COX-2 expression and function in the hyperalgesic response to paw inflammation in mice

Peripheral inflammation and edema are often accompanied by primary and secondary hyperalgesia which are mediated by both peripheral and central mechanisms. The role of cyclooxygenase-2 (COX-2)-mediated prostanoid production in hyperalgesia is a topic of substantial current interest. We have established a murine foot-pad inflammation model in which both pharmacologic and genetic tools can be used to characterize the role of COX-2 in hyperalgesia. Zymosan, an extract from yeast, injected into the plantar surface of the hindpaw induces an edema response and an increase in COX-2 expression in the hindpaw, spinal cord and brain. Zymosan-induced primary hyperalgesia, measured as a decrease in hindpaw withdrawal latency in response to a thermal stimulus, is long-lasting and is not inhibited by pre-treatment with the systemic COX-2 selective inhibitor, parecoxib (20 mg/kg). In contrast, the central component of hyperalgesia, measured as a reduction in tail flick latency in response to heat, is reduced by parecoxib. Zymosan-induced primary hyperalgesia in Cox-2^−/− mice is similar to that of their Cox-2^+/+ littermate controls. However, the central component of hyperalgesia is substantially reduced in Cox-2^−/− versus Cox-2^+/+ mice, and returns to baseline values much more rapidly. Thus pharmacological data suggest, and genetic experiments confirm, (i) that primary hyperalgesia in response to zymosan inflammation in the mouse paw is not mediated by COX-2 function and (ii) that COX-2 function plays a major role in the central component of hyperalgesia in this model of inflammation.     

Sex differences in inflammation and inflammatory pain in cyclooxygenase-deficient mice

There are two cyclooxygenase (COX) genes encoding characterized enzymes, COX-1 and COX-2. Nonsteroidal anti-inflammatory drugs are commonly used as analgesics in inflammatory arthritis, and these often inhibit both cyclooxygenases. Recently, inhibitors of COX-2 have been used in the treatment of inflammatory arthritis, as this isoform is thought to be critical in inflammation and pain. The objective of this study was to determine the effect of COX-1 or COX-2 gene disruption on the development of chronic Freund's adjuvant-induced arthritis and inflammatory pain in male and female mice. The effect of COX-1 or COX-2 gene disruption on inflammatory hyperalgesia, allodynia, inflammatory edema, and arthritic joint destruction was studied. COX-2 knockout mice (COX-2-/-) showed reduced edema and joint destruction in female, but not male, animals. In addition, neither male nor female COX-2-/- mice developed thermal hyperalgesia or mechanical allodynia, either ipsilateral or contralateral to the inflammation. COX-1 gene disruption also reduced inflammatory edema and joint destruction in female, but not male mice, although females of both COX-/- lines did show some bony destruction. There was no difference in ipsilateral allodynia between COX-1 knockout and wild-type animals, but female COX-1-/- mice showed reduced contralateral allodynia compared with male COX-1-/- or wild-type mice. These data show that the gene products of both COX genes contribute to pain and local inflammation in inflammatory arthritis. There are sex differences in some of these effects, and this suggests that the effects of COX inhibitors may be sex dependent.     

美普他酚及其同分异构体对角叉菜胶引起的炎症大鼠具有抗热痛敏作用

实验以清醒大鼠的缩腿潜伏期为指标,观察了腹腔注射美普他酚及其同分异构体112824和112825对角叉菜胶引起的热痛敏的影响.外周炎症由单侧足底注射角叉菜胶(2 mg/100 μl)引起.注射角叉菜胶3 h后,注射侧后肢局部红肿及热痛过敏反应达到高峰,持续数小时.腹腔注射0.1 mg/kg美普他酚对炎症和非炎症侧后肢的缩腿潜伏期无明显影响(P＞0.05,n=8).腹腔注射1mg/kg和10 mg/kg美普他酚对炎症和非炎症侧后肢产生明显的抗痛敏和抗伤害效应,且对炎症侧缩腿反应的抑制(抗痛敏)作用明显强于非炎症侧(抗伤害)(P＜0.05,n=8～11).预先腹腔注射1.5 mg/kg纳洛酮明显阻断美普他酚引起的抗伤害和抗痛敏效应.腹腔注射美普他酚的同分异构体112824(1 mg/kg)和112825(1.5 ms/kg)可产生与美普他酚类似的抗痛敏作用,该效应可被预先腹腔注射1.5 mg/kg纳洛酮完全阻断.提示美普他酚及其同分异构体具有明显抗伤害和抗痛敏作用,且以后者为强.该作用主要通过mu阿片受体介导.本研究为扩展美普他酚及其同分异构体在临床上的应用提供了依据.     

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.     

Warm temperature-sensitive transient receptor potential vanilloid 4 (TRPV4) plays an essential role in thermal hyperalgesia

Animals sense various ranges of temperatures by cutaneous thermal stimuli. Transient receptor potential vanilloid 4 (TRPV4) is a cation channel activated at a warm temperature (over 30 degrees C) in exogenously expressed cells. We found in the present study that TRPV4 is essential in thermal hyperalgesia at a warm temperature in vivo. TRPV4-/- and TRPV4+/+ mice exhibited the same latency of escape from 35-50 degrees C hotplates. Neuronal activity in the femoral nerve, however, revealed that the number and activity level of neurons decreased in response to a warm temperature in TRPV4-/- mice. TRPV4-/- mice displayed a significantly longer latency to escape from the plates at 35- 45 degrees C when hyperalgesia was induced by carrageenan without changes in foot volumes. TRPV4 therefore determines the sensitivity rather than the threshold of painful heat detection and plays an essential role in thermal hyperalgesia.     

Hydrogen sulfide upregulates cyclooxygenase-2 and prostaglandin E metabolite in sepsis-evoked acute lung injury via transient receptor potential vanilloid type 1 channel activation

Hydrogen sulfide (H(2)S) has been shown to promote transient receptor potential vanilloid type 1 (TRPV1)-mediated neurogenic inflammation in sepsis and its associated multiple organ failure, including acute lung injury (ALI). Accumulating evidence suggests that the cyclooxygenase-2 (COX-2)/PGE(2) pathway plays an important role in augmenting inflammatory immune response in sepsis and respiratory diseases. However, the interactions among H(2)S, COX-2, and PGE(2) in inciting sepsis-evoked ALI remain unknown. Therefore, the aim of this study was to investigate whether H(2)S would upregulate COX-2 and work in conjunction with it to instigate ALI in a murine model of polymicrobial sepsis. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in male Swiss mice. dl-propargylglycine, an inhibitor of H(2)S formation, was administrated 1 h before or 1 h after CLP, whereas sodium hydrosulfide, an H(2)S donor, was given during CLP. Mice were treated with TRPV1 antagonist capsazepine 30 min before CLP, followed by assessment of lung COX-2 and PGE(2) metabolite (PGEM) levels. Additionally, septic mice were administrated with parecoxib, a selective COX-2 inhibitor, 20 min post-CLP and subjected to ALI and survival analysis. H(2)S augmented COX-2 and PGEM production in sepsis-evoked ALI by a TRPV1 channel-dependent mechanism. COX-2 inhibition with parecoxib attenuated H(2)S-augmented lung PGEM production, neutrophil infiltration, edema, proinflammatory cytokines, chemokines, and adhesion molecules levels, restored lung histoarchitecture, and protected against CLP-induced lethality. The strong anti-inflammatory and antiseptic actions of selective COX-2 inhibitor may provide a potential therapeutic approach for the management of sepsis and sepsis-associated ALI.     

Carrageenan-induced paw edema in rat elicits a predominant prostaglandin E2 (PGE2) response in the central nervous system associated with the induction of microsomal PGE2 synthase-1

Peripheral inflammation involves an increase in cyclooxygenase-2 (COX-2)-mediated prostaglandin (PG) synthesis in the central nervous system (CNS), which contributes to allodynia and hyperalgesia. In the present study we have determined the changes in prostanoid tissue levels and in expression of terminal prostanoid synthases in both the CNS and inflamed peripheral tissue during carrageenan-induced paw inflammation in the rat. Prostanoid levels were measured by liquid chromatography-mass spectrometry and enzyme expression at the RNA level by quantitative PCR analysis during both the early (1-6 h) and late (12 and 24 h) phases of the inflammatory response. In the paw, the early phase was associated with increases in PGE(2) and thromboxane (TX)B(2) levels and with a peak of COX-2 expression that preceded that of microsomal prostaglandin-E(2) synthase-1 (mPGES-1). COX-2 and mPGES-1 remained elevated during the late phase, and PGE(2) continued to further increase through 24 h. The cytosolic PGE(2) synthase (cPGES) showed a small transient increase during the early phase, whereas mPGES-2 expression was not affected by inflammation. In the cerebrospinal fluid, elevated levels of PGE(2), 6-keto-PGF(1alpha), PGD(2), and TXB(2) were detected during the early phase. PGE(2) levels also increased in the spinal cord and, to a lesser extent, in the brain and remained elevated in both the cerebrospinal fluid and the spinal cord during the late phase. The expression of mPGES-1 was strongly up-regulated in the brain and spinal cord during inflammation, whereas no change was detected for the expression of cPGES, mPGES-2, COX-1, and terminal PGD, TX, or PGI synthases. The results show that the carrageenan-induced edema in the paw elicits an early phase of COX-2 induction in the CNS leading to an increase synthesis in PGD(2), 6-keto-PGF(1alpha), and TXB(2) in addition to the major PGE(2) response. The data also indicate that the up-regulation of mPGES-1 contributes to COX-2-mediated PGE(2) production in the CNS during peripheral inflammation.     

早期干预ERK在脊髓的激活可阻断外周神经损伤引起的神经病理性疼痛的发生

本文采用大鼠坐骨神经慢性压迫损伤引起的神经病理痛模型,研究脊髓背角细胞外信号调节激酶(extracellular signal-regulatedkinase,ERK)在外周神经损伤引起的神经病理疼痛发生中的作用.结果显示,单侧坐骨神经压迫性损伤后1天,大鼠损伤侧脊髓背角ERK的磷酸化(激活)水平显著上调,其下游转录因...     

Serum IL-10 involved in morphine tolerance development during adjuvant-induced arthritis

Opioid receptors play an important role in modulation of hyperalgesia in inflamed tissues, but chronic morphine application induces such side effects as tolerance. There is near communications between cytokines and mu opioid receptor expression. This study was aimed to assess the role of serum IL-10 in morphine tolerance development during adjuvant-induced arthritis. Adjuvant arthritis (AA) was induced on day 0 by single injection of Complete Freund’s Adjuvant (CFA) into the rats’ hindpaw. Hyperalgesia, edema, and spinal mu opioid receptor (mOR) variations were assessed on 0, 7, 14, and 21 days of the study. For assessment of the morphine tolerance development, morphine effective dose (4 mg/kg) was administered from the 14th day after CFA injection and continued until the morphine post-dose paw withdrawal latency (PWL); it did not significantly differ from the baseline. For assessment of the effects of IL-10 on tolerance induction, a neutralizing dose (ND50) of anti-IL-10 was administered daily during different stages of the study. AA induction in the right hindpaw of rats resulted in unilateral inflammation and hyperalgesia within 21 days of the study. Anti-IL-10 antibody administration in the AA rats induced marked elevation of hyperalgesia compared to the AA control group. Our data also indicated that morphine effective anti-hyperalgesic dose significantly decreased in the AA rats compared to the control group, which this symptom was aligned with spinal mu opioid receptor (mOR) expression increase during AA. Moreover, there was a significant difference in morphine tolerance induction between the AA and control rats, and our results also demonstrated that IL-10 played an important role in tolerance-induction process. It can be concluded that morphine tolerance slowly progressed when administered morphine effective dose was reduced during AA chronic inflammation. On the other hand, it seems that increased level of serum IL-10 may affect morphine tolerance development during inflammation.     

鞘内注射孤啡肽对大鼠足底注入蜜蜂毒诱致长时程自发痛、痛敏和炎症的不同效果

为进一步了解孤啡肽在脊髓水平是否具有抗伤害及抗炎作用,本实验在具有多种痛行为表现的蜜蜂毒模型上观察了鞘内注射孤啡肽对大鼠一侧后足底注入蜜蜂毒所诱致的同侧自发缩足反射、原发热和机械性痛敏以及注射部位炎症反应的影响,同时观察了新的高选择性孤啡肽受体拮抗剂CompB的作用.结果表明与生理盐水对照组比较,鞘内注射孤啡肽(3、10、30 nmol/10μl)对蜜蜂毒诱发的自发缩足反射次数的抑制作用随剂量提高而增大,抑制率分别为37±7,43±6and57±11%(三个剂量vs对照,P＜0.05);而对蜜蜂毒诱发的注射部位炎症反应(爪体积、爪背腹厚度和蛋白渗出的增加)无显著影响.CompB(30 nmo1)可完全翻转10 nmol孤啡肽对自发缩足反射的抑制作用.鞘内单次或重复注射孤啡肽(10 nmol/10μl)对蜜蜂毒诱致的原发性热和机械性痛敏的发生和维持均无作用.本实验结果提示,外源性孤啡肽在脊髓通过孤啡肽受体的介导产生一定的镇痛作用,但是它可能仅对持续性自发痛有抑制作用,而对热和机械性痛敏及炎症反应均无影响.     

Decreased expression of the NF-κB family member RelB in lung fibroblasts from Smokers with and without COPD potentiates cigarette smoke-induced COX-2 expression

Background

Heightened inflammation, including expression of COX-2, is associated with COPD pathogenesis. RelB is an NF-κB family member that attenuates COX-2 in response to cigarette smoke by a mechanism that may involve the miRNA miR-146a. There is no information on the expression of RelB in COPD or if RelB prevents COX-2 expression through miR-146a.

Methods

RelB, Cox-2 and miR-146a levels were evaluated in lung fibroblasts and blood samples derived from non-smokers (Normal) and smokers (At Risk) with and without COPD by qRT-PCR. RelB and COX-2 protein levels were evaluated by western blot. Human lung fibroblasts from Normal subjects and smokers with and without COPD, along with RelB knock-down (siRNA) in Normal cells, were exposed to cigarette smoke extract (CSE) in vitro and COX-2 mRNA/protein and miR-146a levels assessed.

Results

Basal expression of RelB mRNA and protein were significantly lower in lung cells derived from smokers with and without COPD, the latter of which expressed more Cox-2 mRNA and protein in response to CSE. Knock-down of RelB in Normal fibroblasts increased Cox-2 mRNA and protein induction by CSE. Basal miR-146a levels were not different between the three groups, and only Normal fibroblasts increased miR-146a expression in response to smoke. There was a positive correlation between systemic RelB and Cox-2 mRNA levels and circulating miR-146a levels were higher only in GOLD stage I subjects.

Conclusions

Our data indicate that RelB attenuates COX-2 expression in lung structural cells, such that loss of pulmonary RelB may be an important determinant in the aberrant, heightened inflammation associated with COPD pathogenesis.     

Disruption of cyclooxygenase-2 prevents downregulation of cortical AQP2 and AQP3 in response to bilateral ureteral obstruction in the mouse

Bilateral ureteral obstruction (BUO) in rats is associated with increased cyclooxygenase type 2 (COX-2) expression, and selective COX-2 inhibition prevents downregulation of aquaporins (AQPs) in response to BUO. It was hypothesized that a murine model would display similar changes in renal COX-2 and AQPs upon BUO and that targeted disruption of COX-2 protects against BUO-induced suppression of collecting duct AQPs. COX-2(-/-) and wild-type littermates (C57BL/6) were employed to determine COX-1, -2, AQP2, and AQP3 protein abundances and localization after BUO. In a separate series, sham and BUO wild-type mice were treated with a selective COX-2 inhibitor, parecoxib. The COX-2 protein level increased in wild-type mice in response to BUO and was not detectable in COX-2(-/-). COX-1 protein abundance was increased in sham-operated and BUO mice. Total AQP2 and -3 mRNA and protein levels decreased significantly after BUO in the cortex+outer medulla (C+OM) and inner medulla (IM). The decrease in C+OM AQP2 and -3 levels was attenuated/prevented in COX-2(-/-) mice, whereas there was no change in the IM. In parallel, inhibition of COX-2 by parecoxib rescued C+OM AQP3 and IM AQP2 protein level in wild-type mice subjected to BUO. In summary, 1) In C57BL/6 mice, ureteral obstruction increases renal COX-2 expression in interstitial cells and lowers AQP2/-3 abundance and 2) inhibition of COX-2 activity by targeted disruption or pharmacological blockade attenuates obstruction-induced AQP downregulation. In conclusion, COX-2-derived prostaglandins contribute to downregulation of transcellular water transporters in the collecting duct and likely to postobstruction diureses in the mouse.     

MyD88 signaling promotes both mucosal homeostatic and fibrotic responses during Salmonella-induced colitis

Salmonella enterica serovar Typhimurium is a clinically important gram-negative, enteric bacterial pathogen that activates several Toll-like receptors (TLRs). While TLR signaling through the adaptor protein MyD88 has been shown to promote inflammation and host defense against the systemic spread of S. Typhimurium, curiously, its role in the host response against S. Typhimurium within the mammalian gastrointestinal (GI) tract is less clear. We therefore used the recently described Salmonella-induced enterocolitis and fibrosis model: wild-type (WT) and MyD88-deficient (MyD88(-/-)) mice pretreated with streptomycin and then orally infected with the ΔaroA vaccine strain of S. Typhimurium. Tissues were analyzed for bacterial colonization, inflammation, and epithelial damage, while fibrosis was assessed by collagen quantification and Masson's trichrome staining. WT and MyD88(-/-) mice carried similar intestinal pathogen burdens to postinfection day 21. Infection of WT mice led to acute mucosal and submucosal inflammation and edema, as well as significant intestinal epithelial damage and proliferation, leading to widespread goblet cell depletion. Impressive collagen deposition in the WT intestine was also evident in the submucosa at postinfection days 7 and 21, with fibrotic regions rich in fibroblasts and collagen. While infected MyD88(-/-) mice showed levels of submucosal inflammation and edema similar to WT mice, they were impaired in the development of mucosal inflammation, along with infection-induced epithelial damage, proliferation, and goblet cell depletion. MyD88(-/-) mouse tissues also had fewer submucosal fibroblasts and 60% less collagen. We noted that cyclooxygenase (Cox)-2 expression was MyD88-dependent, with numerous Cox-2-positive cells identified in fibrotic regions of WT mice at postinfection day 7, but not in MyD88(-/-) mice. Treatment of WT mice with the Cox-2 inhibitor rofecoxib (20 mg/kg) significantly reduced fibroblast numbers and collagen levels without altering colitis severity. In conclusion, MyD88 and Cox-2 signaling play roles in intestinal fibrosis during Salmonella-induced enterocolitis.     

Topical application of a selective cyclooxygenase inhibitor suppresses UVB mediated cutaneous inflammation

Ultraviolet B (UVB) radiation causes much of the cutaneous damage after both acute and long-term exposure, and is also the most important etiologic agent in human skin cancer. UVB exposure initially induces an inflammatory response characterized by edema, dermal infiltration of leukocytes, sunburn cell formation, as well as the induction of cyclooxygenase-2 (COX-2) gene expression and subsequent increase in the production and release of prostaglandins. This process of inflammation induced by UVB exposure has been linked to tumor formation. Recently, a specific COX-2 inhibitor, Celecoxib, was developed, which inhibits COX-2-induced inflammation without inhibiting the cytoprotective function of cyclooxygenase-1 (COX-1). The present study compared the effects of topical treatment with Celecoxib (a specific COX-2 inhibitor) and Ibuprofen (a nonspecific COX inhibitor) on the acute UVB-induced cutaneous inflammatory response. We show that the specific inhibition of COX-2 effectively reduced many parameters of UVB-mediated inflammation, including edema, dermal neutrophil infiltration and activation, prostaglandin E2 (PGE2) levels and the formation of sunburn cells. By inhibiting this inflammatory response, topical Celecoxib treatment may ultimately be effective in preventing UVB-induced tumor development in the skin.     

Local gene expression changes after UV-irradiation of human skin

UV-irradiation is a well-known translational pain model inducing local inflammation and primary hyperalgesia. The mediators and receptor proteins specifically contributing to mechanical or heat hyperalgesia are still unclear. Therefore, we irradiated buttock skin of humans (n = 16) with 5-fold MED of UV-C and assessed the time course of hyperalgesia and axon reflex erythema. In parallel, we took skin biopsies at 3, 6 and 24 h after UVC irradiation and assessed gene expression levels (RT-PCR ) of neurotrophins (e.g. NGF, BDNF, GDNF), ion channels (e.g. NaV1.7, TRPV1), inflammatory mediators (e.g. CCL-2, CCL-3) and enzymes (e.g. PGES, COX2). Hyperalgesia to mechanical impact (12 m/s) and heat (48 °C) stimuli was significant at 6 h (p<0.05 and p<0.01) and 24 h (p<0.005 and p<0.01) after irradiation. Axon reflex erythema upon mechanical and thermal stimuli was significantly increased 3 h after irradiation and particularly strong at 6 h. A significant modulation of 9 genes was found post UV-C irradiation, including NGF (3, 6, 24 h), TrkA (6, 24 h), artemin, bradykinin-1 receptor, COX-2, CCL-2 and CCL-3 (3 and 6 h each). A significant down-regulation was observed for TRPV1 and iNOS (6, 24 h). Individual one-to-one correlation analysis of hyperalgesia and gene expression revealed that changes of Nav1.7 (SCN9A) mRNA levels at 6 and 24 h correlated to the intensity of mechanical hyperalgesia recorded at 24 h post UV-irradiation (Pearson r: 0.57, p<0.04 and r: 0.82, p<0.001). Expression of COX-2 and mPGES at 6 h correlated to the intensity of heat-induced erythema 24 h post UV (r: 0.57, p<0.05 for COX-2 and r: 0.83, p<0.001 for PGES). The individual correlation analyses of functional readouts (erythema and pain response) with local expression changes provided evidence for a potential role of Nav1.7 in mechanical hyperalgesia.