Electroacupuncture improves gout arthritis pain via attenuating ROS-mediated NLRP3 inflammasome overactivation

Abstract

Background: Gout results from disturbed uric acid metabolism, which causes urate crystal deposition in joints and surrounding tissues. Gout pain management is largely limited to colchicine and nonsteroidal anti-inflammatory drugs. Constant usage of these medications leads to severe side effects. We previously showed electroacupuncture (EA) is effective for relieving pain in animal model of gout arthritis. Here we continued to study the mechanisms underlying how EA alleviates gout pain.

Methods: Monosodium urate was injected into ankle joint to establish gout arthritis model in mice. EA or sham EA was applied at ST36 and BL60 acupoints of model animals. Biochemical assays, immunostaining, live cell Ca²⁺ imaging and behavioral assays were applied.

Results: Model mice displayed obvious mechanical allodynia, accompanied with gait impairments. EA attenuated mechanical hypersensitivities and improved gait impairments. EA reduced the overexpression of NLRP3 inflammasome signaling molecules in ankle joints of model animals. EA-induced anti-allodynia, as well as inhibition on NLRP3 inflammasome, were mimicked by antagonizing but abolished by activating NLRP3 inflammasome via pharmacological methods. EA attenuated oxidative stress, an upstream signaling of NLRP3 inflammasome in ankle joints of model mice. Exogenously increasing oxidative stress abolished EA's inhibitory effect on NLRP3 inflammasome and further reversed EA's anti-allodynic effect. EA reduced neutrophil infiltrations in ankle joint synovium, a major mechanism contributing to oxidative stress in gout. Pharmacological blocking NLRP3 inflammasome or EA reduced TRPV1 channel overexpression in dorsal root ganglion (DRG) neurons. Ca²⁺ imaging confirmed that EA could reduce functional enhancement in TRPV1 channel in DRG neurons during gout.

Conclusions: Our results demonstrate that EA reduces gout pain possibly through suppressing ROS-mediated NLRP3 inflammasome activation in inflamed ankle joints and TRPV1 upregulation in sensory neurons, supporting EA as a treatment option for gout pain.

Keywords: Acupuncture; Gout; Inflammasome; Inflammation; Pain; TRPV1.

Fig. 1

The establishment of the gout arthritis mouse model and EA’s intervention on pain in mice. A Representative photos showing ipsilateral ankles injected with sterile PBS (control group) or MSU (+ MSU group). The right panels denote the enlarged ankle joint. B Time courses showing the percent increase in ankle diameter of control and MSU group mice. BL: basal level. **p < 0.01 vs. control group. C Representative photos of pathological sections of ankle joint synovium from control and MSU group mice. The scale bar indicates 20 μm. D Summary of the number of infiltrated cells per observation field. **p < 0.01 vs. control group. E Experimental protocol illustrating time points for model establishment, EA/sham EA interventions and mechanical/thermal pain measurements. F 50% PWT changes of control, MSU, MSU + EA and MSU + sham EA groups. **p < 0.01 vs. control group. ^##p < 0.01 vs. MSU + sham EA. G Normalized AUC analysis of curves shown in panel F. The value from the control group was taken as 100%, and other groups were normalized thereafter. H Time course showing PWL changes of 4 groups. **p < 0.01 vs. control group. ^##p < 0.01 vs. MSU + sham EA. I Normalized AUC analysis of the data in panel H. n = 5–8 mice/group. **p < 0.01, NS no significance

Fig. 2

EA intervention improved gait impairments of gout arthritis model mice. A Schedule for model establishment, gait analysis and EA/sham EA treatment. B Representative pictures illustrating the mice from each group recorded by the gait analysis system at 0 (baseline), 8 or 24 h time points after model establishment. The left panel shows the instant images of the mice on the treadmill. The right panel shows the paw area being analyzed. The letter L or R denote left or right, whereas F or H denote forepaw or hind paw, respectively. C–E Summary of paw area ratio (RH/LH), stride length ratio (RH/LH) and swing ratio (RH/LH) of baseline (− 24 h), 8 h and 24 h time points. n = 7–8 mice/group. *p < 0.05, **p < 0.01. NS no significance

Fig. 3

EA intervention attenuated NLRP3 inflammasome activation in ankle joints of gout arthritis model mice. A–E Western blotting examination of protein expressions of NLRP3 inflammasome signaling components, including NLRP3 (A), Caspase-1 (B) ASC (C) and inflammatory cytokines IL-1β (D), IL-18 (E) in ankle joint tissues from control, MSU, MSU + EA and MSU + sham EA groups of mice. Upper panels showed the representative Western blot gels, whereas lower panels indicated the summarized data. n = 5–6 mice/group. *p < 0.05. NS no significance

Fig. 4

Specific blocking NLRP3 inflammasome activation ameliorates mechanical allodynia of gout arthritis model mice. A Molecular structure of MCC950. B Schedual illustrating model establishment, MCC950 administration, PWT test and tissue collection. C–E Western blotting examination of protein expression of NLRP3 inflammasome components, including NLRP3 (C), Caspase-1 (D) and IL-1β (E) in ankle joints from control, MSU + Veh and MSU + MCC950 groups of mice. *p < 0.05, **p < 0.01. F Time courses showing 50% PWT changes of ipsilateral hindpaws. **p < 0.01 vs. control group. ^##p < 0.01 vs. MSU + Veh group. G Normalized AUC analysis of the data in panel F. *p < 0.05, **p < 0.01. n = 5–7 mice/group

Fig. 5

Pharmacological activation of NLRP3 inflammasome in ankle joints reversed EA-induced anti-allodynia in gout model mice. A Molecular structure of NLRP3 inflammasome activator nigericin. B Schedule illustrating nigericin (NG)/vehicle application, PWT test and tissue collection. (C-E) Western blotting examination of protein expression NLRP3 (C), Caspase-1 (D) and IL-1β (E) in ankle joints of MSU + EA + Veh and MSU + EA + NG groups. F Time courses showing 50% PWT changes of ipsilateral hindpaws of MSU + EA + Veh and MSU + EA + NG group mice. G Normalized AUC analysis of panel F. n = 5–7 mice/group. *p < 0.05, **p < 0.01 vs. MSU + EA + Veh group

Fig. 6

EA intervention attenuates oxidative stress in ankle joint tissues of gout arthritis model mice. A–C Biochemical assays of oxidative stress markers, including GSH-Px activity, SOD activity and H₂O₂ level in control, MSU, MSU + EA and MSU + sham EA groups of mice. D Representative gels of Western blotting examining tissue expressions of 4-HNE-protein adducts. E Summarized data as in panel D. F 8-OHdG immunostaining in periarticular tissues of 4 groups. G Summary of 8-OHdG immunostaining in 4 groups. n = 5–8 mice/group. *p < 0.05, **p < 0.01

Fig. 7

EA-induced antiallodynia and inhibition of NLRP3 inflammasome in model mice are reversed by H₂O₂. A Schedule depicting the time points for model establishment, H₂O₂ and EA application and tissue collection. B 50% PWT changes in MSU + EA + H₂O₂, MSU + EA + Veh and MSU + ShamEA + Veh groups. **p < 0.01 vs. MSU + sham EA + PBS group, ^##p < 0.01 vs. MSU + EA + PBS. C Normalized AUC analysis of curves shown in panel B. (D&E) Western blot of NLRP3 and IL-1β expressions in ankle joint tissues. *p < 0.05, **p < 0.01. n = 6 mice/group

Fig. 8

EA intervention attenuates neutrophil infiltrations in synovium of ankle joints of gout arthritis model mice. A Cartoon showing the design of Ly6G-IRES-EGFP knock-in mouse line, which enables direct visualization of neutrophils by GFP signal. B Representative photos of the fluorescence of Ly6G-EGFP⁺ neutrophils in ankle joint synovarium sections from control, MSU, MSU + EA and MSU + sham EA groups. DAPI was used as a counterstain. Scale bar: 50 μm. C Summarized numbers of Ly6g-EGFP⁺ neutrophils/field in each group. **p < 0.01. n = 5–7 mice/group. NS no significance

Fig. 9

MCC950 or EA intervention reduces pain-sensing TRPV1 channel overexpression in DRG neurons innervating the hindlimbs. A Schedule depicting MCC950/vehicle, EA/sham interventions and DRG tissue collection. B Representative photos of TRPV1 immunostaining (in green) in DRG neurons across all groups. NeuN staining (in red) was used to identify all DRG neurons. C, D Summary of the percentage of TRPV1 positively stained (TRPV1⁺) neurons among all DRG neurons in each group. n = 5 mice/group. E Representative overlaid Ca²⁺ transients recorded in DRG neurons from control (left panel), MSU + shamEA (middle panel) and MSU + EA (right panel) group of mice. Capsaicin (300 nM) was applied to activate TRPV1 channels. KCl (40 mM) was perfused to determine all alive neurons. 90 cells were included in each panel. F Summarized data for delta increase in peak R_340/380 caused by capsaicin of 3 groups. n = 5–6 tests/group. G Summarized data for the percentage of capsaicin positively responding (capsaicin⁺) neurons of 3 groups. > 200 neurons were included in each group. *p < 0.05, **p < 0.01