Electroacupuncture Pretreatment Alleviates Cerebral Ischemic Injury Through α7 Nicotinic Acetylcholine Receptor-Mediated Phenotypic Conversion of Microglia.

Electroacupuncture (EA) pretreatment alleviates cerebral ischemic injury through α7 nicotinic acetylcholine receptor (α7nAChR). We attempted to investigate whether the phenotypic conversion of microglia was involved in the therapeutic effect of EA pretreatment in cerebral ischemia through α7nAChR. Adult male Sprague-Dawley (SD) rats were subjected to middle cerebral artery occlusion (MCAO) after EA or α7nAChR agonist N-(3R)-1-azabicyclo[2.2.2]oct-3-yl-furo[2,3-c]pyridine-5-carboxamide hydrochloride (PHA-543,613 hydrochloride) and antagonist α-bungarotoxin (α-BGT) pretreatment. Primary microglia were subjected to drug pretreatment and oxygen-glucose deprivation (OGD). The expressions of the classical activated phenotype (M1) microglia markers induced nitric oxide synthase (iNOS), interleukin-1ß (IL-1ß), and cluster of differentiation 86 (CD86); the alternative activated phenotype (M2) microglia markers arginase-1 (Arg-1), transforming growth factor-ß1 (TGF-ß1), and cluster of differentiation 206 (CD206); and the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and anti-inflammatory cytokines interleukin-4 (IL-4) and interleukin-10 (IL-10) in the ischemic penumbra or in the supernatant of primary microglia were analyzed. The infarction volume and neurological scores were assessed 72 h after reperfusion. The cell viability and lactate dehydrogenase (LDH) release of neurons co-cultured with microglia were analyzed using cell counting kit-8 (CCK-8) and LDH release assays. EA pretreatment decreased the expressions of M1 markers (iNOS, IL-1ß, and CD86) and pro-inflammatory cytokines (TNF-α and IL-6), whereas it increased the expressions of M2 markers (Arg-1, TGF-ß1, and CD206) and anti-inflammatory cytokines (IL-4 and IL-10) by activating α7nAChR. EA pretreatment also significantly reduced the infarction volume and improved the neurological deficit. The activation of α7nAChR in microglia relieved the inflammatory response of primary microglia subjected to OGD and attenuated the injury of neurons co-cultured with microglia. In conclusion, EA pretreatment alleviates cerebral ischemic injury through α7nAChR-mediated phenotypic conversion of microglia, which may be a new mechanism for the EA pretreatment-induced neuroprotection against cerebral ischemia.

Inhibition of chemokine CX3CL1 in spinal cord mediates the electroacupuncture-induced suppression of inflammatory pain.

PURPOSE: Chemokine CX3CL1 and its receptor CX3CR1 in the lumbar spinal cord play crucial roles in pain processing. Electroacupuncture (EA) is recognized as an alternative therapy in pain treatment due to its efficacy and safety. However, the analgesic mechanism of EA remains unclear. The aim of this study was to investigate whether EA suppressed complete Freund's adjuvant (CFA)-induced pain via modulating CX3CL1-CX3CR1 pathway. MATERIALS AND METHODS: Inflammatory pain was induced by intraplantar injection of CFA to the left hind paw of Sprague-Dawley rats. EA with 2 Hz for 30 mins was given to bilateral Zusanli acupoints (ST36) on the first and third day after CFA injection. Mechanical allodynia and thermal hyperalgesia were tested with von Frey tests and Hargreaves tests, respectively. The expressions of CX3CL1, CX3CR1 and p38 mitogen-activated protein kinase (MAPK) were quantified with Western blots. The release of IL-1ß, IL-6 and TNF-α were evaluated with ELISA. Recombinant CX3CL1 or control IgG were then injected through intrathecal catheters in the EA-treated CFA model rats. The behavioral tests, p38 MAPK activation and cytokine release were then evaluated. RESULTS: EA significantly inhibited inflammatory pain induced by CFA for 3 days. Meanwhile, EA downregulated the expression of CX3CL1 but not CX3CR1 in the lumbar spinal cord of the CFA rats. Besides, activation of p38 MAPK and the release of pain-related cytokines (IL-1ß, IL-6 and TNF-α) were inhibited by EA. Intrathecal injection of CX3CL1 largely reversed the analgesic effect of EA treatment and re-activated p38 MAPK signaling, and resulted in pro-inflammatory cytokines increase in acupuncture-treated rats. CONCLUSION: Our findings indicate that EA alleviates inflammatory pain via modulating CX3CL1 signaling in lumbar spinal cord, revealing a potential mechanism of anti-nociception of EA in inflammatory pain.

Acupoint Sensitization is Associated with Increased Excitability and Hyperpolarization-Activated Current (Ih) in C- But Not Aδ-Type Neurons.

Under pathological conditions, acupoint sensitization is the phenomenon of acupoints transforming from the stable state to the dynamic state. Evidences suggest that hyperpolarization-activated current (Ih), conducted by the hyperpolarization-activated/cyclic nucleotide-gated (HCN) channel, greatly contributes to the peripheral and central sensitization. However, the role of the Ih current in acupoint sensitization has not been explained. In the present study, changes in excitability, Ih density and the HCN channel of dorsal root ganglion (DRG) nociceptive neurons were examined in the later phase of knee osteoarthritis (KOA) rats. To investigate the neuronal specificity of acupoint sensitization, retrograde dyes were injected into the acupoints ST35 and GB37. The results showed that acupoint sensitization occurred in bilateral ST35 but not GB37 acupoints. The excitability and Ih density of C- but not Aδ-type neurons innervating ST35 acupoint increased in bilateral L5 DRG of acupoint sensitized rats than that of sham rats. No obvious changes were found in the excitability or Ih density of C- and Aδ-type neurons innervating the GB37 acupoint in the bilateral L5 DRG. HCN channel subtype 2 (HCN2) expression levels significantly increased after acupoint sensitization. Furthermore, ZD7288, an HCN current (Ih) blocker, attenuated the acupoint sensitization of the ST35 acupoint. Taken together, our findings suggest that the increased excitability of C- but not Aδ-type neurons and the upregulation of Ih/HCN2 channels contribute to the formation of acupoint sensitization.

Spinal Cord Glycine Transporter 2 Mediates Bilateral ST35 Acupoints Sensitization in Rats with Knee Osteoarthritis.

The concept of "acupoint sensitization" refers to the functional status of acupoint switches from silent to active under pathological conditions. In clinic, acupoint sensitization provides important guidance for acupoints selection in different diseases. However, the mechanism behind this phenomenon remains unclear. We generated a model of knee osteoarthritis (KOA) by intra-articular injection of monosodium iodoacetate (MIA) into the left knee of rats. The paw withdrawal mechanical threshold (PWMT) and the total number of mast cells as well as mast cell degranulation rate (MCDR) of acupoint tissue were used to test whether the acupoints were sensitized. The results showed that KOA resulted in a reduced mechanical threshold and elevated total number of mast cell as well as mast cell degranulation rate at bilateral ST35 (Dubi) but not GB37 (Guangming) or nonacupoint area. The acupoint sensitization was accompanied by upregulation of glycine transporter 2 (GlyT2) and reduction of extracellular glycine levels in the bilateral dorsal horns of the spinal cord at L3-5. Selective inhibition of GlyT2 or intrathecal administration of glycine attenuated ST35 acupoint sensitization. The sensitization of bilateral ST35 was blocked after intraspinal GlyT2 short hairpin (sh) RNA (GlyT2-shRNA) microinjection to specifically downregulate GlyT2 expression in the left side (ipsilateral) L3-5 spinal cord dorsal horn before MIA injection. Moreover, electroacupuncture (EA) stimulation at ST35 ameliorated articular pathological lesions and improved KOA-related pain behaviors. GlyT2-shRNA injection reversed EA-induced pain relief but not EA-induced reduction of joint lesions. Overall, this study demonstrated that spinal GlyT2, especially elevated GlyT2 expression in the ipsilateral dorsal horn of the spinal cord, is a crucial mediator of ST35 acupoint sensitization in KOA rats.

A rat model of knee osteoarthritis suitable for electroacupuncture study.

Acupuncture is widely used for knee osteoarthritis (KOA) treatment in clinical practice. In the present study, we aimed to set a standard KOA animal model for electroacupuncture (EA) study and provide an acupuncture recipe for further KOA studies. Rats intra-articularly administered monosodium iodoacetate (MIA, 0.3, 1 or 3 mg respectively, n=12 each) were evaluated for pain-like behavior: paw withdrawal mechanical threshold, weight bearing deficit, and joint pathological changes (OARSI score) until 28 days after injury. Then by using the suitable dose (1 mg MIA), therapeutic effects of EA treatment (bilateral ST36 and ST35 acupoints, 2/10 Hz, 30 min/d, 6d/w, 2w) were evaluated in 3 groups (n=16 each): Early-on EA, Mid-term EA and Delayed EA, in which EA was started on day 1, day 7 or day 14 after MIA injection. Both 1 mg and 3 mg MIA induced significant joint damage and persistent pain behavior. But animals accepted 3 mg MIA rapidly developed cartilage and bone damage within 14 days. Early-on EA treatment provided significant pain relief and joint structure preservation in KOA rats. Mid-term EA treatment only reduced pain, while delayed EA treatment resulted in no effects in both aspects. 1 mg of MIA produces steady pain behavior and progressive joint damage, which was suitable for EA treatment evaluation. Early-on EA treatment provided both joint protection and pain reduction, while Mid-term EA could only be used for studying EA-induced analgesia in KOA.

Electroacupuncture Improves Neurobehavioral Function Through Targeting of SOX2-Mediated Axonal Regeneration by MicroRNA-132 After Ischemic Stroke.

Our previous studies have shown that electroacupuncture (EA) enhances neurobehavioral functional recovery after ischemic stroke, however, the underlying regulatory mechanisms remain unclear. MicroRNAs (miRNAs) are abundant in the brain and are involved in post-transcriptional gene regulation. During cerebral ischemia reperfusion, miRNAs perform numerous biological functions in the central nervous system related to regeneration and repair of damaged nerves. Our previous studies also have shown that the expression of miRNA-132 (miR-132) is obviously down-regulated after stroke by middle cerebral artery occlusion (MCAO), which can be up-regulated by EA. This study aimed to identify whether up-regulation of miR-132 by EA improved the damaged nerves after stroke and to screen the potential target of miR-132. The results showed that EA up-regulated miR-132 thus suppressing SOX2 expression in vivo after MCAO, which obviously ameliorated neurobehavioral functional recovery. Moreover, our results also suggested that up-regulated miR-132 suppressed SOX2 in primary neurons after oxygen-glucose deprivation (OGD), which promoted neurite outgrowth. In conclusion, EA enhances neurobehavioral functional recovery against ischemic stroke through targeting of SOX2-mediated axonal regeneration by miR-132.

Electroacupuncture Alleviates Brain Damage Through Targeting of Neuronal Calcium Sensor 1 by miR-191a-5p After Ischemic Stroke.

Electroacupuncture (EA) administration before or after cerebral ischemia has been shown to afford protection against ischemic injury. However, the underlying mechanism of EA-mediated protection is still unclear. Functional microRNAs (miRNAs) are believed to play important roles in neuroprotection and synaptic plasticity during and after ischemia. In a previous study, we identified 20 miRNAs that are expressed in the penumbra and are significantly changed after EA treatment. Here, we used bioinformatic analysis to predict the biological functions and gene networks of these miRNAs. Consistent with our predictions, downregulation of miR-191a-5p in primary neurons and in cortexes of rats increased cell viability, decreased apoptosis, reduced infarct volumes, and improved neurological scores; whereas upregulation of miR-191a-5p exacerbated neuronal injury and partly reversed the neuroprotective effect of EA treatment after ischemia/reperfusion injury. In silico analysis predicted that miR-191a-5p targets neuronal calcium sensor 1 (NCS-1), brain-derived neurotrophic factor, and growth-associated protein 43 (GAP43), and using luciferase reporter assays, we confirmed that the NCS-1 3'UTR (untranslated region) is targeted by miR-191a-5p. Furthermore, lentivirus-mediated overexpression of NCS-1 in primary neurons and in the cortexes of rats induced neuroprotection, while lentivirus-mediated knockdown had the opposite effect. Taken together, these data suggest that miRNAs participate in the response to EA treatment after cerebral ischemia and further imply that NCS-1 may constitute a miR-191a-5p target gene and a potential therapeutic target for neuroprotection.

The endocannabinoid system, a novel and key participant in acupuncture's multiple beneficial effects.

Acupuncture and its modified forms have been used to treat multiple medical conditions, but whether the diverse effects of acupuncture are intrinsically linked at the cellular and molecular level and how they might be connected have yet to be determined. Recently, an emerging role for the endocannabinoid system (ECS) in the regulation of a variety of physiological/pathological conditions has been identified. Overlap between the biological and therapeutic effects induced by ECS activation and acupuncture has facilitated investigations into the participation of ECS in the acupuncture-induced beneficial effects, which have shed light on the idea that the ECS may be a primary mediator and regulatory factor of acupuncture's beneficial effects. This review seeks to provide a comprehensive summary of the existing literature concerning the role of endocannabinoid signaling in the various effects of acupuncture, and suggests a novel notion that acupuncture may restore homeostasis under different pathological conditions by regulating similar networks of signaling pathways, resulting in the activation of different reaction cascades in specific tissues in response to pathological insults.

Electroacupuncture enhances rehabilitation through miR-181b targeting PirB after ischemic stroke.

Recent studies have demonstrated microRNAs (miRNAs) and proteins are beneficial to axon regeneration, which may be involved in Electroacupuncture (EA) therapy against stroke. In this study, we aimed to determine the pivotal role of PirB in EA-produced rehabilitation against ischemic stroke; and to screen and investigate the potential miRNAs directly regulating PirB expression. The results showed EA treatment enhanced axon regeneration and new projections from the corticospinal tract at 28 d after cerebral ischemic reperfusion injury of rats. Then, we found EA decreased pirb mRNA and PirB protein expression in the penumbra within 28 days after reperfusion. The reduction of PirB expression facilitated neurite outgrowth after oxygen-glucose deprivation injury. The miRNA microarray showed the level of twenty kinds of miRNAs changed in the penumbra after EA administration. The bioinformatics study and luciferase assay verified miR-181b directly regulated pirb mRNA expression. EA increased miR-181b levels in the penumbras, and improved neurobehavioral function rehabilitation through miR-181b direct targeting of pirb mRNA to regulate the expression of PirB, RhoA and GAP43. In conclusion, we provide the first evidence that EA enhances rehabilitation against stroke by regulating epigenetic changes to directly act on its targets, such as the miR-181b/PirB/RhoA/GAP43 axis, which is a novel mechanism of EA therapy.

Involvement of GluR2 up-regulation in neuroprotection by electroacupuncture pretreatment via cannabinoid CB1 receptor in mice.

We investigated whether glutamate receptor subunit 2 (GluR2) is involved in EA pretreatment-induced neuroprotection via cannabinoid CB1 receptors (CB1R) after global cerebral ischemia in mice. Two hours after electric acupuncture (EA) pretreatment, global cerebral ischemia (GCI) was induced by bilateral common carotid artery occlusion (BCCAO) for 20 min. The GluR2 expression was examined in the hippocampus after reperfusion. Cell survival, neuronal apoptosis, the Bax/Bcl-2 ratio and neurological scores were evaluated at 24 h after BCCAO in the presence or absence of the GluR2 inhibitor. Furthermore, the GluR2 was determined in the presence and absence of CB1R inhibitor. Our results showed EA pretreatment enhanced expression of GluR2 in the hippocampus 2 h after reperfusion. Moreover, EA pretreatment improved neurological outcome, promoted cell survival, inhibited neuronal apoptosis, and decreased the Bax/Bcl-2 ratio after reperfusion. GluR2 knockdown by GluR2 siRNA effectively reversed the beneficial effects of EA pretreatment. Furthermore, CB1R siRNA and two CB1R antagonists blocked the elevation of GluR2 expression by EA pretreatment, whereas the two CB1R agonists up-regulated GluR2 expression as EA pretreatment. In conclusion, GluR2 up-regulation is involved in neuroprotection of EA pretreatment against GCI through CB1R, suggesting that GluR2 may be a novel target for stroke intervention.