Antinociceptive actions of honokiol and magnolol on glutamatergic and inflammatory pain

The antinociceptive effects of honokiol and magnolol, two major bioactive constituents of the bark of Magnolia officinalis, were investigated on animal paw licking responses and thermal hyperalgesia induced by glutamate receptor agonists including glutamate, N-methyl-D-aspartate (NMDA), and metabotropic glutamate 5 receptor (mGluR5) activator (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), as well as inflammatory mediators such as substance P and prostaglandin E₂ (PGE₂) in mice. The actions of honokiol and magnolol on glutamate-induced c-Fos expression in the spinal cord dorsal horn were also examined. Our data showed that honokiol and magnolol blocked glutamate-, substance P- and PGE₂-induced inflammatory pain with similar potency and efficacy. Consistently, honokiol and magnolol significantly decreased glutamate-induced c-Fos protein expression in superficial (I-II) laminae of the L4-L5 lumbar dorsal horn. However, honokiol was more selective than magnolol for inhibition of NMDA-induced licking behavioral and thermal hyperalgesia. In contrast, magnolol was more potent to block CHPG-mediated thermal hyperalgesia. These results demonstrate that honokiol and magnolol effectively decreased the inflammatory pain. Furthermore, their different potency on inhibition of nociception provoked by NMDA receptor and mGluR5 activation should be considered.     

Ethyl Pyruvate Attenuates Formalin-induced Inflammatory Nociception by Inhibiting Neuronal ERK Phosphorylation

ABSTRACT: BACKGROUND: Ethyl pyruvate (EP) possesses anti-inflammatory activity. However, the potential anti-nociceptive value of EP for the treatment of the inflammatory nociception is largely unknown. We investigated whether EP could have any anti-nociceptive effect on inflammatory pain, after systemic administration of EP (10, 50, and 100 mg/kg, i.p.), 1 hour before formalin (5%, 50 mul) injection into the plantar surface of the hind paws of rats. RESULTS: EP significantly decreased formalin-induced nociceptive behavior during phase II, the magnitude of paw edema, and the activation of c-Fos in L4-L5 spinal dorsal horn. EP also attenuated the phosphorylation of extracellular signal-regulated kinase (ERK) in the neurons of L4-L5 spinal dorsal horn after formalin injection. Interestingly, the i.t. administration of PD-98059, an ERK upstream kinase (MEK) inhibitor, completely blocked the formalin-induced inflammatory nociceptive responses. CONCLUSIONS: These results demonstrate that EP may effectively inhibit formalin-induced inflammatory nociception via the inhibition of neuronal ERK phosphorylation in the spinal dorsal horn, indicating its therapeutic potential in suppressing acute inflammatory pain.     

CGRP受体拮抗剂CGRP8-37对甲醛炎性痛大鼠自发痛反应及脊髓后角NOS表达和NO含量的影响

目的:探讨CGRP受体拮抗剂CGRP8-37对甲醛炎性痛大鼠自发痛反应及脊髓后角NOS表达和NO含量的影响.方法:大鼠足底注射甲醛制造炎性痛模型;计数缩足反射次数反映自发痛程度;NADPH-d组织化学法观察脊髓后角NOS表达;硝酸还原酶法测定NO-3/NO-2含量以反映NO含量.结果:足底注射甲醛后,动物出现自发痛反应行为.足底注射甲醛后24 h,双侧脊髓后角NOS表达及NO含量明显增加.预先鞘内注射CGRP8-37可使甲醛诱导的自发性缩足反射次数明显减少,并可明显抑制甲醛炎性痛诱导的脊髓后角NOS表达及NO含量的增加.结论:甲醛炎性痛时,脊髓后角CGRP受体激活可促进NOS活性表达及NO的产生.     

Endomorphin-2, deltorphin II and their analogs suppress formalin-induced nociception and c-Fos expression in the rat spinal cord

In this study, we evaluated the effects of intrathecally administered agonists of mu- and delta-opioid receptor and their analogs on the pain-induced behavior and expression of c-Fos immunoreactivity in the spinal cord, elicited by intraplantar injection of 12% formalin to the hindpaw of the rat. Previous report from our laboratory and other author's study indicated that intrathecal administration of mu agonists morphine and endomorphin-2 and delta-opioid agonist deltorphin II produced a dose-dependent antinociceptive effects in acute and inflammatory pain. In this study, intrathecal injection of morphine (10 microg), endomorphin-2 (5 microg) and its analog Dmt-endomorphin-2 (10 microg) significantly decreased the formalin-induced pain behavior, and lowered a number of c-Fos positive neurons in the laminae I, II and III of the spinal cord by about 40%, 30% and 40%, respectively. Significant reduction of formalin-induced behavioral responses was also observed after i.th. administration of deltorphin II (15 microg) and its analog ile-deltorphin II (15 microg). Agonists of delta-opioid receptor significantly reduced a number of c-Fos positive neurons by about 28% and 40%, respectively. Analog of endomorphin-2 and analog of deltorphin II suppressed more potently expression of c-Fos in the dorsal horn of the spinal cord than the parent peptides. Our study indicates that new analogs of mu- and delta-opioid receptor exhibit strong antinociceptive potency similar or even higher than the parent peptides, and that their effect is positively correlated with the inhibition of c-Fos expression.     

Significant changes in mitochondrial distribution in different pain models of mice

Mitochondria play an important role in pathophysiology of inflammatory and neuropathic pain but the mechanism is unclear. So far no comprehensive study exists that evaluates the changes of mitochondrial dynamics following the pain. In this study, we detected the mitochondrial distribution and subcellular morphology by using intrathecal injection of mitochondrial marker, Mitotracker Red® CM-H2XRox (Mito-Red) and confocal microscopic analysis in models of formalin-induced acute inflammatory pain, Complete Freund's Adjuvant (CFA)-induced persistent pain and spared nerve injury (SNI)-induced neuropathic pain. The results demonstrated that subcutaneous formalin injection did not affect the number of Mito-Red cells within the spinal dorsal horn at both acute and tonic phases, but significantly increased the number of cluster type mitochondria in superficial spinal dorsal horn (laminas I–II) at tonic phase. Differently, the number of Mito-Red cells significantly increased in superficial and deep spinal dorsal horn (laminas III–V) following persistent CFA and SNI neuropathic pain. Moreover, both CFA and SNI remarkably increased the number of cluster type mitochondria and decreased the number of granule type mitochondria, in both superficial and deep spinal dorsal horn. So we concluded that abnormal mitochondrial distribution contributes to neuropathic and some forms of inflammatory pain.     

Prevention by celecoxib of secondary hyperalgesia induced by formalin in rats

Administration of formalin in rat paws results in stimulation of nociceptive pathways, which leads to an increase in the excitability of neurons present in dorsal horn. This increased neuron excitability, described as central sensitization, may result in development of inflammatory pain at a distant site of injury application, known as secondary hyperalgesia. The aim of the present study was to verify whether formalin injection in rat paws would lead to secondary hyperalgesia development, as measured by the tail-flick test. We also aimed to investigate whether celecoxib, a specific cyclooxygenase 2 (COX-2) inhibitor, would affect secondary hyperalgesia. Formalin injected into the rat paws significantly reduced the latency for a flick response in the rat tail, which characterized development of secondary hyperalgesia. In addition, formalin-induced secondary hyperalgesia was locally prevented by pre-but not post-celecoxib treatment. However, celecoxib administered spinally inhibited formalin-induced secondary hyperalgesia, either administered previously or following formalin. In contrast, piroxicam, an unspecific COX inhibitor which displays an increased selectivity towards COX-1, only prevented secondary hyperalgesia to formalin at a high dose following spinal administration. Taken together, these results suggest that COX-2 plays an important role both in the central and in the peripheral nerve sensitization following formalin administration in rat paws. They also suggested that once central sensitization starts it can no longer be blocked by a specific COX-2 inhibitor administered locally. Notwithstanding, spinal administration of a specific COX-2 inhibitor still blocks ongoing sensitization and prevents maintenance of central sensitization.     

Increased Sensitivity to Inflammatory Pain Induced by Subcutaneous Formalin Injection in Serine Racemase Knock-Out Mice

D-Serine, an endogenous coagonist of the N-methyl-D-aspartate receptor (NMDAR), is widely distributed in the central nervous system and is synthesized from L-serine by serine racemase (SR). NMDAR plays an important role in pain processing including central sensitization that eventually causes hyperalgesia. To elucidate the roles of D-serine and SR in pain transmission, we evaluated the behavioral changes and spinal nociceptive processing induced by formalin using SR knock-out (KO) mice. We found that SR is mainly distributed in lamina II of the dorsal horn of the spinal cord in wild-type (WT) mice. Although the formalin injected subcutaneously induced the biphasic pain response of licking in SR-KO and WT mice, the time spent on licking was significantly longer in the SR-KO mice during the second phase of the formalin test. The number of neurons immunopositive for c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK), which are molecular pain markers, in laminae I-II of the ipsilateral dorsal horn was significantly larger in the SR-KO mice. Immunohistochemical staining revealed that the distribution of SR changed from being broad to being concentrated in cell bodies after the formalin injection. On the other hand, the expression level of the cytosolic SR in the ipsilateral dorsal horn significantly decreased. Oral administration of 10 mM D-serine in drinking water for one week cancelled the difference in pain behaviors between WT and SR-KO mice in phase 2 of the formalin test. These findings demonstrate that the SR-KO mice showed increased sensitivity to inflammatory pain and the WT mice showed translocation of SR and decreased SR expression levels after the formalin injection, which suggest a novel antinociceptive mechanism via SR indicating an important role of D-serine in pain transmission.     

MK-801抑制福尔马林实验引起的大鼠脊髓背角环氧合酶-2的表达

应用免疫组织化学方法,观察鞘内注射N-methyl—D—aspartate(NMDA)受体拮抗剂MK-801对福尔马林实验引起的大鼠脊髓背角环氧合酶-2(cyclooxygenase-2,COX-2)表达的影响。结果表明：MK-801对福尔马林实验引起的第1相缩足反射仅有一定抑制作用,但对第2相缩足反射有显著的抑制作用,且呈剂量依赖性。与这种行为学的变化相对应,MK-801可显著抑制福尔马林实验引起的脊髓背角COX-2表达的增加,并且这种抑制作用与MK-801的剂量呈正相关。这些结果表明,在福尔马林实验中,NMDA受体的活动是引起脊髓背角COX-2表达增加的原因之一。     

甲醛炎性痛对大鼠脊髓HO-1表达的影响

目的:观察足底注射甲醛引起的外周组织炎性疼痛是否可诱导大鼠脊髓血红素氧合酶-1(HO-1)表达发生改变以及变化的时程特征。方法:健康雄性SD大鼠随机分为7组(n=6):对照组(control组)、甲醛6 h组(F6 h组)、甲醛12 h(F12 h组)、甲醛1 d组(F1 d组)、甲醛2 d组(F2 d组)、甲醛3 d组(F3 d组)和甲醛7 d组(F7 d组)。采用足底注射甲醛溶液复制炎性痛模型,采用免疫组织化学方法检测左、右两侧脊髓后角以及中央管周围灰质HO-1蛋白的表达。结果:Control组大鼠HO-1免疫反应阳性细胞在脊髓后角及中央管周围灰质仅有少量分布,且这些细胞染色较浅。足底注射甲醛后6 h,L5节段双侧脊髓后角和中央管周围灰质HO-1免疫反应阳性细胞数目即有所增多,足底注射甲醛后12 h时,双侧脊髓后角和中央管周围灰质HO-1免疫反应阳性细胞数目进一步增多,阳性细胞染色明显加深,1 d时阳性细胞数目和染色深度均达到高峰,7 d时仍高于control组水平。各时间点双侧脊髓后角比较,阳性细胞数目和阳性细胞染色深度均无明显差异。结论:大鼠足底注射甲醛引起的炎性痛可诱导双侧脊髓后角和中央管周围灰质HO-1表达增多,以注射甲醛后1 d时增多最为明显。     

Comparison of the antinociceptive effect of celecoxib, diclofenac and resveratrol in the formalin test

The peripheral antinociceptive effect of the selective COX-2 inhibitor celecoxib in the formalin-induced inflammatory pain was compared with that of resveratrol (COX-1 inhibitor) and diclofenac (non-selective COX inhibitor). Rats received local pretreatment with saline, celecoxib, diclofenac or resveratrol followed by 50 microl of either 1% or 5% formalin. Peripheral administration of celecoxib did not produce antinociception at either formalin concentration. In contrast, diclofenac and resveratrol produced a dose-dependent antinociceptive effect in the second phase of both 1% and 5% formalin test. The peripheral antinociception produced by diclofenac or resveratrol was due to a local action, as drug administration in the contralateral paw was ineffective. Results indicate that the selective COX-2 inhibitor celecoxib does not produce peripheral antinociception in formalin-induced inflammatory pain. In contrast, selective COX-1 and non-selective COX inhibitors (resveratrol and diclofenac, respectively) are effective drugs in this model of pain.     

Reduced pain behaviors and extracellular signal-related protein kinase activation in primary sensory neurons by peripheral tissue injury in mice lacking platelet-activating factor receptor

Peripheral tissue injury causes the release of various mediators from damaged and inflammatory cells, which in turn activates and sensitizes primary sensory neurons and thereby produces persistent pain. The present study investigated the role of platelet-activating factor (PAF), a phospholipid mediator, in pain signaling using mice lacking PAF receptor (pafr-/- mice). Here we show that pafr-/- mice displayed almost normal responses to thermal and mechanical stimuli but exhibit attenuated persistent pain behaviors resulting from tissue injury by locally injecting formalin at the periphery as well as capsaicin pain and visceral inflammatory pain without any alteration in cytoarchitectural or neurochemical properties in dorsal root ganglion (DRG) neurons and a defect in motor function. However, pafr-/- mice showed no alterations in spinal pain behaviors caused by intrathecally administering agonists for N-methyl-d-aspartate (NMDA) and neurokinin(1) receptors. A PAFR agonist evoked an intracellular Ca(2+) response predominantly in capsaicin-sensitive DRG neurons, an effect was not observed in pafr-/- mice. By contrast, the PAFR agonist did not affect C- or Adelta-evoked excitatory post-synaptic currents in substantia gelatinosa neurons in the dorsal horn. Interestingly, mice lacking PAFR showed reduced phosphorylation of extracellular signal-related protein kinase (ERK), an important kinase for the sensitization of primary sensory neurons, in their DRG neurons after formalin injection. Furthermore, U0126, a specific inhibitor of the ERK pathway suppressed the persistent pain by formalin. Thus, PAFR may play an important role in both persistent pain and the sensitization of primary sensory neurons after tissue injury.     

MK—801降低炎性痛在鼠脊髓NOS表达和NO含量

用NADPH-d组织化学法,观察鞘内注射NMDA受体拮抗剂MK-801对大鼠右后掌皮下注射甲醛诱发的炎症性痛及痛过敏过程中脊髓后角一氧化氮合酶(NOS)表达的影响,同时测定一氧化氮(NO)代谢终产物     

Long-term ovariectomy changes formalin-induced licking in female rats: the role of estrogens

Gonadal hormones have been shown to exert modulatory effects on nociception and analgesia. To investigate the role of gonadal hormones in the response by female rats to both phasic and persistent nociceptive stimulation, we evaluated the effects of long-term ovariectomy (OVX, 6 months) on the thermal pain threshold and on formalin-induced responses. The thermal pain threshold was evaluated with the plantar test apparatus, while persistent pain was induced by a subcutaneous injection of dilute formalin (50 microliter, 10%) in the dorsal hind paw. The formalin test was carried out in an open field apparatus where the animal's spontaneous behavior and formalin-induced responses (licking duration, flinching frequency and flexing duration of the injected paw) were recorded for 60 min. Estradiol and corticosterone plasma levels were determined in blood collected from the anesthetized animals at the end of the test. In OVX females, the duration of formalin-induced licking was longer than in Intact females during both the first and the second phase; flinching and flexing did not differ from Intact. The thermal pain threshold was only slightly affected by OVX. Estradiol and corticosterone were lower in OVX females than Intact ones. These data indicate that long-term depletion of gonadal hormones in female rats modulates the pain-induced behavioral responses related to supraspinal neural circuits (licking of the injected paw) rather than more spinally mediated responses such as formalin-induced flinching and withdrawal latency in the plantar test.     

靶向毁损触液核对大鼠痛行为及脊髓背角5-HT和c-Fos表达的影响

目的：观察缺失触液核（CSF-contacting nucleus）对大鼠痛行为及脊髓背角痛相关物质5-羟色胺（5-HT）和c—Fas表达的影响,为触液核参与疼痛调制及机制提供实验依据。方法：成年雄性SD大鼠随机分为正常组（Control）,假手术组（Sham）,霍乱毒素亚单位B与辣根过氧化酶复合物（CB—HRP）组和毁损触液核组（Damage）。以机械缩足阈值（MWT）和热缩足潜伏期（耶儿）测定大鼠痛行为。免疫荧光法检测脊髓背角5-HT和c—Fos表达,并进行痛行为阈值与物质变化趋势的相关分析。结果：与Control、Sham和CB—HRP组相比,Damage组大鼠MWT和TWL明显降低（P〈0．05）。免疫荧光结果显示,正常大鼠触液核神经元高表达5-HT;Damage组大鼠触液核神经元数量随毁损天数延续逐渐减少,且在给予毁损剂CB—SAP第10天完全消失。与此同时脊髓背角5-HT和c—Fos表达量日趋增加,且与痛行为阈值变化趋势成负相关。结论：CB—SAP能科学可靠靶向毁损触液核,缺失触液核可致大鼠痛行为阈值减低,而脊髓背角5-HT和c—Fos表达量增加。本研究提示触液核参与了疼痛调制,且5-HT和c—Fos在此调制中发挥了重要作用。     

Spinal NTS1 receptors regulate nociceptive signaling in a rat formalin tonic pain model

Central administration of the neuropeptide neurotensin (NT) was shown to induce antinociceptive responses both spinally and supraspinally. Although NTS2 receptors play an important role in modulating the activity of spinal neurons, we have recently implicated NTS1 receptors in NT's analgesic effects in acute spinal pain paradigms. The current experiments were thus designed to examine the antinociceptive effects of intrathecal administration of NTS1 agonists in formalin-induced tonic pain in rats. We first established, using immunoblotting and immunohistochemical approaches, that NTS1 receptors were present in small- and medium-sized dorsal root ganglion cells and localized in the superficial layers of the dorsal horn of the spinal cord. We then examined the effects of intrathecal injection of NT (1–15 μg/kg) or NTS1 preferring agonists on the nocifensive response to intraplantar formalin. Both NTS1-agonists, PD149163 (10–120 μg/kg) and NT69L (1–100 μg/kg), dose-dependently attenuated the formalin-induced behaviors. Accordingly, NTS1 agonists markedly suppressed pain-evoked c- fos expression in the superficial, nucleus proprius and neck regions of the spinal dorsal horn. The concomitant administration of PD149163 with the NTS1 antagonist SR48692 (3 μg/kg) significantly reversed PD149163-induced antinociception, confirming the implication of NTS1 in tonic pain. In contrast, NT69L's analgesic effects were partly abolished by co-administration of SR48692, indicating that NT69L-induced effects may also be exerted through interaction with NTS2. These results demonstrate that NTS1 receptors play a key role in the mediation of the analgesic effects of NT in persistent pain and suggest that NTS1-selective agonists may represent a new line of analgesic compounds.     

神经激肽-1(NK-1)受体介导福尔马林诱发的大鼠脊髓c-fos表达

本文用Ｆｏｓ作为背角伤害性反应神经元活动的一个标志物,结合免疫细胞化学和神经药理学方法,观察了速激肽受体拮抗剂对福尔马林诱发的脊髓ｃ－ｆｏｓ原癌基因表达的影响。一侧大鼠后肢跖部皮下注射福尔马林,仅在同侧脊髓背角有ｃ－ｆｏｓ表达。Ｆｏｓ阳性神经元最密集分布于Ｉ层和Ⅱ层背侧的内侧部,中等量分布于Ⅳ层和Ｖ型,少量定位于Ⅱ层腹侧部、Ⅲ、Ⅵ和Ⅹ层。若预先在一侧大鼠后肢跖部皮下注射福尔马林前,鞘内给予神经激肽     

Fluorocitrate,an Inhibitor of Glial Metabolism,Inhibits the Up-Regulation of NOS Expression,Activity and NO Production in the Spinal Cord Induced by Formalin Test in Rats

Previous experiments have suggested that nitric oxide plays an important role in nociceptive transmission in the spinal cord. In order to explore the involvement of glia in the NO-mediated nociceptive transmission, the present study was undertaken to investigate the effect of fluorocitrate (FC), an inhibitor of glial metabolism, on NOS expression and activity and NO production in the spinal cord during the process of peripheral inflammatory pain and hyperalgesia induced by formalin test in rats. Sixty adult male Sprague–Dawley rats were randomly assigned into sham, formalin, formalin + normal saline (NS), and formalin + FC groups. The NOS expression, NOS activity and NO production was detected by NADPH-d histochemistry staining, NOS and NO assay kit, respectively. It was found that formalin test significantly up-regulated NOS expression and activity and NO production in the laminae I–II of the dorsal horn and the grey matter around the central canal in the lumbar spinal cord at 1 h after the formalin test. Selective inhibition of glia metabolism with intrathecal administration of FC (1 nmol) significantly inhibited the up-regulation in NOS expression and activity and NO production normally induced by the formalin test, which was represented with decreases in the number and density of the NADPH-d positive cells in the dorsal horn and grey matter around the central canal, and decrease in density of NADPH-d positive neuropil in the dorsal horn in formalin + FC group compared with formalin group. The results suggested that glia may be involved in the NO-mediated nociceptive transmission in the spinal cord. X.-C. Sun, W.-N. Chen and S.-Q. Li contributed equally to this work.     

ERK MAP Kinase Activation in Spinal Cord Regulates Phosphorylation of Cdk5 at Serine 159 and Contributes to Peripheral Inflammation Induced Pain/Hypersensitivity

Cyclin-dependent kinase 5 is a proline-directed serine/threonine kinase and its activity participates in the regulation of nociceptive signaling. Like binding with the activators (P35 or P25), the phosphorylation of Cdk5 plays a critical role in Cdk5 activation. However, it is still unclear whether Cdk5 phosphorylation (p-Cdk5) contributes to pain hyperalgesia. The aim of our current study was to identify the roles of p-Cdk5 and its upstream regulator in response to peripheral inflammation. Complete Freund''s adjuvant (CFA) injection induced acute peripheral inflammation and heat hyperalgesia, which was accompanied by sustained increases in phospho-ERK1/2 (p-ERK1/2) and phospho-Cdk5^S159 (p-Cdk5^S159) in the spinal cord dorsal horn (SCDH). CFA-induced p-ERK primarily colocalized with p-Cdk5^S159 in superficial dorsal horn neurons. Levels in p-ERK and p-Cdk5 were also increased in the 2^nd phase of hyperalgesia induced by formalin injection, which can produce acute and tonic inflammatory pain. MAP kinase kinase inhibitor U0126 intrathecal delivery significantly suppressed the elevation of p-Cdk5^S159, Cdk5 activity and pain response behavior (Heat hyperalgesia, Spontaneous flinches) induced by CFA or formalin injection. Cdk5 inhibitor roscovitine intrathecal administration also suppressed CFA-induced heat hyperalgesia and Cdk5 phosphorylation, but did not attenuate ERK activation. All these findings suggested that p-Cdk5^S159 regulated by ERK pathway activity may be a critical mechanism involved in the activation of Cdk5 in nociceptive spinal neurons contributes to peripheral inflammatory pain hypersensitivity.     

Inhibition of Spinal Interlukin-33/ST2 Signaling and Downstream ERK and JNK Pathways in Electroacupuncture Analgesia in Formalin Mice

Although acupuncture is widely used to manage pain, it remains highly controversial, largely due to the lack of a clear mechanism for its benefits. Here, we investigated the role of IL-33, a novel interleukin (IL)-1 family member, and its receptor ST2 in the analgesic effects of electroacupuncture (EA) on formalin-induced inflammatory pain. The results showed that 1) EA stimulation of ipsilateral Zusanli (ST 36) and Yanglingquan (GB 34) acupoints for 30 min remarkably suppressed the two phases of formalin-induced spontaneous pain; 2) subcutaneous or intrathecal administration of recombinant IL-33 (rIL-33) significantly inhibited the analgesic effect of EA, whereas the ST2 antibody potentiated EA analgesia in formalin mice; 3) EA treatment decreased the up-regulation of IL-33 and ST2 protein following formalin injection; and 4) the suppression of the formalin-induced expression of spinal phosphorylated ERK and JNK induced by EA treatment was significantly attenuated following subcutaneous rIL-33 delivery, and was further decreased by the ST2 antibody. These data suggest that EA alleviates formalin-induced inflammatory pain, at least partially, by inhibiting of spinal IL-33/ST2 signaling and the downstream ERK and JNK pathways.