Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic debilitating inflammatory disease that significantly lowers the life quality of its patients. Despite several treatment guidelines, most treatments show variable, short-term improvements, which can vary from one patient to another making IC/BPS difficult to diagnose and treat. Thus, there is an unmet need for more effective therapeutic agents be they naturally derived or synthetic. Whilst the pathophysiology is elusive and multi-factorial, neurogenic inflammation is believed to underlie the chronic bladder inflammation seen in IC/BPS. Histology shows the urothelium in IC/BPS to be thin and denuded with or without ulceration. This allows harmful urinary constituents to leak into the sub-urothelial layer and depolarize bladder sensory nerve endings, resulting in the release of neuroactive substances including substance P (Sub P). Supporting this theory are studies showing that the density of SP-positive nerve endings in IC/BPS is not only significantly higher compared to controls but are located in close proximity to mast cells.
Upon binding to its G protein-coupled receptors (MRGPRX2 and NK-1), Sub P triggers intracellular signalling events resulting in mast cell activation and degranulation. Upon activation, mast cells release of both pre-stored and de-novo synthesized pro-inflammatory mediators, which promote chronic bladder wall inflammation and pain in IC/BPS bladders. The intracellular signalling events underlying mast cell activation and degranulation include phosphorylation of the enzymes phospholipase C-β (PLC-β), phosphoinositide-3 kinase (PI3K), protein kinase B/Akt, extracellular signal-regulated kinase (ERK1/2) and the signal transducer and activator of transcription 3 (STAT3).
Manuka honey is a unique type of honey from New Zealand with a wide range of properties including antimicrobial activity and biofilm inhibition with recent reports highlighting its potential anti-inflammatory and anti-oxidant effects. Interestingly, Medihoney (MH), a medical-grade Manuka honey, has been shown to inhibit the Calcium ionophore-induced degranulation and histamine release in vitro using a human mast cell line (LAD-2).  
The aim of the present study was to investigate the potential anti-inflammatory effect of MH against Sub P-induced mast cell degranulation (as a model for neurogenic inflammation), and to explore the potential cellular mechanisms through which MH might exhibit such properties using an in vitro mast cell model.

For mast cell activation, human LAD2 mast cells were incubated with Sub P at 37 °C and 5% CO2 for 40-min with or without 20-minute pre-incubation with MH at 2, 4 and 6% concentrations. Mast cell degranulation was assessed by quantification of the lysosomal enzyme β-hexosaminidase in the cell culture supernatant. In addition, the effect of MH pre-incubation on the intracellular signalling events underlying mast cell activation was assessed using a specific proteome profiler Phospho MAPK array kit (Cat# ARY003C, R&D Systems, USA) and Phospho(Ser473)/Total Akt Whole Cell Lysate Kit (Cat# K15100D, MSD, USA).  
Lactate dehydrogenase (LDH) activity assay (Cat# MAK066, Sigma) was performed to rule out any cytotoxicity on the LAD2 cells.

(1) The effect of MH on Sub P and compound 48/80 (C48/80)-induced LAD2 cell degranulation:
Both the neuropeptide substance P and the MRGPRX2 agonist C48/80 induced 42% and 61% release, respectively. Pre-incubation with MH at 2%, 4% and 6% induced 79%, 97% and 94% inhibition of β-hexosaminidase release induced by both Sub P or C48/80, respectively (Figure 1). 

(2) The effect of  MH pre-incubation on the Sub P-induced  intracellular signalling pathways in LAD2 cells (Phospho MAPRK array kit, ARY003C, R&D Systems):
Over 5-independent assays, 15-minute incubation of the LAD2 cells with Sub P induced significant increase in the levels of phosphorylated protein kinases Akt, at the serine 473 position, lysine deficient protein kinase 1 (WNK1), at the threonine 60 position, and ERK1/2, in addition to STAT3 at the serine 727 position. Akt, WNK1 and STAT3 phosphorylation was significantly and consistently inhibited upon pre-treatment of the LAD2 cells with 4% MH. However, MH did not induce statistically significant inhibition of ERK1/2 phosphorylation (Figure 2).

(3) The effect of MH and substance P on the viability of LAD2 cells (LDH assay, MAK066, Sigma):
MH at both 2% and 4% concentrations, in addition to substance P 1µM, did not caused any rise of the lactate dehydrogenase activity when compared to the spontaneous levels (controls), suggesting no cytotoxic effect on the LAD2 cells. 
It is worth mentioning that this assay was repeated four times using two different batches of LAD2 cells.

MH strongly and significantly inhibited mast cell degranulation induced by the neuropeptide Sub P or C48/80 in a dose dependent manner. This effect culminated in 97% inhibition of degranulation at 4% MH concentration. Such effect indicates a strong potential for MH as a potent anti-inflammatory agent against neurogenic inflammation. Interestingly, 4% MH inhibited the Sub P-induced phosphorylation of Akt (Serine 473), WNK1 and STAT3 (Serine 727). Besides its crucial role in the positive regulation of mast cell development and survival, PI3K-mediated Akt activation is essential for the pro-inflammatory responses of mast cells upon activation by variable secretagogues, a finding which is consistent with our results. Akt and WNK1 phosphorylation are known to enhance the membrane trafficking of the membrane glucose transporter GLUT1 resulting in increased intracellular glucose uptake and the subsequent metabolic changes, which enhance mast cell activation and pro-inflammatory mediator release. At the same time, STAT3 induces mitochondrial oxidative respiration, thus enhancing mast cell activation. In addition, STAT3 phosphorylation at the position serine 727 position has been reported to be an essential event for mast cell degranulation. This evidence might provide a possible explanation for the mast cell stabilising effects of MH through the inhibition of glucose uptake and subsequent metabolic activity in mast cells. However, this would require further studies investigating GLUT1 membrane trafficking levels, lactic acid and ATP production, and oxygen consumption during LAD-2 activation with or without MH.

Our findings demonstrate for the first time the potential anti-inflammatory properties of MH, through its ability to inhibit mast cell degranulation induced by the neuropeptide Sub P. Moreover, the study provides the first scientific explanation for the stabilisation of mast cells by MH through the inhibition of intracellular Akt, WNK1 and STAT3 phosphorylation. These data support the potential use of MH as an anti-inflammatory agent in neurogenic inflammatory conditions including atopic dermatitis, chronic urticaria, and IC/BPS. Specifically, aqueous solutions of MH might be instilled intra-vesically (via a bladder catheter) to aid the management of patients with IC/BPS.

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Continence 2S2 (2022) 100408
DOI: 10.1016/j.cont.2022.100408