Role And Mechanism Of Spinal IL-33and Its Receptor ST2in Electroacupuncture Analgesia On Inflammatory Pain

Inflammatory pain is a continuous unpleasant sensory and emotional experience associated with tissue damage caused by a variety of reasons, including trauma, bacterial, viral infections and surgical operation. Inflammatory pain, one of the most common pathological pain, is a difficult clinical problem. At present, most of the analgesic drugs for inflammatory pain are not ideal for their side effects. Therefore, researche on the mechanism of inflammatory pain have been hot worldwide. However, the exact mechanism is still not completely clear. A large number of clinical practice and basic research indicate that acupuncture can relieve inflammation pain, while the mechanism is still not very clear, needing further research.IL-33is a new proinflammatory cytokine found by Schmitz et al in2005, which has the similar gene sequence and structure like other IL-1family members such as IL-1β and IL-18. Its receptor ST2is one of the Toll-like family members. IL-33is documented to mediate in a variety of inflammatory diseases. And it is reported that peripheral IL-33is involved in inflammatory pain. Our preliminary experiments showed that IL-33and its receptor ST2can be expressed in the spinal cord of normal rat. Also, the increased expression of IL-33and ST2in arthritic pain could be downregulated by electroacupuncture treatment, suggesting spinal IL-33and its receptor ST2may be involved in inflammatory pain and in electroacupuncture analgesia.IL-33combines with its receptor ST2, and causes the recruitment of MyD88. Subsequently, transcription factors such as nuclear factor-KB (NF-κB) and mitogen-activated protein kinases (MAPKs) are activated, including ERK, p38and JNK. Lots of studies and previous experiments of our lab showed that spinal MAPK signaling pathway mediated the inflammatory pain, neuropathic pain and cancer pain.Documents show that, inflammation can cause the central sensitization for pain in central nervous system.The process of central sensitization is related to many neurotransmitter and glia. In the primary integration center of pain--spinal cord, glia can directly regulate the activity and the excitability of neurons, and play an important role in pain modulation, especially in chronic pain formation and maintenance of central sensitization. Our previous research has shown that, arthritis pain and spinal cord glial cell activation in rats play an important role in electroacupuncture analgesia.It is reported that, astrocyte in the central nervous system (CNS) can express IL-33, and ST2can be expressed in neuron and astrocytes in CNS.Whether the expression of IL-33increased in spinal cord during inflammatory pain? Whether IL-33combines with its receptor ST2on the cell membrane surface in an autocrine or paracrine manner to involve in the development and process of inflammatory pain? Whether they involve in electroacupuncture analgesia in inflammatory pain? Whether their downstream signaling pathway MAPK in spinal cord involved in this process? All above need to be answered in this study.Therefore, in this experiment, we use male BALB/C mice to establish an acute inflammatory pain model by plantar subcutaneous injection of20μl5%formalin, and also a chronic inflammatory pain model by plantar subcutaneous injection of20μl CFA (Complete Freund’s Adjuvant). In the formalin pain model, we observe the spontaneous pain behavior, including spontaneous paw lifting and paw licking in the injected paw, spontaneous ultrasonic vocalization as well. In the CFA pain model, we observe the mechanical threshold by using Von Frey and the paw with latency by using radiant thermal hyperalgesia. Moreover, to explore the underlying mechanism, we also apply Immunofluorescence combined with confocal laser technology, Real-time PCR technology, Western Blot technology. The present study is designed:(1) to observe whether IL-33and its receptor ST2mediate in inflammatory pain, including acute inflammatory pain induced by formalin and chronic inflammatory pain induced by CFA, and the distribution of IL-33and its receptor ST2in spinal cord,and its underlying mechanism;(2) to observe analgesic effects of electroacupuncture in formalin-induced acute inflammatory pain and CFA-induced chronic pain;(3) to observe roles of IL-33and its receptor ST2in electroacupuncture analgesia in inflammatory pain, and also its underlying mechanism.The main results of the present research were as following:I. The mechanism and role of IL-33and its receptor in mice inflammatory painWe established the formalin-induced acute miceinflammatory pain model and the CFA-induced chronic mice inflammatory pain model successfully and we observe the spontaneous behavior, including paw lifting, paw licking and ultrasonic vocalization in the formalin model and mechanical threshold and radiant thermal hyperalgesia (paw withdrawl lantency) in the CFA model to evaluate the inflammatory pain models.In ST2gene knockout mice, the pain behavior of both formalin and CFA mice model are alleviated significantly, compared with the corresponding control group.Both the subcutaneous injection and the intrathecal injection of recombinant IL-33can increase the spontaneous pain behavior in normal mice, including paw lifting time and paw lifting time significantly, compared with PBS control group.Both the subcutaneous injection and the intrathecal injection of recombinant IL-33can aggravate the spontaneous pain behavior (paw lifting and paw licking), compared with Formalin+PBS control group.Moreover, the subcutaneous injection or the intrathecal injection of anti-ST2antibody can alleviate the spontaneous pain of formalin model and even alleviate pain behavior aggravated by recombinant IL-33.Immunofluorescence technology combined with Laser Scanning Confocal Microscopy shows that, IL-33expresses in the astrocyte in mice spinal cord, and its receptor ST2expresses in the astrocyte and neuron of the spinal cord.Subcutaneous injection of naloxone (the antagonist of opioid receptor) had no effects on the spontaneous pain behavior of formalin mice model with the administration of anti-ST2antibody.Western Blot shows that subcutaneous injection of anti-ST2antibody can reduce the expression of pERK in mice spinal cord significantly, compared with Formalin+IgG control group.Real-time PCR shows that the expression of IL-33mRNA in the spinal cord of CFA mice model increases on the1st and7th day after the operation, compared with NS control group.II. The analgesic role of electroacupuncture (EA) in inflammatory painThe behavioral tests show that, electroacupuncture decreases the spontaneous paw lifting and paw licking time of formalin model significantly, compared with Formalin+Sham EA control group.Besides, electroacupuncture decreases the mechanical threshold and paw withdrawal latency of CFA model significantly on the4thh/6th hour,1st day,7th day after the operation, compared with CFA+Sham EAcontrol group.Ⅲ. The mechanism and role of IL-33and its receptor in electroacupuncture (EA) analgesia in inflammatory painBehavioral tests show that:In CFA model of ST2gene knockout mice, the paw withdrawal latency has been decreased compared with electroacupuncture control group.Subcutaneous injection or intrathecal injection of recombinant IL-33can aggravate the pain behavior of formalin mice model alleviated by electoracupuncture; subcutaneous or intrathecal injection of anti-ST2antibody can further alleviate the pain behavior of formalin mice model reduced by electoracupuncture.Western Blot shows that compared with the electroacupuncture control group, subcutaneous injection of anti-ST2antibody can reduce expression of the spinal pERK in formalin mice model.In conclusion, the present study suggests that:1. Injection of recombinant IL-33can increase the paw lifting time and licking time of the injected paw, which suggests that IL-33can work as a proinflammatory cytokine and can induce mild pain behavior in normal animals.2. Spinal IL-33mediates formalin-induced acute inflammatory pain and CFA-induced chronic pain through its receptor ST2.3. Spinal IL-33and its receptor ST2mediate formalin-induce pain through their downstream MAPK ERK signaling pathway.4. Spinal IL-33mediates electroacupuncture analgesia in inflammatory pain of mice through its receptor ST2.5. Spinal IL-33and its receptor ST2mediate electroacupuncture analgesia in formalin-induced inflammatory pain through their downstream MAPK ERK signaling pathway.