Zinc deficiency disrupts pain signaling promoting nociceptive but not inflammatory pain in mice

Abstract Zinc (Zn) is an essential micronutrient involved in the physiology of nervous system and pain modulation. There is little evidence for the role of nutritional Zn alternations to the onset and progression of neuropathic (NP) and inflammatory pain. The study investigated the effects of a zinc restricted diet on the development of pain. Weaned mice were submitted to a regular (38 mg/kg of Zn) or Zn deficient (11 mg/kg of Zn) diets for four weeks, pain responses evaluated (mechanical, cold and heat allodynia; formalin- and carrageenan-induced inflammatory hypernociception), plasma and tissues collected for biochemical and metabolomic analysis. Zn deficient diet inhibited animal growth (37%) and changed mice sensitivity pattern, inducing an intense allodynia evoked by mechanical, cold and heat stimulus for four weeks. The inflammatory pain behavior of formalin test was drastically reduced or absent when challenged by an inflammatory stimulus. Zn restriction also reduce plasma TNF, increase neuronal activation, oxidative stress, indicating a disruption of the immune response. Liver metabolomic analyses suggest a downregulation of lipid metabolism of arachidonic acid. Zn restriction since weaned disrupts pain signaling considerably and reduce inflammatory pain. Zn could be considered a predisposing factor for the onset of chronic pain such as painful neuropathies

Amylin and PEG structures.

<p><b>a)</b> Aminoacid sequence of the human and murine amylin. In bold are the different aminoacid. Notice the similarity in sequence from aminoacid 1 to 17. The N-terminus is the Lys1, which comprises the α- and ε-aminogroup, the two unique primary aminogroups in amylin targeted for PEGylation. <b>b)</b> Chemical structure of the methoxyl PEG N-hydroxylsuccinimide (NHS) carbonate. <b>c)</b> Structure of the human amylin. Human amylin (from NMR structure, PDB ID 2KB8) is represented in ribbons colored from aminoacid 1 (blue) to 37 (red). Notice the two aminogroups at the top left side of the representation.</p

Amylin self-assembly and interaction with co-receptors.

<p>Free murine amylin or PEGylated human amylin were assayed for binding with (<b>A</b>) murine amylin, (<b>B</b>) CTR–1 and (<b>C</b>) RAMP–3 by fluorescence anisotropy of fluorescein labeled molecular partners. Assays were conducted in PBS, pH 7.4, 25°C, in the presence of 50 nM FITC-labelled proteins (amylin, CTR–1 or RAMP–3). Ex 480 nm, Em 520 nm, filter 515 nm. The raw data can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138803#pone.0138803.s005" target="_blank">S2 File</a></b>.</p

Monoconjugation of Human Amylin with Methylpolyethyleneglycol

<div><p>Amylin is a pancreatic hormone cosecreted with insulin that exerts unique roles in metabolism and glucose homeostasis. The therapeutic restoration of postprandial and basal amylin levels is highly desirable in diabetes mellitus. Protein conjugation with the biocompatible polymer polyethylene glycol (PEG) has been shown to extend the biological effects of biopharmaceuticals. We have designed a PEGylated human amylin by using the aminoreactive compound methoxylpolyethylene glycol succinimidyl carbonate (mPEGsc). The synthesis in organic solvent resulted in high yields of monoPEGylated human amylin, which showed large stability against aggregation, an 8 times increase in half-life <i>in vivo</i> compared to the non-conjugated amylin, and pharmacological activity as shown by modulation of cAMP production in MCF–7 cell line, decrease in glucagon and modulation of glycemia following subcutaneous administration in mice. Altogether these data reveal the potential use of PEGylated human amylin for the restoration of fasting amylin levels.</p></div

Amylin-stimulated generation of cAMP in MCF–7 cells.

<p>Non-conjugated or PEGylated amylin were assayed for activity in cell by means of stimulation of production of cAMP in MCF–7 cell line. Lines represent best adjust of experimental data with logistic function. Human amylin EC50 = 35.2 ± 7.5 nM, PEGylated human amylin EC50 = 30.8 ± 6.7 nM. The raw data can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138803#pone.0138803.s005" target="_blank">S2 File</a></b>.</p

Pharmacologic evaluation of PEGylated human amylin.

<p><b>a)</b> Pharmacokinetics. Non-conjugated and PEGylated human amylin were injected to two separated groups of swiss male mice by subcutaneous administration and the decay in plasma concentration was evaluated. Continuous lines are best fitting of single-exponential decay function to data. <b>b)</b> Modulation of glucagon. Swiss male mice were administered with 10 μg PEGylated human amylin and the serum glucagon was monitored over time. * P<0.05. <b>c)</b> Modulation of glycemia. Swiss male mice (8 weeks old; fasting for 6 h before intervention and throughout the experiment) received by subcutaneous injection regular insulin (0.3 IU/kg body weight) alone (closed circles; n = 5) or in combination with free murine amylin (open circles; 400 μg/kg body weight; n = 5) or PEGylated human amylin (closed inverted triangles; 400 μg/kg body weight, expressed as peptide fraction; n = 5) and glycemia was monitored in the tail tip of free, conscientious mice. The activity of PEGylated human amylin showed significant difference from control (Insulin-R): * P<0.05, **P<0.01. The raw data can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138803#pone.0138803.s005" target="_blank">S2 File</a></b>.</p

Physical stability of the free and the PEGylated human amylin.

<p>Human amylin was subjected to aggregation at 25°C and monitored for fibril formation by ThT fluorescence <b>(a)</b> for up to 12h at varying pH (closed symbols, free non-conjugated human amylin; open symbols, PEGylated human amylin) and <b>(b)</b> for up to 7 days in PBS pH 7.4, and the products of these aggregation kinetic isotherms were further evaluated by transmission electron microscopy (TEM) as follow: <b>(c)</b> free human amylin; <b>(d)</b> PEGylated human amylin; <b>(e)</b> free PEG. Scale bar = 200 nm. The raw data can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138803#pone.0138803.s005" target="_blank">S2 File</a></b>.</p

Purification and characterization of monoPEGylated human amylin.

<p><b>a)</b> MonoPEGylated human amylin was synthesized in organic solvent by conjugation human amylin (5 mg/mL) with mPEGsc5k (5:1 molar excess) and purified by C18-RP-HPLC with a 30–70% CH₃CN gradient (in the presence of 0.1% TFA) up to 20 min. <b>b)</b> MALDI-ToF-MS of the fractions from the purification step showing the mono PEGylated human amylin corresponding to the peak comprising the elution time 11 min–13 min. The panel is rotated in order to align the PAGE-like display of the mass spectra with the respective original fractions in the chromatogram (upper panel, A). <b>c)</b> Trypsin-digestion of PEGylated human amylin. Purified monoPEGylated human amylin was subjected to trypsin digestion and submitted to MALDI-ToF-MS for identification of the products. The 2748 m/z ion coincides with the expected monoisotopic mass for the sodium adduct of the non-modified (non-PEGylated) human amylin 12–37 fragment (amylin 12–37, amide at C-terminus; monoisotopic mass 2,725.3 Da)</p

Antiviral, Cytoprotective, and Anti-Inflammatory Effect of <i>Ampelozizyphus amazonicus</i> Ducke Ethanolic Wood Extract on Chikungunya Virus Infection

Chikungunya fever, a debilitating disease caused by Chikungunya virus (CHIKV), is characterized by a high fever of sudden onset and an intense arthralgia that impairs individual regular activities. Although most symptoms are self-limited, long-term persistent arthralgia is observed in 30–40% of infected individuals. Currently, there is no vaccine or specific treatment against CHIKV infection, so there is an urgent need for the discovery of new therapeutic options for CHIKF chronic cases. This present study aims to test the antiviral, cytoprotective, and anti-inflammatory activities of an ethanol extract (FF72) from Ampelozizyphus amazonicus Ducke wood, chemically characterized using mass spectrometry, which indicated the major presence of dammarane-type triterpenoid saponins. The major saponin in the extract, with a deprotonated molecule ion m/z 897 [M-H]−, was tentatively assigned as a jujubogenin triglycoside, a dammarane-type triterpenoid saponin. Treatment with FF72 resulted in a significant reduction in both virus replication and the production of infective virions in BHK-21-infected cells. The viability of infected cells was assessed using an MTT, and the result indicated that FF72 treatment was able to revert the toxicity mediated by CHIKV infection. In addition, FF72 had a direct effect on CHIKV, since the infectivity was completely abolished in the presence of the extract. FF72 treatment also reduced the expression of the major pro-inflammatory mediators overexpressed during CHIKV infection, such as IL-1β, IL-6, IL-8, and MCP-1. Overall, the present study elucidates the potential of FF72 to become a promising candidate of herbal medicine for alphaviruses infections