Activation of acid-sensing ion channel 3 by lysophosphatidylcholine 16:0 mediates psychological stress-induced fibromyalgia-like pain

Abstract

Objectives: Fibromyalgia is commonly considered a stress-related chronic pain disorder, and daily stressors are known triggers. However, the relation between stress and pain development remains poorly defined by clinical approaches. Also, the aetiology remains largely unknown.

Methods: We used a newly developed mouse model and lipidomic approaches to probe the causation and explore the biological plausibility for how perceived stress translates into chronic non-inflammatory pain. Clinical and lipidomic investigations of fibromyalgia were conducted for human validation.

Results: Using non-painful sound stimuli as psychological stressors, we demonstrated that mice developed long-lasting non-inflammatory hyperalgesia after repeated and intermittent sound stress exposure. Elevated serum malondialdehyde level in stressed mice indicated excessive oxidative stress and lipid oxidative damage. Lipidomics revealed upregulation of lysophosphatidylcholine 16:0 (LPC16:0), a product of lipid oxidisation, in stressed mice. Intramuscular LPC16:0 injection triggered nociceptive responses and a hyperalgesic priming-like effect that caused long-lasting hypersensitivity. Pharmacological or genetic inhibition of acid-sensing ion channel 3 impeded the development of LPC16:0-induced chronic hyperalgesia. Darapladib and antioxidants could effectively alleviate the stress-induced hyperalgesia by inhibiting LPC16:0 synthesis. Clinical investigations showed that excessive oxidative stress and LPC16:0 expression also exist in patients with fibromyalgia. Moreover, LPC16:0 expression was correlated with pain symptoms in patients with high oxidative stress and disease severity.

Conclusions: Our study provides experimental evidence for the causal effect of psychological stressors on chronic pain development. The findings identify a possible pathophysiological mechanism of stress-induced chronic non-inflammatory pain at molecular, behavioural and clinical levels that might indicate a new therapeutic approach for fibromyalgia.

Keywords: ASIC3; fibromyalgia; lysophosphatidylcholine; oxidative stress; pain.

Figure 1

RISS induces fibromyalgia-like pain behaviours in mice. (A) Schematic representation of the RISS protocol: mice were placed in a bedding cage under a speaker emitting continuous pure tone stimuli with randomly varied frequencies (5–19 kHz), duration (5 s or 10 s) and amplitude (0–100 dB) for 30 min as a set. In the original sound stress (SS) protocol, the stimulus set was given once a day on days 1, 3 and 4. The RISS protocol intensified the stimulus intensity by repeating the stimulus set every 3 hour for six times overnight (from 16:00 to 10:00) on the same day. (B) Sensory-related and anxiety-related behaviour assessed in mice at day 0 post SS and RISS exposure. Upper left, mechanical hyperalgesia measured by von Frey test. Upper right, muscle hyperalgesia measured as muscle withdrawal threshold. Lower left and right, anxiety state measured as duration of time spent in the centre area of the open field test and in the open arm of the plus maze test (n=6). (C) Time course of pain behaviours after RISS. Left, mechanical hyperalgesia. Middle, thermal hyperalgesia measured by Hargreaves test. Right, muscle hyperalgesia (n=6). (D) Time course of muscle fatigue-like behaviours after RISS measured by grip force test (n=6). (E) Hyperalgesic behaviour assessed in mice of both sexes at day 0 after RISS. Left, mechanical hyperalgesia measured by von Frey test. Right, muscle hyperalgesia measured as muscle withdrawal threshold (n=6). (F) Time course of pain behaviours assessed by von Frey test in female and male mice after RISS (n=6). (G) Analgesic effect of pregabalin, morphine and diclofenac on RISS-induced pain (n=6). Upper panel, mechanical hyperalgesia. Middle, muscle hyperalgesia. Lower, thermal hyperalgesia. Data are mean±SEM. B, basal status. ns, non-significant. Ctrl, control. *p<0.05, **p<0.01, ***p<0.001 compared with B (B and E) or Ctrl (C, D and F). #p<0.05, ##p<0.01, ###p<0.001 compared with RISS-Vehicle injection groups (E). ++p<0.01 compared with Ctrl-Vehicle injection groups (G) (all two-way ANOVA). For complete statistical analyses, please refer to online supplemental results. RISS, repeated and intermittent sound stress.

Figure 2

Upregulated expression of phosphorylated extracellular signal-regulated kinase (pERK) after RISS in lumbar dorsal root ganglia (DRG) of mice at post-RISS day 0. (A) Representative imaging of pERK expression of L4 DRG neurons in control, SS and RISS mice. Arrowheads indicate examples of positive pERK expression. (B) Quantitative analysis of expression of pERK in L4 DRG neurons (n=3) (Kruskal-Wallis test). (C) Representative imaging of pERK expression of cervical (C8), thoracic (T12) and lumbar (L4) DRG neurons in control and RISS mice. Scale bar, 100 µm. (D) Quantification of pERK expression in control and RISS mice (n=3 mice) (Mann-Whitney test). (E) Quantitative analysis of pERK colocalisation with sensory neurons markers. Left, percentage of pERK-positive DRG neurons immunostained for substance P (SP), calcitonin gene-related peptide (CGRP), isolectin B4 (IB4) and neurofilament 200 (N52). Right, percentage of CGRP-positive, SP-positive, IB4-positive and N52-positive neurons expressing pERK (n=3). (F) Representative double immunofluorescence staining showed colocalisation of pERK with SP, CGRP, IB4 and N52 in L4 DRG of RISS mice. Arrowheads indicate examples of colocalised neuronal profiles. Scale bar, 100 µm. (G) Magnified images of (F). Scale bar, 25 µm. Ctrl, control. Data are mean±SEM (n=3 mice). *p<0.05, **p<0.01 compared with controls. SS, sound stress; RISS, repeated and intermittent sound stress.

Figure 3

LPC16:0 resulting from lipid oxidisation inflicts RISS-induced chronic hyperalgesia via activating ASIC3. (A) Serum malondialdehyde (MDA) level assayed by TBARS method in RISS mice compared with controls (n=6). (B) Representative imaging of ROS expression in gastrocnemius cross-sections at P0 in control and RISS groups. Tissue ROS levels were measured by CM-H₂DCFDA staining. Scale bar, 80 µm. (C) Quantification of ROS expression in gastrocnemius muscles of mice. Positive controls were with intramuscular H₂O₂ injection (n=3). (D) RISS effects on blood leucocyte oxidative status (n=11). Intracellular ROS levels were measured with CM-H₂DCFDA staining using flow cytometry. (E) RISS effects on serum H₂O₂ level (n=6). (F) Score plot of OPLS-DA distinguishing basal and RISS (P4) groups based on serum lipidomic profiling (R²Y=0.71, Q²=0.166) (n=6). (G) S-plot of OPLS-DA and the differentiating metabolites identified for P4 (marked with red colour). The fold change analysis and variation trends of the selected lipids were presented in online supplemental figure S4B. (H) Quantitative analysis of LPC16:0 by QqQ MS (n=6). (I) Dose-dependent effects of repeated LPC16:0 injection on mechanical hypersensitivity as assessed by von Frey test in WT mice. Mice received two intramuscular injections (1 day apart; orange arrows) of neutral saline containing LPC16:0 (4.8 nmol, 1.6 nmol and 0.48 nmol) or vehicle (n=6). (J) Effect of repeated intramuscular (left; n=8) and intraplantar (right; n=6) LPC16:0 injection (4.8 nmole; orange arrows) on mechanical hyperalgesia as assessed by von Frey test. (K) Effects of pharmacological (left; n=6) and genetic (right; n=7) inhibition of ASIC3 on the chronification of mechanical hyperalgesia induced by repeated LPC16:0 injection (orange arrows). APETx2, ASIC3 inhibitor. (L) Effect of RISS on anxiety-like (open field test; n=5) and pain behaviours (von Frey test; n=7) in WT and Asic3 ^−/− mice at P0. (M) Schematic presentation of drug administration. (N) Left, effect of darapladib on serum LPC16:0 level in RISS mice measured by QqQ MS at P0 (n=6). Right, effect of darapladib on the RISS-induced hyperalgesia as assessed by von Frey test (n=5). (O) Left, effect of NAC and Tempol on serum LPC16:0 level in RISS mice as assessed by relative quantitative analysis with LC-MS at P0 (n=6). Right, effect of NAC and Tempol on the RISS-induced chronic hyperalgesia (n=6). B, basal status. Ctrl, control; i.p., intraperitoneal injection; LPC, lysophosphatidylcholine; NAC, N-acetylcysteine. Data are mean±SEM. *p<0.05, **p<0.01, ***p<0.001 compared with Ctrl or B. #p<0.05. ##p<0.01, ###p<0.001 compared with LPC injection (K left), WT mice (K right and L) or RISS-Vehicle injection (N, O) (Kruskal-Wallis test or ANOVA). For complete statistical analyses, please refer to online supplemental results. ASIC3, acid sensing ion channel 3; OPLS-DA, orthogonal partial least squares discriminant analysis; PCs, phosphatidylcholines; QqQ MS, triple quadrupole mass spectrometry; RISS, repeated and intermittent sound stress; ROS, reactive oxygen species; SM, sphingomyelin; TBARS, thiobarbituric acid reactive substances.

Figure 4

Patients with fibromyalgia (FM) have higher perceived stress, increased oxidative stress and discriminative LPC expression in plasma. (A) Left, psychological stress levels in FM patients (n=31) and healthy controls (HCs) (n=30) as assessed by the Perceived Stress Scale-10 (PSS-10) (unpaired t test). Right, plasma malondialdehyde (MDA) level assayed by the thiobarbituric acid reactive substances (TBARS) method in FM patients and HCs (unpaired t test). (B) Score plot of principal component analysis based on lipidomic analysis of plasma from FM patients and HCs. (C) Score plot of orthogonal partial least squares discriminant analysis (OPLS-DA) distinguishing FM and HC groups based on plasma lipidomic profiling (R²Y=0.593, Q²=0.149). (D) S-plot of OPLS-DA and the differentiating metabolites identified for FM (marked with red colour). (E) Box-and-whisker plots of discriminative lipids for FM and their fold change in peak intensity (Mann-Whitney test). Whiskers represent minimum and maximum values. (F) Correlation matrix between lipid expression of peak intensity and FM symptom measures (VRS and FIQR Scores) in the HC and FM groups (Spearman’s rank correlation test). Spearman correlation coefficients are labelled in each cell. FIQR, the Revised Fibromyalgia Impact Questionnaire; VRS, Verbal Rating Scale of pain. Data are mean±SEM. *p<0.05, **p<0.01, ***p<0.001 compared with HC. For complete statistical analyses, please refer to online supplemental results. cer, ceramides; LPC, lysophosphatidylcholine; PCs, phosphatidylcholines.

Figure 5

Expression of LPC16:0 and other lipids are correlated with FM symptoms in patients with increased oxidative stress and high disease severity. (A) Subgrouping FM patients based on disease severity by cluster analysis. Patients were classified into two subgroups (cluster 1 and 2) based on FIQR Score with K-means cluster analysis. FIQR Scores were higher in cluster 2 (n=12) than cluster 1 (n=19). Patients of cluster 2 were thus designated as the group with severe symptoms (FM-S), and those of cluster 1 with mild symptoms (FM-M) (unpaired t test). (B) Left, pain intensity assessed by Verbal Rating Scale (VRS; 0–10) among the HC (n=30), FM-M (n=19) and FM-S (n=12) groups. Right, extent of pain diffuseness assessed by Widespread Pain Index (WPI; 0–19) (one-way ANOVA). (C) Left, psychological stress levels among HC, FM-M and FM-S groups as assessed by the Perceived Stress Scale-10 (PSS-10). Right, plasma malondialdehyde (MDA) level assayed by the thiobarbituric acid reactive substances (TBARS) method (one-way ANOVA). (D) Box-and-whisker plots of peak intensity and fold change of lipid expression in the FM-M and FM-S subgroups versus HCs (Kruskal-Wallis test). (E) Correlation matrix between lipid expression of peak intensity and clinical symptom measures (VRS and FIQR Scores) (Spearman’s rank correlation test). Spearman correlation coefficients are labelled in each cell. Statistical details and scatter plots were presented in online supplemental figureS7A, B. Data are mean±SEM *p<0.05, **p<0.01, ***p<0.001 compared with HC (A–D) or with significant relevance (E). #p<0.05, ###p<0.001 comparing FM-S with FM-M. For complete statistical analyses, please refer to online supplemental results. cer, ceramides; FIQR, the Revised Fibromyalgia Impact Questionnaire; FM, fibromyalgia; HC, healthy control; LPC, lysophosphatidylcholine; pc, phosphatidylcholine.

Figure 6

Schematic presentation of the mechanisms of repeated and intermittent sound stress (RISS)-induced chronic hyperalgesia. RISS inflicts excessive oxidative stress and lipid oxidisation, thus resulting in ensuing production of lysophosphatidylcholine (LPC) 16:0 via platelet-activating factor acetylhydrolase (PAF-AH) catalyzation or spontaneous deacylation of ox-PCs. LPC16:0 accordingly evokes nociceptive responses and causes chronic hyperalgesia by activating the acid-sensing ion channel 3 (ASIC3) in muscle tissue. By reducing LPC generation, PAF-AH inhibitor may thus be beneficial in the stress-related pain disorders, such as fibromyalgia. Ox-PCs, oxidised phosphatidylcholines; PCs, phosphatidylcholines; ROS, reactive oxygen species. WBC, white blood cell.