**1. Introduction**

Inflammatory pain is a common clinical symptom that widely exists in various acute and chronic diseases [1]. However, its underlying mechanism is still unclear. At present, anti-inflammatory and analgesic drugs are commonly used in the clinic, but drug treatment is accompanied by gastrointestinal discomfort and other side effects [2].

Electroacupuncture (EA), a modified technique based on the theory of traditional manual acupuncture, has been widely used in clinical and scientific research [3,4]. Acupoints, frequency and the period of stimulation are three main elements that affect the effect of EA. Different frequencies of EA stimulation at the same acupoint may lead to different therapeutic effects, while the same frequency may also exert "pain-type" specific analgesic effects on different pathological conditions [5,6]. Our previous studies compared the analgesic effects of 2, 100 and 2/100 Hz EA on chronic pain models of inflammatory pain and neuropathic pain, e.g., Freund's complete adjuvant (CFA) injection and

spared nerve injury (SNI), respectively. We found that 100 Hz exhibited the best analgesic effect on inflammatory pain [7]. However, whether the analgesic effects of EA are related to the period of stimulation is largely unknown.

P2X3 is purinoceptor with ion channel activity in response to extracellular adenosine triphosphate (ATP) [8,9]. P2X3 is highly expressed in small- and medium-sized nociceptors of dorsal root ganglions (DRGs) and has been associated with pain response in multiple animal pain models, including the bone cancer pain model, chronic constriction nerve injury (CCI) model, spared nerve injury (SNI) model, and the CFA model [10–13]. P2X3 can be found in the peripheral terminals where it senses the ATP leaked from damaged tissues or released from inflammatory cells [14]. In addition, P2X3 is distributed in the central terminals of nociceptors, indicating that P2X3 might play presynaptic roles in the spinal cord dorsal horn [15]. Indeed, blockage of P2X3 at the spinal cord level significantly reduced postsynaptic neuronal hyperexcitability in a model of bone cancer pain [16]. Previous studies demonstrated that the expression of P2X3 was upregulated by CFA induced inflammation and CCI in the DRG and spinal cord dorsal horn (SCDH), respectively [7,17]. Notably, EA stimulation was reported to attenuate CFA (100 Hz) and CCI (15 Hz) induced pain hypersensitivity [18,19]. We speculated that EA stimulation modulated P2X3 levels in pain pathways and then exerted analgesic actions on chronic inflammatory pain.

In this study, the analgesic effects of short-term and long-term 100 Hz EA stimulation on CFA-induced chronic inflammatory pain were detected and compared. The potential involvement of P2X3 underlying EA mediated analgesia was also explored.
