Alkaloids of Abuta panurensis Eichler: In silico and in vitro study of acetylcholinesterase inhibition, cytotoxic and immunomodulatory activities.

Natural products obtained from species of the genus Abuta (Menispermaceae) are known as ethnobotanicals that are attracting increasing attention due to a wide range of their pharmacological properties. In this study, the alkaloids stepharine and 5-N-methylmaytenine were first isolated from branches of Abuta panurensis Eichler, an endemic species from the Amazonian rainforest. Structure of the compounds was elucidated by a combination of 1D and 2D NMR spectroscopic and MS and HRMS spectrometric techniques. Interaction of the above-mentioned alkaloids with acetylcholinesterase enzyme and interleukins IL-6 and IL-8 was investigated in silico by molecular docking. The molecules under investigation were able to bind effectively with the active sites of the AChE enzyme, IL-6, and IL-8 showing affinity towards the proteins. Along with the theoretical study, acetylcholinesterase enzyme inhibition, cytotoxic, and immunomodulatory activity of the compounds were assessed by in vitro assays. The data obtained in silico corroborate the results of AChE enzyme inhibition, the IC50 values of 61.24µM for stepharine and 19.55µM for 5-N-methylmaytenine were found. The compounds showed cytotoxic activity against two tumor cell lines (K562 and U937) with IC50 values ranging from 11.77 µM to 28.48 µM. The in vitro assays revealed that both alkaloids were non-toxic to Vero and human PBMC cells. As for the immunomodulatory activity, both compounds inhibited the production of IL-6 at similar levels. Stepharine inhibited considerably the production of IL-8 in comparison to 5-N-methylmaytenine, which showed a dose dependent action (inhibitory at the IC50 dose, and stimulatory at the twofold IC50 one). Such a behavior may possibly be explained by different binding modes of the alkaloids to the interleukin structural fragments. Occurrence of the polyamine alkaloid 5-N-methylmaytenine was reported for the first time for the Menispermaceae family, as well as the presence of stepharine in A. panurensis.

Function of hesperidin alleviating inflammation and oxidative stress responses in COPD mice might be related to SIRT1/PGC-1α/NF-κB signaling axis.

Purpose: Hesperidin has anti-inflammatory and anti-oxidant stress effects, but its functions in chronic obstructive pulmonary disease (COPD) remains unknown. This study analyzed the role of hesperidin in COPD mice, aiming to provide a basis for the hesperidin application.Materials and methods: Mice were injected with cigarette smoke extract (CSE) to construct COPD models and then treated with budesonide or hesperidin. Hematoxylin-eosin (HE) and TUNEL assays were used to observe the pathological changes and cell death of lung tissue. The levels of interleukin (IL)-6, IL-8, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) in bronchoalveolar lavage fluid (BLAF), as well as myeloperoxidase (MPO) content in lung tissues were confirmed. The expression levels of SIRT1, PGC-1α, and p65 proteins were measured by western blotting (WB) analysis.Results: CSE induced inflammatory cell infiltration and cell death in the lung tissues of mice, whereas budesonide and hesperidin effectively alleviated these pathological changes. The levels of IL-6, IL-8, and MDA in BLAF and pulmonary MPO content in the COPD mice were effectively increased, while the levels of SOD and CAT in BLAF were decreased, which could be reversed by budesonide and hesperidin. Moreover, the addition of budesonide or hesperidin reliably accelerated the expression levels of PGC-1α and SIRT1 but suppressed the phosphorylation of p65 in COPD mice. In general, high-dose hesperidin had a stronger regulatory effect on COPD mice.Conclusions: Hesperidin alleviated inflammation and oxidative stress responses in CES-induced COPD mice, associated with SIRT1/PGC-1α/NF-κB signaling axis, which might become a new direction for COPD treatment.

Designing CXCL8-based decoy proteins with strong anti-inflammatory activity in vivo.

IL (interleukin)-8 [CXCL8 (CXC chemokine ligand 8)] exerts its role in inflammation by triggering neutrophils via its specific GPCRs (G-protein-coupled receptors), CXCR1 (CXC chemokine receptor 1) and CXCR2, for which additional binding to endothelial HS-GAGs (heparan sulphate-glycosaminoglycans) is required. We present here a novel approach for blocking the CXCL8-related inflammatory cascade by generating dominant-negative CXCL8 mutants with improved GAG-binding affinity and knocked-out CXCR1/CXCR2 activity. These non-signalling CXCL8 decoy proteins are able to displace WT (wild-type) CXCL8 and to prevent CXCR1/CXCR2 signalling thereby interfering with the inflammatory response. We have designed 14 CXCL8 mutants that we subdivided into three classes according to number and site of mutations. The decoys were characterized by IFTs (isothermal fluorescence titrations) and SPR (surface plasmon resonance) to determine GAG affinity. Protein stability and structural changes were evaluated by far-UV CD spectroscopy and knocked-out GPCR response was shown by Boyden chamber and Ca2+ release assays. From these experiments, CXCL8(Δ6F17KF21KE70KN71K) emerged with the most promising in vitro characteristics. This mutant was therefore further investigated in a murine model of mBSA (methylated BSA)-induced arthritis in mice where it showed strong anti-inflammatory activity. Based on these results, we propose that dominant-negative CXCL8 decoy proteins are a promising class of novel biopharmaceuticals with high therapeutic potential in inflammatory diseases.

Identification and expression profiles of IL-8 in bighead carp (Aristichthys nobilis) in response to microcystin-LR.

Microcystin-LR (MCLR) is a widespread cyanotoxin and has immunotoxicity to animals, including fish. Chemokines are considered to play important roles in inflammatory response induced by MCLR. In this study, we cloned the full-length cDNA of interleukin-8 (IL-8) from bighead carp (Aristichthys nobilis) for the first time. The full-length IL-8 cDNA was 552 bp and contained a 297-bp open-reading frame that encoded for a 98-amino acid protein. The deduced IL-8 protein had a typical aspartic acid (D)-leucine (L)-arginine (R) and a CXC motif at the N-terminal, which were conserved in most fish species. Phylogenetic analysis showed that bighead carp IL-8 protein was grouped in the teleost IL-8 lineage 2. Under normal conditions, the expression of IL-8 is constitutive and weak in all tested tissues. However, MCLR treatment could significantly increase the transcription of IL-8 in bighead carp in a temporal- and dose-dependent pattern. The present study will help us to understand more about the evolution of IL-8 and its function in the MCLR induced proinflammatory response in bighead carp.

Rapid homogeneous detection of biological assays using magnetic modulation biosensing system.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Identification of novel synthetic toll-like receptor 2 agonists by high throughput screening.

Toll-like receptors (TLRs) play a central role in host defense by inducing inflammatory and adaptive immune responses following infection. Drugs that target TLRs are of considerable interest as potential inflammatory regulators, vaccine adjuvants, and novel immunotherapeutics. TLR2, in cooperation with either TLR1 or TLR6, mediates responses to a wide variety of microbial products as well as products of host tissue damage. In an effort to understand the structural basis of TLR2 recognition and uncover novel TLR2 agonists, a synthetic chemical library of 24,000 compounds was screened using an IL-8-driven luciferase reporter in cells expressing these human receptors. The screening yielded several novel TLR2-dependent activators that utilize TLR1, TLR6, or both as co-receptors. These novel small molecule compounds are aromatic in nature and structurally unrelated to any known TLR2 agonists. The three most potent compounds do not exhibit synergistic activity, nor do they act as pseudoantagonists toward natural TLR2 activators. Interestingly, two of the compounds exhibit species specificity and are inactive toward murine peritoneal macrophages. Mutational analysis reveals that although the central extracellular region of TLR1 is required for stimulation, there are subtle differences in the mechanism of stimulation mediated by the synthetic compounds in comparison with natural lipoprotein agonists. The three most potent compounds activate cells in the nanomolar range and stimulate cytokine production from human peripheral blood monocytes. Our results confirm the utility of high throughput screens to uncover novel synthetic TLR2 agonists that may be of therapeutic benefit.