PGE2 Potentiates Orai1-Mediated Calcium Entry Contributing to Peripheral Sensitization.

Peripheral sensitization is one of the primary mechanisms underlying the pathogenesis of chronic pain. However, candidate molecules involved in peripheral sensitization remain incompletely understood. We have shown that store-operated calcium channels (SOCs) are expressed in the dorsal root ganglion (DRG) neurons. Whether SOCs contribute to peripheral sensitization associated with chronic inflammatory pain is elusive. Here we report that global or conditional deletion of Orai1 attenuates Complete Freund's adjuvant (CFA)-induced pain hypersensitivity in both male and female mice. To further establish the role of Orai1 in inflammatory pain, we performed calcium imaging and patch-clamp recordings in wild-type (WT) and Orai1 knockout (KO) DRG neurons. We found that SOC function was significantly enhanced in WT but not in Orai1 KO DRG neurons from CFA- and carrageenan-injected mice. Interestingly, the Orai1 protein level in L3/4 DRGs was not altered under inflammatory conditions. To understand how Orai1 is modulated under inflammatory pain conditions, prostaglandin E2 (PGE2) was used to sensitize DRG neurons. PGE2-induced increase in neuronal excitability and pain hypersensitivity was significantly reduced in Orai1 KO mice. PGE2-induced potentiation of SOC entry (SOCE) was observed in WT, but not in Orai1 KO DRG neurons. This effect was attenuated by a PGE2 receptor 1 (EP1) antagonist and mimicked by an EP1 agonist. Inhibition of Gq/11, PKC, or ERK abolished PGE2-induced SOCE increase, indicating PGE2-induced SOCE enhancement is mediated by EP1-mediated downstream cascade. These findings demonstrate that Orai1 plays an important role in peripheral sensitization. Our study also provides new insight into molecular mechanisms underlying PGE2-induced modulation of inflammatory pain.Significance Statement Store-operated calcium channel (SOC) Orai1 is expressed and functional in dorsal root ganglion (DRG) neurons. Whether Orai1 contributes to peripheral sensitization is unclear. The present study demonstrates that Orai1-mediated SOC function is enhanced in DRG neurons under inflammatory conditions. Global and conditional deletion of Orai1 attenuates complete Freund's adjuvant (CFA)-induced pain hypersensitivity. We also demonstrate that prostaglandin E2 (PGE2) potentiates SOC function in DRG neurons through EP1-mediated signaling pathway. Importantly, we have found that Orai1 deficiency diminishes PGE2-induced SOC function increase and reduces PGE2-induced increase in neuronal excitability and pain hypersensitivity. These findings suggest that Orai1 plays an important role in peripheral sensitization associated with inflammatory pain. Our study reveals a novel mechanism underlying PGE2/EP1-induced peripheral sensitization. Orai1 may serve as a potential target for pathological pain.

[Effect of heat-reinforcing needling on expression of serum inflammatory factors and autophagy of knee joint synovial tissue in rheumatoid arthritis rabbits with cold syndrome].

OBJECTIVE: To observe the effect of heat-reinforcing needling on the expression of serum inflammatory factors and autophagy of knee synovial tissue in rheumatoid arthritis (RA) rabbits with cold syndrome, so as to explore its mechanism of anti-inflammatory in the treatment of RA. METHODS: Fifty rabbits were randomly divided into normal, model, heat-reinforcing needling, inhibitor and agonist groups (n=10 rabbits in each group). The model of RA with cold syndrome was established by Freund's adjuvant and ovalbumin mixed solution injection combined with freezing and wind-cold dampness method. Heat-reinforcing needling was applied at "Zusanli" (ST36) for 30 min, once a day for 14 days. Rabbits of the inhibitor and agonist groups were given intraperitoneally injected with autophagy inhibitor 3-methyladenine (3-MA) or autophagy agonist rapamycin, once every 2 days for 7 days. The knee circumference and skin temperature of the rabbits in each group were measured. Color doppler ultrasonography was applied to examine the synovial membrane, joint effusion and blood flow signals in the knee joints of the rabbits in each group. Serum tumor necrosis factor (TNF) -α, interleukin (IL)-1ß, IL-6 and C-creactive protein (CRP) were detected by ELISA. Transmission electron microscopy was applied to observe the ultrastructure and autophagosomes of synovial cells. The protein expressions of autophagy-related protein Atg5, serine/threonine protein kinase-dysregulated 51-like kinase 1 (ULK1), microtubule-associated protein light chain 3B (LC3B), and Beclin-1 were detected by Western blot. Fluorescence quantitative PCR was used to detect the mRNA expressions of NOD-like receptor 3 (NLRP3) and nuclear factor-κB (NF-κB). RESULTS: Compared with the normal group, the circumference of the knee joint was increased (P<0.01), the skin temperature was decreased (P<0.01), the knee joint synovium was thickened and the blood flow signal was abundant, the contents of serum TNF-α, IL-1ß, IL-6, and CRP were increased (P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3BⅡ/LC3BⅠof synovial tissue were significantly decreased (P<0.01), the mRNA expressions of NLRP3 and NF-κB were increased (P<0.01) in the model group. In comparison with the model and inhibitor groups, the circumference of the knee joint was decreased (P<0.01), whlie the skin temperature was increased (P<0.01), the synovial membrane became thinner and the blood flow signal was wea-kened, the contents of TNF-α, IL-1ß, IL-6 and CRP were decreased (P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3B Ⅱ/LC3B Ⅰ were increased (P<0.01), and the mRNA expressions of NLRP3 and NF-κB were decreased (P<0.01) in the heat-reinforcing needling and agonist groups. CONCLUSION: Heat-reinforcing needling can alleviate the inflammatory response of the knee joint synovium in RA rabbits with cold syndrome, which may be related to its function in enhancing the autophagy activity of synovial cells and inhibiting the synthesis and release of inflammatory factors TNF-α, IL-1ß, IL-6 and CRP.

Upregulation of RCAN1.4 in spinal dorsal horn is involved in inflammatory pain hypersensitivity.

The calcium/calmodulin-dependent protein phosphase calcineurin (CaN) regulates synaptic plasticity by controlling the phosphorylation of synaptic proteins including AMPA type glutamate receptors. The regulator of calcineurin 1 (RCAN1) is characterized as an endogenous inhibitor of CaN and its dysregulation is implicated in multiple neurological disorders. However, whether RCAN1 is engaged in nociceptive processing in the spinal dorsal horn remains unrevealed. In this study, we found that RCAN1 was predominately expressed in pain-related neurons in the superficial dorsal horn of the spinal cord. Intraplantar injection of complete Freund's adjuvant (CFA) specifically increased the total and synaptic expression of the RCAN1.4 isoform in spinal dorsal horn. The CFA-induced inflammation also caused an increased binding of RCAN1.4 to CaN. Overexpression of RCAN1.4 in spinal dorsal horn of intact mice produced both mechanical allodynia and thermal hyperalgesia, which were accompanied by increased synaptic expression and phosphorylation of GluA1 subunit. Furthermore, the siRNA-mediated knockdown of RCAN1.4 significantly attenuated the development of pain hypersensitivity, meanwhile, decreased the synaptic expression of GluA1 in mice with peripheral inflammation. These data suggested that the increased expression of RCAN1.4 contributed to the development of inflammatory pain hypersensitivity, at least in part by promoting the synaptic recruitment of GluA1-containing AMPA receptor.

TRAF6 Contributes to CFA-Induced Spinal Microglial Activation and Chronic Inflammatory Pain in Mice.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been reported to be expressed in spinal astrocytes and is involved in neuropathic pain. In this study, we investigated the role and mechanism of TRAF6 in complete Freund's adjuvant (CFA)-evoked chronic inflammatory hypersensitivity and the effect of docosahexaenoic acid (DHA) on TRAF6 expression and inflammatory pain. We found that TRAF6 was dominantly increased in microglia at the spinal level after intraplantar injection of CFA. Intrathecal TRAF6 siRNA alleviated CFA-triggered allodynia and reversed the upregulation of IBA-1 (microglia marker). In addition, intrathecal administration of DHA inhibited CFA-induced upregulation of TRAF6 and IBA-1 in the spinal cord and attenuated CFA-evoked mechanical allodynia. Furthermore, DHA prevented lipopolysaccharide (LPS)-caused increase of TRAF6 and IBA-1 in both BV2 cell line and primary cultured microglia. Finally, intrathecal DHA reduced LPS-induced upregulation of spinal TRAF6 and IBA-1, and alleviated LPS-induced mechanical allodynia. Our findings indicate that TRAF6 contributes to pain hypersensitivity via regulating microglial activation in the spinal dorsal horn. Direct inhibition of TRAF6 by siRNA or indirect inhibition by DHA may have therapeutic effects on chronic inflammatory pain.

Synthetic mycobacterial molecular patterns partially complete Freund's adjuvant.

Complete Freund's adjuvant (CFA) has historically been one of the most useful tools of immunologists. Essentially comprised of dead mycobacteria and mineral oil, we asked ourselves what is special about the mycobacterial part of this adjuvant, and could it be recapitulated synthetically? Here, we demonstrate the essentiality of N-glycolylated peptidoglycan plus trehalose dimycolate (both unique in mycobacteria) for the complete adjuvant effect using knockouts and chemical complementation. A combination of synthetic N-glycolyl muramyl dipeptide and minimal trehalose dimycolate motif GlcC14C18 was able to upregulate dendritic cell effectors, plus induce experimental autoimmunity qualitatively similar but quantitatively milder compared to CFA. This research outlines how to substitute CFA with a consistent, molecularly-defined adjuvant which may inform the design of immunotherapeutic agents and vaccines benefitting from cell-mediated immunity. We also anticipate using synthetic microbe-associated molecular patterns (MAMPs) to study mycobacterial immunity and immunopathogenesis.

NMP-7 inhibits chronic inflammatory and neuropathic pain via block of Cav3.2 T-type calcium channels and activation of CB2 receptors.

BACKGROUND: T-type calcium channels and cannabinoid receptors are known to play important roles in chronic pain, making them attractive therapeutic targets. We recently reported on the design, synthesis and analgesic properties of a novel T-type channel inhibitor (NMP-7), which also shows mixed agonist activity on CB1 and CB2 receptors in vitro. Here, we analyzed the analgesic effect of systemically delivered NMP-7 (intraperitoneal (i.p.) or intragstric (i.g.) routes) on mechanical hypersensitivity in inflammatory pain induced by Complete Freund's Adjuvant (CFA) and neuropathic pain induced by sciatic nerve injury. RESULTS: NMP-7 delivered by either i.p. or i.g. routes produced dose-dependent inhibition of mechanical hyperalgesia in mouse models of inflammatory and neuropathic pain, without altering spontaneous locomotor activity in the open-field test at the highest active dose. Neither i.p. nor i.g. treatment reduced peripheral inflammation per se, as evaluated by examining paw edema and myeloperoxidase activity. The antinociception produced by NMP-7 in the CFA test was completely abolished in CaV3.2-null mice, confirming CaV3.2 as a key target. The analgesic action of intraperitoneally delivered NMP-7 was not affected by pretreatment of mice with the CB1 antagonist AM281, but was significantly attenuated by pretreatment with the CB2 antagonist AM630, suggesting that CB2 receptors, but not CB1 receptors are involved in the action of NMP-7 in vivo. CONCLUSIONS: Overall, our work shows that NMP-7 mediates a significant analgesic effect in a model of persistent inflammatory and chronic neuropathic pain by way of T-type channel modulation and CB2 receptor activation. Thus, this study provides a novel therapeutic avenue for managing chronic pain conditions via mixed CB ligands/T-type channel blockers.

Leak K⁺ channel mRNAs in dorsal root ganglia: relation to inflammation and spontaneous pain behaviour.

Two pore domain potassium (K2P) channels (KCNKx.x) cause K⁺ leak currents and are major contributors to resting membrane potential. Their roles in dorsal root ganglion (DRG) neurons normally, and in pathological pain models, are poorly understood. Therefore, we examined mRNA levels for 10 K2P channels in L4 and L5 rat DRGs normally, and 1 day and 4 days after unilateral cutaneous inflammation, induced by intradermal complete Freund's adjuvant (CFA) injections. Spontaneous foot lifting (SFL) duration (spontaneous pain behaviour) was measured in 1 day and 4 day rats <1h before DRG harvest. mRNA levels for KCNK channels and Kv1.4 relative to GAPDH (n=4-6 rats/group) were determined with real-time RT-PCR. This study is the first to demonstrate expression of THIK1, THIK2 and TWIK2 mRNA in DRGs. Abundance in normal DRGs was, in descending order: Kv1.4>TRESK(KCNK18)>TRAAK(KCNK4)>TREK2(KCNK10)=TWIK2(KCNK6)>TREK1 (KCNK2)=THIK2(KCNK12)>TASK1(KCNK3)>TASK2(KCNK5)>THIK1(KCNK13)=TASK3(KCNK9). During inflammation, the main differences from normal in DRG mRNA levels were bilateral, suggesting systemic regulation, although some channels showed evidence of ipsilateral modulation. By 1 day, bilateral K2P mRNA levels had decreased (THIK1) or increased (TASK1, THIK2) but by 4 days they were consistently decreased (TASK2, TASK3) or tended to decrease (excluding TRAAK). The decreased TASK2 mRNA was mirrored by decreased protein (TASK2-immunoreactivity) at 4 days. Ipsilateral mRNA levels at 4days compared with 1 day were lower (TRESK, TASK1, TASK3, TASK2 and THIK2) or higher (THIK1). Ipsilateral SFL duration during inflammation was positively correlated with ipsilateral TASK1 and TASK3 mRNAs, and contralateral TASK1, TRESK and TASK2 mRNAs. Thus changes in K2P mRNA levels occurred during inflammation and for 4 K2P channels were associated with spontaneous pain behaviour (SFL). K2P channels and their altered expression are therefore associated with inflammation-induced pain.

CJ-13610, an orally active inhibitor of 5-lipoxygenase is efficacious in preclinical models of pain.

Zileuton, a redox and iron chelator 5-lipoxygenase (5-LOX) inhibitor and, leukotriene receptor antagonists are presently used clinically in the long term treatment of asthma. Recent data implicate 5-LOX pathway in pain signaling. We report 5-LOX expression in the central nervous system (CNS) and analyze the pain efficacy of a new class of non redox, non iron chelating 5-LOX inhibitor. CJ-13610, 4-(3-(4-(2-methyl-1H-imidazol-1-yl) phenylthio) phenyl)-tetrahydro-2H-pyran-4-carboxamide, demonstrated antihyperalgesic activity in inflammatory pain models including the acute carrageenan model and the chronic inflammatory model using complete Freund's adjuvant. Following complete Freund's adjuvant stimulus leukotrieneB(4) concentration in the brain was elevated (9+/-1 ng/g, mean+/-S.E.M.) by about 3 times that of the control group (3+/-0.11, mean+/-S.E.M.). Hyperalgesia and leukotrieneB(4) concentration were both reversed following CJ-13610 treatment. Furthermore, we demonstrate CJ-13610 efficacy against osteoarthritis like pain using the rat medial meniscal transection model. CJ-13610 at oral doses of 0.6, 2 and 6 mg/kg/day reversed two modalities of pain in this model; tactile allodynia and weight bearing differential. Taken together, these data suggest that 5-LOX pathway and the leukotriene products are important mediators of pain.

Mycobacteria attenuate nociceptive responses by formyl peptide receptor triggered opioid peptide release from neutrophils.

In inflammation, pain is regulated by a balance of pro- and analgesic mediators. Analgesic mediators include opioid peptides which are secreted by neutrophils at the site of inflammation, leading to activation of opioid receptors on peripheral sensory neurons. In humans, local opioids and opioid peptides significantly downregulate postoperative as well as arthritic pain. In rats, inflammatory pain is induced by intraplantar injection of heat inactivated Mycobacterium butyricum, a component of complete Freund's adjuvant. We hypothesized that mycobacterially derived formyl peptide receptor (FPR) and/or toll like receptor (TLR) agonists could activate neutrophils, leading to opioid peptide release and inhibition of inflammatory pain. In complete Freund's adjuvant-induced inflammation, thermal and mechanical nociceptive thresholds of the paw were quantified (Hargreaves and Randall-Selitto methods, respectively). Withdrawal time to heat was decreased following systemic neutrophil depletion as well as local injection of opioid receptor antagonists or anti-opioid peptide (i.e. Met-enkephalin, beta-endorphin) antibodies indicating an increase in pain. In vitro, opioid peptide release from human and rat neutrophils was measured by radioimmunoassay. Met-enkephalin release was triggered by Mycobacterium butyricum and formyl peptides but not by TLR-2 or TLR-4 agonists. Mycobacterium butyricum induced a rise in intracellular calcium as determined by FURA loading and calcium imaging. Opioid peptide release was blocked by intracellular calcium chelation as well as phosphoinositol-3-kinase inhibition. The FPR antagonists Boc-FLFLF and cyclosporine H reduced opioid peptide release in vitro and increased inflammatory pain in vivo while TLR 2/4 did not appear to be involved. In summary, mycobacteria activate FPR on neutrophils, resulting in tonic secretion of opioid peptides from neutrophils and in a decrease in inflammatory pain. Future therapeutic strategies may aim at selective FPR agonists to boost endogenous analgesia.

Immunoadjuvant action of liposomes: comparison with other adjuvants.

Dehydration-rehydration vesicles (DRV liposomes) composed of equimolar phospholipid and cholesterol and containing bovine serum albumin (BSA) were used together with free BSA to immunize Balb/C mice. Primary and secondary immune responses (IgG1) to the liposomal antigen, as measured by ELISA in mouse sera, were similar for egg phosphatidylcholine (PC) and distearoyl phosphatidylcholine (DSPC) DRV, and much greater than those elicited by free BSA. The adjuvanticity of PC DRV was compared with that of aluminium salts (alum), complete Freund's adjuvant (CFA) and N-acetyl muramyl-L-threonyl-D-isoglutamine ([Thr1]MDP), the latter used as such or in a liposome form co-entrapped with the antigen. DRV (with or without co-entrapped [Thr1]MDP), and alum were equally strong in producing primary and secondary immune responses (IgG1) to BSA. Such responses were significantly higher than those achieved with CFA and [Thr1]MDP alone. The implications of these results for the potential role of liposomes as immunological adjuvants in vaccines are discussed.