Xanthine Derivative KMUP-1 Reduces Inflammation and Hyperalgesia

Mar 27, 2014 - Department of Pediatrics, Division of Pediatric Pulmonology and Cardiology, Kaohsiung Medical University Hospital, 100. Shih-Chuan 1st ...
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Xanthine Derivative KMUP‑1 Reduces Inflammation and Hyperalgesia in a Bilateral Chronic Constriction Injury Model by Suppressing MAPK and NFκB Activation Zen-Kong Dai,† Ting-Chun Lin,‡ Jau-Cheng Liou,§ Kuang-I Cheng,∥ Jun-Yih Chen,⊥ Li-Wen Chu,# Ing-Jun Chen,*,‡ and Bin-Nan Wu*,‡ †

Department of Pediatrics, Division of Pediatric Pulmonology and Cardiology, Kaohsiung Medical University Hospital, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan ‡ Department of Pharmacology, School of Medicine, College of Medicine, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan § Department of Biological Sciences, National Sun Yat-Sen University, No. 70, Lianhai Road, Kaohsiung, Taiwan ∥ Department of Anesthesiology, School of Medicine, College of Medicine, Kaohsiung Medical University and Hospital, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan ⊥ Department of Neurosurgery, Kaohsiung Veterans General Hospital, 386 Ta-Chung 1st Road, Kaohsiung, Taiwan # School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan ABSTRACT: Neuropathic pain is characterized by spontaneous pain, hyperalgesia, and allodynia. The aim of this study was to investigate whether KMUP-1 (7-[2-[4-(2chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) could improve pain hypersensitivity and reduce inflammatory mediators, and also explore possible mechanisms in the rat sciatic nerve using bilateral chronic constriction injury (CCI) to induce neuropathic pain. Sprague−Dawley rats were randomly divided into four groups: Sham, Sham+KMUP-1, CCI, and CCI+KMUP-1. KMUP-1 (5 mg/kg/day) was injected intraperitoneally starting at day 1 after surgery. Mechanical and thermal responses were assessed before surgery and at days 3, 7, and 14 after CCI. Sciatic nerves around the injury site were isolated for Western blots and enzyme-linked immunosorbent assay to analyze protein and cytokine levels. The results show that thermal hyperalgesia and mechanical allodynia were reduced in the KMUP-1 treated group as compared to that in the CCI group. Inflammatory proteins (COX2, iNOS, and nNOS) and proinflammatory cytokines (TNF-α and IL-1β) induced by CCI were decreased in the KMUP-1 treated group at day 7 after surgery. KMUP-1 also inhibited neuropathic pain-related mechanisms, including p38 and ERK activation, but not JNK. Furthermore, KMUP-1 blocked IκB phosphorylation (p-IκB) and phospho-nuclear factor κB (p-NF-κB) translocation to nuclei. Double immunofluorescent staining further demonstrated that pIκB (an indicator of activated NFκB) and p-NFκB proteins were almost abolished by KMUP-1 in peripheral macrophages and spinal microglia cells at day 7 after surgery. On the basis of these findings, we concluded that KMUP-1 has antiinflammatory and antihyperalgesia properties in CCI-induced neuropathic pain via decreases in MAPKs and NF-κB activation. KEYWORDS: chronic constriction injury, neuropathic pain, mitogen-activated protein kinases, phospho-nuclear factor κB, neuroinflammation

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INTRODUCTION Neuropathic pain remains the most frequent cause of suffering and disability throughout the world. Hyperalgesia and allodynia associated with neuropathic pain are the hallmark of peripheral nerve injury. Neuropathic pain is a complex disorder that leads to chronic illness and adversely affects the quality of a patient’s life. Although considerable progress has been made, the mechanisms underlying neuropathic pain are poorly understood.1−3 Since current available treatments for neuropathic pain remain inadequate, it is imperative to continue searching for novel targets and improved therapies.1,3,4 The chronic constriction injury (CCI) model has provided a better © 2014 American Chemical Society

understanding of nociception and the pathogenesis of chronic pain.5,6 In the current study, a similar animal model was employed to assess the potential efficacies of KMUP-1 (7-[2[4-(2-chlorobenzene) piperazinyl]ethyl]-1,3-dimethylxanthine), a xanthine derivative, in neuropathic pain. Proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) are released in the spinal Received: Revised: Accepted: Published: 1621

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evoked pain. 31,32 Briefly, rats were anesthetized with pentobarbital sodium (40 mg/kg, i.p.). The sciatic nerve on each side was exposed via blunt dissection through the biceps femoris muscle. CCI was induced using 3 loosely constrictive ligatures of 4−0 chromic catgut placed around the sciatic nerve proximal to the trifurcation. The surgical incision was then closed in layers, and the rats were allowed to recover from anesthesia. In Sham-operated rats, an identical surgery was performed except for the ligation of the sciatic nerve. Experimental Protocol. Rats were randomly divided into four groups: (1) Sham-operated; (2) Sham-operated+ KMUP-1 (5 mg/kg/day, i.p.); (3) CCI; and (4) CCI+KMUP-1 (5 mg/ kg/day, i.p.). Rats were administered with KMUP-1 once daily for both treatment groups (CCI and Sham-operated, n = 6), starting on the next day after surgery (day 1). Animals were sacrificed at days 3, 7, and 14 after CCI. Bilateral sciatic nerves from each rat were collected for further experiments. A dosage of 5 mg/kg/day KMUP-1 was used based on our previous studies.25 Additionally, the acute toxicity of KMUP-1 was evaluated by the Center of Toxicology and Preclinical Sciences, Development Center for Biotechnology (DCB), Taipei, Taiwan. The calculated median lethal dose (LD50) of oral administration of KMUP-1 in male and female rats was found to be 3216 mg/kg and 2503 mg/kg, respectively. The LD50 of intravenous injection of KMUP-1 was 33.8 mg/kg for males and 59.0 mg/kg for females. Thermal Hyperalgesia and Mechanical Allodynia. Hyperalgesia and allodynia were evaluated among the four groups. Paw withdrawal latency (PWL) was used to estimate thermal hyperalgesia.30,33 In brief, rats were put in a plastic chamber and allowed to acclimatize to the environment for 30 min before testing. An infrared radiant heat source was then positioned beneath the plantar surface of the right hind paw. The PWL was recorded using a plantar analgesiometer (IITC, Woodland Hills, CA) and a cutoff latency of 30 s used. The paw withdrawal threshold (PWT) was performed to evaluate mechanical allodynia. The PWT was assessed using calibrated von Frey filaments as previously described.30,34 Rats were placed in a wire mesh cage and habituated for 30 min to the environment. An automated dynamic plantar aesthesiometer (Ugo Basile, Varese, Italy) was used to measure the PWT, which was recorded as the lowest force (g) causing a rapid withdrawal of the right hind paw. Each measurement was repeated three times at intervals of 5 min, and the average force evoking reliable withdrawals was taken as threshold. Protein Extraction and Western Blot Analysis. Sciatic nerves around the injury site were collected, frozen in liquid nitrogen, and stored at −80 °C.30 All specimens were homogenized in ice-cold lysis buffer (Roche protease inhibitor cocktail tablet/10 mL of Thermo Scientific T-PER Tissue Protein Extraction Reagent) and then centrifuged (15,000g) for 15 min at 4 °C. Each protein concentration in the supernatant was measured using bovine serum albumin as the standard. The protein extraction kit (Millipore, Temecula, CA) was employed to separate nuclear and cytoplasmic fractions from sciatic nerves according to the manufacturer’s instructions. The procedures and analyses of Western blot were performed as previously described.28,30 The primary antibodies of COX-2 (1:500 dilution; Abcam, London, UK), iNOS, nNOS (1:1000 dilution; BD Transduction Laboratories, San Jose, CA), phospho-p38 (p-p38), t-p38, p-ERK, t-ERK, p-JNK, t-JNK, pIκB, p-NFκB p65 (1:1000 dilution; Cell Signaling, Boston, MA), GAPDH, PARP (1:2000 dilution; Millipore, Temecula,

cord upon activation of glial cells, which eventually play a central role in pain facilitation. The release of these cytokines during inflammatory processes is regulated transcriptionally by nuclear factor κB (NF-κB).7−12 NF-κB expression and activity are augmented in sciatic nerves and dorsal root ganglia following nerve injury, and during paw inflammation induced by complete Freund’s adjuvant (CFA).13,14 In contrast, inhibition of NF-κB activation suppresses pain hypersensitivity in CCI-induced animals15,16 and reduces inflammatory hypersensitivity in the hind paw caused by CFA-injection.14 These reports reveal that NF-κB is a key regulator of inflammatory processes in reactive glial cells, and activation of glial cells and neuro-glial interactions are emerging as essential mechanisms underlying chronic pain. Thus, suppression of NF-κB is an attractive concept for treating pain and inflammation, and it may serve as a potential target for pain management.7−9 Bulk of evidence indicates that nerve injury proceeds to activation of p38 mitogen-activated protein kinases (MAPKs), followed by inhibition of p38 MAPK, and resulting in protection and/or prevention of nociceptive pain behaviors.17−19 Therefore, MAPKs were also investigated to evaluate their relationship to the neuroprotective effects of a xanthine derivative, KMUP1, in peripheral nerve CCI-induced neuropathic pain. KMUP-1 has been established to increase protein kinase A (PKA), stimulate protein kinase G (PKG), and activate K+ channels, resulting in relaxation in aortic,20 corporeal cavernosa,21 and tracheal smooth muscles.22 KMUP-1 also activates BKCa channels23 but inhibits L-type Ca2+ channels24 in rat basilar arteries. KMUP-1 was further shown to prevent pulmonary arterial hypertension through Rho kinase inhibition and K+-channel activation.25,26 KMUP-1 was capable of decreasing cardiac hypertrophy via the NO/cGMP/PKG pathway.27 Additionally, KMUP-1 has been shown to have value in the control of cerebral vasospasm after subarachnoid hemorrhage due to its K+-channel opening activities.28 Recently, it became clear that opening of the K+-channel is associated with antinociception.29 On the basis of these findings, K+ channels have been viewed as a unique target for the development of antinociceptive drugs. Since KMUP-1 has K+-channel opening activities, the present study was carried out to investigate its antinociceptive activities in an in vivo pain model. To determine if KMUP-1 can be used to control peripheral nerve injury-induced neuropathic pain, our research was focused on investigating the neuroprotective role of KMUP-1 using the CCI model. Molecular pathways related to CCI-induced neuropathic pain and possible mechanisms by which KMUP-1 attenuated this pain behavior were explored.

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MATERIALS AND METHODS Experimental Animals. Male Sprague−Dawley rats weighing 250−300 g were purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan) and housed under constant temperature and controlled illumination. Food and water were available ad libitum. All procedures and protocols were approved by the Animal Care and Use Committee at Kaohsiung Medical University and complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Bilateral Sciatic Nerve Ligation Surgery. Bilateral CCI of the sciatic nerves was performed as described previously.6,30,31 This is a reproducible animal model to explore possible therapeutic interventions in spontaneous or stimulus1622

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Figure 1. Effects of KMUP-1 on paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) evoked by chronic constriction injury (CCI) in rats. The top panel illustrates the experimental design for the CCI model in rats. Rats were anesthetized with pentobarbital (40 mg/kg) for CCI surgery (day 0), and injected with KMUP-1 (5 mg/kg/day, i.p.) once daily starting the next day after surgery (day 1). Beginning 1 h after injection with KMUP-1, rat behaviors were monitored, and rats were sacrificed and the sciatic nerves (SN) removed at days 3, 7, and 14 after surgery. (A) PWL and (B) PWT were estimated by the thermal stimulation and paw pressure test, respectively, applied before CCI and at days 3, 7, and 14 after CCI. Sham-operated (Sham) rats were subjected to the same surgical procedure without manipulation of the nerve. Data represent the mean ± SE for 6 rats per group. ###p < 0.001 compared with the Sham group; *P < 0.05 and ***P < 0.001 compared with the CCI group at the corresponding time points.

CA), and β-actin (1:2000 dilution; Sigma-Aldrich, Saint Louis, MO) were used in this experiment. TNF-α and IL-1β. The TNF-α and IL-1β concentrations were quantified spectrophotometrically using commercially available Quantikine ELISA kits according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). Sciatic nerves were homogenized in lysis buffer comprising protease inhibitors, and the insoluble pellet was removed by centrifugation. Total protein concentration in the supernatant was determined using the Bradford method. This assay employs the sandwich enzyme immunoassay technique. The intensity of color measurement is proportional to the amount of TNF-α and IL-1β. Accordingly, a standard curve is established, and therefore, sample values can be read off the standard curve. Immunohistochemistry Detection of COX-2 and iNOS. The immunohistochemistry of the sciatic nerve was conducted as we previously described.30 Briefly, the sample was embedded in an optimal cutting temperature (OCT) compound and frozen to decrease the variation in experimental procedures.30,35 Ten micrometer thick sections were cut at −20 °C with a Leica cryostat and collected onto gelatin-coated slides. The primary antibodies of COX-2 (1:100 dilution; Abcam, London, UK) and iNOS (1:100 dilution; BD Transduction Laboratories, San Jose, CA) were used. Immunofluorescent Localization of p-IκB and p-NFκB. The lumbar spinal cord segments and sciatic nerves were removed at day 7 after CCI. Fresh-frozen section of specimens were performed the same as immunohistochemistry.9,30 For double immunofluorescent staining, the spinal dorsal horn was

incubated with a mix of monoclonal p-NFκB (1:100 dilution; Cell Signaling, Boston, MA) antibody and monoclonal glial fibrillary acidic protein (GFAP) (astrocyte marker, 1:100 dilution; Millipore, Temecula, CA) or OX-42 (CD11b, microglia/macrophage marker, 1:100 dilution; Millipore) overnight at 4 °C; the section of sciatic nerve was incubated with a mixture of anti-p-IκB (1:100 dilution; Cell Signaling) and anti-OX42 (CD11b, microglia/macrophage marker, 1:100 dilution; Millipore).7−9 The appropriate secondary antibody conjugated with Alexa Fluor 555 (red; Invitrogen, Carlsbad, CA) or Alexa Fluor 488 (green; Invitrogen) was added. Images were acquired using a confocal microscope (Olympus Fluoview FV500, Tokyo, Japan). Data Analysis and Statistics. Data are expressed as the mean ± SE, n = 6. Obtained behavior results and calculated Western blot data were analyzed by one-way analysis of variance (ANOVA). When appropriate, a Tukey−Kramer pairwise comparison was used for post hoc analysis. P < 0.05 was considered statistically significant.

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RESULTS KMUP-1 Reduced CCI-Induced Hyperalgesia and Allodynia. CCI-induced inflammations caused an obvious thermal hyperalgesia and mechanical allodynia with a reduction in the PWL (Figure 1A) and PWT (Figure 1B), respectively. The results exhibited that CCI elicited a rapid-onset and longlasting thermal hyperalgesia and mechanical allodynia. There were no significant changes of PWL (s) and PWT (g) observed in the Sham group as well as in the KMUP-1-treated Sham 1623

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Figure 2. Effects of KMUP-1 (5 mg/kg/day, i.p.) on the expression of inflammatory proteins (A) COX2, (B) iNOS, and (C) nNOS in the sciatic nerve induced by chronic constriction injury (CCI). Western blots analysis of COX2, iNOS, and nNOS proteins in the sciatic nerve at days 3, 7, and 14 after nerve injury. GAPDH was used as the internal control. The intensity of each band was quantified by densitometry and indicated as the relative percentage change compared to the Sham group (100%). Data were presented as the mean ± SE from 6 independent experiments. #P < 0.05, ## P < 0.01, and ###P < 0.001 compared with the Sham group; *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the CCI group at the corresponding time points.

Figure 3. Effects of KMUP-1 (5 mg/kg/day, i.p.) on the expression of MAPKs proteins (A) p38, (B) ERK, and (C) JNK in the sciatic nerve induced by chronic constriction injury (CCI). Western blots analysis of p-p38/t-p38, p-ERK/t-ERK, and p-JNK/t-JNK proteins in the sciatic nerve at days 3, 7, and 14 after nerve injury. The intensity of each band was quantified by densitometry and indicated as the relative percentage change compared to the Sham group (100%). Data were presented as the mean ± SE from 6 independent experiments. ##P < 0.01 and ###P < 0.001 compared with the Sham group; *P < 0.05 and ***P < 0.001 compared with the CCI group at the corresponding time points.

group. However, marked decreases in the PWL and PWT following CCI were noted at day 3 after surgery. After KMUP-1 (5 mg/kg/day, i.p.) was administered, the PWL and PWT increased markedly in rats compared with that of the CCI group at day 7 and day 14. In other words, the antinociceptive effect of the KMUP-1-treated CCI group was strikingly different from day 7 to day 14 compared with that of the CCI group. Taken together, the results demonstrated that KMUP-1 ameliorates CCI-induced pain behaviors such as thermal hyperalgesia and mechanical allodynia. Effects on CCI-Induced COX2, iNOS, and nNOS Protein Activation. CCI induced the expression of large amounts of inflammatory hypernociceptive proteins. COX2,

iNOS, and nNOS were shown in the sciatic nerve at days 3, 7, and 14 compared with that of the Sham group. The KMUP-1treated Sham group showed no significant effects on these proteins compared with that in the Sham group. The inflammatory COX2, iNOS, and nNOS proteins were significantly attenuated by the KMUP-1-treated CCI group at day 7 and day 14 (Figure 2). Effects on CCI-Augmented MAPK Proteins. CCI increased the expression of inflammatory MAPK protein. Higher ratios of p-p38/t-p38, p-ERK/t-ERK, and p-JNK/t-JNK were shown in the sciatic nerve at days 3, 7, and 14 compared with that of the Sham group. The KMUP-1-treated Sham group showed no significant effects on these proteins compared with 1624

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Figure 4. Effects of KMUP-1 (5 mg/kg/day, i.p.) on the expression of p-IκB and p-NFκB proteins in the sciatic nerve induced by chronic constriction injury (CCI). The expression of (A) pIκB/IκB (β-actin as the internal control) and (B) p-NFκB (PARP as the internal control) were determined by cytoplasmic and nuclear fractions from the sciatic nerve, respectively. The intensity of each band was quantified by densitometry and indicates the percentage change relative to the Sham group (100%). Data were presented as the mean ± SE from 6 independent experiments. ##P < 0.01 and ###P < 0.001 compared with the Sham group; *P < 0.05 and **P < 0.01 compared with the CCI group at the corresponding time points.

Figure 5. Effects of KMUP-1 (5 mg/kg/day) on the elevated proinflammatory cytokines TNF-α and IL-1β in the sciatic nerve induced by chronic constriction injury (CCI) at day 7. (A) IL-1β assessment was conducted in triplicate, n = 4. ###p < 0.001 compared with the Sham group; ***p < 0.001 compared with the CCI group. (B) TNF-α assessment was conducted in triplicate, n = 4. ###p < 0.001 compared with the Sham group; ***p < 0.001 compared with the CCI group.

and IL-1β in the sciatic nerve. We observed significant neuropathic pain behaviors and inflammatory proteins at day 7 after CCI, and thus proinflammatory cytokines were measured under this time point. TNF-α and IL-1β levels were markedly increased in the rat sciatic nerve compared to that in the Sham control (Figure 5). The KMUP-1-treated CCI group significantly decreased these elevated cytokine levels. Nevertheless, the KMUP-1-treated Sham group exhibited little influence on these cytokines compared with that in the Sham group. KMUP-1 Decreased COX-2 and iNOS Immunohistochemical Localization. Immunohistochemical staining further confirmed that COX-2 and iNOS activation was involved in peripheral nerve injury after CCI. We found that CCI induced an increase of COX-2 and iNOS proteins in the sciatic nerve at day 3. KMUP-1 (5 mg/kg/day, i.p.) significantly attenuated CCI-induced COX-2 (Figure 6) and iNOS (Figure 7) protein levels from day 7 to day 14.

that of the Sham group. The inflammatory p-p38/t-p38 and pERK/t-ERK proteins, but not p-JNK/t-JNK, were significantly attenuated in the KMUP-1-treated CCI group at day 14 (Figure 3). Effects on CCI-Stimulated Cytoplasmic p-IκB/IκB and Nuclear p-NFκB Proteins. The time-course alterations of inflammatory cytoplasmic p-IκB/IκB and nuclear p-NFκB proteins in the sciatic nerve after CCI are shown in Figure 4. The IκB activity was estimated by the ratio of p-IκB/IκB, a crucial step of NFκB activation, which revealed marked increases after CCI compared with that in the Sham group. KMUP-1 markedly decreased p-IκB/IκB expression from day 7 to day 14 after CCI (Figure 4A). The nuclear p-NFκB protein was also reduced from day 7 to day 14 after the KMUP-1 treated group (Figure 4B). Effects on CCI-Induced TNF-α and IL-1β Accumulations. To explore the possible mechanisms of KMUP-1 effects on neuropathic pain in rats, we quantified the levels of TNF-α 1625

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Figure 6. COX2 immunohistochemistry staining in chronic constriction injury (CCI) induced in the sciatic nerve. The top panel shows high magnification images of the CCI and CCI+KMUP-1 groups at day 7. Red arrows indicate cells positive for COX2 (brown). Expression of COX2 (brown) was increased in the CCI group compared with that in the Sham group. Administration of KMUP-1 (5 mg/kg/day, i.p.) inhibited CCIenhanced COX2 protein at day 7 and day 14.

Figure 7. iNOS immunohistochemistry staining in chronic constriction injury (CCI) induced in the sciatic nerve. The top panel shows high magnification images of the CCI and CCI+KMUP-1 groups at day 7. Red arrows indicate cells positive for iNOS (brown). Expression of iNOS (brown) was significantly increased in the CCI group compared with that in the Sham group. Administration of KMUP-1 (5 mg/kg/day, i.p.) inhibited CCI-enhanced iNOS protein at day 7 and day 14.

KMUP-1 Reduced p-IκB and p-NFκB Proteins in Sciatic Nerve and Spinal Cord. To reveal NFκB activation in sciatic nerves, we performed double immunofluorescent staining for measuring p-IκB (an indicator of activated NFκB) and OX-42 (macrophage maker) proteins.8 In both Sham and CCI groups, the numbers of OX-42-expressing macrophage cells appeared

unaffected by KMUP-1 treatment, indicating that KMUP-1 may not block immune cell infiltration in the sciatic nerve (Figure 8). Similar results were also confirmed by hematoxylin and eosin (H&E) staining (data not shown). Some p-IκB proteins were expressed in the Sham group but showed dramatic 1626

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Figure 8. Double immunofluorescent staining for OX-42 (macrophage marker) and p-IκB in the peripheral sciatic nerve at day 7 after chronic constriction injury (CCI). Expression of p-IκB proteins was significantly increased in the CCI group compared with that in the Sham group. Administration of KMUP-1 (5 mg/kg/day, i.p.) abolished CCI-enhanced pIκB protein.

Figure 9. Double immunofluorescent staining for OX-42 (microglia marker) and p-NFκB in the central spinal dorsal horn at day 7 after chronic constriction injury (CCI). Expression of p-NFκB proteins was significantly increased in the CCI group compared with that in the Sham group. Administration of KMUP-1 (5 mg/kg/day, i.p.) nearly blocked CCI-enhanced p-NFκB protein.

increases in the CCI group. In both groups, the expression of pIκB was abolished by KMUP-1 (Figure 8). To understand KMUP-1 inhibition of the NFκB pathway targeting on central microglia or astrocyte cells, spinal dorsal horn sections were incubated with a mix of anti-p-NFκB and anti-OX-42 (microglia maker) or anti-GFAP (astrocyte maker).7−9 In Figure 9, the morphology of OX-42-expressing microglia cells in the central nerve system between Sham

(resting microglia) and CCI (activated microglia) groups had an apparent difference, from a ramified appearance to an amoeboid form. Few p-NFκB proteins were expressed in the Sham group but showed extreme increases in the CCI group. The expression of p-NFκB was abolished and reduced by KMUP-1 in the Sham and CCI groups, respectively (Figure 9). As shown in Figure 10, the greater reactivity of GFAPexpressing astrocytes was seen in the CCI group than in the 1627

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Figure 10. Double immunofluorescent staining for GFAP (astrocyte marker) and p-NFκB in the central spinal dorsal horn at day 7 after chronic constriction injury (CCI). The total numbers of GFAP-expressing astrocytes in the CCI group appeared to be greater than that in the Sham group. The expression of p-NFκB proteins was little to none in both Sham and CCI groups.

improved treatments for these patients.37 So far, management of neuropathic pain is oriented toward symptoms-directed therapy instead of elucidating the predisposing mechanisms responsible for the pain. This is most likely attributed to poor understanding of the pathophysiology that leads to neuropathic pain. Comprehensive studies of neuropathic pain are making progress in search of better alternatives. In this study, we identified possible mechanisms that involved in neuropathic pain induced by peripheral nerve injury in a CCI rat model. Undoubtedly, the CCI is an excellent model for the investigation of peripheral nerve injury-induced neuropathic pain.6,30−32 CCI-induced rat behavioral alternations closely resemble thermal hyperalgesia and mechanical allodynia under various stimuli. Thermal hyperalgesia is partly mediated via sensitization of the primary afferent nociceptors reflected by enhanced responses to heat stimuli.38 It appears that Aδ and C sensory neurons may participate in thermal stimuli-evoked peripheral nerve injury-induced neuropathic pain.39 Mechanical allodynia is due to the sensitization of the central nervous system, and it is characterized by hyperalgesia to mechanical stimuli.38 Aβ sensory neurons may contribute to mechanical stimuli-evoked peripheral neuropathy in the animal model.39 In our study, KMUP-1 was observed to have a very robust analgesic effect in thermal hyperalgesia but only had a modest effect in mechanical allodynia, suggesting that it could be more sensitive on Aδ and C fibers than on Aβ fibers. Previous reports showed that nerve injury suppresses large-conductance Ca2+activated K+ (BKCa) channel expression40 and activity41 in the dorsal root ganglion (DRG) neurons. Since KMUP-1 has been demonstrated to have BKCa-channel opening activity,23,28 its antineuropathic pain would be partly involved in the modulation of this channel. This hypothesis needs to be investigated further. Preliminary data from this laboratory indicate that KMUP-1 is capable of preventing CCI-inhibited BKCa currents in rat DRG neurons using whole-cell patchclamp recordings. The ionic mechanism between antineur-

Sham group in the dorsal horn of the spinal cord. Notably, few or no p-NFκB proteins were expressed in both Sham and CCI groups (Figure 10). Taken together, we suggested that microglia cells are responsible for the activation of NFκBmediated excessive cytokine production in the spinal dorsal horn after bilateral sciatic nerve injury but not astrocytes.

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DISCUSSION The present study is the first to demonstrate that KMUP-1 lessened CCI-induced upregulation of inflammatory mediators in the sciatic nerve. Peripheral nerve CCI increased the levels of TNF-α and IL-1β and elevated the expression of COX-2, iNOS, nNOS, MAPKs, p-IκB, and p-NFκB in the sciatic nerve; and these increases coincided with the production and persistence of neuropathic pain behaviors. KMUP-1 decreased the levels of proinflammatory cytokines (TNF-α and IL-1β) in sciatic nerve tissues, reduced the expression of COX-2, iNOS, nNOS, MAPKs, p-IκB, and p-NFκB in sciatic nerve tissues, and attenuated neuropathic pain behaviors (thermal hyperalgesia and mechanical allodynia) in a rat bilateral CCI model. Data from immunofluorescence staining showed that KMUP-1 abolished the expression of CCI (day 7)-induced p-IκB protein in the peripheral sciatic nerve. KMUP-1 also nearly abolished the accumulation of p-NFκB in central microglia cells at day 7 after CCI. These findings demonstrate that the antineuroinflammatory response of KMUP-1 was a result of the reduction of NFκB upregulation and the MAPKs pathway in the nerve system. Thus, KMUP-1 could function as a potential therapeutic agent for nerve injury and associated neuropathic pain. Nerve injury-induced neuropathic pain is persistent, and it may never be completely relieved.36 For chronic neuropathic pain, therapeutic intervention using various pharmacological agents remains the most desirable option, but the results are not overwhelmingly successful, and not all patients achieve sufficient pain relief. There is a considerable unmet need for 1628

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NFκB proteins were nearly abolished by KMUP-1 treatment in peripheral macrophage and central microglia at day 7 after CCI. On the basis of these results, it is suggested that KMUP-1 may be a good candidate to control neuroinflammation-induced pain. This study demonstrates the possibility of using KMUP-1 for therapeutic intervention of neuropathic pain based on the bilateral CCI rat model, which closely resembles the clinical peripheral mononeuropathy.6,30−32 We suggest that the antineuroinflammatory actions of KMUP-1 could be attributed to the inhibition of TNF-α release, which would result in the reduction of TNFR1 receptor activation.3,49 Accordingly, the downstream cytoplasmic p-IκB is decreased, and thus, NFκB translocation into nucleus is diminished, resulting in the decreased synthesis and release of inflammatory proteins COX2, iNOS, and nNOS.7−9 KMUP-1 undoubtedly has the ability to disrupt the deleterious cycle of this inflammatory process. In summary, our results reveal that KMUP-1 reduced p-NFκB activation in spinal microglia and attenuated ERK and p38 upregulation in the sciatic nerve. Since activation/upregulation of p-NFκB, ERK, and p38 are the major factors contributing to neuroinflammation, our findings thus provide a biochemical basis for using KMUP-1 as an innovative therapeutic agent for peripheral nerve-injury-induced neuropathic pain.

opathic pain and BKCa currents by KMUP-1 is currently under investigation. Taken together, KMUP-1 attenuated CCIinduced hyperalgesic and allodynic responses and therefore can be developed as a therapeutic agent for neuropathic pain. Inflammatory mediators including prostaglandins produced in injured nerves have been recognized as important factors contributing to the development of neuropathic pain. A dramatic increase has been observed in the prostaglandin synthesizing enzyme cyclooxygenase 2 (COX2)-immunoreactive cells in the injured site and surrounding region.42 One previous report showed that iNOS expression was increased in the sciatic nerve of CCI rats.43 In addition to iNOS, nNOS was reported to be involved in the pathogenesis of neuropathic pain.44 nNOS contribution to pain hypersensitivity has been characterized in neuropathic pain.45,46 KMUP-1 decreased the expression of inflammatory hypernociceptive COX-2, iNOS, and nNOS proteins in CCI rats. These results support the behavioral findings, suggesting that KMUP-1 has therapeutic effects on hyperalgesia and allodynia in neuropathic pain. The proinflammatory cytokine-mediated process during neuroinflammation can be elicited by systemic tissue injury, most often associating with a direct insult to the nervous system.47 Both TNF-α and IL-1β are important cytokines released by innate immune cells during inflammation. They are sensed directly by nociceptive neurons that express the cognate receptors. These in turn activate MAPKs, leading to increased membrane excitability.48 TNF-α signaling occurs via the two functionally diverse receptors, namely, TNFR1 (p55) and TNFR2 (p75).47 TNF-α and TNFR1 are upregulated in glia cells and neurons following peripheral nerve injury.49 TNF-α bound to TNFR1 incites two downstream signaling cascades, the NFκB and MAPKs transduction cascades. The NFκB cascade leads to the release of proinflammatory cytokines. Activation of the NFκB signaling transduction pathway depends upon phosphorylation and degradation of IκB proteins, resulting in the translocation of NFκB to the nucleus. The MAPK kinases activate ERK, p38 MAPK, and JNK. MAPKs play important regulatory roles in neural plasticity and inflammatory responses. Since both ERK and p38 are activated in various populations of spinal microglia, conceivably they can act concomitantly to regulate neuropathic pain.50 KMUP-1 significantly decreased the expression of MAPKs (ERK and p38) but not JNK, cytoplasmic p-IκB/IκB, and nuclear p-NFκB proteins. Since KMUP-1 treatment had no effects in both nuclear p-NFκB and cytoplasmic p-IκB/IκB up to experimental day 3, there might be no direct inhibition of NFκB. Indirect or cumulative mechanisms of action could be involved, which will require further investigation. JNK is preferentially activated in spinal astrocytes and contributes to neuropathic pain and central sensitization.51,52 This study showed that KMUP-1 had little effect on JNK protein following peripheral nerve injury, and so far, we have no explanation for this. However, KMUP-1 also decreased levels of proinflammatory cytokines (TNF-α and IL-1β) from the sciatic nerve of neuropathic pain rats. Nerve injury released pain-related mediators rapidly, which in turn elicited immune and inflammatory responses, sensitized the central nerve system, and facilitated pain processing.53 Since the KMUP-1-treated CCI group could not block peripheral macrophage (sciatic nerve) infiltration and central microglia (spinal dorsal horn) activation, it is not surprising that KMUP-1 could significantly reduce but not entirely abolish CCI-induced TNF-α and IL-1β release. Double immunofluorescent staining also confirmed that p-IκB (an index of activated NFκB) and p-

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AUTHOR INFORMATION

Corresponding Authors

*(I.-J.C.) Fax: 886-7-3234686. E-mail: [email protected]. *(B.-N.W.) Fax: 886-7-3234686. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Ms. Li-Mei An for her excellent technical assistance and ex-tenure Professor Maw-Shung Liu at School of Medicine, Saint Louis University for his editorial assistance with the manuscript. This study was supported by grants NSC 1012320-B-037-032-MY3 from the National Science Council, Taiwan, and NSYSUKMU 101-026 from Kaohsiung Medical University, Kaohsiung, Taiwan.

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ABBREVIATIONS ANOVA, one-way analysis of variance; CCI, chronic constriction injury; CFA, complete Freund’s adjuvant; COX2, cyclooxygenase 2; ERK, extracellular signal-regulated kinase; GFAP, glial fibrillary acidic protein; H&E, hematoxylin and eosin; IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase; JNK, c-Jun N-terminal kinase; MAPKs, mitogenactivated protein kinases; p-NF-κB, phospho-nuclear factor κB; PWL, paw withdrawal latency; PVDF, polyvinylidene fluoride; PWT, paw withdrawal threshold; TBS, Tris-buffered saline; nNOS, neuronal nitric oxide synthase; OCT, optimal cutting temperature compound; TNF-α, tumor necrosis factor-α; TNFR1, TNF receptor 1

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