To observe the effect of electroacupuncture (EA) on mast cell (MC) activity, substance P (SP) and glial fibrillary acidic protein (GFAP) expression in the lumbar spinal dorsal horns (SDHs) and related inflammatory factors in the anterior tibial muscle, lumbar spinal cord and serum of fibromyalgia syndrome (FMS) model rats, so as to explore its mechanisms underlying improvement of FMS.

Thirty-six male SD rats were randomly divided into blank control, model and EA groups, with 12 rats in each group. The FMS model was established by injecting 2-morphine phenol ethanol sulfate into the left anterior tibial muscle. After successful modeling, EA was applied to “Zusanli” (ST36) and “Yanglingquan” (GB34) on the left hind limb for 20 min, once every other day for a total of 7 times. von Frey test was used to detect the mechanical pain threshold (MPT) of both hind limbs. H.E. staining was used to observe morphological changes of the anterior tibial muscle. Immunofluorescence staining was used to determine the expressions of SP, MC, GFAP and 5-hydroxtryptamine (5-HT) in the lumbar SDHs. Suspension chip method was used to detect the contents of inflammatory factors in the lumbar spinal cord, left anterior tibial muscle and serum.

Compared with the blank control group, the difference value of MPT between the bilateral hind-paws, immunofluorescence-positive number of SP and GFAP in the SDHs, contents of TNF-α, macrophage inflammatory protein-1α (MIP-1α), monocyte chemoattractant protein-1 (MCP-1) in the lumbar spinal cord, and the contents of MIP-α, MCP-1, interleukin (IL)-1α, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte-colony stimulating factor (G-CSF) in the left anterior tibial muscle, as well as TNF-α, IL-1β, GM-CSF and 5-HT contents in the serum were significantly increased (P<0.05), while the immunofluorescence-positive number of 5-HT and contents of 5-HT and IL-10 in the lumbar spinal cord, and IL-10 content in the left anterior tibial muscle and serum were strikingly decreased (P<0.05) in the model group. In comparison with the model group, the increase of the difference value of MPT between the bilateral hind-paws, immunofluorescence-positive number of SP and GFAP, contents of TNF-α, MIP-α and MCP-1 in the lumbar spinal cord, and the contents of MIP-α, MCP-1, IL-1α, GM-CSF and G-CSF in the left anterior tibial muscle, as well as the contents of TNF-α, IL-1β, GM-CSF and 5-HT in the serum, and the decrease of the immunofluorescence-positive number of 5-HT and contents of 5-HT and IL-10 in the lumbar spinal cord, and IL-10 content in the left anterior tibial muscle and serum were reversed in the EA group (P<0.05). Immunofluorescence staining displayed that no apparent changes in the number of MC in the lumbar SDHs after modeling and EA, and MCs presented degranulation in both the model group and EA group. H. E. staining showed disordered arrangement of muscle fibers, enlargement of the gaps between the muscle fibers, and obvious inflammatory infiltration of the anterior tibial muscle in the model group. Compared with the model group, the morphology of muscle fibers in the EA group was relatively complete, with orderly arrangement and reduction of the inflammatory infiltration.

EA of ST36 and GB34 can relieve pain and reduce the central and peripheral inflammatory response in FMS rats, which may be related to its functions in suppressing the expressions of SP and GFAP, and astrocytes activation in the lumbar SDHs and up-regulating the expression of 5-HT in the lumbar spinal cord.

To investigate the effect of wrist-ankle acupuncture on the α7 nicotinic acetylcholine receptor (α7nAChR) /Janus kinase 2 (JAK2) /signal transducer and activator of transcription 3 (STAT3) signaling pathway in the spinal dorsal horn of mice with chemotherapy-induced neuropathic pain (CINP), and to explore the mechanisms by which low-intensity wrist-ankle electroacupuncture alleviates pain hypersensitivity in CINP mice.

C57BL/6 mice were randomly divided into a blank group, a model group, a wrist-ankle acupuncture group, and a wrist-ankle acupuncture + antagonist group, with 8 mice in each group. A CINP model was established by intraperitoneal injection of paclitaxel (2 mg/kg). The wrist-ankle acupuncture group received electroacupuncture at the “Lower 4” and “Lower 5” points in the wrist-ankle region for 30 min per session, once every other day, for a total of 7 sessions. The wrist-ankle acupuncture + antagonist group received intrathecal injections of the α 7nAChR antagonist α-BGT (0.5 μg/kg) on day 1, 7, and 13, in addition to acupuncture. The mechanical and thermal pain thresholds were assessed using Von Frey filaments and hot plate tests. The expression level of α7nAChR protein in the spinal dorsal horn (L4-L6) was determined by Western blot. Immunofluorescence staining was used to detect the expression of ionized calcium-binding adapter molecule 1 (Iba-1) in the spinal dorsal horn. ELISA was used to measure the relative levels of phosphorylated (p) -JAK2 and p-STAT3 in the spinal dorsal horn. Quantitative PCR was used to detect the relative mRNA expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6).

Compared with the blank group, the model group exhibited decreased mechanical and thermal pain thresholds (P＜0.001), reduced α7nAChR protein expression (P＜0.05), increased Iba-1 expression (P＜0.05), elevated relative contents of p-JAK2 and p-STAT3 (P＜0.05), and increased mRNA expression levels of TNF-α, IL-1β, and IL-6 (P＜0.05). Compared with the model group, the wrist-ankle acupuncture group showed increased mechanical and thermal pain thresholds (P＜0.05), elevated α7nAChR protein expression (P＜0.05), decreased Iba-1 expression (P＜0.05), reduced relative contents of p-JAK2 and p-STAT3 (P＜0.05), and decreased mRNA expression levels of TNF-α, IL-1β, and IL-6 (P＜0.05). Compared with the wrist-ankle acupuncture group, the wrist-ankle acupuncture + antagonist group showed decreased mechanical and thermal pain thresholds (P＜0.05), lower α7nAChR protein expression (P＜0.05), increased Iba-1 expression (P＜0.05), higher relative contents of p-JAK2 and p-STAT3 (P＜0.05), and elevated mRNA expression levels of TNF-α, IL-1β, and IL-6 (P＜0.05).

Wrist-ankle acupuncture may alleviate pain hypersensitivity in CINP mice by regulating the α7nAChR/JAK2/STAT3 signaling pathway in the spinal dorsal horn, inhibiting microglial activation, and reducing neuroinflammation.

To observe the effect of low-intensity electroacupuncture (EA) at “Zusanli”(ST36) on the hippocampal cholinergic anti-inflammatory pathway and α7 nicotinic acetylcholine receptor (α7nAChR) of hippocampal microglia in mice with systemic inflammatory cognitive impairment, so as to investigate the central mechanism of EA in improving neuroinflammatory response and cognitive function.

C57BL/6 mice were randomly divided into blank, model and EA groups (6 mice/group); another set of C57BL/6 mice were randomly divided into blank, model, EA and EA+antagonist groups (6 mice/group); Cx3Cr1-Cre/ERT2 mice and α7nAChR^fl/fl mice were mated to construct transgenic mice with microglial α7nAChR knockout (EA+ knockout group, 6 mice), with the same litter negative mice used as non-knockout mice and randomly divided into blank, model+non-knockout and EA+non-knockout groups (6 mice/group). The systemic inflammatory cognitive impairment model was established by intraperitoneal injection of lipopolysaccharide (LPS, 1 mg/kg). Mice in the EA group received EA at bilateral ST36 (dense-sparse wave, 1 Hz/20 Hz, 0.5 mA), for 30 minutes each time; mice in the EA+antagonist group received injection of α7nAChR antagonist methyllycaconitine citrate (MLA) into hippocampus. The episodic memory behavioral tests were used to observe the abilities of mice in new object exploration, object location change recognition and object temporal order change memory. Nuclear magnetic resonance spectroscopy was used to observe the choline level in the hippocampal tissue. Western blot was used to detect the protein expression of α7nAChR in the hippocampus. Immunofluorescence staining was used to detect the expression of α7nAChR in hippocampal microglia and the activation of microglia. Real-time quantitative PCR was used to detect the mRNA expression of tumor necrosis factor-α (TNF-α) in hippocampus, and ELISA was used to detect the contents of α7nAChR, inflammatory factors and microglial phenotypic markers in hippocampal tissue.

Compared with the blank group, the abilities of new object exploration, location change recognition and object temporal order change memory, the choline level in the hippocampus, the protein expression level and content of α7nAChR and its co-expression with microglial markers, and the contents of anti-inflammatory factor interleukin (IL)-10 and M2 macrophage marker arginase 1 (Arg1) of mice in the model group were decreased (P<0.001, P<0.01); while the expression of hippocampal ionized calcium-binding adapter molecule 1 (Iba-1), the contents of M1 macrophage marker CD206, inducible nitric oxide synthase (iNOS), the content of pro-inflammatory factor IL-1β and the mRNA expression of TNF-α were increased (P<0.001, P<0.01). Compared with the model group, the above indexes in the EA group were reversed except for the ability of location change recognition (P<0.05, P<0.01, P<0.001). Compared with the EA group, the abilities of new object exploration and object temporal order change memory in the EA+antagonist group were decreased (P<0.05), and the mRNA expression of TNF-α was increased (P<0.01); the abilities of new object exploration and object temporal order change memory in the EA+knockout group were decreased (P<0.05), and the mRNA expression of TNF-α was increased (P<0.01).

EA at ST36 with low-intensity may improve neuroinflammatory response and systemic inflammatory cognitive dysfunction by increasing the choline level in the hippocampus and reducing the release of inflammatory factors through up-regulating the expression of α7nAChR in hippocampal microglia.