Evidence for the involvement of descending pain-inhibitory mechanisms in the attenuation of cancer pain by carvacrol aided through a docking study

Aims

The present study evaluated the carvacrol (CARV) effect on hyperalgesia and nociception induced by sarcoma 180 (S180) in mice.

Main methods

Carvacrol treatment (12.5–50 mg/kg s.c.) once daily for 15 days was started 24 h after injection of the sarcoma cells in the hind paw (s.c.). Mice were evaluated for mechanical sensitivity (von Frey), spontaneous and palpation-induced nociception, limb use and tumor growth on alternate days. CARV effects on the central nervous system were evaluated through immunofluorescence for Fos protein. Molecular docking studies also were performed to evaluate intermolecular interactions of the carvacrol and muscimol, as ligands of interleukin-10 and GABA_A receptors.

Key findings

CARV was able to significantly reduce mechanical hyperalgesia and spontaneous and palpation-induced nociception, improve use paw, decrease the number of positively marked neurons in lumbar spinal cord and activate periaqueductal gray, nucleus raphe magnus and locus coeruleus. CARV also caused significant decreased tumor growth. Docking studies showed favorable interaction overlay of the CARV with IL-10 and GABA_A.

Significance

Together, these results demonstrated that CARV may be an interesting option for the development of new analgesic drugs for the management of cancer pain.     

Aloperine attenuated neuropathic pain induced by chronic constriction injury via anti-oxidation activity and suppression of the nuclear factor kappa B pathway

Objective

To investigate whether aloperine (ALO) has antinociceptive effects on neuropathic pain induced by chronic constriction injury, whether ALO reduces ROS against neuropathic pain, and what are the mechanisms involved in ALO attenuated neuropathic pain.

Methods

Mechanical and cold allodynia, thermal and mechanical hyperalgesia and spinal thermal hyperalgesia were estimated by behavior methods such as Von Frey filaments, cold-plate, radiant heat, paw pressure and tail immersion on one day before surgery and days 7, 8, 10, 12 and 14 after surgery, respectively. In addition, T-AOC, GSH-PX, T-AOC and MDA in the spinal cord (L4/5) were measured to evaluate anti-oxidation activity of ALO on neuropathic pain. Expressions of NF-κB and pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) in the spinal cord (L4/5) were analyzed by using Western blot.

Results

Administration of ALO (80 mg/kg and 40 mg/kg, i.p.) significantly increased paw withdrawal threshold, paw pressure, paw withdrawal latencies, tail-curling latencies, T-AOC, GSH-PX and T-SOD concentration, reduced the numbers of paw lifts and MDA concentration compared to CCI group. ALO attenuated CCI induced up-regulation of expressions of NF-κB, TNF-α, IL-6, IL-1β at the dose of 80 mg/kg (i.p.). Pregabalin produced similar effects serving as positive control at the dose of 10 mg/kg (i.p.).

Conclusion

ALO has antinociceptive effects on neuropathic pain induced by CCI. The antinociceptive effects of ALO against neuropathic pain is related to reduction of ROS, via suppression of NF-κB pathway.     

Particle-Induced Pulmonary Acute Phase Response Correlates with Neutrophil Influx Linking Inhaled Particles and Cardiovascular Risk

Background

Particulate air pollution is associated with cardiovascular disease. Acute phase response is causally linked to cardiovascular disease. Here, we propose that particle-induced pulmonary acute phase response provides an underlying mechanism for particle-induced cardiovascular risk.

Methods

We analysed the mRNA expression of Serum Amyloid A (Saa3) in lung tissue from female C57BL/6J mice exposed to different particles including nanomaterials (carbon black and titanium dioxide nanoparticles, multi- and single walled carbon nanotubes), diesel exhaust particles and airborne dust collected at a biofuel plant. Mice were exposed to single or multiple doses of particles by inhalation or intratracheal instillation and pulmonary mRNA expression of Saa3 was determined at different time points of up to 4 weeks after exposure. Also hepatic mRNA expression of Saa3, SAA3 protein levels in broncheoalveolar lavage fluid and in plasma and high density lipoprotein levels in plasma were determined in mice exposed to multiwalled carbon nanotubes.

Results

Pulmonary exposure to particles strongly increased Saa3 mRNA levels in lung tissue and elevated SAA3 protein levels in broncheoalveolar lavage fluid and plasma, whereas hepatic Saa3 levels were much less affected. Pulmonary Saa3 expression correlated with the number of neutrophils in BAL across different dosing regimens, doses and time points.

Conclusions

Pulmonary acute phase response may constitute a direct link between particle inhalation and risk of cardiovascular disease. We propose that the particle-induced pulmonary acute phase response may predict risk for cardiovascular disease.     

Inhibition of Sirtuin 3 prevents titanium particle-induced bone resorption and osteoclastsogenesis via suppressing ERK and JNK signaling

Implant-derived wear particles can be phagocytosed by local macrophages, triggering an inflammatory cascade that can drive the activation and recruitment of osteoclasts, thereby inducing peri-prosthetic osteolysis. Efforts to suppress pro-inflammatory cytokine release and osteoclastsogenesis thus represent primary approaches to treating and preventing such osteolysis. Sirtuin 3 (SIRT3) is a NAD⁺-dependent deacetylases that control diverse metabolic processes. However, whether SIRT3 could mitigate wear debris-induced osteolysis has not been reported. Herein we explored the impact of the SIRT3 on titanium particle-induced osteolysis. Tartrate resistant acid phosphatase (TRAP) staining revealed that the inhibition of SIRT3 suppressed nuclear factor-κB ligand (RANKL)-mediated osteoclasts activation in a dose-dependent fashion. Notably, inhibition of SIRT3 also suppressed matrix metallopeptidase 9 (MMP9) and nuclear factor of activated T‐cell cytoplasmic 1 (NFATc1) expression at the mRNA and protein levels, while also inhibiting the mRNA expression of dendritic cell-specific transmembrane protein (DC-STAMP), ATPase H⁺ Transporting V0 Subunit D2 (Atp6v0d2), TRAP and Cathepsin K (CTSK) . In addition, inhibition of SIRT3 suppressed titanium particle-induced tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) expression and prevented titanium particle-induced osteolysis and bone loss in vivo. This inhibition of osteoclasts differentiation was found to be linked to the downregulation and reduced phosphorylation of JNK and ERK. Taken together, inhibition of SIRT3 may be a potential target for titanium particle-induced bone loss.     

Investigation of the role of endosomal Toll-like receptors in murine collagen-induced arthritis reveals a potential role for TLR7 in disease maintenance

Introduction

Endosomal toll-like receptors (TLRs) have recently emerged as potential contributors to the inflammation observed in human and rodent models of rheumatoid arthritis (RA). This study aims to evaluate the role of endosomal TLRs and in particular TLR7 in the murine collagen induced arthritis (CIA) model.

Methods

CIA was induced by injection of collagen in complete Freund''s adjuvant. To investigate the effect of endosomal TLRs in the CIA model, mianserin was administered daily from the day of disease onset. The specific role of TLR7 was examined by inducing CIA in TLR7-deficient mice. Disease progression was assessed by measuring clinical score, paw swelling, serum anti-collagen antibodies histological parameters, cytokine production and the percentage of T regulatory (T_reg) cells.

Results

Therapeutic administration of mianserin to arthritic animals demonstrated a highly protective effect on paw swelling and joint destruction. TLR7^-/- mice developed a mild arthritis, where the clinical score and paw swelling were significantly compromised in comparison to the control group. The amelioration of arthritis by mianserin and TLR7 deficiency both corresponded with a reduction in IL-17 responses, histological and clinical scores, and paw swelling.

Conclusions

These data highlight the potential role for endosomal TLRs in the maintenance of inflammation in RA and support the concept of a role for TLR7 in experimental arthritis models. This study also illustrates the potential benefit that may be afforded by therapeutically inhibiting the endosomal TLRs in RA.     

Zonisamide: Antihyperalgesic efficacy,the role of serotonergic receptors on efficacy in a rat model for painful diabetic neuropathy

Aims

The purpose of this study was to compare the changes of antihyperalgesic effectiveness of zonisamide (25 and 50 mg/kg), an antiepileptic drug, on the early and late phases of neuropathy and to investigate the role of serotonergic descending inhibitory pain pathways in antihyperalgesic effectiveness of zonisamide in the streptozotocin-induced rat model for painful diabetic neuropathy.

Main methods

The hot-plate and tail-immersion, to determine thermal thresholds, and paw pressure withdrawal tests, to determine mechanical thresholds, were performed as hyperalgesia tests. To investigate the role of serotonergic pathway, 1 mg/kg ketanserin (5-HT_2A/2C antagonist) and ondansetron (serotonin 5-HT₃ receptor antagonist) were used.

Key findings

Zonisamide enhanced pain thresholds significantly in the 3rd, 6th and 8th weeks as the reference drugs morphine (5 mg/kg) and carbamazepine (32 mg/kg, tested only in the 3rd week). There were no observed differences on the potency of antihyperalgesic effect between weeks and between doses. Each antagonist reversed the effect of zonisamide in the hot-plate and tail-immersion tests significantly, but, relatively in the paw pressure withdrawal tests.

Significance

These results support the role for zonisamide in the management of diabetic neuropathic pain in all phases. Serotonin 5-HT_2A/2C and 5-HT₃ receptors are involved in the antihyperalgesic effect of zonisamide by enhancement of thermal threshold, and partially by mechanical threshold, so they may not mediate mechanical hyperalgesia in diabetic neuropathy.     

Carvacryl acetate,a derivative of carvacrol,reduces nociceptive and inflammatory response in mice

Aims

The present study aimed to investigate the potential anti-inflammatory and anti-nociceptive effects of carvacryl acetate, a derivative of carvacrol, in mice.

Main methods

The anti-inflammatory activity was evaluated using various phlogistic agents that induce paw edema, peritonitis model, myeloperoxidase (MPO) activity, pro and anti-inflammatory cytokine levels. Evaluation of antinociceptive activity was conducted through acetic acid-induced writhing, hot plate test, formalin test, capsaicin and glutamate tests, as well as evaluation of motor performance on rotarod test.

Key findings

Pretreatment of mice with carvacryl acetate (75 mg/kg) significantly reduced carrageenan-induced paw edema (P < 0.05) when compared to vehicle-treated group. Likewise, carvacryl acetate (75 mg/kg) strongly inhibited edema induced by histamine, serotonin, prostaglandin E₂ and compound 48/80. In the peritonitis model, carvacryl acetate significantly decreased total and differential leukocyte counts, and reduced levels of myeloperoxidase and interleukin-1 beta (IL-1β) in the peritoneal exudate. The levels of IL-10, an anti-inflammatory cytokine, were enhanced by carvacryl acetate. Pretreatment with carvacryl acetate also decreased the number of acetic acid-induced writhing, increased the latency time of the animals on the hot plate and decreased paw licking time in the formalin, capsaicin and glutamate tests. The pretreatment with naloxone did not reverse the carvacryl acetate-mediated nociceptive effect.

Significance

In conclusion, the current study demonstrated that carvacryl acetate exhibited anti-inflammatory activity in mice by reducing inflammatory mediators, neutrophil migration and cytokine concentration, and anti-nociceptive activity due to the involvement of capsaicin and glutamate pathways.     

Accumulation of nano-sized particles in a murine model of angiogenesis

Purpose

To evaluate the ability of nm-scaled iron oxide particles conjugated with Azure A, a classic histological dye, to accumulate in areas of angiogenesis in a recently developed murine angiogenesis model.

Materials and methods

We characterised the Azure A particles with regard to their hydrodynamic size, zeta potential, and blood circulation half-life. The particles were then investigated by Magnetic Resonance Imaging (MRI) in a recently developed murine angiogenesis model along with reference particles (Ferumoxtran-10) and saline injections.

Results

The Azure A particles had a mean hydrodynamic diameter of 51.8 ± 43.2 nm, a zeta potential of −17.2 ± 2.8 mV, and a blood circulation half-life of 127.8 ± 74.7 min. Comparison of MR images taken pre- and 24-h post-injection revealed a significant increase in R₂^* relaxation rates for both Azure A and Ferumoxtran-10 particles. No significant difference was found for the saline injections. The relative increase was calculated for the three groups, and showed a significant difference between the saline group and the Azure A group, and between the saline group and the Ferumoxtran-10 group. However, no significant difference was found between the two particle groups.

Conclusion

Ultrahigh-field MRI revealed localisation of both types of iron oxide particles to areas of neovasculature. However, the Azure A particles did not show any enhanced accumulation relative to Ferumoxtran-10, suggesting the accumulation in both cases to be passive.     

Polymorphism distribution and structural conservation in RNA-sensing Toll-like receptors 3, 7, and 8 in pigs

Background

Viral genomic RNA—both single-stranded (ss) and double-stranded (ds)—is recognized by RNA-sensing Toll-like receptors (TLRs), notably TLR3 (dsRNA), TLR7 (ssRNA), and TLR8 (ssRNA). However, our knowledge of the roles of porcine TLR3, 7, and 8 in antiviral immunity is inadequate.

Methods

From information on exon–intron boundaries obtained through comparisons of the genomic and cDNA sequences, polymorphisms in the coding sequences of each gene were detected in 84 male pigs of 11 breeds.

Results

Genomic structures are conserved between pigs and humans. The RNA-sensing TLR genes had fewer polymorphisms causing amino acid alterations than did the cell-surface TLR genes, but the alterations were distributed with a similar bias toward ectodomains.

Conclusions

The low level of diversity of substitutive polymorphisms in RNA-sensing TLRs than cell-surface ones implies that polymorphisms severely affecting function have been eliminated by selection pressure during longstanding pig breeding.

General significance

Recognition of virus-derived RNA is critical in host defense against infection. These results should provide a useful clue to analysis of the association between polymorphisms in RNA-sensing TLRs and disease resistance.     

Pyrroloquinoline Quinine Inhibits RANKL-Mediated Expression of NFATc1 in Part via Suppression of c-Fos in Mouse Bone Marrow Cells and Inhibits Wear Particle-Induced Osteolysis in Mice

The effects of pyrroloquinoline quinine (PQQ) on RANKL-induced osteoclast differentiation and on wear particle-induced osteolysis were examined in this study. PQQ inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages (BMMs) in a dose-dependent manner without any evidence of cytotoxicity. The mRNA expression of c-Fos, NFATc1, and TRAP in RANKL-treated BMMs was inhibited by PQQ treatment. Moreover, RANKL-induced c-Fos and NFATc1 protein expression was suppressed by PQQ. PQQ additionally inhibited the bone resorptive activity of differentiated osteoclasts. Further a UHMWPE-induced murine calvaria erosion model study was performed to assess the effects of PQQ on wear particle-induced osteolysis in vivo. Mice treated with PQQ demonstrated marked attenuation of bone erosion based on Micro-CT and histologic analysis of calvaria. These results collectively suggested that PQQ demonstrated inhibitory effects on osteoclast differentiation in vitro and may suppress wear particle-induced osteolysis in vivo, indicating that PQQ may therefore serve as a useful drug in the prevention of bone loss.     

Preclinical evaluation of zoledronate using an in vitro mimetic cellular model for breast cancer metastatic bone disease

Background

The interactions between metastatic breast cancer cells and host cells of osteoclastic lineage in bone microenvironment are essential for osteolysis. In vitro studies to evaluate pharmacological agents are mainly limited to their direct effects on cell lines. To mimic the communication between breast cancer cells and human osteoclasts, a simple and reproducible cellular model was established to evaluate the effects of zoledronate (zoledronic acid, ZOL), a bisphosphonate which exerts antiresorptive properties.

Methods

Human precursor osteoclasts were cultured on bone-like surfaces in the presence of stimuli (sRANKL, M-CSF) to ensure their activation. Furthermore, immature as well as activated osteoclasts were co-cultured with MDA-MB-231 breast cancer cells. TRAP5b and type I collagen N-terminal telopeptide (NTx) were used as markers. Osteoclasts’ adhesion to bone surface and subsequent bone breakdown were evaluated by studying the expression of cell surface receptors and certain functional matrix macromolecules in the presence of ZOL.

Results

ZOL significantly suppresses the precursor osteoclast maturation, even when the activation stimuli (sRANKL and M-SCF) are present. Moreover, it significantly decreases bone osteolysis and activity of MMPs as well as precursor osteoclast maturation by breast cancer cells. Additionally, ZOL inhibits the osteolytic activity of mature osteoclasts and the expression of integrin β3, matrix metalloproteinases and cathepsin K, all implicated in adhesion and bone resorption.

Conclusions

ZOL exhibits a beneficial inhibitory effect by restricting activation of osteoclasts, bone particle decomposition and the MMP-related breast cancer osteolysis.

General significance

The proposed cellular model can be reliably used for enhancing preclinical evaluation of pharmacological agents in metastatic bone disease.     

IL-29 enhances Toll-like receptor-mediated IL-6 and IL-8 production by the synovial fibroblasts from rheumatoid arthritis patients

Introduction

We previously reported that IL-29, a newly described member of interferon (IFN) family, was overexpressed in blood and synovium of rheumatoid arthritis (RA) patients and triggered proinflammatory cytokine IL-6 and IL-8 mRNA expression in RA synovial fibroblasts (RA-FLS). This suggests that IL-29 has an important role in synovial inflammation. Toll-like receptors (TLRs) also activate RA-FLS to produce inflammatory mediators including tumor necrosis factor α (TNF-α) and IL-1β in RA-FLS. Since the TLR family plays an early role in the innate immune response and the subsequent induction of the adaptive immune response, we hypothesize that IL-29 interacts with TLRs in RA inflammation. This study aimed to investigate the effect of IL-29 on TLR-mediated proinflammatory cytokine production in RA-FLS.

Methods

The mRNA level of IL-29 receptors (IL-28Rα and IL-10R2) in RA-FLS was determined by semi-quantitative RT- PCR. IL-6 and IL-8 mRNA expressions in RA-FLS were evaluated by real-time PCR after pre-incubation with IL-29 and subsequent stimulation with peptidoglycan (PGN, TLR2 ligand), or polycytidylic acid (poly(I:C), TLR3 ligand), or lipopolysaccharide (LPS, TLR4 ligand) . The production of TLR2, 3, and 4 in RA-FLS after IL-29 stimulation was also assessed by real-time PCR and flow cytometry. IL-29 mRNA and protein expression in RA-FLS after stimulation with PGN, poly(I:C), or LPS were measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively.

Results

The IL-29 receptor complex (IL-28Rα and IL-10R2) was identified in RA-FLS. IL-29 enhanced TLR-mediated IL-6 and IL-8 expression in RA-FLS. IL-29 upregulated expression of TLR2, 3 and 4 in RA-FLS. Exposure to PGN, poly(I:C) or LPS triggered IL-29 production by RA-FLS.

Conclusions

We show for the first time that IL-29 enhances TLR-induced proinflammatory cytokine production in RA-FLS via upregulation of TLRs.     

Mannose binding lectin and lung collectins interact with Toll-like receptor 4 and MD-2 by different mechanisms

Background

We have previously shown that lung collectins, surfactant protein A (SP-A) and surfactant protein D, interact with Toll-like receptor (TLR) 2, TLR4, or MD-2. Bindings of lung collectins to TLR2 and TLR4/MD-2 result in the alterations of signaling through these receptors, suggesting the immunomodulatory functions of lung collectins. Mannose binding lectin (MBL) is another collectin molecule which has structural homology to SP-A. The interaction between MBL and TLRs has not yet been determined.

Methods

We prepared recombinant MBL, and analyzed its bindings to recombinant soluble forms of TLR4 (sTLR4) and MD-2.

Results

MBL bound to sTLR4 and MD-2. The interactions were Ca²⁺-dependent and inhibited by mannose or monoclonal antibody against the carbohydrate-recognition domain of MBL. Treatment of sTLR4 or MD-2 by peptide N-glycosidase F significantly decreased the binding of MBL. SP-A bound to deglycosylated sTLR4, and this property did not change in chimeric molecules of SP-A/MBL in which Glu¹⁹⁵–Phe²²⁸ or Thr¹⁷⁴–Gly¹⁹⁴ of SP-A were replaced with the corresponding MBL sequences.

General Significance

These results suggested that MBL binds to TLR4 and MD-2 through the carbohydrate-recognition domain, and that oligosaccharide moieties of TLR4 and MD-2 are important for recognition by MBL. Since our previous studies indicated that lung collectins bind to the peptide portions of TLRs, MBL and lung collectins interact with TLRs by different mechanisms. These direct interactions between MBL and TLR4 or MD-2 suggest that MBL may modulate cellular responses by altering signals through TLRs.     

Modifications of mesenteric adipose tissue during moderate experimental colitis in mice

Aims

Adipose tissue secretes various proteins referred to as adipokines, being involved in inflammation. It was recognized that mesenteric adipose tissue (MAT) is altered by inflammation, and pathologies such as inflammatory bowel disease (IBD). The aim of this study was to investigate the alterations of the mesenteric adipose tissue in two experimental colitis models in mice adapted to obtain moderate colonic inflammation.

Main methods

Colonic inflammation was obtained using two models, either DSS dissolved in drinking water or intra-colonic instillation of DNBS. The expression of adipokines (leptin and adiponectin) and inflammatory markers (IL-6, MCP-1, F4/80) was studied by qRT-PCR in the MAT of treated and control mice.

Key findings

Observations of the colon and IL-6 plasma level determination demonstrated that DNBS treatment led to stronger inflammation. Colitis induced a decrease of mRNA encoding to leptin and adiponectin in MAT. In contrast, colonic inflammation led to an increase of mRNA encoding to IL-6, MCP-1 and F4/80, a specific marker of macrophages.

Significance

The mesenteric adipose tissue, in two models of moderate colitis, shows a loss of adipose profile and a strong increase of inflammatory pattern, close to the observations made in MAT of IBD patients. These data suggest that these pro-inflammatory modifications of MAT have to be taken into account in the pathophysiology of IBD.     

Differential effect of molecular size HA in mouse chondrocytes stimulated with PMA

Background

Hyaluronan (HA) fragments elicit the expression of inflammatory mediators through a mechanism involving the CD44 receptor. This study investigated the effects of HA at different molecular weights on PMA-induced inflammation in mouse chondrocytes.

Methods

mRNA and related protein levels were measured for CD44, PKCδ, PKC?, TNF-α, IL-1β, MMP-13, and iNOS in chondrocytes, untreated or PMA treated, with and without the addition of HA. The level of NF-kB activation was also assayed.

Results

CD44, PKCδ, and PKC? mRNA expression resulted higher than controls in chondrocytes treated with PMA. PMA also induced NF-kB up-regulation and increased TNF-α, IL-1β, MMP-13, and iNOS expression. HA treatment produced different effects: low MW HA up-regulated CD44 expression, increased PKCδ and PKC? levels, and enhanced inflammation in untreated chondrocytes; while in PMA-treated cells it increased CD44, PKCδ, PKC?, NF-kB, TNF-α, IL-1β, MMP-13, and iNOS expression and enhanced the effects of PMA; medium MW HA did not exert action; high MW HA had no effect on untreated chondrocytes; however, it reduced PKCδ, PKC?, NF-kB activation and inflammation in PMA-stimulated cells. Specific CD44 blocking antibody was utilised to confirm CD44 as the target of HA modulation.

General Significance

These data suggest that HA via CD44 may modulate inflammation via its different molecular mass.     

Association of three SNPs in interleukin-28B with graft hepatic dysfunction after liver transplantation in Chinese Han population

Background

Recurrent graft infection limited the effect of LT, early recognition and prophylaxis of HBV recurrence are very important, and interleukin 28B (IL‐28B) gene was reported to be associated with HBV infection.

Aims

To explore the association between IL-28B single-nucleotide polymorphisms (SNPs) and graft re-infection after liver transplantation(LT).

Methods

21 recipients with hepatitis B virus(HBV) recurrence and 157 recipients without HBV recurrence were included. We studied three SNPs in the promoter region of IL-28B gene at the positions rs12979860, rs12980275 and rs8099917 by HRM analysis (high-resolution melting curve analysis).

Results

Hepatic allograft dysfunction was more likely to be associated with IL-28B SNPs. However, there was no significant difference in the frequencies of IL-28B gene distribution in recipients with or without HBV recurrence.

Conclusion

IL-28B gene polymorphism may be associated with the prognosis of LT recipients but it needs more experiments.     

Small molecule inhibitors of protein interaction with glycosaminoglycans (SMIGs), a novel class of bioactive agents with anti-inflammatory properties

Background

Small molecule inhibitors of biologically important protein–glycosaminoglycan (GAG) interactions have yet to be identified.

Methods

Compound libraries were screened in an assay of L-selectin–IgG binding to heparin (a species of heparan sulfate [HS-GAG]). Hits were validated, IC-50s established and direct binding of hits to HS-GAGs was investigated by incubating compounds alone with heparin. Selectivity of inhibitors was assessed in 11 different protein-GAG binding assays. Anti-inflammatory activity of selected compounds was evaluated in animal models.

Results

Screening identified a number of structurally-diverse planar aromatic cationic amines. Scaffolds similar to known GAG binders, chloroquine and tilorone, were also identified. Inhibitors displayed activity also against bovine kidney heparan sulfate. Direct binding of compounds to GAGs was verified by incubating compounds with heparin alone. Selectivity of inhibitors was demonstrated in a panel of 11 heparin binding proteins, including selectins, chemokines (IL-8, IP-10), Beta Amyloid and cytokines (VEGF, IL-6). A number of selected lead compounds showed dose-dependent efficacy in peritonitis, paw edema and delayed type hypersensitivity.

Conclusions

A new class of compounds, SMIGs, inhibits protein–GAG interaction by direct binding to GAGs. Although their IC-50s were in the low micro-molar range, SMIGs binding to HS-GAGs appeared to be stable in physiological conditions, indicating high avidity binding. SMIGs may interfere with major checkpoints for inflammatory and autoimmune events.

General significance

SMIGs are a class of structurally-diverse planar aromatic cationic amines that have an unusual mode of action — inhibiting protein–GAG interactions via direct and stable binding to GAGs. SMIGs may have therapeutic potential in inflammatory and autoimmune disorders.     

Antinociceptive effects of lacosamide on spinal neuronal and behavioural measures of pain in a rat model of osteoarthritis

Introduction

Alterations in voltage-gated sodium channel (VGSC) function have been linked to chronic pain and are good targets for analgesics. Lacosamide (LCM) is a novel anticonvulsant that enhances the slow inactivation state of VGSCs. This conformational state can be induced by repeated neuronal firing and/or under conditions of sustained membrane depolarisation, as is expected for hyperexcitable neurones in pathological conditions such as epilepsy and neuropathy, and probably osteoarthritis (OA). In this study, therefore, we examined the antinociceptive effect of LCM on spinal neuronal and behavioural measures of pain, in vivo, in a rat OA model.

Methods

OA was induced in Sprague Dawley rats by intraarticular injection of 2 mg of monosodium iodoacetate (MIA). Sham rats received saline injections. Behavioural responses to mechanical and cooling stimulation of the ipsilateral hind paw and hindlimb weight-bearing were recorded. In vivo electrophysiology experiments were performed in anaesthetised MIA or sham rats, and we recorded the effects of spinal or systemic administration of LCM on the evoked responses of dorsal horn neurones to electrical, mechanical (brush, von Frey, 2 to 60 g) and heat (40°C to 50°C) stimulation of the peripheral receptive field. The effect of systemic LCM on nociceptive behaviours was assessed.

Results

Behavioural hypersensitivity ipsilateral to knee injury was seen as a reduced paw withdrawal threshold to mechanical stimulation, an increase in paw withdrawal frequency to cooling stimulation and hind limb weight-bearing asymmetry in MIA-treated rats only. Spinal and systemic administration of LCM produced significant reductions of the electrical Aβ- and C-fibre evoked neuronal responses and the mechanical and thermal evoked neuronal responses in the MIA group only. Systemic administration of LCM significantly reversed the behavioural hypersensitive responses to mechanical and cooling stimulation of the ipsilateral hind paw, but hind limb weight-bearing asymmetry was not corrected.

Conclusions

Our in vivo electrophysiological results show that the inhibitory effects of LCM were MIA-dependent. This suggests that, if used in OA patients, LCM may allow physiological transmission but suppress secondary hyperalgesia and allodynia. The inhibitory effect on spinal neuronal firing aligned with analgesic efficacy on nociceptive behaviours and suggests that LCM may still prove worthwhile for OA pain treatment and merits further clinical investigation.     

Serglycin secretion is part of the inflammatory response in activated primary human endothelial cells in vitro

Background

Endothelial cells have important functions in e.g. regulating blood pressure, coagulation and host defense reactions. Serglycin is highly expressed by endothelial cells, but there is limited data on the roles of this proteoglycan in immune reactions.

Methods

Cultured primary human endothelial cells were exposed to proinflammatory agents lipopolysaccharide (LPS) and interleukin 1β (IL-1β). The response in serglycin synthesis, secretion and intracellular localization and effect on the proteoglycan binding chemokines CXCL-1 and CXCL-8 were determined by qRT-PCR, Western blotting, immunocytochemistry, ELISA and serglycin knockdown experiments.

Results

Both LPS and IL-1β increased the synthesis and secretion of serglycin, while only IL-1β increased serglycin mRNA expression. Stimulation increased the number of serglycin containing vesicles, with a greater portion of large vesicles after LPS treatment. Also, increased intracellular and secreted levels of CXCL-1 and CXCL-8 were observed. The increase in CXCL-8 secretion was unchanged in serglycin knockdown cells. However, the increase in CXCL-1 secretion from IL-1β stimulation was reduced 27% in serglycin knockdown cells; while the LPS-induced secretion was not affected. In serglycin expressing cells CXCL-1 positive vesicles were evenly distributed throughout the cytoplasm, while confided to the Golgi region in serglycin knockdown cells. This was the case only for IL-1β stimulated cells. LPS-induced CXCL-1 distribution was unaffected by serglycin expression.

Conclusions

These results suggest that different signaling pathways are involved in regulating secretion of serglycin and partner molecules in activated endothelial cells.

General significance

This knowledge increases our understanding of the roles of serglycin in immune reactions. This article is part of a Special Issue entitled: Matrix-mediated cell behaviour and properties.