Xiaomei Shao

IL-33/ST2 signaling excites sensory neurons and mediates itch response in a mouse model of poison ivy contact allergy

Significance In the United States, the most common cause of allergic contact dermatitis (ACD) is contact with poison ivy. Severe itch and skin inflammation are the major manifestations of poison ivy-induced ACD. In this study, we have established a critical role of IL-33/ST2 (interleukin 33/growth stimulation expressed gene 2) signaling in both itch and skin inflammation of poison ivy-induced ACD and revealed a previously unidentified interaction of IL-33/ST2 signaling with primary sensory neurons that may underlie the pruritic mechanisms of poison ivy-induced ACD. Blocking IL-33/ST2 signaling may represent a therapeutic approach to ameliorate itch and skin inflammation related to poison ivy dermatitis and, possibly, other chronic itch conditions in which IL-33/ST2 signaling may participate. Poison ivy-induced allergic contact dermatitis (ACD) is the most common environmental allergic condition in the United States. Case numbers of poison ivy ACD are increasing due to growing biomass and geographical expansion of poison ivy and increasing content of the allergen, urushiol, likely attributable to rising atmospheric CO2. Severe and treatment-resistant itch is the major complaint of affected patients. However, because of limited clinical data and poorly characterized models, the pruritic mechanisms in poison ivy ACD remain unknown. Here, we aim to identify the mechanisms of itch in a mouse model of poison ivy ACD by transcriptomics, neuronal imaging, and behavioral analysis. Using transcriptome microarray analysis, we identified IL-33 as a key cytokine up-regulated in the inflamed skin of urushiol-challenged mice. We further found that the IL-33 receptor, ST2, is expressed in small to medium-sized dorsal root ganglion (DRG) neurons, including neurons that innervate the skin. IL-33 induces Ca2+ influx into a subset of DRG neurons through neuronal ST2. Neutralizing antibodies against IL-33 or ST2 reduced scratching behavior and skin inflammation in urushiol-challenged mice. Injection of IL-33 into urushiol-challenged skin rapidly exacerbated itch-related scratching via ST2, in a histamine-independent manner. Targeted silencing of neuronal ST2 expression by intrathecal ST2 siRNA delivery significantly attenuated pruritic responses caused by urushiol-induced ACD. These results indicate that IL-33/ST2 signaling is functionally present in primary sensory neurons and contributes to pruritus in poison ivy ACD. Blocking IL-33/ST2 signaling may represent a therapeutic approach to ameliorate itch and skin inflammation related to poison ivy ACD.

Oxidized Phospholipid OxPAPC Activates TRPA1 and Contributes to Chronic Inflammatory Pain in Mice.

Oxidation products of the naturally occurring phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphatidylcholine (PAPC), which are known as OxPAPC, accumulate in atherosclerotic lesions and at other sites of inflammation in conditions such as septic inflammation and acute lung injury to exert pro- or anti-inflammatory effects. It is currently unknown whether OxPAPC also contributes to inflammatory pain and peripheral neuronal excitability in these conditions. Here, we observed that OxPAPC dose-dependently and selectively activated human TRPA1 nociceptive ion channels expressed in HEK293 cells in vitro, without any effect on other TRP channels, including TRPV1, TRPV4 and TRPM8. OxPAPC agonist activity was dependent on essential cysteine and lysine residues within the N-terminus of the TRPA1 channel protein. OxPAPC activated calcium influx into a subset of mouse sensory neurons which were also sensitive to the TRPA1 agonist mustard oil. Neuronal OxPAPC responses were largely abolished in neurons isolated from TRPA1-deficient mice. Intraplantar injection of OxPAPC into the mouse hind paw induced acute pain and persistent mechanical hyperalgesia and this effect was attenuated by the TRPA1 inhibitor, HC-030031. More importantly, we found levels of OxPAPC to be significantly increased in inflamed tissue in a mouse model of chronic inflammatory pain, identified by the binding of an OxPAPC-specific antibody. These findings suggest that TRPA1 is a molecular target for OxPAPC and OxPAPC may contribute to chronic inflammatory pain through TRPA1 activation. Targeting against OxPAPC and TRPA1 signaling pathway may be promising in inflammatory pain treatment.

Electroacupuncture exerts an anti-inflammatory effect in a rat tissue chamber model of inflammation via suppression of NF-κB activation

Objective Electroacupuncture (EA) has beneficial effects in patients with various inflammatory diseases. However, the underlying mechanism remains unclear. As the kappa B inhibitor/nuclear factor-kappa B (IκB/NF-κB) pathway exerts a pivotal role in the mammalian immune response, we examined the involvement of the IκB/NF-κB pathway in EA-induced anti-inflammation. Methods Ninety tissue chamber implanted rats were randomly divided into control (C), model (M) and EA (E) groups. Physiological saline and human recombinant interleukin-1β (hr IL-1β) were injected into the rats in groups C and M, respectively, and EA treatment was applied to the rats in group E after IL-1β injection. Nuclear staining of p65 (a subunit of NF-κB) was quantified in the exudate cells by immunohistochemical analysis and IκBα expression in the cytoplasm was quantified by western blot analysis. Results Our results showed that, compared with group C, the percentage of cells with nuclear-localised p65 was increased in group M by 71.3%, 50.7% and 33.1% at 1, 5 and 24 h time points (p<0.01), respectively. This increase was fully inhibited in group E at 5 and 24 h time points (p<0.01). The expression of IκBα was stably enhanced in group M (p<0.05) during the test period. Compared with group M, greater expression of IκBα in group E was only observed at the 1 h time point (p<0.01). Conclusions Collectively, our data suggest that EA inhibits the nuclear translocation of p65 and increases the expression of IκBα, which leads to the suppression of NF-κB activation in a rat tissue chamber model of inflammation.

Inhibition of the cAMP/PKA/CREB Pathway Contributes to the Analgesic Effects of Electroacupuncture in the Anterior Cingulate Cortex in a Rat Pain Memory Model

Pain memory is considered as endopathic factor underlying stubborn chronic pain. Our previous study demonstrated that electroacupuncture (EA) can alleviate retrieval of pain memory. This study was designed to observe the different effects between EA and indomethacin (a kind of nonsteroid anti-inflammatory drugs, NSAIDs) in a rat pain memory model. To explore the critical role of protein kinase A (PKA) in pain memory, a PKA inhibitor was microinjected into anterior cingulate cortex (ACC) in model rats. We further investigated the roles of the cyclic adenosine monophosphate (cAMP), PKA, cAMP response element-binding protein (CREB), and cAMP/PKA/CREB pathway in pain memory to explore the potential molecular mechanism. The results showed that EA alleviates the retrieval of pain memory while indomethacin failed. Intra-ACC microinjection of a PKA inhibitor blocked the occurrence of pain memory. EA reduced the activation of cAMP, PKA, and CREB and the coexpression levels of cAMP/PKA and PKA/CREB in the ACC of pain memory model rats, but indomethacin failed. The present findings identified a critical role of PKA in ACC in retrieval of pain memory. We propose that the proper mechanism of EA on pain memory is possibly due to the partial inhibition of cAMP/PKA/CREB signaling pathway by EA.

Effects of Electroacupuncture with Dominant Frequency at SP 6 and ST 36 Based on Meridian Theory on Pain-Depression Dyad in Rats.

Epidemic investigations reveal an intimate interrelationship between pain and depression. The effect of electroacupuncture (EA) on pain or depression has been demonstrated individually, but its effect on pain-depression dyad is unknown. Our study aimed to screen a dominant EA frequency on pain-depression dyad and determine the validity of acupoint selection based on meridian theory. The pain-depression dyad rat model was induced by reserpine and treated using EA with different frequencies at identical acupoints to extract a dominant frequency and then administrated dominant-frequency EA at different acupoints in the above models. Paw withdrawal latency (PWL), emotional behavior of elevated zero maze (EZM) test, and open field (OF) test were conducted. We found that 100 Hz EA at Zusanli (ST 36) and Sanyinjiao (SP 6) (classical acupoints for spleen-deficiency syndrome) were the most effective in improving PWL, travelling distance in the EZM, and maximum velocity in OF compared to EA with other frequencies; ST 36 and SP 6 were proved more effective than other acupoints beyond the meridian theory and nonacupoints under the same administration of EA. Therefore, we concluded that 100 Hz is the dominant frequency for treating the pain-depression dyad with EA, and acupoints on spleen and stomach meridians are preferable choices.

Strong Manual Acupuncture Stimulation of “Huantiao” (GB 30) Reduces Pain-Induced Anxiety and p-ERK in the Anterior Cingulate Cortex in a Rat Model of Neuropathic Pain

Persistent neuropathic pain is associated with anxiety. The phosphorylation of extracellular signal-regulated kinase (p-ERK) in the anterior cingulate cortex (ACC) plays an important role in pain-induced anxiety. Acupuncture is widely used for pain and anxiety. However, little is known about which acupuncture technique is optimal on pain-induced anxiety and the relationship between acupuncture effect and p-ERK. The rat model was induced by L5 spinal nerve ligation (SNL). Male adult SD rats were randomly divided into control, SNL, strong manual acupuncture (sMA), mild manual acupuncture (mMA), and electroacupuncture (EA) group. Bilateral “Huantiao” (GB 30) were stimulated by sMA, mMA, and EA, respectively. The pain withdrawal thresholds (PWTs) and anxiety behavior were measured, and p-ERK protein expression and immunoreactivity cells in ACC were detected. PWTs increased significantly in both sMA and EA groups. Meanwhile, anxiety-like behavior was improved significantly in the sMA and mMA groups. Furthermore, the overexpression of p-ERK induced by SNL was downregulated by strong and mild manual acupuncture. Therefore, strong manual acupuncture on bilateral “Huantiao” (GB 30) could be a proper therapy relieving both pain and pain-induced anxiety. The effect of different acupuncture techniques on pain-induced anxiety may arise from the regulation of p-ERK in ACC.

Electroacupuncture alleviates retrieval of pain memory and its effect on phosphorylation of cAMP response element-binding protein in anterior cingulate cortex in rats

BackgroundRecent evidence suggests that persistent pain and recurrent pain are due to the pain memory which is related to the phosphorylation of cAMP response element-binding protein (p-CREB) in anterior cingulate cortex (ACC). Eletroacupuncture (EA), as a complementary Chinese medical procedure, has a significant impact on the treatment of pain and is now considered as a mind-body therapy.MethodsThe rat model of pain memory was induced by two injections of carrageenan into the paws, which was administered separately by a 14-day interval, and treated with EA therapy. The paw withdrawal thresholds (PWTs) of animals were measured and p-CREB expressions in ACC were detected by using immunofluorescence (IF) and electrophoretic mobility shift assay (EMSA). Statistical comparisons among different groups were made by one-way, repeated-measures analysis of variance (ANOVA).ResultsThe second injection of carrageenan caused the decrease of PWTs in the non-injected hind paw. EA stimulation applied prior to the second injection, increased the values of PWTs. In ACC, the numbers of p-CREB positive cells were significantly increased in pain memory model rats, which were significantly reduced by EA. EMSA results showed EA also down-regulated the combining capacity of p-CREB with its DNA. Furthermore, the co-expression of p-CREB with GFAP, OX-42, or NeuN in ACC was strengthened in the pain memory model rats. EA inhibited the co-expression of p-CREB with GFAP or OX-42, but not NeuN in ACC.ConclusionsThe present results suggest the retrieval of pain memory could be alleviated by the pre-treatment of EA, which is at least partially attributed to the down-regulated expression and combining capacity of p-CREB and the decreased expression of p-CREB in astrocytes and microglia cells.

The Effect of Electroacupuncture on PKMzeta in the ACC in Regulating Anxiety-Like Behaviors in Rats Experiencing Chronic Inflammatory Pain

Chronic inflammatory pain can induce emotional diseases. Electroacupuncture (EA) has effects on chronic pain and pain-related anxiety. Protein kinase Mzeta (PKMzeta) has been proposed to be essential for the maintenance of pain and may interact with GluR1 to maintain CNS plasticity in the anterior cingulate cortex (ACC). We hypothesized that the PKMzeta-GluR1 pathway in the ACC may be involved in anxiety-like behaviors of chronic inflammatory pain and that the mechanism of EA regulation of pain emotion may involve the PKMzeta pathway in the ACC. Our results showed that chronic inflammatory pain model decreased the paw withdrawal threshold (PWT) and increased anxiety-like behaviors. The protein expression of PKCzeta, p-PKCzeta (T560), PKMzeta, p-PKMzeta (T560), and GluR1 in the ACC of the model group were remarkably enhanced. EA increased PWT and alleviated anxiety-like behaviors. EA significantly inhibited the protein expression of p-PKMzeta (T560) in the ACC, and only a downward trend effect for other substances. Further, the microinjection of ZIP remarkably reversed PWT and anxiety-like behaviors. The present study provides direct evidence that the PKCzeta/PKMzeta-GluR1 pathway is related to pain and pain-induced anxiety-like behaviors. EA treatment both increases pain-related somatosensory behavior and decreases pain-induced anxiety-like behaviors by suppressing PKMzeta activity in the ACC.

Alleviating Mechanical Allodynia and Modulating Cellular Immunity Contribute to Electroacupuncture’s Dual Effect on Bone Cancer Pain

Hypothesis: Electroacupuncture (EA) has been used as an alternative analgesic therapy for hundreds of years, yet its analgesic potency and therapeutic advantage against bone cancer pain (BCP) in comparison with morphine remains unclear. This study aimed to investigate the effects of EA on mechanical allodynia and cellular immunity of BCP rats, and to further explore the potential mechanism. Methods: The BCP model was established by implanting Walker 256 mammary gland carcinoma cells into the left tibia of adult female Sprague-Dawley rats. EA (dilatational wave, 2/100 Hz, 0.5 mA–1mA–1.5 mA for 10 minutes each intensity) was applied bilaterally to Zusanli (ST 36) and Kunlun (BL 60) for 30 minutes. Both EA stimulation and morphine (10 mg/kg, intraperitoneally) was given once every other day. Naloxone (0.3 mg/kg, intraperitoneally) was injected at 30 minutes prior to EA. Mechanical allodynia were demonstrated by paw withdrawal thresholds (PWTs) which measured by dynamic plantar aesthesiometer. T cell proliferation, percentage of CD3+, CD4+ and CD8+ T lymphocytes in spleen as well as expression of interleukin-2 (IL-2) in plasma were detected by WST-8, flow cytometry, and enzyme-linked immunosorbent assay technique, respectively. Results: An intratibial inoculation of Walker 256 mammary gland carcinoma cells significantly decreased PWTs to mechanical stimuli. EA stimulation alleviated mechanical allodynia in BCP rats, and the analgesic potency of EA was weaker than that of morphine. In contrast to morphine, EA stimulation of BCP rats increased splenic concanavalin A (Con A)-induced T cell proliferation and plasma IL-2 content, as well as increased the percentages of splenic CD3+CD4+ and CD3+CD8+ T cell subsets. Moreover, both the analgesic effect and the partial immunomodulation of EA were suppressed by an intraperitoneal injection of naloxone. Conclusion: EA could significantly alleviate BCP-induced mechanical allodynia. Although the analgesic effect of EA was weaker than that of morphine, EA had an immunomodulation effect on cellular immunity. Both analgesic and immunomodulatory effect of EA might share the same mechanism via the opioid-mediated pathway, which needs further investigation.

Electroacupuncture Alleviates Pain Responses and Inflammation in a Rat Model of Acute Gout Arthritis

Acute gout arthritis is one of the most painful inflammatory conditions. Treatments for gout pain are limited to colchicine, nonsteroidal anti-inflammatory drugs, and corticosteroids, which oftentimes result in severe adverse effects. Electroacupuncture (EA) has been proved to be effective in relieving many inflammatory pain conditions with few side effects. Here, we aim to investigate the therapeutic potentials of EA on pain and inflammation of a rat model of acute gout arthritis and underlying mechanisms. We found that 2/100 Hz EA produced the most robust analgesic effect on mechanical hyperalgesia of acute gout arthritis rat model compared with 2 and 100 Hz. EA produced similar analgesic effect compared with indomethacin. 2/100 Hz EA also significantly alleviates the ongoing pain behavior, thermal hyperalgesia, and ankle edema. Locally applied μ and κ-opioid receptor antagonists but not adenosine A1 receptor antagonist significantly abolished the analgesic effect of EA. Locally applied μ and κ-opioid receptor agonists produced significant antiallodynia on acute gout arthritis rats, mimicking EA. Furthermore, 2/100 Hz EA upregulated β-endorphin expression in inflamed ankle skin tissue. Our results demonstrated, for the first time, that EA can be used for relieving acute gout arthritis with effect dependent on peripheral opioid system and comparable with the one obtained with indomethacin.