P38 MAPK Protein Research Articles

Effects of Fluoride on the Expression of p38MAPK Signaling Pathway-Related Genes and Proteins in Spleen Lymphocytes of Mice.

This study investigated the effects of sodium fluoride on the expression of p38MAPK signaling pathway-related genes and proteins in the spleen lymphocytes of mice, revealing the mechanism of the toxicity of fluoride to the immune system. The spleen lymphocytes, isolated from mice consuming different NaF doses (0, 50, 100, and 150mg/L) for 60days, were cultured in medium with bacteria lipopolysaccharide, and the cells' proliferation ability was analyzed through MTT; real-time PCR detected the change of MLK3/MKK6/p38MAPK/MSK1/ATF1 on mRNA, and the difference of protein expression of MKK6/p38MAPK were detected through the Western blotting. The results suggested that the proliferation ability of spleen lymphocytes isolated from mice consuming different NaF doses is lower, and the expression of genes and proteins of MKK6/p38MAPK showed a decreasing trend. These results demonstrate that fluoride can suppress the activation of p38MAPK pathway in mice spleen lymphocytes and further influences the function of the immune system.

TGF-beta-induced early gene-1 overexpression promotes oxidative stress protection and actin cytoskeleton rearrangement in human skin fibroblasts

BackgroundTransforming growth factor beta inducible early gene-1 (TIEG-1), a member of the Krüppel-like factor, was identified as a primary response gene for TGF-β. The role of TIEG-1 in skin repair has been mainly addressed in vivo on TIEG-1 null mice model and the mechanism remains unexplored. MethodsWe investigated the modulation of TIEG-1 expression in normal human skin fibroblasts by either down-expressing or overexpressing the gene. We evaluated reactive oxygen species production and the cell viability of treated cells. The effect of TIEG-1 overexpression was monitored by wound healing assay and immunofluorescence staining of actin fibers organization and alpha-smooth muscle actin (α-SMA). Western blots were carried out to identify the level of expression or phosphorylation of key proteins such as cofilin, Rho GTPases, and p38 mitogen-activated protein kinase (p38 MAPK). ResultsTIEG-1 down-regulation had a deleterious effect on the cell viability. It was significantly reduced (65±5%) and exposure to ultraviolet further increased this effect (47±3%). By contrast, cells overexpressing TIEG-1 had a reduced reactive oxygen species production (75%) compared to control and mock-transfected cells. This overexpression also resulted in formation of actin stress fibers and increased α-SMA expression and an enhanced wound healing feature. RhoB GTPase was upregulated and phosphorylation of cofilin and p38 MAPK was observed. ConclusionTIEG-1 overexpression in normal human skin fibroblasts results in improved resistance to oxidative stress, myofibroblast-like conversion that involved RhoB signaling pathway with cofilin and p38 MAPK proteins activation. General significanceThis study enlightens the role of TIEG-1 role in skin biology.

Effects of total flavone of hawthorn leaf on expression of p38MAPK signaling pathway and inflammation factors in rats brain with chronic cerebral ischemia

Objective To investigate the therapeutic effect and mechanism of total flavone of hawthorn leaf (TFHL) on p38MAPK signaling pathway and inflammation factors in rats brain with chronic cerebral ischemia. Methods SPF class healthy male SD rats were randomly divided into sham group, model group, TFHL group and Ginkgo leaf group(12 rats in each group). Permanent bilateral carotid artery ligation was used to prepare chronic cerebral ischemia model. Morris water maze method was used to evaluate learning and memory abilities of rats. Immunohistochemistry and Western blot methods were used to measure the expression of caspase-3 and p38MAPK proteins. ELISA method was used to measure the amounts of TNF-α and IL-1β in hippocampal tissue. Results Compared with the model group, TFHL treatment (36 d) can improve learning and memory capabilities of vascular dementia rats, shorten the escape latency (TFHL group (10.01±2.85) s vs Model group (19.54±6.12) s, P<0.05) and the course of searching platform(TFHL group(2.6044±0.3219)m vs model group(3.3502±0.6231)m, P<0.05), increase the numbers of crossing the platform (TFHL group(5.17±2.12) times vs Model group (3.96±1.34) times, P<0.05) and the platform quadrant swimming distance percentage (TFHL group(48.22±7.39)% vs model group (33.42±5.32) %, P<0.01). The number of caspase-3 positive cells in the hippocampus significantly reduced (TFHL group(1.677±0.164) vs Model group (2.387±0.171), P<0.05), the expression level of P38MAPK protein (TFHL group(0.0161±0.0003) vs Model group (0.0254±0.0018), P<0.05), TNF-α(TFHL group(19.61±3.61) ng/10 mg vs Model group (27.82±6.57) ng/10 mg, P<0.01)and IL-1β(TFHL group(24.41±2.56) ng/10 mg vs Model group (29.43±5.26) ng/10 mg, P<0.05) were significantly decreased. Conclusion TFHL plays a protective role in nerve function of the chronic cerebral ischemia rats. The mechanism of its anti-apoptosis might be associated with the activation of P38MAPK signaling pathway, inflammation and the apoptosis of neurons in the brain. Key words: Total flavone of hawthorn leaf; Chronic cerebral ischemia; Learning and memory; p38MAPK; TNF-α; IL-1β

Sodium thiosulfate attenuates glial-mediated neuroinflammation in degenerative neurological diseases.

BackgroundSodium thiosulfate (STS) is an industrial chemical which has also been approved for the treatment of certain rare medical conditions. These include cyanide poisoning and calciphylaxis in hemodialysis patients with end-stage kidney disease. Here, we investigated the anti-inflammatory activity of STS in our glial-mediated neuroinflammatory model.MethodsFirstly, we measured glutathione (GSH) and hydrogen sulfide (H2S, SH−) levels in glial cells after treatment with sodium hydrosulfide (NaSH) or STS. We also measured released levels of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) from them. We used two cell viability assays, MTT and lactate dehydrogenase (LDH) release assays, to investigate glial-mediated neurotoxicity and anti-inflammatory effects of NaSH or STS. We also employed Western blot to examine activation of intracellular inflammatory pathways.ResultsWe found that STS increases H2S and GSH expression in human microglia and astrocytes. When human microglia and astrocytes are activated by lipopolysaccharide (LPS)/interferon-γ (IFNγ) or IFNγ, they release materials that are toxic to differentiated SH-SY5Y cells. When the glial cells were treated with NaSH or STS, there was a significant enhancement of neuroprotection. The effect was concentration-dependent and incubation time-dependent. Such treatment reduced the release of TNFα and IL-6 and also attenuated activation of P38 MAPK and NFκB proteins. The compounds tested were not harmful when applied directly to all the cell types.ConclusionsAlthough NaSH was somewhat more powerful than STS in these in vitro assays, STS has already been approved as an orally available treatment. STS may therefore be a candidate for treating neurodegenerative disorders that have a prominent neuroinflammatory component.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0488-8) contains supplementary material, which is available to authorized users.

Inhibitory gene expression of the Cav3.1 T-type calcium channel to improve neuronal injury induced by lidocaine hydrochloride

Cav3.1 is a low-voltage-activated (LVA) calcium channel that plays a key role in regulating intracellular calcium ion levels. In this study, we observed the effects of lidocaine hydrochloride on the pshRNA-CACNA1G-SH-SY5Y cells that silenced Cav3.1 mRNA by RNA interference, and investigated the roles of p38 MAPK in these effects. We constructed the pNC-puro-CACNA1G-SH-SY5Y cells and pshRNA-CACNA1G –SH-SY5Y cells by the RNA interference. All the cells were cultured with or without 10mM lidocaine hydrochloride for 24h. The cell morphology, cell viability, Cav3.1 and p38 protein expression, cell apoptosis rate and intracellular calcium ion concentration were detected. We found that all cells treated with 10mM lidocaine hydrochloride for 24h showed cellular rounding, axonal regression, and cellular floating. Compared with the cells in SH-SY5Y+Lido group and NC+Lido group, those in the RNAi+Lido group showed similar changes, but of smaller magnitude. Additionally, following lidocaine hydrochloride all cells displayed increased Cav3.1 and p38 MAPK protein, apoptosis rate, and intracellular calcium ion levels; however,these changes in the RNAi+Lido group were less pronounced than in the SH-SY5Y+Lido and NC+Lido groups. The cell viability decreased following lidocaine hydrochloride treatment, but viability of the cells in the RNAi+Lido group was higher than in the SH-SY5Y+Lido and NC+Lido groups. The results showed that Cav3.1 may be involved in neuronal injury induced by lidocaine hydrochloride and that p38 MAPK phosphorylation was reduced upon Cav3.1 gene silencing.

Low-dose occupational exposure to benzene and signal transduction pathways involved in the regulation of cellular response to oxidative stress

AimsBenzene metabolism seems to modulate NF-κB, p38-MAPK (mitogen-activated protein kinase) and signal transducer and activator of transcription 3 (STAT3) signalling pathways via the production of reactive oxygen species. This study aims to evaluate the effects of low-dose, long-term exposure on NF-κB, STAT3, p38-MAPK and stress-activated protein kinase/Jun amino-terminal kinase (SAPK/JNK) signal transduction pathways in peripheral blood mononuclear cells in gasoline station attendants. The influence of consumption of vegetables and fruits on these pathways has also been evaluated. Main methodsA total of 91 men, employed in gasoline stations located in eastern Sicily, were enrolled for this study and compared with a control group of 63 male office workers with no history of exposure to benzene. The exposure was assessed by measuring urinary trans,trans-muconic acid (t,t-MA) concentration. Quantitative analyses were performed for proteins NF-κB p65, phospho-NF-κB p65, phospho-IκB-α, phospho-SAPK/JNK, phospho-p38 MAPK and phospho-STAT3 using an immunoenzymatic assay. Key findingsThe results of this study indicate significantly higher t,t-MA levels in gasoline station attendants. With regard to NF-κB, phospho-IκB-α and phospho-STAT3 proteins, statistically significant differences were observed in workers exposed to benzene. However, no differences were observed in SAPK/JNK and p38-MAPK activation. These changes were positively correlated with t,t-MA levels, but only phospho-NF-κB p65 was associated with the intake of food rich in antioxidant active principles. SignificanceChronic exposure to low-dose benzene can modulate signal transduction pathways activated by oxidative stress and involved in cell proliferation and apoptosis. This could represent a possible mechanism of carcinogenic action of chronic benzene exposure.

Tumor Necrosis Factor-Like Weak Inducer of Apoptosis Accelerates the Progression of Renal Fibrosis in Lupus Nephritis by Activating SMAD and p38 MAPK in TGF-β1 Signaling Pathway.

This study aim was to explore the effects of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) in lupus nephritis and its potential underlying mechanisms. MRL/lpr mice were used for in vivo experiments and human proximal tubular cells (HK2 cells) were used for in vitro experiments. Results showed that MRL/lpr mice treated with vehicle solution or LV-Control shRNA displayed significant proteinuria and severe renal histopathological changes. LV-TWEAK-shRNA treatment reversed these changes and decreased renal expressions of TWEAK, TGF-β1, p-p38 MAPK, p-Smad2, COL-1, and α-SMA proteins. In vitro, hTWEAK treatment upregulated the expressions of TGF-β1, p-p38 MAPK, p-SMAD2, α-SMA, and COL-1 proteins in HK2 cells and downregulated the expressions of E-cadherin protein, which were reversed by cotreatment with anti-TWEAK mAb or SB431542 treatment. These findings suggest that TWEAK may contribute to chronic renal changes and renal fibrosis by activating TGF-β1 signaling pathway, and phosphorylation of Smad2 and p38 MAPK proteins was also involved in this signaling pathway.

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.

Contribution of p38 MAPK to the Ameliorating Effect of Enriched Environment on the Cognitive Deficits Induced by Chronic Cerebral Hypoperfusion

Background/Aims: An enriched environment (EE) ameliorates learning and memory impairments induced by chronic cerebral hypoperfusion, and the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway exerts both beneficial and deleterious effects on the nervous system during the progression of ischemia. Methods: The present study investigated whether p38 MAPK participates in the process by which EE exposure ameliorates the cognitive deficits induced by chronic cerebral hypoperfusion. Results: EE exposure significantly enhanced the cognitive performance of vascular dementia (VD) model rats, and p38 MAPK protein decreased in parallel with cognitive improvements. Inhibition of p38 MAPK function by its selective inhibitor SB203580 improved the cognition index of VD rats and upregulated p38 MAPK expression with p38 MAPK antisense oligodeoxynucleotides. This impaired cognition in VD rats could not be rescued by EE exposure. Conclusion: p38 MAPK participates in the process by which EE exposure ameliorates cognitive deficits induced by chronic cerebral hypoperfusion.

Decreased hepatic phosphorylated p38 mitogen-activated protein kinase contributes to attenuation of thioacetamide-induced hepatic necrosis in diet-induced obese mice.

We previously reported that thioacetamide (TA)-induced hepatocellular necrosis was attenuated in mice fed a high-fat diet (HFD mice) compared with mice fed a normal rodent diet (ND mice). In this study, we investigated whether p38 mitogen-activated protein kinase (p38 MAPK) was involved in this attenuation. Western blot analysis revealed that hepatic phosphorylated p38 MAPK protein decreased at 8 and 24 hours (hr) after TA dosing in the HFD mice, while it decreased only at 24 hr in the ND mice in comparison to the time- and diet-matched, vehicle-treated mice. p38 MAPK regulates various biological functions including inflammation, therefore, hepatic metabolomics analysis focusing on pro-inflammatory lipid mediators was performed. At 24 hr after TA dosing, only one pro-inflammatory mediator, 12-hydroxyeicosatetraenoic acid (HETE), was higher in the HFD mice. On the other hand, in addition to 12-HETE, 15-HETE and 12-hydroxyeicosapentaenoic acid (HEPE) were higher and omega-3/omega-6 polyunsaturated fatty acids ratios were lower in the ND mice at 24 hr. These results of metabolomics indicated that less pro-inflammatory state was seen in HFD mice than in ND mice at 24 hr. Finally, to confirm whether the observed decrease in phosphorylated p38 MAPK could attenuate TA-induced hepatocellular necrosis, we showed that SB203580 hydrochloride, an inhibitor of p38 MAPK, partially attenuated TA-induced hepatic necrosis in ND mice. Collectively, these results suggest that a prompt decrease in phosphorylation of p38 MAPK after TA administration is one of the factors that attenuate TA-induced hepatic necrosis in HFD mice.

BMP9/p38 MAPK is essential for the antiproliferative effect of resveratrol on human colon cancer.

Colon cancer is one of the most common malignancies of the digestive system. Although more effective therapeutic strategies have been developed in the last decades, there is still a great clinical need to explore new treatment regimens for colon cancer due to the undesirable prognosis. In the present study, we investigated the anticancer activity of resveratrol (Res) in human colon cancer cells, and the possible mechanism underlying this effect. We employed crystal violet staining, flow cytometry and western blotting to test the antiproliferation- and apoptosis-inducing effects of Res in LoVo cells. A xenograft tumor model was also introduced to confirm the in vivo anticancer effect of Res. Using PCR, western blotting, a recombinant adenovirus and a specific inhibitor of p38 MAPK or bone morphogenetic protein receptor (BMPR) to explore the possible molecular mechanisms. We found that Res markedly inhibited the proliferation and promoted the apoptosis of LoVo cells, and suppressed the in vivo tumor growth of colon cancer. Res substantially upregulated the expression of bone morphogenetic protein 9 (BMP9). Exogenous expression of BMP9 enhanced the anticancer effect of Res in LoVo cells, while BMP9 knockdown partly reduced this activity. Res increased the activation of p38 MAPK, which was enhanced by the exogenous expression of BMP9. The anticancer activity of Res, or Res combined with BMP9, was reduced partly by the p38 MAPK inhibitor. The BMPR inhibitor almost abolished the Res-induced activation of p38 MAPK, and attenuated the antiproliferative effect of Res in the LoVo cells. Our findings strongly suggest that the anticancer effect of Res in human colon cancer cells may be partly mediated by upregulation of BMP9 to activate p38 MAPK in a BMPR-dependent manner.

Leukotriene B4receptor type 2 (BLT2) enhances skin barrier function by regulating tight junction proteins

GPCRs are involved in numerous physiologic functions and are important drug targets. Although the epithelial barrier is important for protection from invading pathogens, the correlation between GPCRs and epithelial barrier function remains unknown. Leukotriene B4 (LTB4) receptor type 2 (BLT2), mainly expressed in epithelial cells, is a GPCR for 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) and LTB4. In our study, BLT2 localized at the lateral membrane in BLT2-overexpressing Madin-Darby canine kidney (MDCK) II cells and in the small intestine of BLT2-transgenic mice. BLT2-deficient mice exhibited higher transepidermal water loss and were more sensitive to epicutaneous sensitization. MDCK-BLT2 cells recovered transepithelial electrical resistance (TER) after a calcium switch faster than did MDCK-Mock cells, and 12-HHT stimulation accelerated TER recovery only in MDCK-BLT2 cells. Quantitative PCR and immunoblot analyses revealed that the 12-HHT/BLT2 axis up-regulated claudin-4 (CLDN4) expression in MDCK-BLT2 cells and human primary keratinocytes, and CLDN4 knockdown abolished 12-HHT-dependent TER recovery. Acceleration of TER recovery and induction of CLDN4 expression by 12-HHT stimulation were abolished by inhibition of Gαi protein or p38 MAPK. These results show that 12-HHT/BLT2 enhances epithelial barrier function by increasing CLDN4 expression via the Gαi protein-p38 MAPK pathway.

Effects of Candida albicans on the expression of tumor necrosis factor-α and activation of the intracellular signaling molecule p38MAPK in a human acute monocytic leukemia cell line THP-1

Objective To investigate the effects of Candida albicans on the expression of tumor necrosis factor-α (TNF-α) and activation of the intracellular signaling molecule p38 mitogen-activated protein kinase (p38MAPK) in a human acute monocytic leukemia cell line THP-1. Methods Some THP-1 cells were divided into several groups in vitro: two C. albicans groups treated with 105 CFU/ml and 106 CFU/ml heat-killed C. albicans respectively, a lipopolysaccharide (LPS) group treated with 100 μg/L LPS, a blank control group treated with RPMI 1640 medium, two dexamethasone-inhibited groups pretreated with 40 μg/L dexamethasone for 30 minutes followed by treatment with 106 CFU/ml heat-killed C. albicans and LPS respectively. After treatment for 1, 3 and 6 hours, real-time fluorescence-based quantitative PCR was performed to measure TNF-α mRNA expression in THP-1 cells in the above groups. Enzyme-linked immunosorbent assay (ELISA) was conducted to determine the level of TNF-α protein in the supernatant of THP-1 cells treated with 106 CFU/ml heat-killed C. albicans, 100 μg/L LPS or RPMI 1640 medium (blank control group) for 24 hours. Western blot was performed to measure the protein expression of p38MAPK and phosphorylated p38MAPK in THP-1 cells after treatment with 106 CFU/ml heat-killed C. albicans or RPMI 1640 medium (blank control group) for 30 and 60 minutes. Statistical analysis was carried out by using two-way analysis of variance, one-way analysis of variance and the least significant difference (LSD)-t test. Results Significant differences were observed in the mRNA expression level of TNF-α among the C. albicans groups, LPS group and blank control group (F=110.98, P< 0.001). The mRNA expression level of TNF-α in THP-1 cells increased over time in a time-dependent manner after C. albicans treatment, with significant differences among different time points (F=701.680, P< 0.001). Compared with the blank control group, both 106-CFU/ml C. albicans group and LPS group showed a significant increase in TNF-α protein expression (6385.70±533.99 ng/L and 3212.06±353.00 ng/L vs. 147.10±0.53 ng/L, P< 0.001 and 0.005, respectively). An obvious increase was observed in the expression level of phosphorylated p38MAPK protein, but no significant changes were noted in that of p38MAPK protein, in THP-1 cells treated with 106 CFU/ml C. albicans for 30 and 60 minutes compared with the blank control group. The mRNA expression level of TNF-α significantly decreased in dexamethasone-pretreated 106-CFU/ml C. albicans group and LPS group compared with those without dexamethasone pretreatment (3.77±0.62 vs. 208.50±10.50, 6.20±1.93 vs. 161.35±1.65, both P< 0.001). Conclusions Heat-killed C. albicans can induce the activation of p38MAPK in and secretion of TNF-α by human THP-1 cells, which then participate in the innate immune response against C. albicans. Key words: Candida albicans; Leukemia, monocytic, acute; Tumor necrosis factor-alpha; p38 Mitogen-activated protein kinases; Cell line, tumor

Autoregulatory loop between TGF-β1/miR-411-5p/SPRY4 and MAPK pathway in rhabdomyosarcoma modulates proliferation and differentiation.

The origin of rhabdomyosarcoma (RMS) remains controversial. However, specific microRNAs (miRNAs) are downregulated in RMS and it is possible that re-expression of these miRNAs may lead to differentiation. Transforming growth factor-β1 (TGF-β1) is known to block differentiation of RMS. We therefore analyzed miRNA microarrays of RMS cell lines with or without TGF-β1 knockdown and identified a novel anti-oncogene miR-411-5p. Re-expression of miR-411-5p inhibited RMS cell proliferation in vitro and tumorigenicity in vivo. Using a luciferase reporting system and sequence analysis, the potential target of miR-411-5p was identified as sprouty homolog 4 (SPRY4), which inhibits protein kinase Cα-mediated activation of mitogen-activated protein kinases (MAPKs), especially p38MAPK phosphorylation. These results revealed an inverse correlation between TGF-β1/SPRY4 and miR-411-5p levels. SPRY4 small interfering RNA and miR-411-5p both activated p38MAPK phosphorylation and also promoted apoptosis and myogenic differentiation, indicated by increased caspase-3, myosin heavy chain, and myosin expression. SPRY4 and miR-411 mRNA levels correlated with TGF-β1 expression levels in RMS tissues, which was confirmed by immunohistochemical staining for TGF-β1, SPRY4, and phosphorylated p38MAPK proteins. Overall, these results indicate that miR-411-5p acts as an RMS differentiation-inducing miRNA prompting p38MAPK activation via directly downregulating SPRY4. These results establish an autoregulatory loop between TGF-β1/miR-411-5p/SPRY4 and MAPK in RMS, which governs the switch between proliferation and differentiation.

Effect of different concentrations of oxygen on inflammatory response in lipopolysaccharide-induced alveolar typeIIcells

Objective To investigate the secretion of inflammatory cytokines in lipopolysaccharide(LPS)-induced aveolar epithelium typeⅡcells(AT-Ⅱ) cells exposed to various oxygen concentrations, and the possible mechanism. Methods Primary cultured AT-Ⅱcells were randomly divided into five groups: A, B, C, D, and E, and then placed in sterilized incubators: 37℃, 21% O2, 5% CO 2; 37℃, 21% O2, 5% CO 2; 37℃, 40% O2, 55% N2, 5% CO 2; 37℃, 60% O 2, 35% N2, 5% CO2; 37℃, 90% O 2, 5% N 2, 5% CO 2.Towenty-four h later, the supernatant fluid was used to assay the content of interleukin-8(IL-8) by enzyme linked immunosorbent assay(ELISA).The expression of p38 mitogen-activated protein kinase(p38 MAPK) m RNA and protein was detected in AT-Ⅱcells by reverse transcription-polymerase chain reaction and Western blotting. Results ELISA, reverse transcriptase-polymerase chain reaction(RT-PCR) and Western blotting showed that the content of IL-8 was(40. 22±7. 25),(109. 35±10. 19),(213. 98 ±30. 51),(369. 71±21. 73)and(489. 32±42. 35) ng/L, p38MAPK mRNA expression level was 0. 066±0. 010, 0. 163±0. 008, 0. 228±0. 027, 0. 268±0. 016 and 0. 292±0. 010, and p38MAPK protein expression level was 0. 055±0. 005, 0. 124±0. 006, 0. 182±0. 009, 0. 227±0. 013 and 0. 303±0. 010 in groups A, B, C, D and E respectively.As compared with group A, the content of IL-8 in the supernatant fluid,and the expression of p38MAPK m RNA and protein were significantly increased in groups B, C, D and E(P< 0. 05).As compared with groups B, the content of IL-8 in the supernatant fluid, and the p38 MAPK m RNA and protein expression level in groups C, D and E were gradually increased with the difference being statistically significant(P< 0. 05).In different oxygen concentration groups, with the increases in oxygen concentrations, the content of IL-8 in the supernatant fluid, and the expression of p38MAPK m RNA and protein expression level were gradually increased with the difference being statistically significant(P< 0. 05). Conclusion 40%-90% oxygen could concentrasion-dependently increase the LPS-induced secretion of IL-8 in AT-Ⅱcells probably by up-regulating the intracellular p38 MAPK expression. Key words: Hyperoxia-induced lung injury; Aveolar epithelium typeⅡcells; Lipopolysaccharide; Interleukin-8; p38 mitogen-activated protein kinase

GLP-1 receptor activation effects the p38MAPK signal pathway in hepatic stellate cells

To investigate the effects of activation of the GLP-1 receptor on the p38MAPK signaling pathway in hepatic stellate cells (HSCs). HSCs were isolated and identified according to morphological features; the levels of GLP-1R protein were determined by western blotting.The HSCs were randomly divided into a control grouP (normal saline treatment) and experimental grouP(liraglutide treatment); after 120 hours, the expression of p38MAPK mRNA was examined by RT-PCR and of phosphorylated (p)-p38MAPK protein was detected by western blotting. GLP-1R proteins were detected in the HSCs. Compared with the control group, the experimental group showed significantly decreased p38MAPK mRNA and p-p38MAPK protein (both P < 0.01). The p38MAPK signaling pathway could be down-regulated when GLP-1R is activated in HSCs.

Activation of the cardiac proteasome promotes angiotension II-induced hypertrophy by down-regulation of ATRAP

Proteasomal degradation is critical to maintaining cardiac function and is altered in various diseases. Angiotensin II (Ang II) acts as a growth factor to induce cardiac growth. Here we aimed to test whether proteasome is involved in the development of Ang II-induced cardiac hypertrophy and dissect its molecular mechanisms. Cardiac hypertrophy was induced by Ang II infusion (1000ng/kg/min) using mini-osmotic pumps. Starting 1day before implantation, the mice were injected with the proteasome inhibitor bortezomib (BTZ, 50μg/kg, 3 times per week) or with vehicle. After 14days, the pool of ubiquitinated proteins was reduced but the protein expression of proteasome subunits (including β1i, β2i and β5/β5i) was markedly up-regulated in left ventricular hypertrophy versus control, which was accompanied by a significant increase in proteasome activities. Furthermore, Ang II-treated mice showed a significant increase in blood pressure but decrease in cardiac contractile function, and significant left ventricular hypertrophy, fibrosis and inflammation, which were all attenuated in BTZ-treated mice. Mechanistically, these beneficial effects were associated with the inhibition of degradation of angiotensin II type 1 receptor-associated protein (ATRAP) and inactivation of AT1R-mediated p38 MAPK and STAT3 signaling pathways. In conclusion, our data indicate that the activation of proteasome is required for the Ang II-induced cardiac hypertrophy, and suggest that the inhibition of proteasome activity by BTZ could be a potential therapeutic strategy for the treatment of cardiac hypertrophy and other heart diseases.

Sigma 1 Receptor antagonist potentiates the anti-cancer effect of p53 by regulating ER stress, ROS production, Bax levels, and caspase-3 activation

Over the last years, many improvements have been made in the treatment of breast cancer; however, novel and less toxic therapies are still needed, especially for relapsing and chemo-resistant patients. Here, we analyzed the therapeutic potential of p53 and Rimcazole, a Sigma 1 Receptor antagonist. Rimcazole and p53 are being evaluated in preclinical and clinical trials, respectively. While p53 is a promising antitumor therapeutic agent, antagonists of Sigma 1 Receptor also inhibit tumor cell survival and induce apoptosis. Our current study demonstrates for the first time the synergistic effect of p53 in combination with the Sigma 1 Receptor antagonist Rimcazole. Furthermore, we show that shRNA knockdown of Sigma 1 Receptor in combination with p53, lead to a similar synergistic effect, and that this synergistic effect, in breast cancer growth suppression occurs independent of p53 status. Furthermore, this combination treatment induced ER stress, p38 MAPK activation, ROS production, and proteins involved in apoptosis (caspases-3, Bax) in breast cancer cells. Combining these therapeutic anti-cancer molecules provides an innovative approach for potentially treating human breast cancer.

Cytotoxic effects of cytoplasmic-targeted and nuclear-targeted gold and silver nanoparticles in HSC-3 cells – A mechanistic study

Nanoparticles (NPs), in particular noble metal nanoparticles, have been incorporated into many therapeutic and biodiagnostic applications. While these particles have many advantageous physical and optical properties, little is known about their intrinsic intracellular effects in biological environments. Here, we report the possible cell death mechanisms triggered in human oral squamous cell carcinoma (HSC-3) cells after exposure to extracellular, cytoplasm, and nuclear localized AuNPs and AgNPs. NP uptake and localization, cell viability, ATP levels, modes of cell death, ROS generation, mitochondrial depolarization, and the levels and/or translocation of caspase-dependent and caspase-independent proteins were assessed under control and localized metal nanoparticle exposure. Exposure to AuNPs resulted the adoption of a quiescent cellular state, as AuNPs caused a decrease in intracellular ATP, but no change in viability or cell death populations. However, AgNP exposure significantly reduced HSC-3 cell viability and increased apoptotic populations, especially when localized at the cytoplasm and nucleus. Increased cell death populations were linked to an increase in intracellular ROS generation. Western blot analysis indicated cytoplasm localized AgNPs and nuclear localized AgNPs utilized a caspase-independent apoptotic pathway that involved the nuclear translocation of AIF and p38 MAPK proteins. These results demonstrate that the degree of cytotoxicity increases as AgNPs move from extracellular localization to nuclear localization, whereas changing AuNP localization does not trigger any significant cytotoxicity.

ISAV infection promotes apoptosis of SHK-1 cells through a ROS/p38 MAPK/Bad signaling pathway

The impact that the infectious salmon anemia virus (ISAV) has on the immune response of Salmo salar, from the perspective of activating/inactivating cellular processes, is currently unknown. Therefore, the present study evaluated this interaction and found that SHK-1 cells infected with ISAV resulted in respiratory burst activation and the induction of a strong pro-apoptotic imbalance through an increased expression of the Bad protein and decreased transcripts of Bcl-xl. Interestingly, the pharmacological inhibition of the p38 MAPK protein through SB203580 blocked the production of reactive oxygen species, the activity of caspase 3, and the formation of apoptotic nuclei in SHK-1 cells. Additionally, when the NADPH oxidase complex, a producer of superoxide anions, was blocked through apocynin, decreased apoptotic activity was observed in infected cells without significant modifications to viral amplification. These results, together with bioinformatics analysis performed for the Bad gene of fugu, suggest that the ISA virus triggers a strong production of oxygen radicals capable of activating transduction signaling pathways and mediating the expression and activation of pro-apoptotic proteins through the p38 MAPK pathway, all of which results in the apoptosis of ISAV infected cells.