Phosphorylated p38 MAP kinase expression by leucocytes is increased in allergic humans and associated with IgE responses.

Mitogen-activated protein kinases (MAPK) activate cascades that regulate cell proliferation, differentiation and death. Phosphorylated (phos-)p38 MAPK is a cell-signalling pathway associated with Th2 cytokine responses, which is required for immunoglobulin (Ig)E production. It is unknown whether MAPK are associated with IgE production. We examine the evidence linking p38 MAPK to inflammatory responses. Phos-p38, extracellular signal-related kinase (ERK) and c-JUN-n terminal (JNK) MAPK expression by blood leucocyte subsets and levels of serum Igs were measured in blood from adults with asthma and/or rhinoconjunctivitis (N = 28) and non-asthma (N = 10) (flow cytometry, microfluorenzymeimmunoassay). Peripheral blood mononuclear cells (PBMC) from allergic subjects were cultured for 10 days ± anti-CD40/recombinant IL-4 ± inhibitor of phos-P38. Culture supernatants were assayed for IgE (ELISA). Phos-p38 MAPK expression by all leucocyte subsets of allergic subjects was associated with serum IgE levels (p ≤ 0.01), after adjusting for cell counts, age, sex, race and smoking status (p ≤ 0.04). Leucocyte expression of phos-ERK and JNK did not correlate with IgE (p = 0.09-0.99). Instead, phos-ERK expression was associated with serum IgG. When PBMC from atopic subjects were cultured for 10 days with anti-CD40/rhIL-4, IgE levels were 26.2 ± 18 ng/mL. Inclusion of SB202190 (5-20 µg/mL), a specific inhibitor of phos-p38 MAPK, in culture suppressed IgE production in dose-dependent manner, with peak suppression obtained with SB202190 at 20 µg/mL (82.1% ± 11.8) (p = 0.0001), with virtually no cytotoxicity (<5%). Different MAPK pathways may be associated with IgE (p38) and IgG (ERK) responses. Phos-p38 MAPK can be a potential anti-allergy drug target.

CXCR3 signalling partially contributes to the pathogenesis of neuropathic pain in male rodents.

BACKGROUND: Currently, there is a lack of effective therapy for chronic pain. Increasing evidence has shown that chemokines and their correlative receptors involved in the neuron-glial cell cross-talk could contribute to the pathogenesis of neuropathic pain. Our previous studies suggested that CXCR3 expression was elevated in the spinal dorsal horn after nerve injury. OBJECTIVES: In this study, we aimed to explore the role of CXCR3 signalling in chronic pain modulation. METHODS: Reverse transcription quantitative PCR and Western blotting were used to measure the expression of CXCR3 and its ligands in the spinal cord following chronic constriction injury (CCI) of the sciatic nerve. Cxcr3 -knockout mice were used to observe the effect of the receptor on pain-related behaviour and microglial activation. Immunohistochemistry was used to investigate the expression of two activation markers for spinal microglia, Iba-1 and phosphorylated-p38 (p-p38) in these mice. RESULTS: The expression of CXCR3 and its ligand CXCL11 was upregulated in the lumbar dorsal horn of the spinal cord in CCI models. In Cxcr3 -knockout mice, CCI-induced tactile allodynia and thermal hyperalgesia were observed to be alleviated during the early stage of pain processing. Meanwhile, the expression of the glial activation markers, namely, Iba-1 and p-p38, was decreased. CONCLUSION: Our results demonstrate that CXCR3 could be a key modulator involved in pain modulation of the spinal cord; therefore, CXCR3-related signalling pathways could be potential targets for the treatment of intractable pathological pain.

Modification of ischemia/reperfusion induced infarct size by ischemic preconditioning in hypertrophied hearts.

This study examined the effects of ischemic preconditioning (IP) on the ischemia/reperfusion (I/R) induced injury in normal and hypertrophied hearts. Cardiac hypertrophy in rabbits was induced by L-thyroxine (0.5 mg/kg/day for 16 days). Hearts with or without IP (3 cycles of 5 min ischemia and 10 min reperfusion) were subjected to I/R (60 min ischemia followed by 60 min reperfusion). IP reduced the I/R-induced infarct size from 68% to 24% and 57% to 33% in the normal and hypertrophied hearts, respectively. Leakage of creatine phosphokinase in the perfusate from the hypertrophied hearts due to I/R was markedly less than that form the normal hearts; IP prevented these changes. Although IP augmented the increase in phosphorylated p38-mitogen-activated protein kinase (p38-MAPK) content due to I/R, this effect was less in the hypertrophied than in the normal heart. These results suggest that reduced cardioprotection by IP of the I/R-induced injury in hypertrophied hearts may be due to reduced activation of p38-MAPK in comparison with normal hearts.

Inhibition of HDAC6 alleviating lipopolysaccharide-induced p38MAPK phosphorylation and neuroinflammation in mice.

Context: Researchers in a variety of fields have extensively focused on histone deacetylase 6 (HDAC6) due to its aggravation of inflammatory reaction. However, relevant studies examining whether HDAC6 could exacerbate lipopolysaccharide (LPS)-induced inflammation are still lacking. Objective: We assessed the role of HDAC6 in LPS-induced brain inflammation and used the HDAC6-selective inhibitor Tubastatin A (TBSA) to investigate the potential mechanisms further. Materials and methods: Brain inflammation was induced in Kunming (KM) mice via intraperitoneal (I.P.), injection of Lipopolysaccharide (LPS) (1 mg/kg), the TBSA (0.5 mg/kg) was delivered via intraperitoneal. The phosphorylated p38 (p-p38) Mitogen-activated protein kinases (MAPK) and expression of typical inflammatory mediators, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in both the hippocampus and cortex, were examined by immunoblotting. Nissl staining was used to detect the neuronal damage in the hippocampus and the cortex. Results: About 1 mg/kg LPS via daily intraperitoneal (I.P.) injections for 12 days significantly increased p38 MAPK phosphorylation, TNF-α and IL-6 expression, and neuronal loss. However, 0.5 mg/kg TBSA (three days before LPS treatment) by I.P. injections for 15 days could reverse the above results. Conclusions: This present study provided evidence that TBSA significantly suppressed LPS-induced neuroinflammation and the expression of p-p38. Results derived from our study might help reveal the effective targeting strategies of LPS-induced brain inflammation through inhibiting HDAC6.

Atorvastatin Pretreatment Attenuates Ischemic Brain Edema by Suppressing Aquaporin 4.

BACKGROUND: Cerebral edema, a serious complication of acute cerebral infarction, has a crucial impact on morbidity and mortality in the early stage of cerebral infarction. And aquaporin 4 (AQP4), a bidirectional water transporting protein, plays a pivotal role in edema formation. At experimental model, it has proven that atorvastatin could exert pleiotropic neuroprotection on acute cerebral infarction independent of its cholesterol-lowering action. It was a common protective manifestation that atorvastatin can reduce the infarct volume and cerebral edema. However, little is known about atorvastatin improving ischemic brain edema by regulating AQP4 expression. This study intended to investigate the neuroprotection effects of atorvastatin pretreatment in rats with cerebral ischemia and further explore the potential relationship between atorvastatin and AQP4 expression. METHODS: Fifty-one adult male Sprague Dawley rats were randomly divided into 3 groups: sham, middle cerebral artery occlusion (MCAO), and atorvastatin pretreatment (Ator) group. For Ator group, 20 mg/kg of atorvastatin injectable suspension was administered once for 7days by gavage before operation, whereas the others were administered the same volume of saline matching. Except for sham group, MCAO and Ator groups were subjected to permanent MCAO by modified intraluminal suture method. Infarct volume, neurological deficit, brain water content (BWC), immunohistochemistry, western blot, and polymerase chain reaction (PCR) were measured at 24 hours after MCAO. RESULTS: Compared with sham group, the mNSS, infarct volume, and BWC of ischemic hemisphere were significantly increased (P < 0.001) in MCAO group. Positive cells and protein levels of p-p38MAPK and AQP4 in peri-infarction were significantly increased (P < 0.01). The mRNA levels of p38MAPK and AQP4 were also prominently upregulated (P < 0.01). Interestingly, preadministration of atorvastatin dramatically decreased infarct volume and the BWC of ischemic hemisphere compared with MCAO group (P < 0.05). The overexpressions of p-p38MAPK and AQP4 in peri-infarction were significantly decreased (P < 0.05) and their mRNA levels were downregulated by atorvastatin pretreatment (P < 0.05). Neurological deficits were also dramatically improved (P < 0.001). CONCLUSION: To the best of our knowledge, this is the first study that demonstrates an effect of atorvastatin on expression of AQP4, and we propose that decreased AQP4 expression through a p38MAPK-suppression pathway may be the mechanism of atorvastatin alleviating ischemic cerebral edema.

Vacuolar protein sorting 4B regulates apoptosis of intestinal epithelial cells via p38 MAPK in Crohn's disease.

Vacuolar protein sorting 4B (VPS4B), a member of ATPase family proteins, reportedly possesses multiple biological functions, such as regulating the development of breast cancer and non-small-cell lung cancer, participating in Parkinson's disease, and modulating neuronal apoptosis after cerebral ischemia. However, its expression and potential functions in Crohn's disease (CD) has not been understood. In this study, we reported for the first time that VPS4B was over-expressed in intestinal epithelial cell (IECs) of patients with CD. In TNBS-induced mouse colitis models, we observed the up-regulation of VPS4B was accompanied with the elevated levels of IEC apoptotic markers (active caspase-3 and cleaved PARP) and phosphorylated p38 in colitis IECs. Co-localization of VPS4B and active caspase-3 in IECs of the TNBS group further indicated the possible involvement of VPS4B in IEC apoptosis. Employing the TNF-α-treated HT29 cells as an in vitro IEC apoptosis model, we confirmed the positive correlation of VPS4B with caspase-dependent cellular apoptosis. Knocking VPS4B down by siRNA significantly alleviated TNF-α-induced p38 phosphorylation and cellular apoptosis in HT29 cells. Taken together, our findings suggested that VPS4B may facilitate the IEC apoptosis in CD via p38 MAPK signaling pathway.

Effect of acupoint catgut embedding on p38 MAPK pathway in the lung tissue of asthmatic rats. / 穴位埋线通过调节p38ä¸è£‚åŽŸæ´»åŒ–è›‹ç™½æ¿€é ¶é€šè·¯å¯¹å“®å–˜å¤§é¼ æ°”é“ä¸Šçš®çš„å½±å“.

OBJECTIVES: To observe the effect of catgut embedding at "Feishu"(BL13), "Dingchuan" (EX-B1) and "Danzhong" (CV17) on expression of phosphorylated p38 mitogen activated protein kinase (p-p38 MAPK), interleukin-4 (IL-4), interferon-γ (IFN-γ) and changes of airway epithelial cells (AEC) in the lung tissue of bronchial asthma (BA) rats, so as to explore its mechanisms underlying improvement of BA. METHODS: Forty male Wistar rats were randomly and equally divided into blank control, model, dexamethasone (DEX) and catgut embedding groups. The BA model was established by intraperitoneal injection of suspension of ovalbumin and aluminum hydroxide. Rats of the DEX group received intraperitoneal injection of DEX (1.5 mg/kg), once daily for 2 weeks, and those of the catgut embedding group received catgut embedding at BL13, EX-B1 and CV17 only one time. The rats' sneezing times per miniute in each group were recorded. H.E. staining was used to observe the histopathological changes of the lung tissue under light microscope. A transmission electron microscope (TEM) was used to observe the ultrastructural changes of AEC in the lung tissue, including the thickness of bronchial wall and bronchial smooth muscle by using an image analysis software. The protein expressions of p-p38 MAPK, IL-4 and INF-γ in the lung tissue were determined using Western blot. RESULTS: Morphological observation revealed that in the model group, light microscope showed deformed and swollen bronchial tube wall with increased folds and thickened bronchial smooth muscle；and TEM showed a large number of autophagy vesicles containing swollen and deformed organelles in the AEC, and apparent reduction of intracellular mitochondria, these situations were obviously milder in both DEX and catgut embedding groups. Compared with the blank control group, the sneezing times, thickness of bronchial wall and bronchial smooth muscle in the model group were significantly increased (P<0.01), and the expressions of p-p38 MAPK and IL-4 in lung tissue were significantly increased (P<0.01), while the expression of IFN-γ was significantly decreased (P<0.01) in the model group. In comparison with the model group, the sneezing times, thickness of bronchial wall and bronchial smooth muscle, protein expressions of p-p38 MAPK and IL-4 were significantly decreased (P<0.01), while the expression of IFN-γ was obviously increased (P<0.01) in both the DEX and catgut embedding groups. CONCLUSIONS: Acupoint catgut embedding can reduce the expression of IL-4 and increase the expression of IFN-γ by inhibiting p38 MAPK signal pathway of lung tissues in BA rats, which may contribute to its effect in alleviating the degree of airway epithelial cells damage.

Intestinal inflammatory biomarkers and outcome in pediatric Clostridium difficile infections.

OBJECTIVES: To identify specific fecal biomarkers for symptomatic Clostridium difficile infection and predictors of poor outcomes. STUDY DESIGN: We enrolled 65 children with positive C difficile testing (cases) and 37 symptomatic controls. We also analyzed stool samples from colonized and non-colonized asymptomatic children. We performed enzyme immunoassays to determine fecal interleukin (IL)-8, lactoferrin, and phosphorylated-p38 protein concentrations, and quantitative polymerase chain reaction to determine IL-8 and chemokine ligand (CXCL)-5 RNA relative transcript abundances, and C difficile bacterial burden. RESULTS: Of 68 asymptomatic controls, 16 were colonized with C difficile. Phosphorylated-p38 was specific for C difficile infection but lacked sensitivity. Fecal cytokines were elevated in samples from symptomatic children, whether cases or controls. In children with C difficile infection, fecal CXCL-5 and IL-8 messenger RNA abundances at diagnosis correlated with persistent diarrhea after 5 days of C difficile infection therapy and with treatment with vancomycin. When children with concomitant viral gastroenteritis were excluded, these correlations persisted. Time-to-diarrhea resolution was significantly longer in patients with elevated fecal cytokines at diagnosis. A logistic regression model identified high CXCL-5 messenger RNA abundance as the only predictor of persistent diarrhea. Conversely, fecal C difficile bacterial burden was not different in symptomatic and asymptomatic children and did not correlate with any clinical outcome measure. CONCLUSIONS: Fecal inflammatory cytokines may be useful in distinguishing C difficile colonization from disease and identifying children with C difficile infection likely to have prolonged diarrhea.

[Acupuncture affects the apoptosis of eosinophilic granulocytes in lung tissue of asthmatic rats by regulating the P38MAPK signaling pathway].

OBJECTIVE: To investigate the effects of acupuncture on phosphorylated P38 mitogen-activated protein kinase (p-P38MAPK), intercellular adhesion molecule-1 (ICAM-1), interferon γ (IFN-γ), and eosinophilic granulocytes (EOS) in lung tissue of asthmatic rats, and to explore the mechanism of acupuncture in regulating the apoptosis of EOS. METHODS: Clean-grade male Wistar rats were randomly divided into normal, model, dexamethasone and acupuncture groups, 8 rats in each group. The asthmatic model was established by intraperitoneal injection of mixture suspension (1 mL) of 10% ovalbumin and 10% Al(OH)_3+ normal saline, followed by inhalation of atomized 1% ovalbumin solution for 30 min, once daily for 2 weeks to trigger occurrence of asthmatic symptoms. The rats in dexamethasone group were intraperitoneally injected with 0.9 mg/kg dexamethasone since day 15 once a day for two consecutive weeks. In the acupuncture group, bilateral "Feishu" (BL13), "Pishu" (BL20), "Shenshu" (BL23), "Dingchuan" (EX-B1), and "Danzhong" (CV17) were selected for acupuncture treatment once every other day since day 15 for two consecutive weeks. Uniform reinforcing and reducing manipulation was carried out, and the needles were retained for 30 min. The pathological changes of the lung tissue were observed by hematoxylin-eosin (HE) staining. The apoptosis of EOS in the lung tissue of rats was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. The expression of p-P38MAPK in the lung tissue was detected by Western blot. The protein and mRNA levels of ICAM-1 and IFN-γ in the lung tissue were determined by immunohistochemistry and real-time PCR, respectively. RESULTS: The results of HE staining showed that the pulmonary alveoli and surrounding tissues were intact with no inflammatory cell infiltration in the normal group. The model group showed massive exudation of inflammatory materials and thickened pulmonary interstitium. The dexamethasone group and acupuncture group showed less damage of the alveolar structure and only a small number of inflammatory cells around the airway. Compared with the normal group, the apoptosis rate of EOS in lung tissue was decreased (P<0.01), the expression levels of p-P38MAPK and ICAM-1 proteins and mRNAs in the lung tissue were up-regulated (P<0.01), while the expression levels of IFN-γ protein and mRNA in the lung tissue were down-regulated (P<0.01) in the model group. Compared with the model group, the apoptosis rate of EOS in the lung tissue was increased (P<0.05), the expression levels of p-P38MAPK and ICAM-1 proteins and mRNAs in the lung tissue were down-regulated (P<0.01, P<0.05), while the expression levels of IFN-γ protein and mRNA were up-regulated (P<0.05, P<0.01) in the dexamethasone and acupuncture groups. CONCLUSION: Acupuncture may inhibit the P38MAPK signaling pathway, down-regulate ICAM-1 expression, and up-regulate IFN-γ expression to promote the apoptosis of EOS and reduce EOS aggregation, thus alleviating the inflammatory response of airway in asthma.

Ibudilast produces anti-allodynic effects at the persistent phase of peripheral or central neuropathic pain in rats: Different inhibitory mechanism on spinal microglia from minocycline and propentofylline.

Microglia exhibit various activation phenotypes in the spinal cord after peripheral nerve injury, and promote neuropathic pain. Ibudilast is a phosphodiesterase inhibitor with anti-inflammatory activity, but its effect on activated microglia in chronic neuropathic pain is poorly understood. We investigated whether ibudilast was effective on established allodynia associated with activated microglial phenotypes in two rat models of peripheral and central neuropathic pain. A single intrathecal injection of ibudilast (25 µg) inhibited established allodynia on days 7-21 after sciatic nerve injury in rats. Repeated injections of ibudilast (25 µg/day) reduced the numbers of phosphorylated p38-positive cells without changing hypertrophic microglia, whereas minocycline (100 µg/day) decreased the numbers of hypertrophic microglia associated with phosphorylated p38 levels in the spinal cord. Gene analysis revealed that minocycline, but not ibudilast, increased the expression of anti-inflammatory cytokine genes Il10 and Tgfß1 in the spinal cord. Propentofylline (100 µg/day) was less effective on microglial phenotypes and established allodynia. Ibudilast inhibited persistent allodynia after the recovery of motor deficits in experimental autoimmune encephalomyelitis rats. Therefore, ibudilast might be effective for chronic neuropathic pain after peripheral and central nerve damage. Ibudilast mediated these effects on activated microglia using a different mechanism compared with minocycline and propentofylline.

Toll-like receptor-4/p38 MAPK signaling in the dorsal horn contributes to P2X4 receptor activation and BDNF over-secretion in cancer induced bone pain.

Our previous research suggested that the P2X4 receptor (P2X4R) expression in microglia was involved in the activation of toll-like receptor-4 (TLR4) in the dorsal horn in the rat model of cancer induced bone pain (CIBP). In this study, we focused on whether TLR4- mitogen-activated protein kinases, p38 (p38 MAPK) contributes to P2X4R activation and brain-derived neurotrophic factor (BDNF) over-secretion in CIBP. In in vitro experiment, the results showed that BDNF expression evoked by ATP stimulation was dependent on TLR4-p38. In in vivo experiment, the results demonstrated that an intrathecal injection of TLR4 siRNA alleviated nociception induced by lipopolysaccharide (LPS) plus ATP or CIBP with decreased expression of P2X4R, TLR4, BDNF, interleukin-6 (IL-6) and phosphorylated-p38 MAPK (p-p38 MAPK). Moreover, injection with p38MAPK inhibitor SB203580 resulted in an identical pattern compared with treatment with TLR4 siRNA. Our results demonstrate that the activation of TLR4-p38MAPK-P2X4R signaling in microglial possibility plays an important role in the process of nociceptive transmission in CIBP, suggesting new mechanism and potential therapeutic targets for CIBP.

A Central Role for Phosphorylated p38α in Linking Proteasome Inhibition-Induced Apoptosis and Autophagy.

Autophagy and the ubiquitin proteasome system (UPS), as two major protein degradation pathways, coordinate with each other in regulating programmed cell death. Autophagy can compensate for the UPS impairment-induced cell dysfunction and apoptosis. However, it is not clear how cells maintain the delicate balance between UPS-related apoptosis and autophagy. Here, we showed that proteasome inhibition-mediated UPS impairment can activate the phosphorylated p38α (p-p38α)-dependent apoptotic pathway and autophagy pathway in both neuroblastoma cell line N2a and primary cortical neuronal cells. Multiple indices were utilized for the autophagy detection including LC3II transition, acidic vesicle formation, lysosomal accumulation, and p62 reduction. Blockade of autophagy flux with autophagy inhibitor 3-methyladenine or bafilomycin A1 resulted in further phosphorylation of p38α, polyubiquitinated protein aggregation, and greater apoptotic cell death. On the contrary, enhancement of autophagy by rapamycin attenuated the cell loss by lowering p-p38α level and degrading protein aggregates, indicating a protective role of autophagy in cell stress and apoptosis. Moreover, de-activation of p38α with pharmaceutical p38α inhibitor BIRB796 greatly increased autophagy activation, reduced protein aggregates, and attenuated cell loss, suggesting a bidirectional regulation between p-p38α and autophagy. In addition, manipulation of p-p38α by BIRB796 or p38α knockdown decreased the phosphorylation of key components of the mammalian target of rapamycin (mTOR)-dependent pathway, indicating that the mTOR pathway mediates the p-p38α regulation on autophagy. Overall, our data emphasize p-p38α as a key mediator in the antagonistic interaction between apoptosis and autophagy in response to UPS impairment. Centering p-p38α as a potential regulatory target may provide a dual advantage of proteostasis maintenance and cell survival for simultaneous inhibition of apoptosis and activation of autophagy.

Vacuolar Protein Sorting 4B (VPS4B) Regulates Apoptosis of Chondrocytes via p38 Mitogen-Activated Protein Kinases (MAPK) in Osteoarthritis.

To aim of this study is to investigate the expression of VPS4B (vacuolar protein sorting 4B) in articular cartilage with osteoarthritis (OA) and to analyze the relationship between VPS4B and chondrocyte apoptosis. We established an OA rat model by the MLI (meniscal/ligamentous injury) modeling method, and we observed the expression of VPS4B in articular cartilage through immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). Human SW1353 chondrosarcoma cells were treated with IL-1ß to mimic the OA-like chondrocyte injury in vitro, and Western blot was employed to examine the IL-1ß-induced expression of VPS4B, phosphorylated p38, and apoptotic markers, namely active caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). The co-localization of VPS4B and active caspase 3 was confirmed through immunofluorescence. We knocked down VPS4B expression through RNA interference. Western blot was carried out to detect the knockdown efficiency of VPS4B and evaluate its effects on IL-1ß-stimulated expression of apoptotic markers and phosphorylated p38 in SW1353 cells. Annexin V/propidium iodide (PI) staining was used to detect chondrocyte apoptosis. VPS4B expression was significantly upregulated in articular cartilage of OA rat model. IL-1ß stimulation increased the expression of VPS4B, apoptotic markers, and phosphorylated p38 in SW1353 cells. VPS4B co-localized with active caspase 3 in IL-1ß-treated SW1353 cells. VPS4B inhibition significantly reduced IL-1ß-stimulated expression of apoptotic markers and phosphorylated p38 in SW1353 cells. Moreover, flow cytometry assay demonstrated that VPS4B knockdown alleviated IL-1ß-induced apoptosis. Our results suggested that VPS4B might facilitate chondrocyte apoptosis in OA via p38 MAPK signaling pathway. This study may provide a novel insight into the pathophysiology of OA and a potential therapeutic target for its treatment.

Electroacupuncture reduces apoptotic index and inhibits p38 mitogen-activated protein kinase signaling pathway in the hippocampus of rats with cerebral ischemia/reperfusion injury.

Electroacupuncture attenuates cerebral hypoxia and neuronal apoptosis induced by cerebral ischemia/reperfusion injury. To further identify the involved mechanisms, we assumed that electroacupuncture used to treat cerebral ischemia/reperfusion injury was associated with the p38 mitogen-activated protein kinase (MAPK) signaling pathway. We established rat models of cerebral ischemia/reperfusion injury using the modified Zea-Longa's method. At 30 minutes before model establishment, p38 MAPK blocker SB20358 was injected into the left lateral ventricles. At 1.5 hours after model establishment, electroacupuncture was administered at acupoints of Chize (LU5), Hegu (LI4), Zusanli (ST36), and Sanyinjiao (SP6) for 20 minutes in the affected side. Results showed that the combination of EA and SB20358 injection significantly decreased neurologic impairment scores, but no significant differences were determined among different interventional groups. Hematoxylin-eosin staining also showed reduced brain tissue injuries. Compared with the SB20358 group, the cells were regularly arranged, the structures were complete, and the number of viable neurons was higher in the SB20358 + electroacupuncture group. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling assay showed a decreased apoptotic index in each group, with a significant decrease in the SB20358 + electroacupuncture group. Immunohistochemistry revealed reduced phosphorylated p38 expression at 3 days in the electroacupuncture group and SB20358 + electroacupuncture group compared with the ischemia/reperfusion group. There was no significant difference in phosphorylated p38 expression between the ischemia/reperfusion group and SB20358 group. These findings confirmed that the electroacupuncture effects on mitigating cerebral ischemia/reperfusion injury are possibly associated with the p38 MAPK signaling pathway. A time period of 3 days could promote the repair of ischemic cerebral nerves.

Analysis of the protective effects of a neuronal Cav2.1 calcium channel in brain injury.

We previously reported that rolling Nagoya mice carrying a mutation in the α1 subunit of the Cav2.1 channel protective from ischemia- and kainate-induced neuronal damage. However, the protective effect of this mutation and its relationship to brain injury recovery have not been examined. To examine the relationship between Cav2.1 channel function and brain injury, we induced cryogenic brain damage in homozygous rolling Nagoya (rol/rol), control wild-type (+/+), ω-agatoxin IVA-pretreated +/+ (ω-aga +/+), and ω-agatoxin IVA-post-treated +/+ (ω-aga-post-treated +/+) mice. We measured the lesion area, blood brain-barrier permeability and performed immunohistochemistry and western blot analysis. The lesions of rol/rol and ω-aga +/+ mice were significantly smaller than those observed in +/+ mice at both day 1 and day 7 after injury. Similar results were shown in blood-brain barrier permeability. We observed more reactive astrogliosis in +/+ mice than in rol/rol or ω-aga +/+ mice. rol/rol and ω-aga +/+ mice had fewer degenerating cells due to cryogenic injury than did +/+ mice at both day 1 and day 7. ω-Aga-post-treated +/+ mice 24h after injury were sacrificed on day 7. The lesions were smaller in ω-aga-post-treated +/+ mice than those in vehicle-treated +/+ mice. We also examined phosphorylated p38 (pp38) at the injured site. ω-Aga-post-treated +/+ mouse brain slices showed weak pp38 signal; vehicle-treated +/+ mouse brain slices were pp38-positive. These findings demonstrate that the mutant Cav2.1 channel exerts a protective effect against cryogenic brain injury in rolling Nagoya mice. Our results indicate that inhibitors of the Cav2.1-dependent p38 signaling cascade would be useful as therapeutic agents in the treatment of brain injury.

Spinal interleukin-33 and its receptor ST2 contribute to bone cancer-induced pain in mice.

Cancer pain, particularly bone cancer pain, affects the quality of life of cancer patients, and current treatments are limited. Interleukin (IL)-33, a new member of the IL-1 super family, has been reported to be involved in the modulation of inflammatory pain. However, studies focused on its role in the modulation of cancer pain have been rare. The present study was designed to investigate whether spinal IL-33/ST2 signaling was involved in bone cancer-induced pain in mice. Bone cancer was induced via intra-femoral inoculation of 4T1 mammary carcinoma cells. The mice inoculated with carcinoma cells showed mechanical allodynia, heat hyperalgesia and a reduction in limb use, whereas phosphate-buffered saline or heat-killed cells-injected mice showed no significant difference compared to non-treated mice. The pain hypersensitive behaviors worsened over time and with bone destruction. Both the mRNA and the protein levels of IL-33 and relative cytokines (IL-1ß, IL-6, TNF-a) were significantly increased in the spinal cord after the inoculation of carcinoma cells. Intrathecal administration of ST2 antibody to block IL-33/ST2 signaling alleviated pain behaviors in a dose-dependent manner in bone cancer pain mice compared with vehicle-injected mice. Moreover, the ST2(-/-) mice showed a significant amelioration of limb use and heat hyperalgesia compared to wild-type mice. Meanwhile, concentrations of spinal IL-1ß, IL-6 and TNF-a in the cancer-bearing ST2(-/-) mice had no significant changes. These data further suggested that IL-33/ST2 signaling played a vital role in cancer pain. Our results provided evidence that IL-33 and its receptor ST2 may be a potential therapeutic target for the treatment of pain in bone cancer patients.

Identification and characterization of an alternative splice variant of Mpl with a high affinity for TPO and its activation of ERK1/2 signaling.

The thrombopoietin receptor is a crucial element in thrombopoietin-initiated signaling pathways, which stimulates the differentiation of normal hematopoietic progenitor cells, the maturation of megakaryocytes, and the generation of platelets. In this study, we identified a novel activating variant of thrombopoietin receptor, termed Mpl-D, in human megakaryoblastic leukemia Dami cells and demonstrated that the binding affinity of the Mpl-D receptor for thrombopoietin is enhanced. Cell cycle analysis revealed that in the presence of thrombopoietin, most Mpl-D expressing NIH3T3 (NIH3T3/Mpl-D) cells were prevalent in G1 phase while the S and G2/M populations were less frequently observed. Unexpectedly, thrombopoietin induced strong and prolonged ERK1/2 signaling in NIH3T3/Mpl-D cells compared with its receptor wild-type expressing NIH3T3 (NIH3T3/Mpl-F) cells. Further analysis of the mRNA levels of cyclin D1/D2 in NIH3T3/Mpl-D cells demonstrated markedly down-regulated expression compared to NIH3T3/Mpl-F cells in the presence of thrombopoietin. Thus, the prolonged activation of ERK1/2 by Mpl-D might lead to G1 cell cycle arrest through a profound reduction of cyclin D1/D2 in order to support cell survival without proliferation. We also provided tertiary structural basis for the Mpl-D and thrombopoietin interaction, which might provide insights into how Mpl-D effectively increases binding to thrombopoietin and significantly contributes to its specific signaling pathway. These results suggest a new paradigm for the regulation of cytokine receptor expression and function through the alternative splicing variant of Mpl in Dami cells, which may play a role in the pathogenesis of megakaryoblastic leukemia.

Topical alpha-selective p38 MAP kinase inhibition reduces acute skin inflammation in guinea pig.

Certain skin pathologies, including psoriasis, are thought to be immune-mediated inflammatory diseases. Available literature clearly indicates the involvement of inflammatory cells (neutrophils, T cells, and macrophages), their cytokines, and the p38 mitogen-activated protein kinase (MAPK) signaling pathway in the pathophysiology of psoriasis. Neutrophils play an important role in the formation of acute inflammatory changes in psoriasis. Acute inflammation or acute flares in psoriasis remain poorly addressed in clinical medicine. In this communication, we first establish a simple and reproducible model for studying neutrophil-mediated acute skin inflammation. Using the hairless guinea pig, due to the similarity of skin architecture to that of human, acute inflammation was induced with an intradermal injection of 50 µg/mL lipopolysaccharide (LPS) in 50 µL solution. Myeloperoxidase (MPO) activity was measured by MPO-positive neutrophils and shown to increase for 24-hours post-injection. Simultaneously, the level of phosphorylated p38 MAPK was documented for 48-hours post-LPS injection in the skin. Next, we used this model to examine the therapeutic potential of an α-selective p38 MAPK inhibitor, SCIO-469. A comparison of topical application of SCIO-469 at 5 mg/mL or 15 mg/mL to vehicle revealed that SCIO-469 dose-dependently reduces acute skin inflammation and that this effect is statistically significant at the higher dose. Further examination of tissues that received this dose also revealed statistically significant reduction of MPO activity, phosphorylated p38 MAPK, interleukin-6, and cyclooxygenase-2. These data suggest that the α-selective p38 MAPK inhibitor, SCIO-469, acts as a topical anti-inflammatory agent via the p38 MAPK pathway to reduce neutrophil induced acute inflammation in the skin. These observations suggest that α-selective p38 MAPK inhibition may be an effective therapeutic strategy to manage acute skin inflammation.