The Pros1/Tyro3 axis protects against periodontitis by modulating STAT/SOCS signalling.

Periodontitis, an oral inflammatory disease caused by periodontal pathogen infection, is the most prevalent chronic inflammatory disease and a major burden on healthcare. The TAM receptor tyrosine kinases (Tyro3, Axl and Mertk) and their ligands (Gas6 and Pros1) play a pivotal role in the resolution of inflammation and have been associated with chronic inflammatory and autoimmune diseases. In this study, we evaluated the effects of exogenous Pros1 in in vitro and in vivo models of periodontitis. We detected higher Pros1 but lower Tyro3 levels in inflamed gingival specimens of periodontitis patients compared with healthy controls. Moreover, Pros1 was mostly localized in the gingival epithelium of all specimens. In cultured human gingival epithelial cells (hGECs), Porphyromonas gingivalis LPS (p.g-LPS) stimulation down-regulated Pros1 and Tyro3. Exogenous Pros1 inhibited p.g-LPS-induced production of TNF-α, IL-6, IL-1ß, MMP9/2 and RANKL in a Tyro3-dependent manner as revealed by PCR, Western blot analysis, ELISA and gelatin zymography. Pros1 also restored Tyro3 expression down-regulated by p.g-LPS in hGECs. In rats treated with ligature and p.g-LPS, administration of Pros1 attenuated periodontitis-associated gingival inflammation and alveolar bone loss. Our mechanistic studies implicated SOCS1/3 and STAT1/3 as mediators of the in vitro and in vivo anti-inflammatory effects of Pros1. Collectively, the findings from this work supported Pros1 as a novel anti-inflammatory therapy for periodontitis.

Fusion to Tetrahymena thermophila granule lattice protein 1 confers solubility to sexual stage malaria antigens in Escherichia coli.

A transmission-blocking vaccine targeting the sexual stages of Plasmodium species could play a key role in eradicating malaria. Multiple studies have identified the P. falciparum proteins Pfs25 and Pfs48/45 as prime targets for transmission-blocking vaccines. Although significant advances have been made in recombinant expression of these antigens, they remain difficult to produce at large scale and lack strong immunogenicity as subunit antigens. We linked a self-assembling protein, granule lattice protein 1 (Grl1p), from the ciliated protozoan, Tetrahymena thermophila, to regions of the ectodomains of either Pfs25 or Pfs48/45. We found that resulting protein chimera could be produced in E. coli as nanoparticles that could be readily purified in soluble form. When produced in the E. coli SHuffle strain, fusion to Grl1p dramatically increased solubility of target antigens while at the same time directing the formation of particles with diameters centering on 38 and 25 nm depending on the antigen. In a number of instances, co-expression with chaperone proteins and induction at a lower temperature further increased expression and solubility. Based on Western blotting and ELISA analysis, Pfs25 and Pfs48/45 retained their transmission-blocking epitopes within E. coli-derived particles, and the particles themselves elicited strong antibody responses in rabbits when given with an aluminum-based adjuvant. Antibodies against Pfs25-containing nanoparticles blocked parasite transmission in standard membrane-feeding assays. In conclusion, fusion to Grl1p can act as a solubility enhancer for proteins with limited solubility while retaining correct folding, which may be useful for applications such as the production of vaccines and other biologics.

Nesfatin-130-59 Injected Intracerebroventricularly Increases Anxiety, Depression-Like Behavior, and Anhedonia in Normal Weight Rats.

Nesfatin-1 is a well-established anorexigenic peptide. Recent studies indicated an association between nesfatin-1 and anxiety/depression-like behavior. However, it is unclear whether this effect is retained in obesity. The aim was to investigate the effect of nesfatin-130-59-the active core of nesfatin-1-on anxiety and depression-like behavior in normal weight (NW) and diet-induced (DIO) obese rats. Male rats were intracerebroventricularly (ICV) cannulated and received nesfatin-130-59 (0.1, 0.3, or 0.9 nmol/rat) or vehicle 30 min before testing. Nesfatin-130-59 at a dose of 0.3 nmol reduced sucrose consumption in the sucrose preference test in NW rats compared to vehicle (⁻33%, p < 0.05), indicating depression-like/anhedonic behavior. This dose was used for all following experiments. Nesfatin-130-59 also reduced cookie intake during the novelty-induced hypophagia test (-62%, p < 0.05). Moreover, nesfatin-130-59 reduced the number of entries into the center zone in the open field test (-45%, p < 0.01) and the visits of open arms in the elevated zero maze test (-39%, p < 0.01) in NW rats indicating anxiety. Interestingly, DIO rats showed no behavioral alterations after the injection of nesfatin-130-59 (p > 0.05). These results indicate an implication of nesfatin-130-59 in the mediation of anxiety and depression-like behavior/anhedonia under normal weight conditions, while in DIO rats, a desensitization might occur.

Intraperitoneal injection of nesfatin-1 primarily through the CCK-CCK1R signal pathway affects expression of appetite factors to inhibit the food intake of Siberian sturgeon (Acipenser baerii).

Nesfatin-1 is an 82-amino acid protein derived from nucleobindin 2 (NUCB2), which could inhibit food intake in fish and mammals. However, the neuroendocrine mechanism of nesfatin-1 in animal appetite regulation is unclear. To explore the feeding mechanism of nesfatin-1 in Siberian sturgeon (Acipenser baerii), intraperitoneal injections of nesfatin-1 and sulfated cholecystokinin octapeptide (CCK8), Lorglumide (CCK1R selective antagonist), or LY 225,910 (CCK2R selective antagonist) were performed. Co-injection of nesfatin-1 and CCK8 synergistically significantly decreased the food intake in 1 h. Lorglumide reversed the anorectic effect of nesfatin-1, but LY 225,910 had no effect. Moreover, Lorglumide could also reverse the expressions of appetite factors including nucb2, cck, unc3, cart, apelin, pyy, and npy induced by nesfatin-1 in the brain, stomach, and liver, while LY 225,910 partially reversed these changes. These results indicate that nesfatin-1 inhibits the appetite of Siberian sturgeon mainly through the CCK-CCK1R signaling pathway.

Nesfatin-1 Improve Spatial Memory Impairment Following Transient Global Cerebral Ischemia/Reperfusion via Inhibiting Microglial and Caspase-3 Activation.

Nesfatin-1, a recently discovered peptide, is involved in important functions such as food intake regulation and energy homeostasis. Previous studies have demonstrated that it has protective effects following myocardial injury and also protects dopaminergic cells against neurotoxicity with the anti-inflammatory and anti-apoptotic mechanisms. In this study, we aimed to assay the neuroprotective effects of Nesfatin-1 after brain ischemia/reperfusion. Twenty-eight male Wistar rats were randomly selected and allocated in the form of four groups (sham, Nesfatin-1, ischemia, ischemia+Nesfatin-1). Ischemia was created by obstruction couple common carotid arteries in 20-min period. Saline as a vehicle and Nesfatin-1 (20 µg/kg, intraperitoneally) were injected at the time of reperfusion. Spatial memory performances were evaluated by the Morris water maze. The level of protein expression was determined by immunohistochemical and immunofluorescence staining. Nesfatin-1 significantly reduced caspase-3 (P < 0.01) and microglial activation (P < 0.01) and improved spatial memory impairments (P < 0.05) induced by brain ischemia. Nesfatin-1 has significant neuroprotective effects and can be introduced as a therapeutic agent against cerebral ischemia-induced injuries.

Modulation of nesfatin-1-induced cardiovascular effects by the central cholinergic system.

Nesfatin-1, a peptide whose receptor is yet to be identified, has been shown to be involved in the modulation of feeding, stress, and metabolic responses. Recently, increasing evidence has supported a modulatory role of nesfatin-1 in cardiovascular activity. We have previously reported that nesfatin-1 causes an increase in blood pressure in normotensive and hypotensive rats by increasing plasma catecholamine, vasopressin, and renin levels. Recent reports suggest that nesfatin-1 may activate the central cholinergic system. However, there is no evidence showing an interaction between central nesfatin-1 and the cholinergic system. Therefore, this study aimed to determine whether the central cholinergic system may have a functional role in the nesfatin-1-induced cardiovascular effect observed in normotensive rats. Intracerebroventricular injection of nesfatin-1 caused short-term increases in mean arterial pressure and heart rate responses including bradycardic/tachycardic phases in normotensive animals. Central injection of nesfatin-1 increased the acetylcholine and choline levels in the posterior hypothalamus, as shown in microdialysis studies. Central pretreatment with the cholinergic muscarinic receptor antagonist atropine and/or nicotinic receptor antagonist mecamylamine blocked nesfatin-1-induced cardiovascular effects. In conclusion, the results show that centrally administered nesfatin-1 produces a pressor effect on blood pressure and heart rate responses including bradycardic/tachycardic phases in normotensive rats. Moreover, according to our findings, the central cholinergic system can modulate nesfatin-1-evoked cardiovascular activity.

Cardioprotective effect of nesfatin-1 against isoproterenol-induced myocardial infarction in rats: Role of the Akt/GSK-3ß pathway.

The present study was designed to evaluate the cardioprotective effects of nesfatin-1, a novel peptide with anorexigenic properties, in rats with isoproterenol (ISO)-induced myocardial infarction (MI), and to further investigate the role of Akt/GSK-3ß signaling pathway in the protective effect of nesfatin-1. To induce MI, ISO was subcutaneously injected into the rats for two consecutive days at a dosage of 85mg/kg/day. ISO-induced myocardial damage was indicated by elevated levels of cardiac specific troponin-T, enhanced myocardial expression of proinflammatory cytokines (interleukin-1ß, interleukin-6 and tumor necrosis factor-α), and increased number of cells with apoptotic and necrotic appearance in the myocardial tissue. Levels of p-Akt/Akt and p-GSK-3ß/GSK-3ß significantly decreased in heart tissue after ISO-induced MI. However, intraperitoneal administration of nesfatin-1 (10µg/kg/day) elicited a significant cardioprotective activity by lowering the levels of cardiac troponin-T and proinflammatory cytokines, indicating the protective effect of nesfatin-1 against ISO-induced MI. The biochemical findings were further confirmed by histopathological examination, which was demonstrated by reduced number of apoptotic and necrotic cells. Moreover, expressions of p-Akt/Akt and p-GSK-3ß/GSK-3ß in the myocardium of MI group rats were significantly increased by nesfatin-1 administration, suggesting that nesfatin-1, which appears to possess anti-apoptotic and anti-inflammatory properties, may confer protection against ISO-induced MI via an Akt/GSK-3ß-dependent mechanism.

Nesfatin-1 inhibits voltage gated K+ channels in pancreatic beta cells.

The anorexigenic neuropeptide NEFA/nucleobindin 2 (NUCB2)/nesfatin-1-containing neurons are distributed in the brain regions involved in feeding regulation. In spite of the growing knowledge of its physiological functions through extensive studies, its molecular mechanism of reaction, including its receptor, remains unknown. NUCB2/nesfatin-1 is also involved in various peripheral regulations, including glucose homeostasis. In pancreatic beta-cells, NUCB2/nesfatin-1 is reported to enhance glucose-stimulated insulin secretion (GSIS) but its exact mechanism remains unknown. To clarify this mechanism, we measured the effect of nesfatin-1 on the electrical activity of pancreatic beta-cells. Using mouse primary beta cells, we measured changes in the ATP-sensitive K+ (KATP) channel current, the voltage-gated K+ (Kv) channel current, and insulin secretion upon application of nesfatin-1. Nesfatin-1 inhibited the Kv channel, but KATP channel activity was unaffected. Nesfatin-1 enhanced insulin secretion to a same level as Kv channel blocker tetraethylammonium (TEA). The effect was not further enhanced when nesfatin-1 and TEA were applied simultaneously. The inhibition binding assay with [125I]nesfatin-1 in Kv2.1 channels, major contributor of Kv current in beta cell, expressing HEK239 cells indicated the binding of nesfatin-1 on Kv2.1 channel. Because Kv channel inhibition enhances insulin secretion under high glucose conditions, our present data suggest a possible mechanism of nesfatin-1 on enhancing GSIS through regulation of ion channels rather than its unidentified receptor.

Study of the effects of nesfatin-1 on gastric function in obese rats.

AIM: To investigate the effects of nesfatin-1 on gastric function in obese rats. METHODS: The obese rat model was induced by a high-fat diet. The gastric emptying rate and gastric acid secretory capacity of the rats were determined after treatment with different drug concentrations of nesfatin-1 and administration routes. Based on this, the expression of H+/K+-ATPase was measured using RT-PCR and western blot to preliminarily explore the mechanism of gastric acid secretion changes. RESULTS: Body weight, body length, and Lee's index of the rats significantly increased in the high-fat diet-induced obese rat model. Two hours after lateral intracerebroventricular injection of nesfatin-1, the gastric emptying rate and gastric acid secretory capacity of rats decreased. Four hours after injection, both were restored to normal levels. In addition, the expression of H+/K+-ATPase decreased and moved in line with changes in gastric acid secretory capacity. This in vivo experiment revealed that intracerebroventricular injection of nesfatin-1, rather than intravenous injection, could suppress gastric function in obese rats. Moreover, its effect on the gastric emptying and gastric acid secretory capacity of rats is dose-dependent within a certain period of time. CONCLUSION: Through this research, we provide a theoretical basis for further studies on nesfatin-1, a potential anti-obesity drug.

Enhanced sublingual immunotherapy by TAT-fused recombinant allergen in a murine rhinitis model.

Allergen-specific sublingual immunotherapy (SLIT) is well known as an effective and non-invasive route to induce allergy desensitization. The goal of this study was to investigate whether a TAT-fused recombinant allergen could enhance SLIT efficacy. BALB/c mice sensitized to the main allergen (Che a 3) of Chenopodium album pollen were treated sublingually either with rChe a 3 (100µg/dose) or rTAT-Che a 3 (100µg/dose), two times per week for eight weeks. SLIT with rTAT-Che a 3 led to significantly greater allergen-specific IgG2a than rChe a 3; however, neither rTAT-Che a 3 nor rChe a 3 affected allergen-specific IgE or IgG1 antibody levels. In addition, interleukin 4 (IL-4) levels in re-stimulated splenocytes from the rTAT-Che a 3 mice were significantly lower than in those from the rChe a 3 mice, while interferon-γ (IFN-γ) was significantly greater in the rChe a 3 mice than in the rTAT-Che a 3 mice. Furthermore, sublingual administration of rTAT-Che a 3 induced significantly greater TGF-ß secretion in re-stimulated splenocytes than administration of rChe a 3. Accordingly, SLIT with rTAT-Che a 3 led to significantly greater expression of TGF-ß- and Foxp3-specific mRNAs in the splenocytes than in those from the rChe a 3 mice. Our findings demonstrate that TAT-fused rChe a 3 suppressed the allergic response through preferential enhancement of systemic regulatory T-cell (Treg)-mediated immunity responses, likely by facilitating allergen capture and presentation by sublingual Langerhans-like dendritic cells.

Combining Smoothened Agonist and NEL-Like Protein-1 Enhances Bone Healing.

BACKGROUND: Nonhealing bone defects represent an immense biomedical burden. Despite recent advances in protein-based bone regeneration, safety concerns over bone morphogenetic protein-2 have prompted the search for alternative factors. Previously, the authors examined the additive/synergistic effects of hedgehog and Nel-like protein-1 (NELL-1) on the osteogenic differentiation of mesenchymal stem cells in vitro. In this study, the authors sought to leverage their previous findings by applying the combination of Smoothened agonist (SAG), hedgehog signal activator, and NELL-1 to an in vivo critical-size bone defect model. METHODS: A 4-mm parietal bone defect was created in mixed-gender CD-1 mice. Treatment groups included control (n = 6), SAG (n = 7), NELL-1 (n = 7), and SAG plus NELL-1 (n = 7). A custom fabricated poly(lactic-co-glycolic acid) disk with hydroxyapatite coating was used as an osteoinductive scaffold. RESULTS: Results at 4 and 8 weeks showed increased bone formation by micro-computed tomographic analyses with either stimulus alone (SAG or NELL-1), but significantly greater bone formation with both components combined (SAG plus NELL-1). This included greater bone healing scores and increased bone volume and bone thickness. Histologic analyses confirmed a significant increase in new bone formation with the combination therapy SAG plus NELL-1, accompanied by increased defect vascularization. CONCLUSIONS: In summary, the authors' results suggest that combining the hedgehog signaling agonist SAG and NELL-1 has potential as a novel therapeutic strategy for the healing of critical-size bone defects. Future directions will include optimization of dosage and delivery strategy for an SAG and NELL-1 combination product.

Nesfatin-1/NUCB2 in the amygdala influences visceral sensitivity via glucocorticoid and mineralocorticoid receptors in male maternal separation rats.

BACKGROUND: Nesfatin-1, a recently identified satiety molecule derived from nucleobindin 2 (NUCB2), is associated with visceral hypersensitivity in rats and is expressed in the amygdala. We tested the hypothesis that nesfatin-1 expression in the amygdala is involved in the pathogenesis of irritable bowel syndrome (IBS) visceral hypersensitivity. METHODS: An animal model of IBS-like visceral hypersensitivity was established using maternal separation (MS) during postnatal days 2-16. The role of nesfatin-1 in the amygdala on visceral sensitivity was evaluated. KEY RESULTS: Rats subjected to MS showed a significantly increased mean abdominal withdrawal reflex (AWR) score and electromyographic (EMG) activity at 40, 60, and 80 mmHg colorectal distension. Plasma concentrations of nesfatin-1 and corticosterone were significantly higher than in non-handled (NH) rats. mRNA and protein expression of nesfatin-1/NUCB2 in the amygdala were increased in MS rats, but not in NH rats. In MS rats, AWR scores and EMG activity were significantly decreased after anti-nesfatin-1/NUCB2 injection. In normal rats, mean AWR score, EMG activity, and corticosterone expression were significantly increased after nesfatin-1 injection into the amygdala. Nesfatin-1-induced visceral hypersensitivity was abolished following application of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) antagonists. CONCLUSIONS & INFERENCES: Elevated expression of nesfatin-1/NUCB2 in the amygdala in MS rats suggests a potential role in the pathogenesis of visceral hypersensitivity, which could potentially take place via activation of GR and MR signaling pathways.

Toxin-neutralizing antibodies protect against Clostridium perfringens-induced necrosis in an intestinal loop model for bovine necrohemorrhagic enteritis.

BACKGROUND: Bovine necrohemorrhagic enteritis is caused by Clostridium perfringens type A. Due to the rapid progress and fatal outcome of the disease, vaccination would be of high value. In this study, C. perfringens toxins, either as native toxins or after formaldehyde inactivation, were evaluated as possible vaccine antigens. We determined whether antisera raised in calves against these toxins were able to protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis. RESULTS: Alpha toxin and perfringolysin O were identified as the most immunogenic proteins in the vaccine preparations. All vaccines evoked a high antibody response against the causative toxins, alpha toxin and perfringolysin O, as detected by ELISA. All antibodies were able to inhibit the activity of alpha toxin and perfringolysin O in vitro. However, the antibodies raised against the native toxins were more inhibitory to the C. perfringens-induced cytotoxicity (as tested on bovine endothelial cells) and only these antibodies protected against C. perfringens challenge in the intestinal loop model. CONCLUSION: Although immunization of calves with both native and formaldehyde inactivated toxins resulted in high antibody titers against alpha toxin and perfringolysin O, only antibodies raised against native toxins protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis.

Nesfatin-1 stimulates cholecystokinin and suppresses peptide YY expression and secretion in mice.

Nesfatin-1 is an 82 amino acid secreted peptide encoded in the precursor, nucleobindin-2 (NUCB2). It is an insulinotropic anorexigen abundantly expressed in the stomach and hypothalamus. Post-prandial insulin secretion is predominantly regulated by incretins glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Nesfatin-1 was previously reported to modulate GLP-1 and GIP secretion in vitro in an enteroendocrine (STC-1) cell line. Intestine is a source of additional hormones including cholecystokinin (CCK) and peptide YY (PYY) that regulate metabolism. We hypothesized that nesfatin-1 modulates CCK and PYY secretion. Immunofluorescence histochemistry showed NUCB2/nesfatin-1 co-localizing CCK and PYY in the intestinal mucosa of mice. Static incubation of STC-1 cells with nesfatin-1 upregulated both CCK mRNA expression (1 and 10 nM) and secretion (0.1, 1 and 10 nM) at 1 h post-incubation. In contrast, nesfatin-1 treatment for 1 h downregulated PYY mRNA expression (all doses tested) and secretion (0.01 and 0.1 nM) in STC-1 cells. Continuous infusion of nesfatin-1 using osmotic mini-pumps for 12 h upregulated CCK mRNA expression in large intestine, and downregulated PYY mRNA expression in both large and small intestines of male C57BL/6J mice. In these tissues, Western blot analysis found a corresponding increase in CCK and a decrease in PYY content. Collectively, we provide new information on the cell specific localization of NUCB2/nesfatin-1 in the intestinal mucosa, and a novel function for nesfatin-1 in modulating intestinal CCK and PYY expression and secretion in mice.

AMPK-dependent modulation of hepatic lipid metabolism by nesfatin-1.

The aim of this study was to characterize the mechanism by which peripheral nesfatin-1 regulates hepatic lipid metabolism. Continuous peripheral infusion of nesfatin-1 reduced adiposity and plasma levels of triglyceride and cholesterol. In mice fed high fat diet, peripheral nesfatin-1 significantly decreased hepatic steatosis measured by triglyceride content and oil red staining area and diameter. These alterations were associated with a significant reduction in lipogenesis-related transcriptional factors PPARγ and SREBP1, as well as rate-limited enzyme genes such as acaca, fasn, gpam, dgat1 and dgat2. In primary hepatocytes, nesfatin-1 inhibited both basal and oleic acid stimulated triglyceride accumulation, which was accompanied by a decrement in lipogenesis-related genes and an increase in ß-oxidation-related genes. In cultured hepatocytes, nesfatin-1 increased levels of AMPK phosphorylation. Inhibition of AMPK by compound C blocked the reduction of triglyceride content elicited by nesfatin-1. Our studies demonstrate that nesfatin-1 attenuates lipid accumulation in hepatocytes by an AMPK-dependent mechanism.

The role of centrally injected nesfatin-1 on cardiovascular regulation in normotensive and hypotensive rats.

This study investigated the cardiovascular effects of nesfatin-1 in normotensive rats and animals subjected to hypotensive hemorrhage. Hemorrhagic hypotension was induced by withdrawal 2 mL blood/100 g body weight over a period of 10 min. Acute hemorrhage led to a severe and long-lasting decrease in mean arterial pressure (MAP) and heart rate (HR). Intracerebroventricularly (i.c.v.) administered nesfatin-1 (100 pmol) increased MAP in both normotensive and hemorrhaged rats. Nesfatin-1 also caused bradycardia in normotensive and tachycardia in hemorrhaged rats. Centrally injected nesfatin-1 (100 pmol, i.c.v.) also increased plasma catecholamine, vasopressin and renin concentrations in control animals and potentiated the rise in all three cardiovascular mediators produced by hemorrhage. These findings indicate that centrally administered nesfatin-1 causes a pressor response in conscious normotensive and hemorrhaged rats and suggest that enhanced sympathetic activity and elevated vasopressin and renin concentrations mediate the cardiovascular effects of the peptide.

Rational design of gold nanocarrier for the delivery of JAG-1 peptide.

BACKGROUND: Unique properties exhibited by nanoparticles makes them great candidates for applications in physics, chemistry, biology, material science and medicine. The biological applications of water-soluble gold nanoparticles range from contrast agents, delivery vehicles to therapeutics. Notch signaling is a complex network that orchestrates cell fate decisions, which involves proliferation, migration, differentiation and cell death in organisms ranging from insects to humans. Studies have showed that a correct orientation of the Jag-1 signalling protein on the substrates proves to be of great importance when promoting Jagged-1 Notch interactions, also the availability of the ligands, super cedes the importance of their concentration. RESULTS: The aim of the present study was to synthetize a Jag-1 functionalized nanocarrier, which would promote an efficient interaction between the Jag-1 peptide and the Notch receptor. To this end, two routes for gold nanoparticle-peptide assembly were investigated, and the synthetized bio-nanostructures were characterized and compared by means of UV-Vis, FT-IR, DLS and AFM techniques. CONCLUSIONS: We have obtained a stable, monodisperse, hetero-functionalized GNP-PEG-JAG-1 bio-nanostructure for Notch pathway activation applications.

Nesfatin-1 antagonized rotenone-induced neurotoxicity in MES23.5 dopaminergic cells.

Nesfatin-1 is a recently identified brain-gut peptide involved in feeding and energy homeostasis. Recently, it has been proved that nesfatin-1 could exert its neuroprotective effect against subarachnoid hemorrhage-induced injury via its anti-apoptotic and anti-inflammatory properties. However, whether it has neuroprotective effect on dopamine neurons is largely unknown. In the present study, we investigated the neuroprotective effect of nesfatin-1 on rotenone-treated MES23.5 dopaminergic cells and illustrated the underlying mechanisms. Our results showed that nesfatin-1 pretreatment could significantly attenuate rotenone-induced cell loss. Further studies showed that the neuroprotective effect of nesfatin-1 against rotenone was mediated by reversing rotenone-induced mitochondrial dysfunction. Nesfatin-1 could rescue rotenone-induced mitochondrial transmembrane potential collapse and restore the function of mitochondrial respiratory chain complex I. In addition, rotenone-induced release of cytochrome C from mitochondria, ROS production and the subsequent caspase-3 activation were also attenuated by nesfatin-1 pretreatment. Our data suggested that nesfatin-1 exerted its neuroprotective effect on dopaminergic cells against rotenone by ameliorating mitochondrial dysfunction and its anti-apoptotic property. This suggested that nesfatin-1 had the potential to be considered as an aid for prevention of Parkinson's disease.

Pharmacokinetics and osteogenic potential of PEGylated NELL-1 in vivo after systemic administration.

Osteoporosis is a skeletal disorder attributable to an imbalance in osteoblast and osteoclast activity. NELL-1, a secretory protein that promotes osteogenesis while suppressing osteoclastic activity, holds potential as an osteoporosis therapy. Recently, we demonstrated that PEGylation of NELL-1 significantly improves its thermostability while preserving its bioactivity in vitro. However, the effect of PEGylation on the pharmacokinetics and osteogenic potential of NELL-1 in vivo have yet to be investigated. The present study demonstrated that PEGylation of NELL-1 significantly increases the elimination half-life time of the protein from 5.5 h to 15.5 h while distributing more than 2-3 times the amount of protein to bone tissues (femur, tibia, vertebrae, calvaria) in vivo when compared to naked NELL-1. In addition, microCT and DXA analyses demonstrated that systemic NELL-PEG therapy administered every 4 or 7 days significantly increases not only femoral and lumbar BMD and percent bone volume, but also new bone formation throughout the overall skeleton after four weeks of treatment. Furthermore, immunohistochemistry revealed increased osteocalcin expression, while TRAP staining showed reduced osteoclast numbers in NELL-PEG groups. Our findings suggest that the PEGylation technique presents a viable and promising approach to further develop NELL-1 into an effective systemic therapeutic for the treatment of osteoporosis.

Nesfatin-1 inhibits proliferation and enhances apoptosis of human adrenocortical H295R cells.

NUCB2/nesfatin and its proteolytically cleaved product nesfatin-1 are recently discovered anorexigenic hypothalamic neuroproteins involved in energy homeostasis. It is expressed both centrally and in peripheral tissues, and appears to have potent metabolic actions. NUCB2/nesfatin neurons are activated in response to stress. Central nesfatin-1 administration elevates circulating ACTH and corticosterone levels. Bilateral adrenalectomy increased NUCB2/nesfatin mRNA levels in rat paraventricular nuclei. To date, studies have not assessed the effects of nesfatin-1 stimulation on human adrenocortical cells. Therefore, we investigated the expression and effects of nesfatin-1 in a human adrenocortical cell model (H295R). Our findings demonstrate that NUCB2 and nesfatin-1 are expressed in human adrenal gland and human adrenocortical cells (H295R). Stimulation with nesfatin-1 inhibits the growth of H295R cells and promotes apoptosis, potentially via the involvement of Bax, BCL-XL and BCL-2 genes as well as ERK1/2, p38 and JNK1/2 signalling cascades. This has implications for understanding the role of NUCB2/nesfatin in adrenal zonal development. NUCB2/nesfatin may also be a therapeutic target for adrenal cancer. However, further studies using in vivo models are needed to clarify these concepts.