Biology subjects

Lwaleed, B. A.

Publications and source records attributed to Lwaleed, B. A..

3 recordsLinked to original sources

bioRxiv2026

Medical-Grade Manuka Honey and Manuka Honey Extract Inhibit Mast Cell Degranulation through inhibition of MRGPRX2 expression: Potential Intravesical Agent for the Management of Interstitial Cystitis/Bladder Pain Syndrome?

RationaleNeurogenic inflammation is recognised as an important contributor to the pathophysiology of Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS). Substance P (Sub P), a neuropeptide released from sensory nerves, is a potent inducer of mast cell degranulation through the Mas-related G protein-coupled receptor member X2 (MRGPRX2), resulting in the release of pro-inflammatory mediators that perpetuate chronic bladder inflammation. Medihoney, a medical-grade M[a]nuka honey, possesses well-established antimicrobial and anti-inflammatory properties, and we have recently demonstrated its ability to stabilise mast cells through inhibition of histamine release. However, its effects on Sub P-induced mast cell activation and MRGPRX2-mediated neurogenic inflammation have not previously been investigated. Aim of the studyWe aimed to investigate the inhibitory effects of Medihoney and a sugar-free M[a]nuka honey extract on Substance P-induced mast cell degranulation and MRGPRX2 activation as potential therapeutic approaches for chronic neurogenic inflammation associated with IC/BPS. In addition, we examined the expression of MRGPRX2 in bladder biopsies from patients with IC/BPS. Materials and methodsHuman LAD2 mast cells were stimulated with Substance P (1 M) for 40 minutes following 20-minute pre-incubation with Medihoney or a sugar-free M[a]nuka honey extract. Mast cell degranulation was quantified by measuring {beta}-hexosaminidase release. MRGPRX2 activation was assessed by intracellular calcium imaging using Fluo-4 in MRGPRX2-expressing HEK-293 cells. Bladder biopsies obtained from patients with IC/BPS and healthy controls were immunostained for mast cell tryptase, chymase and MRGPRX2. ResultsMedihoney at 2% and 4% markedly inhibited Substance P-induced mast cell degranulation in LAD2 cells by approximately 90%, an effect that was similarly observed with the sugar-free M[a]nuka honey extract. Both preparations produced a dose-dependent inhibition of Substance P-induced intracellular signalling in MRGPRX2-expressing HEK-293 cells, demonstrating suppression of MRGPRX2 activation. Furthermore, immunohistochemical analysis of bladder biopsies revealed that approximately 66% of tryptase-positive mast cells expressed MRGPRX2 in patients with IC/BPS, which was significantly higher than that observed in healthy control tissues (25%). ConclusionThe present study demonstrates that mast cells within IC/BPS bladder tissue express increased levels of MRGPRX2, suggesting enhanced responsiveness to Substance P and supporting a role for neurogenic inflammation in the pathophysiology of IC/BPS. Medihoney and the sugar-free M[a]nuka honey extract significantly inhibit Substance P-induced mast cell degranulation through modulation of MRGPRX2-mediated intracellular signalling, highlighting their potential as novel therapeutic agents for reducing neurogenic bladder inflammation associated with IC/BPS. ImpactThis study provides evidence that MRGPRX2-mediated neurogenic mast cell activation is enhanced in IC/BPS and demonstrates, for the first time, that Medihoney and a sugar-free M[a]nuka honey extract effectively inhibit Substance P-induced mast cell degranulation through modulation of MRGPRX2 signalling. These findings provide new mechanistic insight into the anti-inflammatory actions of M[a]nuka honey-derived preparations and identify MRGPRX2 as a potential therapeutic target in IC/BPS. The observed inhibition of neurogenic mast cell activation suggests that these naturally derived preparations may offer a novel strategy for limiting chronic bladder inflammation. Overall, this work provides a foundation for future preclinical and clinical studies evaluating the safety and therapeutic efficacy of Medihoney and M[a]nuka honey-derived compounds in patients with IC/BPS.

immunology↗

bioRxiv2026

Medical Grade Manuka Honey Inhibits A23187 Induced Mast Cell Degranulation and Cytokine Release via Downregulation of the ERK Signalling Pathways

RationaleMast degranulation is a driver of several pathologies. Several endogenous peptides have been established as stimulators of mast cell degranulation. Compounds that stabilise mast cells have in part demonstrated inhibitory effect on the inflammatory cascade. Manuka honey has been widely used for the treatments of various inflammatory diseases. However, its effect on A23187 (Calcium ionophore) stimulated mast cell degranulation, cytokine release and cellular signalling pathways has not yet been investigated. Aim of the studyWe aim to investigate the effect of Medical Grade Manuka Honey (MGMH) on A23187 induced mast cell degranulation, cytokine release and downstream signalling pathways in a human mast cell lines LAD2 model. Materials and methodsThe cytotoxic effect of MGMH on LAD2 cells was assessed using LDH assay. LAD2 cells were pre-incubated with MGMH and challenged with A23187.Mast cell degranulation was measured by {beta}-hexosaminidase release whilst histamine and cytokines released were measured by ELISA. The effect of MGMH on downstream signalling of Mitogen Activated Protein Kinase (MAPK) pathways was determined and quantified by SDS-PAGE western blotting. ResultsMGMH at 2% and 4% was well tolerated by LAD2 cells. MGMH at all concentrations tested significantly inhibited the A23187 triggered release of {beta}-hexosaminidase but failed to inhibit the release of histamine. MGMH 4% significantly inhibited the release of GM-CSF and IL-8. MGMH (2% and 4%) significantly down regulated the expression of both ERK I and ERK II. However, MGMH at all the doses tested had no effect on the expressions of JNK and p38. On the contrary, an increase expression of these p38 and JNK were noted with MGMH pre-incubation. ConclusionOur present study provides evidence that MGMH inhibition of A23187 stimulated degranulation of mast cells and cytokine release through down regulation of ERK I and II signalling. The results suggest potential use as a mast cell stabiliser and effective treatments of mast cell mediated inflammatory diseases. ImpactThis study provides the first evidence that Medical Grade Manuka Honey (MGMH) can attenuate A23187-induced mast cell activation and pro-inflammatory cytokine release in human LAD2 mast cells through modulation of ERK1/2 signalling pathways. The findings advance our understanding of the cellular and molecular mechanisms underlying the anti-inflammatory properties of Manuka honey. The demonstration that MGMH inhibits {beta}-hexosaminidase release suggests a mast cell-stabilising effect, highlighting its potential as a novel natural therapeutic agent for mast cell-mediated disorders, including allergic diseases, interstitial cystitis, asthma, chronic inflammatory skin conditions, and mast cell activation syndromes. The observed reduction in GM-CSF and IL-8 production further indicates that MGMH may help limit the amplification and persistence of inflammatory responses. Mechanistically, the selective downregulation of ERK1/2 signalling provides new insight into how MGMH exerts its biological effects and identifies a potential molecular target through which its anti-inflammatory activity is mediated. These findings contribute to the growing evidence base supporting the therapeutic value of Manuka honey beyond its established antimicrobial and wound-healing properties. Overall, this work lays the foundation for future preclinical and clinical studies investigating MGMH as a safe, naturally derived mast cell stabiliser and anti-inflammatory intervention, with potential applications across a broad spectrum of allergic and inflammatory diseases.

immunology↗

bioRxiv2026

Neutrophils-Nitroblue Tetrazolium staining: A potential novel marker of women infertilely?

RationaleOxidative stress, resulting from an imbalance between the production of reactive oxygen species (ROS) and antioxidant defence mechanisms, disrupts cellular redox homeostasis and contributes to damage of biomolecules, including nucleic acids, proteins and lipids. Such disturbances can impair intracellular signalling pathways and have been implicated in the pathophysiology of female reproductive disorders. Nitroblue Tetrazolium (NBT) is a distinctive dye that assesses cellular redox activity in neutrophils through the detection of superoxide anion (O2-). However, the relationship between neutrophil-derived oxidative stress and women infertility remains to be fully elucidated. Aim of the study: We aimed to investigate the role of neutrophil oxidative stress in women infertility using a novel Nitroblue Tetrazolium (NBT) method developed in our laboratory. Materials and methodsBlood and serum specimens were obtained from a total of 100 Libyan women, comprising healthy fertile women (n = 21; controls) and infertile women (n = 79). Superoxide anion (O2-) generation in neutrophils was assessed using the novel NBT method, while malondialdehyde (MDA), a marker of lipid peroxidation, was determined using the thiobarbituric acid reactive substances (TBARS) assay. ResultsA significant increase in both NBT-reactivity levels and the percentage of NBT-positive neutrophils was observed in infertile women compared with healthy fertile controls (P < 0.0001). In addition, MDA levels were significantly higher in infertile women than in the control group, indicating enhanced lipid peroxidation. MDA levels were positively correlated with NBT-reactivity levels (r = 0.410, P = 0.0001) and the percentage of NBT-positive neutrophils (r = 0.21, P = 0.047). A significant positive correlation was also observed between NBT-reactivity levels and the percentage of NBT-positive neutrophils (r = 0.510, P = 0.0001), demonstrating a close association between neutrophil oxidative activity and lipid peroxidation in women infertility. ConclusionThe present study demonstrates the utility of a novel NBT method for detecting reactive oxygen species (ROS), a marker of oxidative stress, in neutrophils from both blood and serum specimens. The findings demonstrate increased neutrophil-derived oxidative stress in infertile women and suggest that this method may have potential as a diagnostic tool for the assessment of women infertility. ImpactThis study provides evidence that neutrophil-derived oxidative stress is significantly increased in women infertility and demonstrates the application of a novel Nitroblue Tetrazolium (NBT) assay for assessing oxidative stress in both blood and serum specimens. The significant associations between NBT-reactivity, NBT-positive neutrophils and malondialdehyde levels provide further insight into the contribution of oxidative stress to female reproductive pathophysiology. These findings support the potential utility of the novel NBT method as a simple and reliable diagnostic approach for evaluating oxidative stress in women infertility and provide a foundation for future studies investigating oxidative stress biomarkers in reproductive medicine.

pathology↗