Menthol, a unique urinary volatile compound, is associated with chronic inflammation in interstitial cystitis

Abstract

Chronic inflammation is a potential systemic risk factor for many bladder dysfunctions, including interstitial cystitis (IC). However, the underlying mechanism through which a healthy bladder protects itself from inflammatory triggers remains unknown. In this study, we identified odor compounds in urine obtained from IC patients and healthy controls. Using comprehensive solid-phase microextraction-gas chromatography-time-of-flight-mass spectrometry (SPME-GC-TOF-MS) profiling and bioinformatics, we found that levels of urinary volatile metabolites, such as menthol, were significantly reduced in IC patients, compared to healthy controls. In an attempt to understand the mechanistic meaning of our volatile metabolites data and the role of menthol in the immune system, we performed two independent experiments: (a) cytokine profiling, and (b) DNA microarray. Our findings suggest that lipopolysaccharide (LPS)-stimulated inflammatory events, such as the production and secretion of inflammatory cytokines (e.g., TNF-α, IL-6, and IL-1β) and the activation of NF-κB and associated proteins within a large signaling network (e.g., Akt, TLR1, TNFAIP3, and NF-κB), are suppressed by the presence of menthol. These findings broaden our knowledge on the role of urinary menthol in suppressing inflammatory events and provide potential new strategies for alleviating both the odor and inflammation associated with IC.

Figure 1

The analysis workflow of volatile metabolome identification, conversion to KEGG IDs, and pathway mapping.

Figure 2

Reduced production of LPS-induced inflammatory cytokines by menthol in RAW 264.7 cells (cell lysates) (A) Inflammatory cytokine array analysis of CCL3, CXCL10 and TNF-α. The expression of these inflammatory cytokines is highly induced by LPS, but downregulated by menthol. (B) Quantification of array band intensity of CCL3, CXCL10 and TNF-α with ImageJ-analysis software.

Figure 3

Decreased secretion of LPS-induced inflammatory cytokines by menthol in RAW 264.7 cells (conditioned media) (A) Inflammatory cytokine array analysis of IL-1β, IL-6, CCL5, CCL12 and G-CSF. The expression of these inflammatory cytokines is highly induced by LPS, but downregulated by menthol. (B) Quantification of array band intensity of IL-1β, IL-6, IL-1 and G-CSF with imageJ-analysis software.

Figure 4

Menthol inhibits LPS-induced cytokine production in RAW 264.7 cells. (A) RAW 264.7 cells were pretreated with menthol, followed by stimulation with LPS for 6 hrs. Expression level of TNF-α, IL-6, IL-1β and CCL3 were induced by LPS and reduced in LPS+M compared to LPS. (B) Pretreatment of LPS (100 ng/mL) or control for 3 hrs and followed by induction with different concentration of menthol (50, 100 and 500 μmol/mL) for 6 hrs. Expression level of TNF-α, IL-6 and IL-1β were reduced by menthol treatment in a dose-dependent manner. β-actin was used as a loading control for western blot analysis.

Figure 5

Differentially expressed genes in LPS, LPS+Menthol (LPS+M), and menthol (M) only conditions. (A) The number of DEGs perturbed by LPS or menthol treatment. (B) Venn diagram depicts shared and different DEGs. (C,D) Gene Ontology analysis suggested functional annotations (biological process) that were associated with up- (C) and downregulated (D) genes. Bar graph shows significantly enriched biological processes, which were up- and downregulated genes in test group. The inflammation events induced by LPS were inhibited by menthol in LPS mediated signaling pathway.

Figure 6

Menthol inhibits LPS-induced activation of NF-κB and Akt signaling pathways. (A) Phosphorylation levels of NF-κB and Akt were reduced by menthol treatment. (B) Phosphorylation of NF-κB and Akt were suppressed by menthol treatment in dose-dependent manner. β-actin was used as the loading control in western blot analysis.

Figure 7

Menthol regulates the LPS-induced inflammatory response. (A) Heatmap image of DNA microarray data. RAW 264.7 cells were treated with LPS (100 ng/mL) with or without menthol (500 μmol/mL) for 6 hrs. The list of the genes in the enlarged box was sorted in order of gene symbol. (B) Cells were treated with LPS (100 ng/mL) with or without menthol (500 μmol/mL) for 6 hrs. Few candidates from (Figure A, Box): IFIT1, TNFA1P3, TLR1, and viperin were repressed by menthol treatment. (C) Hypothetical diagram showing how menthol may be attenuating the activation of the TLR1 and TNFAIP3-NF-κB signaling pathways; thereby, reducing downstream cytokine expression.