Electroacupuncture Reduces Fibromyalgia Pain via Neuronal/Microglial Inactivation and Toll-like Receptor 4 in the Mouse Brain: Precise Interpretation of Chemogenetics

Abstract

Fibromyalgia (FM) is a complex, chronic, widespread pain syndrome that can cause significant health and economic burden. Emerging evidence has shown that neuroinflammation is an underlying pathological mechanism in FM. Toll-like receptors (TLRs) are key mediators of the immune system. TLR4 is expressed primarily in microglia and regulates downstream signaling pathways, such as MyD88/NF-κB and TRIF/IRF3. It remains unknown whether electroacupuncture (EA) has therapeutic benefit in attenuating FM pain and what role the TLR4 pathway may play in this effect. We compared EA with sham EA to eliminate the placebo effect due to acupuncture. We demonstrated that intermittent cold stress significantly induced an increase in mechanical and thermal FM pain in mice (mechanical: 2.48 ± 0.53 g; thermal: 5.64 ± 0.32 s). EA but not sham EA has an analgesic effect on FM mice. TLR4 and inflammatory mediator-related molecules were increased in the thalamus, medial prefrontal cortex, somatosensory cortex (SSC), and amygdala of FM mice, indicating neuroinflammation and microglial activation. These molecules were reduced by EA but not sham EA. Furthermore, a new chemogenetics method was used to precisely inhibit SSC activity that displayed an anti-nociceptive effect through the TLR4 pathway. Our results imply that the analgesic effect of EA is associated with TLR4 downregulation. We provide novel evidence that EA modulates the TLR4 signaling pathway, revealing potential therapeutic targets for FM pain.

Keywords: TLR4; chemogenetics; electroacupuncture; fibromyalgia; microglia; thalamus.

Figure 1

Mechanical withdrawal, thermal latency, and the concentration of inflammatory mediators in normal, FM, EA, and sham EA mice. (A) Mechanical threshold from the von Frey tests. (B) Thermal latency from the Hargreaves’ test. (C) IL-1β, IL-2, IL-5, IL-6, and IL-9 and (D) IL-12, IL-17A, TNF-α, IFN-γ, and MCP-1 in mice plasma. * means statistical difference when compared to the normal mice. ^# indicates statistical significance when compared to the FM groups. IL = Interleukin; IFN = Interferon; TNF = Tumor necrosis factor; MCP = Monocyte chemoattractant protein. n = 6 in all groups.

Figure 2

The levels of Iba1, TLR4, and related molecules in the mouse thalamus. Western blot bands contain four lanes of protein expression in the following order: normal, FM, 2 Hz EA, and sham EA. (A) Iba1, TLR4, MyD88, and TRAF6. (B) pERK, pp38, pJNK, and pNFκB. * indicates statistical significance when compared with the normal group. ^# indicates statistical significance when compared to the FM group. n = 6 in all groups. (C) Immunofluorescence staining of TLR4, Iba1, and double staining to assess protein expression in the mouse thalamus. Scale bar represents 100 μm. n = 4 in all groups.

Figure 3

The levels of Iba1, TLR4, and related molecules in the mouse mPFC. Western blot bands contain four lanes of protein expression in the following order: normal, FM, 2 Hz EA, and sham EA. (A) Iba1, TLR4, MyD88, and TRAF6. (B) pERK, pp38, pJNK, and pNFκB. * indicates statistical significance when compared with the normal group. ^# indicates statistical significance when compared to the FM group. n = 6 in all groups. (C) Immunofluorescence staining of TLR4, Iba1, and double staining to assess protein expression in the mouse mPFC. Scale bar represents 100 μm. n = 4 in all groups.

Figure 4

The levels of Iba1, TLR4, and related molecules in the mouse SSC. Western blot bands contain four lanes of protein expression in the following order: normal, FM, 2 Hz EA, and sham EA. (A) Iba1, TLR4, MyD88, and TRAF6. (B) pERK, pp38, pJNK, and pNFκB. * indicates statistical significance when compared with the normal group. ^# indicates statistical significance when compared to the FM group. n = 6 in all groups. (C) Immunofluorescence staining of TLR4, Iba1, and double staining to assess protein expression in the mouse SSC. Scale bar represents 100 μm. n = 4 in all groups.

Figure 5

The levels of Iba1, TLR4, and related molecules in the mouse amygdala. Western blot bands contain four lanes of protein expression in the following order: normal, FM, 2 Hz EA, and sham EA. (A) Iba1, TLR4, MyD88, and TRAF6. (B) pERK, pp38, pJNK, and pNFκB. * indicates statistical significance when compared with the normal group. ^# indicates statistical significance when compared to the FM group. n = 6 in all groups. (C) Immunofluorescence staining of TLR4, Iba1, and double staining for protein expression in the mouse amygdala. Scale bar represents 100 μm. n = 4 in all groups.

Figure 6

Pain behavior of FM and FM mice treated with the chemogenetics technique. Black column: FM group, red column: FM treated with chemogenetics. * p < 0.05 compared with the FM group. (A) Mechanical hyperalgesia (von Frey test). (B) Thermal hyperalgesia (Hargreaves’ test). (C) Protein levels of Iba1, TLR4, MyD88, TRAF6, pERK, pJNK, pp38, and pNFkB were measured in the mouse SSC.

Figure 7

Toll-like receptor (TLR) signaling pathway with a focus on TLR4. Abbreviations: ERK = Extracellular signal-regulated kinase; IFN = Interferon; JNK = c-Jun N-terminal kinase; MyD88 = Myeloid differentiation primary-response protein 88; TLR, Toll-like receptors; TRAF = Tumor necrosis factor receptor-associated factor.