receptor 173 (GPR173) in M17 neuronal cells. Therefore, we speculated that phoenixin-14, as a ligand for GPR173, may be involved in the morphine-mediated response in M17 cells. Further, we found that phoenixin-14 mitigated morphine-induced oxidative stress by reducing the reactive oxygen species (ROS) production and increasing superoxide dismutase (SOD) activity in M17 neuronal cells. The morphine-induced cellular senescence in M17 neuronal cells was prevented by phoenixin-14. Phoenixin-14 resolved the morphine-caused cell cycle arrest with significant changes in the expression levels of p21, cyclin-dependent kinases 6 (CDK6), and p-Rb. It also elevated the telomerase activity and restored the expressions of human telomerase reverse transcriptase (hTERT) and TERF2 in morphine-induced M17 neuronal cells. Furthermore, phoenixin-14 restored the yes-associated protein (YAP) expression against morphine in M17 neuronal cells. Knockdown of YAP abolished the beneficial effects of phoenixin-14 on cellular senescence against morphine induction. Taken together, these aggregate data demonstrate that phoenixin-14 prevented cellular senescence against morphine induction in M17 neuronal cells via regulating YAP expression.

Protective Effects of Phoenixin-14 Peptide in the Indomethacin-Induced Duodenal Ulcer: An Experimental Study.

Phoenixin-14 (PNX -14 ) is a newly identified neuropeptide with potential anti-inflammatory effects in the gastrointestinal tract. In this study, we evaluated the protective effect of PNX-14 against the formation of experimental indomethacin (IND)-induced duodenal ulcer. Thirty-two male Sprague-Dawley rats were randomly assigned to the four following study groups: (1) negative control (2) IND (7.5 mg/kg subcutaneous IND), (3) famotidine (FA) (7.5 mg/kg subcutaneous IND followed by 40 mg/kg intraperitoneal FA), and (4) PNX-14 (7.5 mg/kg subcutaneous IND followed by 50 µ/kg intraperitoneal PNX-14). Outcome measures included macroscopic evaluation of duodenal lesion, serum levels of IL-1ß, TNF-α, IL-6, and IL-12, and tissue biochemical parameters of oxidative stress, including malondialdehyde (MDA) , myeloperoxidase (MPO) activity, superoxide dismutase (SOD) activity, and catalase activity. Results The macroscopic grade of duodenal lesions were significantly smaller in the PNX-14 group than in the IND group (p < 0.001). Serum inflammatory cytokines were significantly increased in the IND group. PNX-14 treatment significantly decreased the serum levels of inflammatory cytokines (p < 0.0001). Oxidative contents (MDA and MPO activity) were significantly smaller in the PNX-14 group compared with the IND group (p < 0.0001), while anti-oxidative contents (SOD and catalase activity) were significantly more (p < 0.0001). PNX-14 was superior to FA in several anti-inflammatory properties, such as inhibiting the release of inflammatory cytokines and increasing the catalase activity. PNX-14 showed significant protective effects against the formation of IND-induced duodenal ulcers. These results suggest a promising therapeutic implication for PNX-14 in the treatment of gastrointestinal inflammatory disorders.

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-14 protects cardiac damages in a streptozotocin-induced diabetes mice model through SIRT3.

BACKGROUND: Type I diabetes is a metabolic syndrome that severely impacts the normal lives of patients through its multiple complications, such as diabetic cardiomyopathy (DCM). Phoenixin-14 is a peptide found to be widely expressed in eukaryons with multiple protective properties, including anti-oxidative stress and anti-inflammatory effects. The present study aims to explore the potential therapeutic impacts of Phoenixin-14 on DCM. METHODS: Type I diabetes was induced by treatment with a single dose of STZ (40 mg/kg body weight) intraperitoneally for 5 consecutive days. Mice were divided into four groups: the Control, Phoenixin-14, T1DM, and Phoenixin-14 +T1DM groups. The levels of myocardial injury markers were measured. Cardiac hypertrophy was assessed using wheat germ agglutinin (WGA) staining. RESULTS: Phoenixin-14 was significantly downregulated in the cardiac tissue of diabetic mice. The myocardial injury and deteriorated cardiac function in diabetic mice induced by STZ were significantly ameliorated by Phoenixin-14, accompanied by the alleviation of cardiac hypertrophy. In addition, the severe oxidative stress and inflammation in diabetic mice were dramatically mitigated by Phoenixin-14. Lastly, the downregulated SIRT3 and upregulated p-FOXO3 in diabetic mice were pronouncedly reversed by Phoenixin-14. It is worth mentioning that compared to the Control, no significant changes to any of the investigated parameters in the present study were found in the Phoenixin-14-treated normal mice, suggesting that treatment with it has no side effects. CONCLUSION: Our data revealed that Phoenixin-14 protected against cardiac damages in STZ-induced diabetes mice models.

Phoenixin-14 regulates proliferation and apoptosis of vascular smooth muscle cells by modulation of KCNQ1OT1/miR-183-3p/CTNNB1 axis.

Phoenixin-14 has been reported to be implicated in the process of blood glucose metabolism, reproduction, lipid deposition and cardioprotection. However, the role of phoenixin-14 in vascular smooth muscle cells (VSMCs) remains unkown. In this study, we focused on the effects of phoenixin-14 on VSMCs under oxidized low-density lipoprotein (ox-LDL) treatment. The experimental results demonstrated that phoenixin-14 inhibited mRNA level and nuclear translocation of ß-catenin. Functionally, phoenixin-14 inhibited cell proliferation and facilitated apoptosis of VSMCs under ox-LDL stimulation, and CTNNB1 overexpression reversed these effects. Mechanistically, KCNQ1OT1 interacted with miR-183-3p to upregulate CTNNB1 in VSMCs. Furthermore, CTNNB1 expression was negatively correlated with miR-183-3p but positively associated with KCNQ1OT1. Rescue assays indicated that KCNQ1OT1 overexpression or Lithium chloride (LiCl) treatment reversed the effects of phoenixin-14 on proliferation and apoptosis of ox-LDL-stimulated VSMCs. In summary, phoenixin-14 regulates proliferation and apoptosis of ox-LDL-treated VSMCs by regulating the KCNQ1OT1/miR-183-3p/CTNNB1 axis.

Phoenixin 14 Inhibits High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease in Experimental Mice.

INTRODUCTION: Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases. The development of NAFLD is closely associated with hepatic lipotoxicity, inflammation, and oxidative stress. The new concept of NAFLD treatment is to seek molecular control of lipid metabolism and hepatic redox hemostasis. Phoenixin is a newly identified neuropeptide with pleiotropic effects. This study investigated the effects of phoenixin 14 against high-fat diet (HFD)-induced NAFLD in mice. MATERIALS AND METHODS: For this study, we used HFD-induced NAFLD mice models to analyze the effect of phonenixin14. The mice were fed on HFD and normal diet and also given phoenixin 14 (100 ng/g body weight) by gastrogavage for 10 weeks. The peripheral blood samples were collected for biochemical assays. The liver tissues were examined for HFD-induced tissue fibrosis, lipid deposition and oxidative activity including SOD, GSH, and MDA. The liver tissues were analyzed for the inflammatory cytokines and oxidative stress pathway genes. RESULTS: The results indicate that phoenixin 14 significantly ameliorated HFD-induced obesity and fatty liver. The biochemical analysis of blood samples revealed that phoenixin 14 ameliorated HFD-induced elevated circulating alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol, and triglyceride levels, suggesting that phoenixin 14 has a protective role in liver function and lipid metabolism. Hematoxylin-eosin (HE) and Oil Red O staining of the liver showed that phoenixin 14 alleviated HFD-induced tissue damage and lipid deposition in the liver. Furthermore, the mice administered with phoenixin 14 had increased hepatic SOD activity, increased production of GSH and reduced MDA activity, as well as reduced production of TNF-α and IL-6 suggesting that phoenixin 14 exerts beneficial effects against inflammation and ROS. The findings suggest an explanation of how mechanistically phoenixin 14 ameliorated HFD-induced reduced activation of the SIRT1/AMPK and NRF2/HO-1 pathways. CONCLUSION: Collectively, this study revealed that phoenixin 14 exerts a protective effect in experimental NAFLD mice. Phoenixin could be of the interest in preventive modulation of NAFLD.

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.

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.

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.