Role of spinal cord alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in complete Freund's adjuvant-induced inflammatory pain

Background

Honeybee's sting on human skin can induce ongoing pain, hyperalgesia and inflammation. Injection of bee venom (BV) into the intraplantar surface of the rat hindpaw induces an early onset of spontaneous pain followed by a lasting thermal and mechanical hypersensitivity in the affected paw. The underlying mechanisms of BV-induced thermal and mechanical hypersensitivity are, however, poorly understood. In the present study, we investigated the role of mitogen-activated protein kinase (MAPK) in the generation of BV-induced pain hypersensitivity.

Results

We found that BV injection resulted in a quick activation of p38, predominantly in the L4/L5 spinal dorsal horn ipsilateral to the inflammation from 1 hr to 7 d post-injection. Phosphorylated p38 (p-p38) was expressed in both neurons and microglia, but not in astrocytes. Intrathecal administration of the p38 inhibitor, SB203580, prevented BV-induced thermal hypersensitivity from 1 hr to 3 d, but had no effect on mechanical hypersensitivity. Activated ERK1/2 was observed exclusively in neurons in the L4/L5 dorsal horn from 2 min to 1 d, peaking at 2 min after BV injection. Intrathecal administration of the MEK inhibitor, U0126, prevented both mechanical and thermal hypersensitivity from 1 hr to 2 d. p-ERK1/2 and p-p38 were expressed in neurons in distinct regions of the L4/L5 dorsal horn; p-ERK1/2 was mainly in lamina I, while p-p38 was mainly in lamina II of the dorsal horn.

Conclusion

The results indicate that differential activation of p38 and ERK1/2 in the dorsal horn may contribute to the generation and development of BV-induced pain hypersensitivity by different mechanisms.     

脊髓p38丝裂原活化蛋白激酶激活参与坐骨神经压迫性损伤所致神经病理性痛

本研究旨在观察脊髓p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase,p38 MAPK）在坐骨神经压迫性损伤所致神经病理性痛中的作用。雄性Sprague-Dawley大鼠鞘内置管后,4-0丝线松结扎左侧坐骨神经制作慢性压迫性损伤（chronic constriction injury,CCI）模型。CCI后第5天,鞘内注射不同剂量的p38 MAPK特异性抑制剂SB203580,并在给药前及给药后不同时间点,分别用von Frey机械痛敏监测仪和热辐射刺激仪监测大鼠损伤侧后爪机械和热刺激反应闽值,用免疫印迹技术（Western blot）观察给药前后脊髓磷酸化p38 MAPK（p-p38 MAPK）和磷酸化环磷酸腺苷反应元件结合蛋白（phosphorylated cAMP response element binding protein,pCREB）表达变化。结果发现：坐骨神经压迫性损伤引起脊髓p-p38 MAPK蛋白表达明显增加;鞘内注射SB203580能剂量依赖性逆转CCI引起的机械性痛觉异常和热痛觉过敏及脊髓水平p-p38 MAPK表达的增加,也明显抑制CCI引起的脊髓pCREB表达的增加。结果提示,脊髓水平p38 MAPK激活参与坐骨神经压迫性损伤所致神经病理性痛的发展,其作用可能通过pCREB介导。     

Activation of p38 mitogen-activated protein kinase in spinal microglia is a critical link in inflammation-induced spinal pain processing

We examined the effect of p38 mitogen-activated protein kinase (MAPK) inhibitors in models of nociception and correlated this effect with localization and expression levels of p38 MAPK in spinal cord. There was a rapid increase in phosphorylated p38 MAPK in spinal cord following intrathecal administration of substance P or intradermal injection of formalin. Immunocytochemistry revealed that phosphorylated p38 MAPK-immunoreactive cells were predominantly present in laminae I-IV of the dorsal horn. Double-staining with markers for neurons, microglia, astrocytes and oligodendrocytes unexpectedly revealed co-localization with microglia but not with neurons or other glia. Pretreatment with p38 MAPK inhibitors (SB20358 or SD-282) had no effect on acute thermal thresholds. However, they attenuated hyperalgesia in several nociceptive models associated with spinal sensitization including direct spinal activation (intrathecal substance P) and peripheral tissue inflammation (intraplantar formalin or carrageenan). Spinal sensitization, manifested by enhanced expression of cyclo-oxygenase-2 and inflammation-induced appearance of Fos-positive neurons, was blocked by pretreatment, but not post-treatment, with p38 MAPK inhibitors. Taken together, these results indicate that spinal p38 MAPK is involved in inflammation-induced pain and that activated spinal microglia play a direct role in spinal nociceptive processing.     

CREB 和NF-κB在p38MAPK所致脊髓星形胶质细胞活化中的作用

目的:探讨CREB和NF-κB在p38MAPK所致脊髓星形胶质细胞活化中的作用,明确脊髓星形胶质细胞活化中p38MAPK细胞信号转导途径的作用。方法:分离培养SPF大鼠脊髓星形胶质细胞,设正常组、SP刺激组(SP组,10-7mol/L)、SP刺激+SB203580(10μmol/L)阻断p38MAPK组(SP+SB组)、SP刺激+PD98059(10μmol/L)阻断CREB组(SP+PD组)、SP刺激+SN50(10μmol/L)阻断NF-κB(SP+SN组)。WB法、免疫荧光法、ELISA法检测12 h和24 h时p-p38、p-CREB、NF-κBp65水平及GFAP、TNF-、IL-1β水平变化。结果:SP组脊髓星形胶质细胞p-p38、p-CREB、NF-κBp65显著升高,GFAP水平显著增高,同时TNF-和IL-1β水平显著增高。与SP组比较,用SB203580阻断p38MAPK通路后,SP+SB组p-p38、p-CREB、NF-κBp65显著降低,GFAP、TNF-和IL-1β水平显著降低。用PD98059阻断CREB通路后,SP+PD组p-p38、NF-κBp65无显著变化,p-CREB显著降...     

CREB和NF-κB在p38MAPK所致脊髓星形胶质细胞活化中的作用（英文）

目的：探讨CREB和NF-κB在p38MAPK所致脊髓星形胶质细胞活化中的作用,明确脊髓星形胶质细胞活化中p38MAPK细胞信号转导途径的作用。方法：分离培养SPF大鼠脊髓星形胶质细胞,设正常组、SP刺激组（SP组,10-7mol/L）、SP刺激＋SB203580（10μmol/L）阻断p38MAPK组（SP＋SB组）、SP刺激＋PD98059（10μmol/L）阻断CREB组（SP＋PD组）、SP刺激＋SN50（10μmol/L）阻断NF-κB（SP＋SN组）。WB法、免疫荧光法、ELISA法检测12 h和24 h时p-p38、p-CREB、NF-κBp65水平及GFAP、TNF-、IL-1β水平变化。结果：SP组脊髓星形胶质细胞p-p38、p-CREB、NF-κBp65显著升高,GFAP水平显著增高,同时TNF-和IL-1β水平显著增高。与SP组比较,用SB203580阻断p38MAPK通路后,SP＋SB组p-p38、p-CREB、NF-κBp65显著降低,GFAP、TNF-和IL-1β水平显著降低。用PD98059阻断CREB通路后,SP＋PD组p-p38、NF-κBp65无显著变化,p-CREB显著降低,GFAP水平降低,同时TNF-和IL-1β水平降低。用SN50阻断NF-κB通路后,SP＋SN组p-p38、p-CREB无显著变化,NF-κBp65显著降低,GFAP水平降低,同时TNF-和IL-1β水平降低。结论：体外培养中,SP刺激后脊髓星形胶质细胞显著活化,p38MAPK活化后通过CREB及NF-κB信号途径导致胶质细胞炎性因子水平显著升高。     

Rapid co-release of interleukin 1beta and caspase 1 in spinal cord inflammation

Mounting evidence supports the hypothesis that pro-inflammatory cytokines secreted by astrocytes and microglia modulate nociceptive function in the injured CNS and following peripheral nerve damage. Here we examine the involvement of interleukin-1beta (IL-1beta) and microglia activation in nociceptive processing in rat models of spinal cord inflammation. Following application of lipopolysaccharide (LPS) to an ex vivo dorsal horn slice preparation, we observed rapid secretion of IL-1beta which was prevented by inhibition of glial cell metabolism and by inhibitors of either p38 mitogen-activated protein kinase (MAPK) or caspase 1. LPS superfusion also induced rapid secretion of active caspase 1 and apoptosis-associated speck-like protein containing a caspase recruitment domain from the isolated dorsal horn. Extensive microglial cell activation in the dorsal horn, as determined by immunoreactivity for phosphorylated p38 MAPK, was found to correlate with the occurrence of IL-1beta secretion. In behavioural studies, intrathecal injection of LPS in the lumbar spinal cord produced mechanical hyperalgesia in the rat hind-paws which was attenuated by concomitant injections of a p38 MAPK inhibitor, a caspase 1 inhibitor or the rat recombinant interleukin 1 receptor antagonist. These data suggest a critical role for the cytokine IL-1beta and caspase 1 rapidly released by activated microglia in enhancing nociceptive transmission in spinal cord inflammation.     

Toll-like Receptor 2 Mediates Peripheral Nerve Injury-induced NADPH Oxidase 2 Expression in Spinal Cord Microglia

We have previously reported that NADPH oxidase 2 (Nox2) is up-regulated in spinal cord microglia after spinal nerve injury, demonstrating that it is critical for microglia activation and subsequent pain hypersensitivity. However, the mechanisms and molecules involved in Nox2 induction have not been elucidated. Previous studies have shown that Toll-like receptors (TLRs) are involved in nerve injury-induced spinal cord microglia activation. In this study, we investigated the role of TLR in Nox2 expression in spinal cord microglia after peripheral nerve injury. Studies using TLR knock-out mice have shown that nerve injury-induced microglial Nox2 up-regulation is abrogated in TLR2 but not in TLR3 or -4 knock-out mice. Intrathecal injection of lipoteichoic acid, a TLR2 agonist, induced Nox2 expression in spinal cord microglia both at the mRNA and protein levels. Similarly, lipoteichoic acid stimulation induced Nox2 expression and reactive oxygen species production in primary spinal cord glial cells in vitro. Studies on intracellular signaling pathways indicate that NF-κB and p38 MAP kinase activation is required for TLR2-induced Nox2 expression in glial cells. Conclusively, our data show that TLR2 mediates nerve injury-induced Nox2 gene expression in spinal cord microglia via NF-κB and p38 activation and thereby may contribute to spinal cord microglia activation.     

Pro-survival effects of JNK and p38 MAPK pathways in LPS-induced activation of BV-2 cells

Identifying MAPK pathways and understanding their role in microglial cells may be crucial for understanding the pathogenesis of neurodegenerative diseases since activated microglia could contribute to the progressive nature of neurodegeneration. In this study we show that the JNK pathway plays an important role in the survival of resting microglia BV-2 cells, as evidenced by Annexin-V positive staining and caspase-3 activation in cells treated with the specific JNK inhibitor SP600125. During LPS-induced activation of BV-2 cells inhibition of the p38 and JNK pathways with SB203580 and SP600125, respectively, results in apoptosis as detected by apoptotic markers. In the presence SP600125 the phosphorylation of p38 was significantly increased both in control and LPS-activated BV-2 cells. This suggests that the pro-survival role of JNK is possible due to its abrogation of a potentially apoptotic signal mediated by p38 MAPK pathway. Furthermore, inhibition of the p38 MAPK pathway during LPS-induced activation of BV-2 cells resulted in an increased phosphorylation of c-Jun, suggesting that the pro-survival effect of p38 MAPK during inflammatory conditions involves the JNK pathway. In conclusion, the results of this study demonstrate that both the JNK and p38 MAPK pathways possess anti-apoptotic functions in the microglial cell line BV-2 during LPS-induced activation.     

Hydrogen sulfide attenuates lipopolysaccharide-induced inflammation by inhibition of p38 mitogen-activated protein kinase in microglia

The present study attempts to investigate the effect of H(2)S on lipopolysaccharide (LPS)-induced inflammation in both primary cultured microglia and immortalized murine BV-2 microglial cells. We found that exogenous application of sodium hydrosulfide (NaHS) (a H(2)S donor, 10-300 micro mol/L) attenuated LPS-stimulated nitric oxide (NO) in a concentration-dependent manner. Stimulating endogenous H(2)S production decreased LPS-stimulated NO production, whereas lowering endogenous H(2)S level increased basal NO production. Western blot analysis showed that both exogenous and endogenous H(2)S significantly attenuated the stimulatory effect of LPS on inducible nitric oxide synthase expression, which is mimicked by SB 203580, a specific p38 mitogen-activated protein kinase (MAPK) inhibitor. Exogenously applied NaHS significantly attenuated LPS-induced p38 MAPK phosphorylation in BV-2 microglial cells. Moreover, both NaHS (300 micro mol/L) and SB 203580 (1 micro mol/L) significantly attenuated LPS-induced tumor necrosis factor-alpha secretion, another inflammatory indicator. In addition, NaHS (10-300 micro mol/L) dose-dependently decreased LPS-stimulated NO production in primary cultured astrocytes, suggesting that the anti-neuroinflammatory effect of H(2)S is not specific to microglial cells alone. Taken together, H(2)S produced an anti-inflammatory effect in LPS-stimulated microglia and astrocytes, which may be due to inhibition of inducible nitric oxide synthase and p38 MAPK signaling pathways. These findings may have important implications in the treatment of neuroinflammation-related diseases.     

Cannabinoid-mediated modulation of neuropathic pain and microglial accumulation in a model of murine type I diabetic peripheral neuropathic pain

Background

Despite the frequency of diabetes mellitus and its relationship to diabetic peripheral neuropathy (DPN) and neuropathic pain (NeP), our understanding of underlying mechanisms leading to chronic pain in diabetes remains poor. Recent evidence has demonstated a prominent role of microglial cells in neuropathic pain states. One potential therapeutic option gaining clinical acceptance is the cannabinoids, for which cannabinoid receptors (CB) are expressed on neurons and microglia. We studied the accumulation and activation of spinal and thalamic microglia in streptozotocin (STZ)-diabetic CD1 mice and the impact of cannabinoid receptor agonism/antagonism during the development of a chronic NeP state. We provided either intranasal or intraperitoneal cannabinoid agonists/antagonists at multiple doses both at the initiation of diabetes as well as after establishment of diabetes and its related NeP state.

Results

Tactile allodynia and thermal hypersensitivity were observed over 8 months in diabetic mice without intervention. Microglial density increases were seen in the dorsal spinal cord and in thalamic nuclei and were accompanied by elevation of phosphorylated p38 MAPK, a marker of microglial activation. When initiated coincidentally with diabetes, moderate-high doses of intranasal cannabidiol (cannaboid receptor 2 agonist) and intraperitoneal cannabidiol attenuated the development of an NeP state, even after their discontinuation and without modification of the diabetic state. Cannabidiol was also associated with restriction in elevation of microglial density in the dorsal spinal cord and elevation in phosphorylated p38 MAPK. When initiated in an established DPN NeP state, both CB1 and CB2 agonists demonstrated an antinociceptive effect until their discontinuation. There were no pronociceptive effects demonstated for either CB1 or CB2 antagonists.

Conclusions

The prevention of microglial accumulation and activation in the dorsal spinal cord was associated with limited development of a neuropathic pain state. Cannabinoids demonstrated antinociceptive effects in this mouse model of DPN. These results suggest that such interventions may also benefit humans with DPN, and their early introduction may also modify the development of the NeP state.     

Role of Lipocalin-2-Chemokine Axis in the Development of Neuropathic Pain following Peripheral Nerve Injury

Lipocalin 2 (LCN2), which is also known as 24p3 and neutrophil gelatinase-associated lipocalin (NGAL), binds small, hydrophobic ligands and interacts with cell surface receptor 24p3R to regulate diverse cellular processes. In the present study, we examined the role of LCN2 in the pathogenesis of neuropathic pain using a mouse model of spared nerve injury (SNI). Lcn2 mRNA levels were significantly increased in the dorsal horn of the spinal cord after SNI, and LCN2 protein was mainly localized in neurons of the dorsal and ventral horns. LCN2 receptor 24p3R was expressed in spinal neurons and microglia after SNI. Lcn2-deficient mice exhibited significantly less mechanical pain hypersensitivity during the early phase after SNI, and an intrathecal injection of recombinant LCN2 protein elicited mechanical pain hypersensitivity in naive animals. Lcn2 deficiency, however, did not affect acute nociceptive pain. Lcn2-deficient mice showed significantly less microglial activation and proalgesic chemokine (CCL2 and CXCL1) production in the spinal cord after SNI than wild-type mice, and recombinant LCN2 protein induced the expression of these chemokines in cultured neurons. Furthermore, the expression of LCN2 and its receptor was detected in neutrophils and macrophages in the sciatic nerve following SNI, suggesting the potential role of peripheral LCN2 in neuropathic pain. Taken together, our results indicate that LCN2 plays a critical role in the development of pain hypersensitivity following peripheral nerve injury and suggest that LCN2 mediates neuropathic pain by inducing chemokine expression and subsequent microglial activation.     

Antioxidant effect of quercetin against acute spinal cord injury in rats and its correlation with the p38MAPK/iNOS signaling pathway

AimsThe present study aimed to investigate the correlation between quercetin (Que) and the p38 mitogen-activated protein kinase (p38MAPK)/inducible nitric oxide synthase (iNOS) signaling pathway and to explore its regulating effect on secondary oxidative stress following acute spinal cord injury (SCI), so as to elucidate the protective effects and mechanism associated with Que treatment during acute SCI.Main methodsSprague–Dawley rats were randomly divided into sham-surgery, SCI, Que, methylprednisolone (MP), and specific p38MAPK inhibitor SB203580 treatment groups. Acute SCI models were established in rats by a modified Allen's method. Real-time PCR analysis, western blot assay and immunohistochemistry for molecular changes in the p38MAPK/iNOS signaling pathway, determination of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity, reflecting the levels of secondary oxidative stress, and functional or behavioral data, reflecting changes induced by Que and control treatments post-SCI were performed.Key findingsQue significantly increased Basso, Beattie and Bresnahan scores and inclined plane test scores in SCI rats similar to the positive control drug, MP. Que significantly inhibited increases in phosphorylated-p38MAPK (p-p38MAPK) and iNOS expression and reduced the rate of iNOS-positive cells in rats with SCI, similar to the effects of SB203580. In addition, both Que and SB203580 reduced MDA content and enhanced SOD activity in SCI rats, with Que effects being stronger.SignificanceThese experimental findings indicate that in SCI rats, Que has protective effects on the spinal cord by the potential mechanism of inhibiting the activation of p38MAPK/iNOS signaling pathway and thus regulating secondary oxidative stress.     

FMLP刺激的HL-60中p38 MAPK对NADPH氧化酶p47~(phox)成分的磷酸化作用

为了解p38促分裂原活化蛋白激酶 (MAPK)参与NADPH氧化酶激活的机理 ,利用p38MAPK抑制剂SB2 0 35 80 ,在甲酰甲硫氨酰 亮氨酰 苯丙氨酸 (FMLP)刺激的分化为中性粒细胞样的HL 6 0细胞中研究p38MAPK对O·2 产生和NADPH氧化酶胞浆成分p4 7phox 的磷酸化作用 .实验发现 ,p38MAPK的激活过程与NADPH氧化酶的激活过程一致 .5 0 μmol LSB2 0 35 80抑制 5 0 % O·2 产生 ,完全抑制p38MAPK激活和部分抑制p4 7phox 体外磷酸化 .结果表明 ,在FMLP刺激的HL 6 0细胞中 ,p38MAPK可以通过磷酸化p4 7phox而参与NADPH氧化酶激活 .     

Pyridinylimidazole compound SB 203580 inhibits the activity but not the activation of p38 mitogen-activated protein kinase.

p38 MAPK is a Ser/Thr protein kinase activated by various inflammatory cytokines and a variety of stress stimuli. It is involved in many physiological processes, including the production of inflammatory cytokines. We have previously reported the design and synthesis of a series of pyridinylimidazole compounds that are selective inhibitors of p38 MAPK. These compounds, exemplified by SB 203580, are exceptionally effective in cell-based assays, including the inhibition of inflammatory cytokine production. SB 203580 is widely used as a tool to dissect the role of p38 MAPK in various physiological processes. It has previously been established that SB 203580 acts primarily to block the catalytic activity of p38 MAPK. However, it has been suggested that in cells, the compounds could also inhibit p38 MAPK activation by virtue of their ability to bind to the inactive enzyme. We undertook careful studies to definitively demonstrate that treatment with SB 203580 had no effect on Thr(180) and Tyr(182) phosphorylation, and hence activation of p38 in vivo. SB 203580, however, potently inhibited the activity of p38 MAPK as demonstrated by the inhibition of the activation of MAPKAP K2, a specific physiological substrate of p38 MAPK. This was observed regardless of stimuli or cell type. Identical results were obtained when the p38 MAPK cascade was partially reconstituted in vitro. Thus, our data clearly indicate that SB 203580 specifically inhibits the activity of p38 MAPK but not its activation by upstream MAPKK.     

The role of RIP2 in p38 MAPK activation in the stressed heart

The activation of p38 MAPK by dual phosphorylation aggravates myocardial ischemic injury and depresses cardiac contractile function. SB203580, an ATP-competitive inhibitor of p38 MAPK and other kinases, prevents this dual phosphorylation during ischemia. Studies in non-cardiac tissue have shown receptor-interacting protein 2 (RIP2) lies upstream of p38 MAPK, is SB203580-sensitive and ischemia-responsive, and aggravates ischemic injury. We therefore examined the RIP2-p38 MAPK signaling axis in the heart. Adenovirus-driven expression of wild-type RIP2 in adult rat ventricular myocytes caused robust, SB203580-sensitive dual phosphorylation of p38 MAPK associated with activation of p38 MAPK kinases MKK3, MKK4, and MKK6. The effect of SB203580 was recapitulated by unrelated inhibitors of RIP2 or the downstream MAPK kinase kinase, TAK1. However, overexpression of wild-type, kinase-dead, caspase recruitment domain-deleted, or kinase-dead and caspase recruitment domain-deleted forms of RIP2 had no effect on the activating dual phosphorylation of p38 MAPK during simulated ischemia. Similarly, p38 MAPK activation and myocardial infarction size in response to true ischemia did not differ between hearts from wild-type and RIP2 null mice. However, both p38 MAPK activation and the contractile depression caused by the endotoxin component muramyl dipeptide were attenuated by SB203580 and in RIP2 null hearts. Although RIP2 can cause myocardial p38 MAPK dual phosphorylation in the heart under some circumstances, it is not responsible for the SB203580-sensitive pattern of activation during ischemia.     

ERK1/2 and p38 mitogen-activated protein kinase mediate iNOS-induced spinal neuron degeneration after acute traumatic spinal cord injury

The enhanced production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of neuronal apoptosis after acute traumatic spinal cord injury (SCI). In the present study, to further characterize the pathways mediating the synthesis and release of NO, we examined activation of extracellular signal regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinases (p38 MAPK) in microglia/macrophages in the injured area of adult rats subjected to a complete transection at the T10 vertebrae level and assessed their role in NO production and survival of neurons by using immunohistochemistry, Western blot, RT-PCR and pharmacological interventions. Results showed activation of microglia/macrophages featured by morphological changes, as visualized immunohistochemically with the marker OX-42, in the areas adjacent to the lesion epicenter 1 h after surgery. Concomitantly, iNOS mRNA and its protein in the activated microglia/macrophages were also significantly upregulated at early hours after surgery. Their levels were maximal at 6 h, persisted for at least 24 h, and returned to basal level 72 h after SCI. Furthermore, phosphorylated ERK1/2 and p38 MAPK were activated as well in microglia/macrophages in injured area with a similar time course as iNOS. With administration of L-NAME, a NOS inhibitor, the number of apoptotic neurons was clearly decreased, as assessed with TUNEL method at 24 h after SCI. In parallel, loss of neurons induced by SCI, assessed with NeuN immunohistochemistry, was also diminished. Moreover, the effect of inhibition of phosphorylation ERK1/2 and p38 MAPK by corresponding inhibitors PD98059 and SB203580 administered before and after SCI was also investigated. Inhibition of p38 effectively reduced iNOS mRNA expression and rescued neurons from apoptosis and death in the area adjacent to the lesion epicenter; whereas the inhibition of ERK1/2 had a smaller effect on decrease of iNOS mRNA and no long-term protective effect on cell loss. These results indicate the ERK1/2 and p38 MAPK signaling pathway, especially the latter, play an important role in NO-mediated degeneration of neuron in the spinal cord following SCI. Strategies directed to blocking the initiation of this cascade prove to be beneficial for the treatment of acute SCI.     

Induction of tumor necrosis factor-alpha (TNF-alpha) by interleukin-12 p40 monomer and homodimer in microglia and macrophages

The present study was undertaken to explore the role of interleukin-12 (IL-12) p40 in the expression of TNF-alpha in microglia. Interestingly, we have found that IL-12 p70, p402 (the p40 homodimer) and p40 (the p40 monomer) dose-dependently induced the production of TNF-alpha and the expression of TNF-alpha mRNA in BV-2 microglial cells. In addition to BV-2 microglial cells, p70, p402 and p40 also induced the production of TNF-alpha in mouse primary microglia and peritoneal macrophages. As the activation of both NF-kappaB and CCAAT/enhancer binding protein beta (C/EBPbeta) is important for the expression of TNF-alpha in microglial cells, we investigated the effect of p40 on the activation of NF-kappaB as well as C/EBPbeta. Activation of NF-kappaB as well as C/EBPbeta by p40 and inhibition of p40-induced expression of TNF-alpha by Deltap65, a dominant-negative mutant of p65, and DeltaC/EBPbeta, a dominant-negative mutant of C/EBPbeta, suggests that p40 induces the expression of TNF-alpha through the activation of NF-kappaB and C/EBPbeta. In addition, we show that p40 induced the activation of both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Interestingly, PD98059, an inhibitor of ERK, inhibited p40-induced expression of TNF-alpha through the inhibition of C/EBPbeta, but not that of NF-kappaB, whereas SB203580, an inhibitor of p38 MAPK, inhibited p40-induced expression of TNF-alpha through the inhibition of both NF-kappaB and C/EBPbeta. This study delineates a novel biological function of p40 in inducing TNF-alpha in microglia and macrophages.     

Induction of autophagy in hepatocellular carcinoma cells by SB203580 requires activation of AMPK and DAPK but not p38 MAPK

SB203580 is a well-known inhibitor of p38 mitogen-activated protein kinase (MAPK). However, it can suppress cell proliferation in a p38 MAPK independent manner. The inhibitory mechanism remains unknown. Here, we showed that SB203580 induced autophagy in human hepatocellular carcinoma (HCC) cells. SB203580 increased GFP-LC3-positive cells with GFP-LC3 dots, induced accumulation of autophagosomes, and elevated the levels of microtubule-associated protein light chain 3 and Beclin 1. It stimulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and p53, but inhibited the phosphorylation of death-associated protein kinase (DAPK). Inhibition of AMPK, p53, or DAPK attenuated SB203580-induced autophagy. AMPK activation appeared to predate the DAPK signal. The activation of both AMPK and DAPK prompted the phosphorylation of p53 and enhanced Beclin 1 expression. Neither the downregulation of p38 MAPK by its siRNA or chemical inhibitor nor the upregulation of p38 MAPK by p38 MAPK DNA transfection affected B203580-induced autophagy. Collectively, the findings demonstrate a novel function of SB203580 to induce autophagy via activating AMPK and DAPK but independent of p38 MAPK. The induction of autophagy can thus account for the antiproliferative effect of SB203580 in HCC cells.