Phosphorylated P38 MAPK Expression Research Articles

Abdominal surgery plus sevoflurane exposure induces abnormal emotional changes and cognitive dysfunction in aged rats

BackgroundCognitive impairment, which includes perioperative psychological distress and cognitive dysfunction, can be determined by preoperative and post-operative neuropsychological tests. Several mechanisms have been proposed regarding the two-way communication between the immune system and the brain after surgery. We aimed to understand the mechanisms underlying perioperative neurocognitive disorders (PND) in elderly rats using an experimental abdominal surgery model. Methods24-month-old SD rats were exposed to the abdominal surgery model (AEL) under 3% anesthesia. On day 15 and day 30 post-surgery, fractional anisotropy (FA) using diffusion kurtosis imaging (DKI) was measured. From day 25 to day 30 post-surgery, behavioral tests, including open field test (OFT), Morris water maze (MWM), novel object recognition (NOR), force swimming test (FST), and elevated plus maze (EPM), were performed. Then, the rats were euthanized to perform pathological analysis and western blot measurement. ResultsThe rats exposed to AEL surgical treatment demonstrated significantly decreased time crossing the platform in the MWM, decreased recognition index in the NOR, reduced time in the open arm in the EPM, increased immobility time in the FST, and increased number of crossings in the OFT. Aged rats, after AEL exposure, further demonstrated decreased FA in the mPFC, nucleus accumbens (NAc), and hippocampus, together with reduced MAP2 intensity, attenuation of GAD65, VGlut2, CHAT, and phosphorylated P38MAPK expression, and increased reactive astrocytes and microglia. ConclusionsIn this study, the aged rats exposed to abdominal surgery demonstrated both emotional changes and cognitive dysfunction, which may be associated with neuronal degeneration and reduced phosphorylated P38MAPK.

Hydrogen gas post-conditioning alleviates cognitive dysfunction and anxiety-like behavior in a rat model of subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) results in high rates of mortality and lasting disability. Hydrogen gas (H2) is an antioxidant with demonstrated neuroprotective efficacy. The present study examined the therapeutic efficacy of H2 inhalation on early brain injury following experimental SAH in rats and the potential underlying molecular mechanisms. The rats were randomly separated into three groups (n=36 per group): Sham, SAH and SAH + H2. Endovascular perforation of the right internal carotid artery was used to establish SAH. After perforation, rats in the SAH + H2 group inhaled 2.9% H2 with regular oxygen for 2 h. Then, 24 h post-SAH, TUNEL staining was used to detect apoptotic neurons, and both immunostaining and western blotting were conducted to examine changes in p38 MAPK activity and the expression levels of apoptotic regulators (Bcl-2, Bax and cleaved caspase-3) in the ventromedial prefrontal cortex. Then, 30 day post-SAH, Nissl staining was performed to detect neuronal injury, brain MRI was conducted to detect gross changes in brain structure and metabolism, the open field test was used to assess anxiety and the novel object recognition test was performed to assess memory. H2 inhalation following experimental SAH stabilized brain metabolites, improved recognition memory and reduced anxiety-like behavior, the neuronal apoptosis rate, phosphorylated p38 MAPK expression, cleaved caspase-3 expression and the Bax/Bcl-2 ratio. Collectively, the present results suggested that H2 inhalation can alleviate SAH-induced cognitive impairment, behavioral abnormalities and neuronal apoptosis in rats, possibly via inhibition of the p38 MAPK signal pathway.

Effects of curcumin on pulmonary inflammation and p38 expression in an infant murine model of pneumococcal pneumonia

Objective To investigate the effects of curcumin on pneumococcal pneumonia-induced pneumonia, apoptosis and p38 MAPK expression in infant mice. Methods A total of 60 male infant C57BL/6 mice at three weeks of age were randomly divided into 6 groups: control group, model group, high-dose curcumin treatment group, middle-dose curcumin treatment group, low-dose curcumin treatment group and SB203580 treatment group. The Curcumin and SB203580 were intraperitoneally applied at doses of 200, 60, and 20 mg/kg (for curcumin) and 100 mg/kg (for SB203580) from two days before bacterial infection to three days post-infection. The control group and model group were intraperitoneally injected with an equal volume of saline. The model group, curcumin treatment groups and SB203580 treatment group were transnasally inoculated with approximately 106 CFU/ml of pneumococcal pneumonia in 50 μl of PBS applied to the tip of the nose to establish the experimental pneumococcal pneumonia. Subsequently, all the mice were killed and lung tissues were harvested for hematoxylin-eosin staining, calculation of lung score indexes, measurement of IL-1β and TNF-α contents by ELISA, and measurement of Bax, Bcl-2and p38 MAPK expression by Western blot. Results Compared to the model group, the edema score (0.50 ± 0.10, 1.51 ± 0.16, 1.38±0.11, vs. 2.50 ± 0.20), hemorrhage score (0.32 ± 0.09, 1.01 ± 0.11, 0.85±0.09 vs. 1.80 ± 0.20), inflammatory cell infiltrate score (0.35 ± 0.09, 1.61 ± 0.16, 1.52±0.10 vs. 3.21 ± 0.22), small airway damage score (0.12 ± 0.03, 0.53 ± 0.14, 0.50±0.04 vs. 1.12 ± 0.19) in the medium-, high-dose group and SB203580 treatment group significantly decreased (P<0.01). Compared to the model group, the contents of IL-1β (20.38 ± 1.69 pg/ml, 25.73 ± 2.08 pg/ml vs. 40.22 ± 5.70 pg/ml) and TNF-α (160.39 ± 15.81 pg/ml, 198.67 ± 18.97 pg/ml vs. 282.22 ± 25.30 pg/ml), Bax/Bcl-2 (0.31 ± 0.05, 0.53 ± 0.06 vs. 1.79 ± 0.17) and expression of phosphorylated p38 MAPK (0.69 ± 0.05, 0.81 ± 0.07 vs. 1.71 ± 0.14) in the high-dose group and SB203580 treeatment group significantly decreased (P<0.01). Conclusions Curcumin can inhibit the inflammatory response and cellular apoptosis in the lungs of mice with pneumococcal pneumonia, and the mechanisms maybe related to its inhibition of p38 MAPK expression. Key words: Curcumin; Pneumonia; Pneumococcal infections; Apoptosis; Inflammation; p38 Mitogen-activated protein kinases; Mice

Relationship between p38MAPK and GRK2 in spinal cord in development of persistent postoperative pain in rats

Objective To evaluate the relationship between p38 mitogen-activated protein kinase (p38MAPK) and G protein-coupled receptor kinase 2 (GRK2) in the development of persistent postoperative pain in rats. Methods Pathogen-free healthy male Sprague-Dawley rats, weighing 200-250 g, aged 2 months, were used in this study.Sixty rats in which intrathecal catheters were successfully implanted were divided into 6 groups (n=10 each) using a random number table method: sham operation group (group S), sham operation plus dimethyl sulfoxide (DMSO) group (group D), sham operation plus GRK2 degradation inhibitor MDL28170 group (group M), persistent postoperative pain group (group PPP), persistent postoperative pain plus DMSO group (group PPP+ D) and persistent postoperative pain plus MDL28170 group (group PPP+ M). Persistent postoperative pain was evoked by skin/muscle incision and retraction (SMIR). Immediately after operation and at 1, 2 and 3 days after operation, normal saline 20 μl was intrathecally injected once a day in S and PPP groups, 5% DMSO 10 μl was intrathecally injected once a day in D and PPP+ D groups, and MDL28170 10 μl (50 μg) was intrathecally injected once a day in M and PPP+ M groups.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before operation and 3, 7, 14 and 21 days after operation (T1-4). Four rats in each group were selected after behavioral testing at T3 and sacrificed, and the L4-6 segments of the spinal cord were removed for determination of the expression of phosphorylated p38MAPK (p-p38MAPK) by Western blot. Results There was no significant difference in MWT at each time point or expression of p-p38MAPK among group S, group D and group M (P>0.05). Compared with group S, the MWT was significantly decreased, and the expression of p-p38MAPK was up-regulated in PPP, PPP+ D and PPP+ M groups (P 0.05). Conclusion Down-regulated expression of spinal GRK2 can promote the activation of p38MAPK in the spinal cord and is involved in the development of persistent postoperative pain in rats. Key words: G protein-coupled receptor kinase 2; p38 Mitogen-actived protein kinases; Pain, postoperative; Spinal cord

Role of TLR4-p38MAPK-NF-κB signaling pathway in sevoflurane-induced decrease in cognitive function of aged rats

Objective To evaluate the role of Toll-like receptor 4(TLR4)-p38 mitogen-assoliated protein kinase (p38MAPK)-nuclear factor kappa B(NF-κB) signaling pathway in sevoflurane-induced decrease in cognitive function of aged rats. Methods Sixty SPF healthy male Sprague-Dawley rats, aged 20 months, weighing 550-750 g, were divided into 5 groups (n=12 each) using a random number table method: control group(C group), sevoflurane group (S group), TAK242 plus sevoflurane group (TS group), SB202190 plus sevoflurane group (SS group), and PDTC plus sevoflurane group (PS group). All the rats were intubated after anesthesia and connected to an animal ventilator.TAK242, SB202190 and PDTC 10 μl were injected into the lateral cerebral ventricle in TS, SS and PS groups, respectively, and normal saline containing the equal volume of DMSO was given in C and S groups.Starting from 10 min after lateral cerebral ventricle injection, 4% sevoflurane was inhaled for 6 h via the tracheal tube, with the inhaled oxygen concentration 30% and oxygen flow rate 2 L/min.The mixture of air and oxygen was inhaled in C group.The learning and memory ability was assessed by Morris water maze test at 7 days after the end of sevoflurane anesthesia, and the escape latency and swimming distance were recorded.Animals were sacrificed after the end of Morris water maze test, and brains were removed and hippocampi were isolated for determination of neural apoptosis (by TUNEL), contents of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) in hippocampal tissues (by enzyme-linked immunosorbent assay), and expression of caspase-3, phosphorylated p38MAPK (p-p38MAPK), total p38MAPK (t-p38MAPK) and NF-κB in nucleus (by Western blot). The apoptosis rate and p-p38MAPK/t-p38MAPK ratio were calculated. Results Compared with C group, the escape latency and swimming distance were significantly prolonged at each time point, the apoptosis rate and contents of TNF-α and IL-1β were increased, the expression of caspase-3, p-p38MAPK and NF-κB was up-regulated, and p-p38MAPK/t-p38MAPK ratio was increased in the other four groups (P<0.05). Compared with S group, the escape latency and swimming distance were significantly shortened at each time point, the apoptosis rate and contents of TNF-α and IL-1β were decreased, and the expression of caspase-3 was down-regulated in TS, SS and PS groups, the expression of NF-κB was significantly down-regulated in TS and SS groups, and the expression of p-p38MAPK was significantly down-regulated, and p-p38MAPK/t-p38MAPK ratio was decreased in TS group (P<0.05). Compared with TS group, the escape latency and swimming distance were significantly prolonged at each time point, the apoptosis rate and contents of TNF-α and IL-1β were increased, the expression of caspase-3 and p-p38MAPK was up-regulated, and p-p38MAPK/t-p38MAPK ratio was increased in SS and PS groups, and the expression of NF-κB was significantly up-regulated in PS group (P<0.05). The expression of NF-κB was significantly up-regulated in PS group when compared with SS group (P<0.05). Conclusion TLR4-p38MAPK-NF-κB signaling pathway is involved in sevoflurane-induced decrease in cognitive function of aged rats. Key words: Toll-like receptor 4; p38 Mitogen-activated protein kinases; NF-κB; Anesthetics, inhalation; Aged; Cognition disorders

Influences of donor and host age on human muscle-derived stem cell-mediated bone regeneration

BackgroundHuman muscle-derived stem cells (hMDSCs) have been shown to regenerate bone efficiently when they were transduced with Lenti-viral bone morphogenetic protein 2 (LBMP2). However, whether the age of hMDSCs and the animal host affect the bone regeneration capacity of hMDSCs and mechanism are unknown which prompted the current study.MethodsWe isolated three gender-matched young and old populations of skeletal muscle stem cells, and tested the influence of cells’ age on in vitro osteogenic differentiation using pellet culture before and after Lenti-BMP2/green fluorescent protein (GFP) transduction. We further investigated effects of the age of hMDSCs and animal host on hMDSC-mediated bone regeneration in a critical-size calvarial bone defect model in vivo. Micro-computer tomography (CT), histology, and immunohistochemistry were used to evaluate osteogenic differentiation and mineralization in vitro and bone regeneration in vivo. Western blot, quantitative polymerase chain reaction (PCR), and oxidative stress assay were performed to detect the effects of age of hMDSCs on cell survival and osteogenic-related genes. Serum insulin-like growth factor 1 (IGF1) and receptor activator of nuclear factor-kappa B ligand (RANKL) were measured with an enzyme-linked immunosorbent assay (ELISA).ResultsWe found LBMP2/GFP transduction significantly enhanced osteogenic differentiation of hMDSCs in vitro, regardless of donor age. We also found old were as efficient as young LBMP2/GFP-transduced hMDSCs for regenerating functional bone in young and old mice. These findings correlated with lower phosphorylated p38MAPK expression and similar expression levels of cell survival genes and osteogenic-related genes in old hMDSCs relative to young hMDSCs. Old cells exhibited equivalent resistance to oxidative stress. However, both young and old donor cells regenerated less bone in old than young hosts. Impaired bone regeneration in older hosts was associated with high bone remodeling due to higher serum levels of RANKL and lower level of IGF-1.ConclusionhMDSC-mediated bone regeneration was not impaired by donor age when hMDSCs were transduced with LBMP2/GFP, but the age of the host adversely affected hMDSC-mediated bone regeneration. Regardless of donor and host age, hMDSCs formed functional bone, suggesting a promising cell resource for bone regeneration.

Sulbactam Protects Hippocampal Neurons Against Oxygen-Glucose Deprivation by Up-Regulating Astrocytic GLT-1 via p38 MAPK Signal Pathway.

Sulbactam is an atypical β-lactam medication and reported to be neuroprotective by up-regulating glial glutamate transporter-1 (GLT-1) in rats. The present study was undertaken to study the role of p38 MAPK signal pathway in sulbactam induced up-regulation of GLT-1 expression in astrocytes and anti-ischemic effect. Neuron-astrocyte co-cultures and astrocyte cultures from neonatal Wistar rats were used. Cerebral ischemia was mimicked by oxygen-glucose deprivation (OGD). Hoechst (HO)/propidium iodide (PI) double fluorescence staining and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay were used to evaluate neuronal death and cell viability, respectively. Immunocytochemistry and Western blot were used to detect protein expressions. Sulbactam pre-incubation significantly and dose-dependently prevented neuronal death and decline in cell viability induced by OGD in neuron-astrocyte co-cultures, and upregulated GLT-1 expression in astrocyte cultures endured OGD, which suggested that sulbactam might protect neurons against OGD by up-regulating astrocytic GLT-1 expression. It was further shown that the phosphorylated-p38 MAPK expression in astrocytes was up-regulated after the sulbactam pre-incubation and this up-regulation was moderate in amplitude. Especially, the time course of the up-regulation of phosphorylated-p38 MAPK was obviously earlier than that of GLT-1, which suggested possibility that p38 MAPK might be an upstream signal for GLT-1 up-regulation induced by sulbactam. We further found that SB203580, the specific inhibitor of p38 MAPK, dose-dependently inhibited the GLT-1 up-regulation induced by sulbactam either in non- or OGD-treated astrocytes and the protective effect of sulbactam on co-cultured neurons against OGD. Taken together, it might be concluded that sulbactam protects cerebral neurons against OGD by up-regulating astrocytic GLT-1 expression via p38 MAPK signal pathway.

Ghrelin protects against depleted uranium-induced bone damage by increasing osteoprotegerin/RANKL ratio

Depleted uranium (DU) is widely used in military and civil activities, and bone is the main target organ of chronic DU toxicity. The aim of this study was to evaluate the effects of ghrelin on rats implanted with DU and explore the underlying mechanisms. The results showed that ghrelin could increase the expression of ghrelin receptor in bone tissue, thus alleviate the apoptosis of bone tissue after 3 months of 0.3 g DU embedded in the tibia. Micro-computed tomography examination showed that after DU implantation, the density of cortical bone showed no significant difference, but the trabecular bone decreased in amount, density and connectivity. Ghrelin treatment can significantly reduce the changes caused by DU. Moreover, ghrelin can inhibit the increase of serum tartrate resistant acid phosphatase and the decrease of alkaline phosphatase and osteocalcin. Furthermore, ghrelin can also significantly reduce the receptor activator of nuclear factor κB ligand (RANKL) and phosphorylated p38-MAPK expression, and increase the level of osteoprotegerin (OPG) in tissues after exposure to DU. Based on cell experimental research, p38-MAPK specific agonist can reverse the function of ghrelin, significantly inhibit the level of OPG and increase the level of RANKL. On the contrary, the use of p38-MAPK specific inhibitor or p38-MAPK siRNA can enhance the function of ghrelin. These results suggest that ghrelin may inhibit p38 MAPK activation induced by DU, and increase the OPG/RANKL ratio caused by DU exposure, hence alleviating the bone damage caused by long-term DU exposure.

Regulation of p38 mitogen-activated protein kinase signal pathway-mediated thrombocytopenia in septic mice

Objective To discuss whether p38 mitogen-activated protein kinase (p38MAPK) signal pathway participates in the modulation of thrombocytopenia in septic mice. Methods Mice models of sepsis were established by intraperitoneal injection of lipopolysaccharide (LPS) in the lethal dose. Sixty mice were randomly divided into three groups: control group, sepsis group and the p38MAPK inhibitor group (SB203580 treatment group). Platelet counts, and the expression of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) were detected continuously in the first four days. Twenty-four h after the injection of LPS, the morphological changes in platelets were observed, the expression of p-p38MAPK was detected, and platelet sequestration in the lung was examined through immunohistochemistry. Results The concentrations of IL-6 and TNF-α were higher in LPS group than in control group. In LPS group, large number of swelling and deformative platelets were observed, showing significant status of activation. The expression of phosphorylated p38MAPK (p-p38MAPK, ratio of band density was 0.603 1±0.032 5, P<0.01) in platelets and sequestration of platelets in the lung increased obviously in LPS group (mean density value was 206.18±5.14, P<0.05). Conclusion p38MAPK may play an important role in the activation of platelets. Inhibition of p38MAPK can reduce activation of platelets, sequestration of platelets in the lung, and finally prevent thrombocytopenia of septic mice. Key words: Sepsis; Platelet; p38 mitogen-activated protein kinase

Caffeine prevents LPS-induced inflammatory responses in RAW264.7 cells and zebrafish

Caffeine is a white crystalline xanthine alkaloid found in the seeds of coffee plants and leaves of the tea bush. In this study, we evaluated whether caffeine exerts anti-inflammatory effects on lipopolysaccharide (LPS)-induced inflammation both in vitro and in vivo. RAW264.7 cells were treated with various concentrations of caffeine in the presence or absence of LPS. Caffeine decreased the LPS-induced inflammatory mediator, nitric oxide (NO). Caffeine treatment also reduced the expression of pro-inflammatory genes, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-3, IL-6 and IL-12, and decreased both IL-6 secretion and phosphorylated p38MAPK expression in LPS-treated RAW264.7 cells. Caffeine inhibited nuclear translocation of nuclear factor κB (NF-κB) via IκBα phosphorylation. In addition, caffeine inhibited LPS-induced NO production in zebrafish. These results suggest that caffeine may suppress LPS-induced inflammatory responses in RAW264.7 cells by regulating NF-κB activation and MAPK phosphorylation.

Radix Astragali Stimulates p38 MARK Phosphorylation in Pediatric Patients with β-Thalassemia.

A previous study conducted by our group demonstrated that Radix Astragali compounded with Codonopsis pilosula and Plastrum testudinis was effective in treating pediatric β-thalassemia in a randomized, controlled clinical trial. However, the mechanism of action that underpins this treatment remains to be elucidated. Blood was collected from patients participating in this clinical trial and nucleated red blood cell-enriched mononuclear cells were isolated to facilitate the extraction of RNA and protein. RT-PCR was used to monitor the expression of globin genes and p38 MAPK, and total and phosphorylated p38 MAPK expression was assessed using Western blot analysis. Expression of α-, β-, and Aγ-globin mRNAs was not significantly affected following treatment with R. Astragali or the compounded formulation. However, Gγ-globin mRNA levels increased significantly in both treatment groups (when compared with pretreatment levels) following 12 weeks of treatment. Moreover, posttreatment Gγ-globin expression was significantly higher in both treatment groups compared with the control group. Although neither p38 MAPK mRNA nor protein levels were affected by the treatments, posttreatment phosphorylation of p38 MAPK was significantly increased in the R. Astragali and compounded formulation groups compared with the control group. These data suggest that the molecular mechanisms that underpin the efficacious use of R. Astragali (and its compounded formulation) in pediatric β-thalassemia treatment facilitate the induction of Gγ-globin expression following activation of p38 MAPK.

C5a Mediated Induction of Phosphorylated P38 MAPK Expression By Blood T Lymphocytes in Adults with Allergic Asthma

C5a Mediated Induction of Phosphorylated P38 MAPK Expression By Blood T Lymphocytes in Adults with Allergic Asthma

Sorbitol induces apoptosis of human colorectal cancer cells via p38 MAPK signal transduction.

Sorbitol has been reported to have anticancer effects in several tumor models, however its effects on colorectal cancer remain elusive. In the present study, the effects of sorbitol on growth inhibition and apoptosis in the colorectal cancer HCT116 cell line were evaluated and its mechanism of action was examined. An MTT assay was utilized to determine the effect of sorbitol on HCT116 cell proliferation at different time points and variable doses. Western blot analysis was used to examine the effect of sorbitol on apoptosis-related protein expression and the p38 MAPK signaling pathway. The results revealed that sorbitol may inhibit the growth of HCT116 cells in a time- and dose-dependent manner. Following treatment with sorbitol for 3 h, western blotting demonstrated cleavage of the caspase-3 zymogen protein and a cleavage product of poly (ADP-ribose) polymerase (PARP), a known substrate of caspase-3, was also evident. During sorbitol-induced apoptosis, the mitochondrial pathway was activated by a dose-dependent increase in Bax expression and cytochrome c release, while the expression of anti-apoptotic protein Bcl-2 was significantly decreased in a dose-dependent manner. The investigation for the downstream signal pathway revealed that sorbitol-induced apoptosis was mediated by an increase in phosphorylated p38 MAPK expression. Overall, the observations from the present study imply that sorbitol causes increased levels of Bax in response to p38 MAPK signaling, which results in the initiation of the mitochondrial death cascade. Therefore, sorbitol is a promising candidate as a potential chemotherapeutic agent for the treatment of colorectal cancer HCT116 cells.

Effects of SB203580 on Homer1a expression in hippocampus and learning-memory after diffuse brain injury in rats

Objective To explore the effects of SB203580(a MAPK inhibitor) on the expression of Homer1a in hippocampus and learning-memory after diffuse brain injury in rats. Method Male Sprague-Dawlley rats were divided randomly into three groups: control group, diffuse brain injury(DBI)group and DBI+ SB203580 group (peritoneal injection, 0.01 μg/kg). Morphological changes of neuronal cells were observed by electron microscope and the expression of Homer1a and phosphorylated p38MAPK was detected by immunohistochemistry and learning and memory functions were performed with Morris water maze (MWM). Results Compared with control group, ultrastructure of neuronal cells and synapses were significantly.The levels of phosphorylated p38MAPK(76.98±16.64, 2.28±0.40, P<0.05) and Homer1a (62.96±12.74, 1.28±0.10, P<0.05)respectively were increased after injury impaired.MWM test showed that the escaping latency was prolonged((74.64± 8.96)s, (24.96±4.98)s, P<0.05) and the frequency of crossing the platform was decreased(4.48±1.12, 12.65±2.36, P<0.05). Compared with the model group, SB203580 decreased ultrastructure impariment in neuronal cells and synapses and decreased phosphorylated p38MAPK expression(54.82±12.48, 76.98±16.64, P<0.05) and increased Homer1a expression(54.82±12.48, 76.98±16.64, P<0.05). MWM test showed that the escaping latency was shorten ((46.72±6.58)s, (74.64±8.96)s, P<0.05), and the frequency of crossing the platform was increased(7.56±1.20, 4.48±1.12, P<0.05). Conclusion SB203580 improves the learning-memory recovery after DBI, which is related to inhibition of p38MAPK activation and increasing Homer1a expression. Key words: Diffuse brain injury; Homer1a; Mitogen-activated protein kinases; learning-memory

Early activation of nSMase2/ceramide pathway in astrocytes is involved in ischemia-associated neuronal damage via inflammation in rat hippocampi

BackgroundCeramide accumulation is considered a contributing factor to neuronal dysfunction and damage. However, the underlying mechanisms that occur following ischemic insult are still unclear.MethodsIn the present study, we established cerebral ischemia models using four-vessel occlusion and oxygen-glucose deprivation methods. The hippocampus neural cells were subjected to immunohistochemistry and immunofluorescence staining for ceramide and neutral sphingomyelinase 2 (nSMase2) levels; immunoprecipitation and immunoblot analysis for nSMase2, receptor for activated C kinase 1 (RACK1), embryonic ectoderm development (EED), p38 mitogen-activated protein kinase (p38MAPK) and phosphorylated p38MAPK expression; SMase assay for nSMase and acid sphingomyelinase (aSMase) activity; real-time reverse transcription polymerase chain reaction for cytokine expression; and Nissl, microtubule-associated protein 2 and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining.ResultsWe found considerable production of ceramide in astrocytes, but not in neurons, during early cerebral ischemia. This was accompanied by the induction of nSMase (but not aSMase) activity in the rat hippocampi. The inhibition of nSMase2 activity effectively reduced ceramide accumulation in astrocytes and alleviated neuronal damage to some extent. Meanwhile, the expression levels of proinflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and IL-6, were found to be upregulated, which may have played an import role in neuronal damage mediated by the nSMase2/ceramide pathway. Although enhanced binding of nSMase2 with RACK1 and EED were also observed after cerebral ischemia, nSMase2 activity was not blocked by the TNF-α receptor inhibitor through RACK1/EED signaling. p38MAPK, but not protein kinase Cζ or protein phosphatase 2B, was able to induce nSMase2 activation after ischemia. p38MAPK can be induced by A2B adenosine receptor (A2BAR) activity.ConclusionsThese results indicate that the inhibition of ceramide production in astrocytes by targeting A2BAR/p38MAPK/nSMase2 signaling may represent a viable approach for attenuating inflammatory responses and neuronal damage after cerebral ischemia.

Effect and influence of mitogen-activated protein kinases pathway on airway inflammation and epithefium apoptosis in asthmatic guinea pigs

Objective To study the airway inflammation, epithefium apoptosis, p38 MAPK expression level in asthmatic guinea pigs and the effect of dexmethasone therapy. Methods Forty-eight guinea pigs were randomly divided into 4 groups: control group, asthma group, dexmethasone-treated group and MAPK pathway inhibitor SB203580 trea-ted group, 12 guinea pigs in each group.Hematoxylin and eosin staining, Massong Trichrome stain, Periodic Acid Schifgtaining were performed.Histological changes were detected under the optical or the electron microscope.Immunohistochemistry assays were used to determine the status of IL-5 and phosphorylated p38 MAPK in airway epitheliam.Inflammatory factor level was detected by enzyme linked immunosorbent assay(ELISA). Immunohistochemistry assays of cleaved caspase 3 was used to detect the level of epithefium apoptosis. Results Compared with the control group, guinea pigs in the asthmatic group showed significantly higher airway epithelium apoptosis(detected as the cleaved caspase-3 imumunoreactive cells), lung tissue phosphorylated p38 MAPK expression level, IL-5 level in BALF(P 0.05). Compared with SB203580 group, guinea pigs in the DEX group showed significantly lower airway epithelium apoptosis, lung tissue phosphorylated p38 MAPK expression level, and IL-5 level in BALF(P<0.01). Conclusions Airway inflammation is involved in epithefium apoptosis in the asthmatic guinea pigs.The interfering effect of apoptosis by DEX may be partly related to inhibition of lung tissue phosphorylated p38 MAPK expression level, and p38 MAPK may participate in the asthmatic inflammation and induce airway epithelium apoptosis. Key words: Bronchial asthma; Apoptosis; Inflammation; Mitogen-activated protein kinase; Dexmethasone; Interleukin

Activation of P38MAP kinase in estrogen-positive invasive breast cancers: Relation with HER2 and downstream signaling phosphorylated proteins expression.

e11560 Background: P38 kinases are members of the mitogen-activated protein kinase (MAPK) family. In breast cancers MAPK, as well as PI3 kinase-AKT pathway signaling proteins have major implication in molecular oncogenesis and are extensively investigated as putative targets for therapy. The present study reports the investigation of the expression of P38MAPK and its phosphorylated form (p-P38MAPK) in clinical specimens of invasive breast carcinomas in relation with estrogen receptor and HER2 expression, as well as MAPK and PI3K signaling phosphorylated proteins. Methods: The expression of P38MAPK and p-P38MAPK as well as p-AKT, p-GSK3β, p-S6 kinase, p-MEK1, p-ERK1/2 were semi-quantitatively assessed using multiplex bead immuno-assay. The analyses were performed retrospectively in frozen specimens from 46 invasive breast tumors classified according to estrogen receptor (ER) and HER2 status. Results: All specimens were taken at diagnosis and validated for tumor content &gt;50%. Twenty-nine were ER+, 17 were HER2+, 10 were triple negative (TN) tumors. Analyses were performed in triplicate from total protein extracts and were achievable in all specimens. P38MAPK was found to be expressed in all tumor specimen and significantly (P=0.002) overexpressed in ER+ tumors. P38MAPK was lower in TN tumors than in all others. The median expression of phosphorylated-P38MAPK was also higher in ER+ than in ER- tumors and lower in TN tumors than in all others. HER2 status had no influence on P38MAPK and p-P38MAPK expression. No variation in the phosphorylation rate of P38MAPK was observed in relation with ER, HER2 or TN status. Significantly higher (P=0.0048) expression of p-AKT tumors was observed in HER2+ tumors. No significant difference in p-MEK1, p-GSK3β and p-S6K expression was evidenced in any other comparisons based on ER and HER2 expression subtypes. Conclusions: Investigation of the expression of multiple phosphorylated signaling proteins can be used as molecular biomarkers for personalized targeted therapy. In ER+ invasive breast cancer, the overexpression of P38MAPK could serve as biomarker for evaluation of P38MAPK inhibitors.

Calbindin‐D28k and calretinin in chicken inner retina during postnatal development and neuroplasticity by dim red light

Members of the family of calcium binding proteins (CBPs) are involved in the buffering of calcium (Ca2+) by regulating how Ca2+ can operate within synapses or more globally in the entire cytoplasm and they are present in a particular arrangement in all types of retinal neurons. Calbindin D28k and calretinin belong to the family of CBPs and they are mainly co-expressed with other CBPs. Calbindin D28k is expressed in doubles cones, bipolar cells and in a subpopulation of amacrine and ganglion neurons. Calretinin is present in horizontal cells as well as in a subpopulation of amacrine and ganglion neurons. Both proteins fill the soma at the inner nuclear layer and the neuronal projections at the inner plexiform layer. Moreover, calbindin D28k and calretinin have been associated with neuronal plasticity in the central nervous system. During pre and early postnatal visual development, the visual system shows high responsiveness to environmental influences. In this work we observed modifications in the pattern of stratification of calbindin immunoreactive neurons, as well as in the total amount of calbindin through the early postnatal development. In order to test whether or not calbindin is involved in retinal plasticity we analyzed phosphorylated p38 MAPK expression, which showed a decrease in p-p38 MAPK, concomitant to the observed decrease of calbindin D28k. Results showed in this study suggest that calbindin is a molecule related with neuroplasticity, and we suggest that calbindin D28k has significant roles in neuroplastic changes in the retina, when retinas are stimulated with different light conditions.

EphA2 mediated vascular endothelial growth factor expression via the p38 MAPK signaling pathway in squamous cell carcinoma of the head and neck

To investigate the regulatory effect of erythropoietin-producing hepatocellular receptor (EphA2) on the expression of VEGF protein, a pro-angiogenic factor, via p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway in squamous cell carcinoma of the head and neck(SCCHN) in vitro. SCCHN Tu686 cells were transfected with EphA2 overexpression vector pEGFP-N1-EphA2. Western blot was used to detect the expression of p38 MAPK and enzyme-linked immunosorbent assay (ELISA) was applied to assay of VEGF. SB203580 as a inhibitor of p38 MAPK signaling pathway was used. The expression of VEGF protein was significantly up-regulated in Tu686 cells transfected with EphA2 overexpression vector (535.31 ± 45.71) pg/ml, when compared with Tu686 cells transfected with empty vector (400.99 ± 33.50) pg/ml and Tu686 cells with no transfection (385.30 ± 33.50) pg/ml (F = 17.091, P < 0.01). The expression of phosphorylated p38 MAPK was obviously increased in Tu686 cells with EphA2 overexpression. SB203580 inhibited the expressions of VEGF and phosphorylated p38 MAPK proteins in Tu686 cells with EphA2 overexpression. EphA2 can regulate the expression of VEGF protein and stimulate p38 MAPK signaling pathway.

P38 MAP Kinase Mediates Apoptosis After Genipin Treatment in Non–Small-Cell Lung Cancer H1299 Cells via a Mitochondrial Apoptotic Cascade

Genipin, an active constituent of Gardenia fruit, has been reported to show an anti-tumor effect in several cancer cell systems. Here, we demonstrate how genipin exhibits a strong apoptotic cell death effect in human non-small-cell lung cancer H1299 cells. Genipin-mediated decrease in cell viability was observed through apoptosis as demonstrated by induction of a sub-G1 peak through flow cytometry, DNA fragmentation measured by TUNEL assay, and cleavage of poly ADP-ribose-polymerase. During genipin-induced apoptosis, the mitochondrial execution pathway was activated by caspase-9 and -3 activation as examined by a kinetic study, cytochrome c release, and a dose-dependent increase in Bax/Bcl-2 ratio. A search for the downstream pathway reveals that genipin-induced apoptosis was mediated by an increase in phosphorylated p38MAPK expression, which further activated downstream signaling by phosphorylating ATF-2. SB203580, a p38MAPK inhibitor, markedly blocked the formation of TUNEL-positive apoptotic cells in genipin-treated cells. Besides, the interference of p38MAPK inhibited Bax expression and cytochrome c release. Altogether, our observations imply that genipin causes increased levels of Bax in response to p38MAPK signaling, which results in the initiation of mitochondrial death cascade, and therefore it holds promise as a potential chemotherapeutic agent for the treatment of H1299 cells.