From root extract to antiarthritic facts: trip across a novel therapeutic approach to inflammation and pain

Osteoarthritis (OA) is a chronical-degenerative and inflammatory disease affecting joints and involving several cellular and molecular processes in different cell types, as chondrocytes, osteoblasts, synoviocytes and immune cells. In OA the cartilage, a specialized tissue that allows the sliding between the two joint heads, is degraded, and, in the joint, the bones have friction each other causing pain and their own remodeling. Synovial membrane cells secrete inflammatory mediators and a greater amount of synovial fluid, resulting in synovial membrane thickening and contributing to the joint damage. To date, there is no specific anti-OA therapy, and so painkillers, anti-inflammatory drugs and intra-articular injections of corticosteroids are administered. However, considering painkilling and anti-inflammatory drug contraindications, in addition to chondroprotectors that preserve cartilage from degeneration, nutraceuticals are often introduced in therapies for their efficacy and less side effects than traditional drugs. Among them, Harpagophytum procumbens, known as Devil’s claw, is one of the most used herbs as an anti-OA remedy. Its secondary roots contain several bioactive compounds able to relieve patient symptoms decreasing inflammation and joint pain. In order to understand its activity on pain and inflammatory pathways, in the present study, a H. procumbens root extract (HPE) was characterized for its phytochemical composition and effects on Fibroblast- like synoviocytes (FLSs) from OA patients. The dry HPE was solubilized in different solvents, deionized water (HPEH2O), DMSO (HPEDMSO), 100% v/v ethanol (HPEEtOH100), and 50% v/v ethanol (HPEEtOH50), chosen based on their biocompatibility and ability to dissolve different classes of phytochemical compounds. Preliminary phytochemical analyses have shown that the highest polyphenol levels were found in HPEDMSO and HPEEtOH50, whereas different volatile bioactive compounds, mainly β-caryophyllene and eugenol, were detected in all the extracts except for HPEH2O. Since these compounds have been described to mitigate pain through an agonism on endocannabinoid type 2 (CB2) receptors and considering the involvement of CB2 in OA pathogenesis demonstrated through our histochemical analyses, it was decided to analyse the HPE analgesic effect on OA FLSs. However, even if both HPEH2O and HPEDMSO were able to enhance CB2 receptor expression, only the latter has been used for subsequent experiments. Considering the CB2 receptor association with Gi protein in cell membrane, the HPEDMSO effectiveness to affect CB2 pathways was studied by analysing the cAMP modulation, the protein kinase A (PKA) and Extra-regulated kinase (ERK) activation and the correlated matrix metalloproteinase (MMP) production. In line with what expected, HPEDMSO was able to inhibit cAMP production and PKA activation, also showing a reduction in ERK1/2 phosphorylation that surprisingly disagreed with the extract effect on PKA. Interestingly, although it is not due to CB2 receptor stimulation, the effect on the MAPK pathway generates a decrease in some extracellular matrix degrading enzyme and pro-inflammatory interleukin expression, suggesting the HPE antiarthritic role. Moreover, to clarify the effects of bioactive constituents and the possible interactions occurring in the phytocomplex, harpagoside, the H. procumbens root extract biomarker, and the main volatile compounds detected at the phytochemical analysis, have been studied in comparison to the whole extract. It was observed that β-caryophyllene, α-humulene, eugenol and harpagoside alone were always less effective than the H. procumbens whole extract and the Mix that contained all the individual compounds. However, the surprising result is that the mixture was also always less effective than HPEDMSO whole extract. This evidence suggested the existence of synergistic interactions between each analysed compound and other molecules not identified in phytochemical analyses that contribute to the entire extract bioactivity. In our most recent analyses, HPEDMSO has demonstrated a role in the inhibition of the phosphoinositide specific-phospholipase C (PI-PLC) γ1 mRNA splicing process. Sanger sequencing analysis revealed some retained introns in HPEDMSO treated FLSs. However, studies describing the H. procumbens ability to interfere with the splicing process were not available in scientific literature. On the other hand, the accurate analysis of the PI-PLC γ1 gene, only carried out by the Human and Vertebrate Analysis and Annotation (HAVANA) group, identified the intron retention phenomenon in PI-PLC γ1 gene. Our results were perfectly in line with what described by the HAVANA group. However, during our analysis we highlighted a lack of HPEDMSO effect on intron retention phenomenon in some FLS samples from different OA patients. This inter-individual variability led us to consider more carefully some data obtained in our previous experiments. Surprisingly, it was shown that HPEDMSO was ineffective in inhibiting MMP gene expression in those samples where the intron retention phenomenon in PI-PLC γ1 mRNA was absent. This result suggested that the decrease of PI-PLC γ1 protein, due to lack of splicing, could be associated with the decrease of MMP production

The formulation of the N-Acetylglucosamine as nanoparticles increases its anti-inflammatory activities: an in vitro study

Nanomedicine can represent a new strategy to treat several types of diseases such as those with inflammatory aetiology. Through this strategy, it is possible to obtain nanoparticles with controlled shape, size, and eventually surface charge. Moreover, the use of molecules in nanoform may allow more effective delivery into the diseased cells and tissues, reducing toxicity and side effects of the used compounds. The aim of the present manuscript was the evaluation of the effects of N-acetylglucosamine in nanoform (GlcNAc NP) in an in vitro model of osteoarthritis (OA). Human primary chondrocytes were treated with Tumor Necrosis Factor (TNF)-α to simulate a low-grade inflammation and then treated with both GlcNAc and GlcNAc NP, in order to find the lowest concentrations able to counteract the inflammatory state of the cells and ensure a chondroprotective action. The findings showed that GlcNAc NP was able to decrease the pro-inflammatory mediators, IL-6 and IL-8, which are among the main effectors of inflammation; moreover, the nanoparticles downregulated the production of metalloprotease enzymes. GlcNAc NP was effective at a very low concentration compared to GlcNAc in its native form. Furthermore, GlcNAc NP stimulated an increase in collagen type II synthesis. In conclusion, the GlcNAc in nanoform showed better performance than GlcNAc, at concentrations lower than those reached in the joints after oral administration to patients of 1.5 g/die of glucosamine

Repositioned natural compounds and nanoformulations: a promising combination to counteract cell damage and inflammation in respiratory viral infections

Respiratory viral diseases are among the most important causes of disability, morbidity, and death worldwide. Due to the limited efficacy or side effects of many current therapies and the increase in antiviral-resistant viral strains, the need to find new compounds to counteract these infections is growing. Since the development of new drugs is a time-consuming and expensive process, numerous studies have focused on the reuse of commercially available compounds, such as natural molecules with therapeutic properties. This phenomenon is generally called drug repurposing or repositioning and represents a valid emerging strategy in the drug discovery field. Unfortunately, the use of natural compounds in therapy has some limitations, due to their poor kinetic performance and consequently reduced therapeutic effect. The advent of nanotechnology in biomedicine has allowed this limitation to be overcome, showing that natural compounds in nanoform may represent a promising strategy against respiratory viral infections. In this narrative review, the beneficial effects of some promising natural molecules, curcumin, resveratrol, quercetin, and vitamin C, which have been already studied both in native form and in nanoform, against respiratory viral infections are presented and discussed. The review focuses on the ability of these natural compounds, analyzed in in vitro and in vivo studies, to counteract inflammation and cellular damage induced by viral infection and provide scientific evidence of the benefits of nanoformulations in increasing the therapeutic potential of these molecules

Structural and dynamical modeling of WINGS clusters. I. The distribution of cluster galaxies of different morphological classes within regular and irregular clusters

[Abridged] We use the WINGS database to select a sample of 67 nearby galaxy clusters with at least 30 spectroscopic members each. 53 of these clusters do not show evidence of substructures in phase-space, while 14 do. We estimate the virial radii and circular velocities of the 67 clusters by a variety of proxies (velocity dispersion, X-ray temperature, and richness) and use these estimates to build stack samples from these 53 and 14 clusters ('Reg' and 'Irr' stacks, respectively). We determine the number-density and velocity-dispersion profiles (VDPs) of E, S0, and Sp+Irr (S) galaxies in the Reg and Irr samples, separately, and fit models to these profiles. The number density profiles of E, S0, and S galaxies are adequately described by either a NFW or a cored King model, both for the Reg and Irr samples, with a slight preference for the NFW model. The spatial distribution concentration increases from the S to the S0 and to the E populations, both in the Reg and the Irr stacks, reflecting the well-known morphology-radius relation. Reg clusters have a more concentrated spatial distribution of E and S0 galaxies than Irr clusters, while the spatial distributions of S galaxies in Reg and Irr clusters are similar. We propose a new phenomenological model that provides acceptable fits to the VDP of all our galaxy samples. The VDPs become steeper and with a higher normalization from E to S0 to S galaxies. The S0 VDP is close to that of E galaxies in Reg clusters, and intermediate between those of E and S galaxies in Irr clusters. Our results suggest that S galaxies are a recently accreted cluster population, that take less than 3 Gyr to evolve into S0 galaxies after accretion, and in doing so modify their phase-space distribution, approaching that of cluster ellipticals. While in Reg clusters this evolutionary process is mostly completed, it is still ongoing in Irr clusters.Comment: A&A, in press - 11 pages, 9 figures, 4 table

In vitro antiviral and anti-inflammatory activities of N-Acetylglucosamine: development of an alternative and safe approach to fight viral respiratory infections

Viral respiratory tract infections (RTIs) are responsible for significant morbidity and mortality worldwide. A prominent feature of severe respiratory infections, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is the cytokine release syndrome. Therefore, there is an urgent need to develop different approaches both against viral replication and against the consequent inflammation. N-acetylglucosamine (GlcNAc), a glucosamine (GlcN) derivative, has been developed as an immunomodulatory and anti-inflammatory inexpensive and non-toxic drug for non-communicable disease treatment and/or prevention. Recent studies have suggested that GlcN, due to its anti-inflammatory activity, could be potentially useful for the control of respiratory virus infections. Our present study aimed to evaluate in two different immortalized cell lines whether GlcNAc could inhibit or reduce both viral infectivity and the inflammatory response to viral infection. Two different viruses, frequent cause of upper and lower respiratory tract infections, were used: the H1N1 Influenza A virus (IAV) (as model of enveloped RNA virus) and the Human adenovirus type 2 (Adv) (as model of naked DNA virus). Two forms of GlcNAc have been considered, bulk GlcNAc and GlcNAc in nanoform to overcome the possible pharmacokinetic limitations of GlcNAc. Our study suggests that GlcNAc restricts IAV replication but not Adv infection, whereas nano-GlcNAc inhibits both viruses. Moreover, GlcNAc and mainly its nanoformulation were able to reduce the pro-inflammatory cytokine secretion stimulated by viral infection. The correlation between inflammatory and infection inhibition is discussed

A Systemic and Severe Infection via Cytomegalovirus and Other Herpesviruses in a Young Apparently Immunocompetent Patient: A Case Report

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Biochemical and computational studies of the interaction between a glucosamine derivative, NAPA, and the IKKα kinase

The glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose (NAPA), was shown to inhibit the kinase activity of IKKα, one of the two catalytic subunits of IKK complex, decreasing the inflammatory status in osteoarthritis chondrocytes. In the present work we have investigated the inhibition mechanism of IKKα by NAPA by combining computational simulations, in vitro assays and Mass Spectrometry (MS) technique. The kinase in vitro assay was conducted using a recombinant IKKα and IKKtide, a 20 amino acid peptide substrate derived from IkBα kinase protein and containing the serine residues Ser32 and Ser36. Phosphorylated peptide production was measured by Ultra Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS), and the atomic interaction between IKKα and NAPA has been studied by molecular docking and Molecular Dynamics (MD) approaches. Here we report that NAPA was able to inhibit the IKKα kinase activity with an IC50 of 0.5 mM, to decrease the Km value from 0.337 mM to 0.402 mM and the Vmax from 0.0257 mM·min-1 to 0.0076 mM·min-1. The computational analyses indicate the region between the KD, ULD and SDD domains of IKKα as the optimal binding site explored by NAPA. Biochemical data indicate that there is a non-significant difference between Km and Ki whereas there is a statistically significant difference between the two Vmax values. This evidence, combined with computational results, consistently indicates that the inhibition is non-competitive, and that the NAPA binding site is different than that of ATP or IKKtide

Dissecting the intracellular signalling and fate of a DNA nanosensor by super-resolution and quantitative microscopy

DNA nanodevices have been developed as platforms for the manipulation of gene expression, delivery of molecular payloads, and detection of various molecular targets within cells and in other complex biological settings. Despite efforts to translate DNA nanodevices from the test tube (in vitro) to living cells, their intracellular trafficking and functionality remain poorly understood. Herein, quantitative and super-resolution microscopy approaches were employed to track and visualise, with nanometric resolution, the molecular interactions between a synthetic DNA nanosensor and transcription factors in intracellular compartments. Specifically, fluorescence resonance energy transfer microscopy, fluorescence correlation spectroscopy, fluorescence lifetime imaging microscopy and multicolour single-molecule localisation microscopy were employed to probe the specific binding of the DNA nanosensor to the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B). We monitored the mobility, subcellular localisation and degradation of the DNA nanosensor inside living prostate cancer PC3 cells. Super-resolution imaging enabled the direct visualisation of the molecular interactions between the synthetic DNA nanosensors and the NF-kappa B molecules in cells. This study represents a significant advance in the effective detection as well as understanding of the intracellular dynamics of DNA nanosensors in a complex biological milieu

Platelet rich fibrin (PRF) and its related products: biomolecular characterization of the liquid fibrinogen

Liquid fibrinogen is an injectable platelet concentrate rich in platelets, leukocytes, and fibrinogen obtained by blood centrifugation. The aim of this study was to analyze the release of different growth factors in the liquid fibrinogen at different times and to assess possible correlations between growth factors and cell counts. The concentration of transforming growth factor beta 1 (TGF-β1), platelet-derived growth factor-AB (PDGF-AB), platelet-derived growth factor-BB (PDGF-BB), bone morphogenetic protein 2 (BMP-2), fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor (VEGF) released by liquid fibrinogen were examined with ELISA at three time points (T0, time of collection; T7, 7 days; T14, 14 days). The cellular content of the liquid fibrinogen and whole blood was also calculated for each volunteer. A mean accumulation of platelets of almost 1.5-fold in liquid fibrinogen compared to whole blood samples was found. An increase of TGF-β1, PDGF-AB, FGF-2, and VEGF levels was detected at T7. At T14, the level of TGF-β1 returned to T0 level; PDGF-AB amount remained high; the levels of FGF-2 and VEGF decreased with respect to T7, but remained higher than the T0 levels; PDGF-BB was high at all time points; BMP-2 level was low and remained constant at all time points. TGF-β1, PDGF-AB, and PDGF-BB showed a correlation with platelet amount, whereas BMP-2, FGF-2, and VEGF showed a mild correlation with platelet amount. Due to the high concentration of platelets, liquid fibrinogen does contain important growth factors for the regeneration of both soft and hard tissue. The centrifugation protocol tested in this study provides a valid solution to stimulate wound healing in oral and periodontal surgery

Short-term multidisciplinary non-pharmacological intervention is effective in reducing liver fat content assessed non-invasively in patients with nonalcoholic fatty liver disease (NAFLD)

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