Metformin hydrochloride and wound healing: from nanoformulation to pharmacological evaluation

Abstract

Niosomes as drug delivery systems have the ability to decrease drugs' side effects and increase their therapeutic effectiveness. Metformin HCl is an oral antihyperglycemic agent belonging to biguanides. It is the most commonly chosen drug as a startup therapy for patients newly diagnosed with type 2 diabetes. This study aims to encapsulate metformin HCl inside niosomes to be used as a transdermal formulation helping to prolong its antidiabetic effect and investigate its ability to enhance wound healing in diabetic patients. Thin film hydration method was used to prepare metformin HCl niosomes using different proportions of Span 60, Span 40, Tween 80, and cholesterol. All formulations were characterized using transmission electron microscope, zeta potential, and vesicle size. In vitro release studies, stability studies and in vivo evaluation were conducted on selected niosomal formulations. The results of entrapment efficiency ranged from 13% to 32%. Vesicle sizes were determined in nano-range. The in vitro release profile of metformin HCl from niosomes occurred in two consecutive phases. Biological evaluation on diabetic rats revealed that metformin HCl niosomal gel given every 2 days showed a better sustained antidiabetic effect than oral doses given daily. It also showed an improvement in wound healing for diabetic rats given metformin formulations compared to nontreated ones.     

Mitogen-activated protein kinase phosphorylates and targets inducible cAMP early repressor to ubiquitin-mediated destruction

Inducible cAMP early repressor (ICER) is an important mediator of cAMP antiproliferative activity that acts as a putative tumor suppressor gene product. In this study, we examined the regulation of ICER protein by phosphorylation and ubiquitination in human choriocarcinoma JEG-3 and mouse pituitary AtT20 cells. We found that cAMP stabilized ICER protein by inhibiting the mitogen-activated protein kinase (MAPK) cascade. Activation of the MAPK pathway increased ICER phosphorylation. ICER phosphorylation was abrogated by inhibition of the MAPK pathway either by cAMP or directly by the MAPK inhibitor PD098059. The MAPKs extracellular signal-regulated kinases 1 and 2 physically interact with ICER and mediated the phosphorylation of ICER on a critical serine residue (Ser-41). A mutant form of ICER in which Ser-41 was substituted by alanine had a half-life 4-5 h longer than its wild-type counterpart. This alteration in stability was due to the inability of the Ser-41-mutant ICER to be efficiently ubiquitinated and degraded via the ubiquitin-proteasome pathway. These results present a novel cell signaling cross-talk mechanism at the cell nucleus between the MAPK and cAMP pathways, whereby MAPK targets a repressor of the cAMP-dependent gene expression for ubiquitination and proteasomal degradation.     

Changes in glycosylation of human bile-salt-stimulated lipase during lactation

Bile-salt-stimulated lipase (BSSL) is an enzyme in human milk, which is important for the fat digestion in the newborn infant. BSSL is highly glycosylated and includes one site for N-glycosylation and several sites for O-glycosylation. BSSL has previously been found to express Lewis a, Lewis b, and Lewis x carbohydrate antigens. In this study, glycosylation of BSSL was studied at different times during lactation. BSSL was purified from milk collected individually from four donors at several different times during the first 6 months of lactation. The BSSL glycans were characterized through monosaccharide analysis, high-pH anion-exchange chromatography, matrix-assisted laser desorption-ionization mass spectrometry, and ELISA. Both total carbohydrate content and relative amount of sialic acid were higher in BSSL from the first lactation month as compared to BSSL from milk collected later in lactation. BSSL from the first lactation month also showed a different composition of sialylated O-linked glycans and the N-linked oligosaccharides consisted of lower amounts of fucosylated structures compared to later in lactation. We also found a gradual increase in the expression of the carbohydrate epitope Lewis x on BSSL throughout the lactation period. This study shows that glycosylation of BSSL is dependent on blood group phenotype of the donor and changes substantially during the lactation period.     

Mitogenic action of LPA in prostate

The lipid growth factor lysophosphatidic acid (LPA) elicits multiple cellular responses, including cell growth and survival. LPA acts upon target cells by activating its cognate receptors, which belong to the G protein-coupled endothelial differentiation gene (EDG) family. To date, three known LPA receptors, termed LPA1, LPA2 and LPA3, have been molecularly characterized and cloned. Here, we review recent data describing the molecular steps involved in the LPA receptor-mediated activation of mitogenic extracellular signal-regulated kinase (ERK) pathway in prostate cancer. Induction of ERK by LPA proceeds via Gbetagamma-dependent activation of tyrosine kinases, including the epidermal growth factor (EGF) receptor and c-Src. Further, LPA-induced ERK activation involves matrix metalloproteinases (MMPs), which cause the release of active EGFR ligands. Finally, we present data demonstrating a correlation between the mitogenic effects of LPA and expression of the lp(A1) gene in the prostate cancer cells.     

The stress response neuropeptide CRF increases amyloid‐β production by regulating γ‐secretase activity

The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid‐β (Aβ) production. In cells, CRF treatment increases Aβ production and triggers CRF receptor 1 (CRFR1) and γ‐secretase internalization. Co‐immunoprecipitation studies establish that γ‐secretase associates with CRFR1; this is mediated by β‐arrestin binding motifs. Additionally, CRFR1 and γ‐secretase co‐localize in lipid raft fractions, with increased γ‐secretase accumulation upon CRF treatment. CRF treatment also increases γ‐secretase activity in vitro, revealing a second, receptor‐independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ‐secretase activity. Unexpectedly, CRFR1 antagonists also increased Aβ. These data collectively link CRF to increased Aβ through γ‐secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aβ and in some cases preferentially increase Aβ42 via complex effects on γ‐secretase.     

Biosynthesis and characterization of silver nanoparticles produced by Pleurotus ostreatus and their anticandidal and anticancer activities

The biosynthesis of nanoparticles has received increasing interest because of the growing need to develop safe, cost-effective and environmentally friendly technologies for the synthesis of nano-materials. In this study, silver nanoparticles (AgNPs) were synthesized using a reduction of aqueous Ag⁺ ions with culture supernatant from Pleurotus ostreatus. The bioreduction of AgNPs was monitored by ultra violet-visible spectroscopy and the obtained AgNPs were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy techniques. TEM studies showed the size of the AgNPs to be in the range of 4–15 nm. The formation of AgNPs might be an enzyme-mediated extracellular reaction process. Furthermore, the antifungal effect of AgNPs against Candida albicans as compared with commercially antifungal drugs was examined. The effect of AgNPs on dimorphic transition of C. albicans was tested. The anticancer properties of AgNPs against cells (MCF-7) were also evaluated. AgNPs caused a significant decrease in cell viability of an MCF-7 cell line (breast carcinoma). Exposure of MCF-7 cells with AgNPs resulted in a dose-dependent increase in cell growth inhibition varying from 5 to 78 % at concentrations in the range of 10–640 μg ml^?1. The present study demonstrated that AgNPs have potent antifungal, antidimorphic, and anticancer activities. The current research opens a new avenue for the green synthesis of nano-materials.     

Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC)

Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer''s disease, and autism spectrum disorder—neurological disorders that exhibit elevated mTORC1 activity. Through a protein–protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson''s disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes with the postsynaptic marker postsynaptic density-95. Our work offers a comprehensive view of mTORC1 and its role in regulating regional protein expression in normal and diseased states.The mechanistic/mammalian target of rapamycin complex 1 (mTORC1)¹ is a serine/threonine protein kinase that is highly expressed in many cell types (1). In the brain, mTORC1 tightly coordinates different synaptic plasticities — long-term potentiation (LTP) and long-term depression (LTD) — the molecular correlates of learning and memory (2–5). Because mTORC1 is at the core of many synaptic signaling pathways downstream of glutamate and neurotrophin receptors, many hypothesize that dysregulated mTORC1 signaling underlies cognitive deficits observed in several neurodegenerative diseases (3, 6–17). For example, mTORC1 and its downstream targets are hyperactive in human brains diagnosed with Alzheimer''s disease (AD) (18–20). Additionally in animal models of autism spectrum disorder (ASD), altered mTORC1 signaling contributes to the observed synaptic dysfunction and aberrant network connectivity (13, 15, 21–27). Furthermore, epilepsy, which is common in AD and ASD, has enhanced mTORC1 activity (28–32).Phosphorylation of mTORC1, considered the active form, is generally regarded to promote protein synthesis (33). Thus, many theorize that diseases with overactive mTORC1 arise from excessive protein synthesis (14). Emerging data, however, show that suppressing mTORC1 activation can trigger local translation in neurons (34, 35). Pharmacological antagonism of N-methyl-d-aspartate (NMDA) receptors, a subtype of glutamate receptors that lies upstream of mTOR activation, promotes the synthesis of the voltage-gated potassium channel, Kv1.1, in dendrites (34, 35). Consistent with these results, in models of temporal lobe epilepsy there is a reduction in the expression of voltage-gated ion channels including Kv1.1 (30, 31, 36). Interestingly in a model of focal neocortical epilepsy, overexpression of Kv1.1 blocked seizure activity (37). Because both active and inactive mTORC1 permit protein synthesis, we sought to determine the proteins whose expression is altered when mTORC1 phosphorylation is reduced in vivo.Rapamycin is an FDA-approved, immunosuppressive drug that inhibits mTORC1 activity (38). We capitalized on the ability of rapamycin to reduce mTORC1 activity in vivo and the unbiased approach of mass spectrometry to identify changes in protein expression. Herein, we provide evidence that mTORC1 activation bidirectionally regulates protein expression, especially in the PSD where roughly an equal distribution of proteins dynamically appear and disappear. Remarkably, using protein–protein interaction networks facilitated the novel discovery that PARK7, a protein thus far only implicated in Parkinson''s disease, (1) is up-regulated by increased mTORC1 activity, (2) resides in the PSD only when mTORC1 is active, and (3) is aberrantly expressed in a rodent model of TSC, an mTORC1-related disease that has symptoms of epilepsy and autism. Collectively, these data provide the first comprehensive list of proteins whose abundance or subcellular distributions are altered with acute changes in mTORC1 activity in vivo.     

In vitro antifungal efficacy of <Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 9642 chitosan-AgNPs composite against post-harvest disease of citrus fruits

Green and blue mold postharvest diseases are the most vital negative components influencing the local market of citrus fruits. Citrus fruits were collected, and fungi were isolated. Among the fungal isolates identified, Penicillium digitatum and Penicillium italicum recorded the highest occurrence of 39.5 and 25.6%, respectively. In this work, we extracted chitosan from Aspergillum niger ATCC 9642. Fourier transform infrared spectroscopy was utilized to confirm the functional groups of the obtained compound, which exhibited the main characteristic bands of O–H stretching at 3302 cm^-1, and C–O–C band at 1125 cm^-1. A. niger ATCC 9642 chitosan had the degree of deacetylation of 88.5%, a molecular weight of 1.8 × 10⁵ Da, and viscosity of 7.3 centipoises; these values were comparable to those for standard shrimp chitosan. Ultraviolet-visible light spectra revealed the presence of A. niger ATCC 9642 chitosan-AgNPs composite. Using antifungal and spore germination assays, it was found that this composite exhibited effective antifungal action against P. digitatum and P. italicum compared with a chitosan standard. In a comet assay, the percentage of tail DNA was considered as a parameter that indicated DNA damage. The comet parameter increased significantly (P < 0.05) with A. niger ATCC 9642 chitosan–AgNPs composite, and the increase was dose-dependent. The increase in the DNA damage positively correlated with the inhibition performance of the A. niger ATCC 9642 chitosan–AgNPs composite.     

Matrix-assisted laser desorption/ionization mass spectrometry of neutral and acidic oligosaccharides with collision-induced dissociation

Using ribonuclease B and human alpha 1-acid glycoprotein (AGP) as model glycoproteins, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry with collision-induced dissociation (CID) is validated here as an effective tool for oligosaccharide sequencing. The spectra acquired for high-mannose and complex oligosaccharide structures show characteristic fragments resulting from cleavages of the glycosidic bonds and a few cross-ring cleavages. Esterification of the sialic acid residues is essential in stabilizing the acidic N-linked oligosaccharides. An important analytical feature observed in all acquired spectra is the occurrence of cleavages on the same antenna up to the branching point, as deduced from the absence of fragmentation due to the simultaneous cleavages on two or more antennas.