P38 MAPK Protein Levels Research Articles

Low-Medium Polarity Ginsenosides from Wild Ginseng Improves Immunity by Activating the AhR/MAPK Pathway through Tryptophan Metabolism Driven by Gut Microbiota.

The gut microbiota contribute significantly to the immune system. Low-medium polarity ginsenosides from wild ginseng (LWG) have potential immunomodulatory effects. However, how the LWG regulates gut microbiota to enhance immunity remains unclear. To explore the interaction between gut microbes and metabolites mediating LWG's immunomodulatory effects, this study examined LWG's impact on splenocytes and CTX-induced immunosuppressed mice. Metabolomic and metagenomic analyses were conducted in vivo to explore the mechanism by which LWG regulates gut microbiota to enhance immunity. In vitro data suggest that LWG at 4 μg/mL enhances the splenocyte activity. Furthermore, LWG effectively reduces symptoms in immunocompromised mice, including weight loss and intestinal mucosal damage. LWG alleviated gut microbiota disturbance, restored tryptophan metabolites (IA, IAA, and IPA), and significantly increased JNK, ERK, and p38MAPK protein levels, which were downstream of AhR. Our study demonstrated that LWG improves the immunity by reshaping gut microbiota, restoring intestinal mucosa, and boosting the gut microbiota-related metabolism of tryptophan to activate the AhR/MAPK pathway. This research offers new insights into the mechanism by which LWG regulates immune function.

Effect of a Plateau Environment on the Oxidation State of the Heart and Liver through AMPK/p38 MAPK/Nrf2-ARE Signaling Pathways in Tibetan and DLY Pigs.

Simple SummaryPlateau stress is a main factor limiting pig production. There are great differences in oxidation state and antioxidant defense between Tibetan pigs (TPs) and DLY pigs exposed to a plateau environment. However, there are limited reports on the mechanism of adaptation of TPs and DLY pigs to the plateau environment involving a signal pathway related to oxidation state. In the present study, we found that TPs exhibit a stronger heart and liver antioxidant stress capacity than DLY pigs through AMPK/p38 MAPK/Nrf2-ARE signaling pathways under a plateau hypoxic environment.This study evaluated the effect of a plateau environment on the heart and liver oxidation state of Tibetan pigs (TPs) and DLY pigs through analyzing AMPK, p38 MAPK, and Nrf2 signaling pathways. Twelve 120-day-old TPs and twelve 120-day-old DLY pigs were randomly divided into two groups in a plateau environment for three weeks. Exposed to a plateau environment, TPs exhibited a lower (p < 0.05) malondialdehyde level but higher (p < 0.05) glutathione, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and total antioxidant capacity (T-AOC) activities in the liver and heart than those observed in DLY pigs. TPs also showed higher (p < 0.05) mRNA levels of SOD and GSH-Px in the liver and heart compared with those of DLY pigs. The TPs showed higher (p < 0.05) mRNA and protein levels of AMPK and Nrf2 in the liver and heart compared with those of DLY pigs. Furthermore, TPs showed higher (p < 0.05) mRNA and protein levels of p38 MAPK in the heart and higher mRNA levels of p38 MAPK in the liver compared with those of DLY pigs under a plateau environment. In summary, TPs possess a stronger antioxidant capacity in the heart and liver than that of DLY pigs in a plateau environment through AMPK/p38 MAPK/Nrf2-ARE signaling pathways.

Lipocalin-2 Regulates Hippocampal Microglial Activation in Poststroke Depression.

Background and Purpose: Microglia play important role in poststroke depression (PSD), however, the exact mechanism was still unclear. The purpose of the study was to study the mechanism of microglial activation in PSD.Methods: 24 rats were randomly divided into three groups: the PSD group (n = 10), the poststroke (PS) group (n = 7), and the sham group (n = 7). Primary hippocampal microglia were isolated and cultured, and recombined LCN2 protein was used to stimulate the cultured microglia. The protein expression of Iba1, P38 MAPK and PP38 MAPK was analyzed by western blotting; the LCN2 expression was measured by RT-qPCR, the serum LCN2 level and the NO level were analyzed by ELISA.Results: Open field test scores (horizontal score, vertical score, and self-grooming score) and the serum LCN2 level were significantly decreased in the PSD group compared with the other two groups (P < 0.05). The serum LCN2 level was positively correlated with the horizontal score and negatively correlated with the self-grooming score in the open field test (P < 0.05). The relative protein level of Iba1 and the LCN2 mRNA level were significantly increased in the hippocampal region compared with other brain regions (P < 0.05), while the relative protein level of Iba1 and the LCN2 mRNA level were significantly increased in the PSD group compared with the other two groups (P < 0.05). The length, supernatant NO level, phagocytic ability and migration ability of LCN2-treated microglia were significantly increased compared with those of untreated microglia (P < 0.05). The relative protein levels of P38 MAPK and the PP38 MAPK significantly increased in hippocampal region in the PSD group and LCN2-treated hippocampal microglia (P < 0.05).Conclusion: Hippocampal microglia are activated during PSD; LCN2 may regulate hippocampal microglial activation by the P38 MAPK pathway in the process of PSD.

SARS-CoV-2 Spike Glycoprotein S1 Induces Neuroinflammation in BV-2 Microglia

In addition to respiratory complications produced by SARS‐CoV‐2, accumulating evidence suggests that some neurological symptoms are associated with the disease caused by this coronavirus. In this study, we investigated the effects of the SARS‐CoV‐2 spike protein S1 stimulation on neuroinflammation in BV-2 microglia. Analyses of culture supernatants revealed an increase in the production of TNF-α, IL-6, IL-1β and iNOS/NO. S1 also increased protein levels of phospho-p65 and phospho-IκBα, as well as enhanced DNA binding and transcriptional activity of NF-κB. These effects of the protein were blocked in the presence of BAY11-7082 (1 µM). Exposure of S1 to BV-2 microglia also increased the protein levels of NLRP3 inflammasome and enhanced caspase-1 activity. Increased protein levels of p38 MAPK was observed in BV-2 microglia stimulated with the spike protein S1 (100 ng/ml), an action that was reduced in the presence of SKF 86,002 (1 µM). Results of immunofluorescence microscopy showed an increase in TLR4 protein expression in S1-stimulated BV-2 microglia. Furthermore, pharmacological inhibition with TAK 242 (1 µM) and transfection with TLR4 small interfering RNA resulted in significant reduction in TNF-α and IL-6 production in S1-stimulated BV-2 microglia. These results have provided the first evidence demonstrating S1-induced neuroinflammation in BV-2 microglia. We propose that induction of neuroinflammation by this protein in the microglia is mediated through activation of NF-κB and p38 MAPK, possibly as a result of TLR4 activation. These results contribute to our understanding of some of the mechanisms involved in CNS pathologies of SARS-CoV-2.

Efficacy of Low Molecular Heparin on Preeclampsia by Inhibiting Apoptosis of Trophoblasts via the p38MAPK Signaling Pathway.

Objective To explore the efficacy of low molecular heparin on preeclampsia by inhibiting apoptosis of trophoblasts via the p38MAPK signaling pathway. Methods A preeclampsia rat model was established, and the effects of low molecular heparin on preeclampsia via the p38MAPK signaling pathway were analyzed based on intervention of the rats with different combinations of low molecular heparin and p38MAPK signaling pathway activator. Furthermore, a hypoxia/reoxygenation model of trophoblasts in vitro was established to explore the effects of low molecular heparin on trophoblasts via the p38MAPK signaling pathway. Results After treatment with low molecular heparin, pregnant rats in the heparin group showed significantly decreased blood pressure, 24 h proteinuria, and p38MAPK protein levels in placenta tissues and decreased apoptosis rate of placenta tissue cells (all P < 0.05) and showed more fetal rats and lowered weight of them (both P < 0.05) but showed no significant change in the weight of placenta (all P > 0.05). Pregnant rats treated with low molecular heparin and p38MAPK activator showed significantly higher blood pressure, 24 h proteinuria, and p38MAPK protein levels in placenta tissues and apoptosis rate of placenta tissue cells than those of pregnant rats in the heparin group (all P < 0.05) and also showed less fetal rats and lighter fetal rats than those in the heparin group (both P < 0.05) but showed no difference with them in the weight of placenta (P > 0.05). Further analysis revealed that low molecular heparin could protect the survival and migration of trophoblasts under hypoxia/reoxygenation conditions and reduce apoptosis of them (all P < 0.05). Conclusion Low molecular heparin can alleviate preeclampsia by inhibiting the p38MAPK signaling pathway and can inhibit apoptosis of trophoblasts and promote proliferation and migration of them.

Long non-coding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) promotes the inflammation and apoptosis of otitis media with effusion through targeting microRNA (miR)-495 and activation of p38 MAPK signaling pathway

ABSTRACT Long non-coding RNA (lncRNA) plays a vital role in human inflammatory diseases. Our study aimed to investigate the function of lncRNA nuclear-enriched abundant transcript 1 (NEAT1) in otitis media with effusion (OME). The mRNA levels of NEAT1 and miR-495 were measured by RT-qPCR. The protein levels of p38 MAPK were detected by western blot. The levels of inflammatory cytokines were examined by ELISA. CCK-8 and flow cytometry assays were used to evaluate the cell viability and apoptosis, respectively. The interaction between NEAT1 and miR-495 was determined by luciferase reporter and RIP assays. NEAT1 was highly expressed in OME, and silencing of NEAT1 facilitated the cell proliferation and suppressed levels of inflammatory cytokines and cell apoptosis in LPS-induced HMEECs. Moreover, miR-495 was confirmed as a downstream target of NEAT1. Functional assays revealed that NEAT1 promoted the OME by targeting miR-495. It was further demonstrated that NEAT1 could activate the p38 MAPK signaling pathway by regulating miR-495, and the p38 MAPK inhibitor restored the effects of NEAT1 overexpression on the inflammation levels, cell proliferation, and apoptosis. Our study revealed that lncRNA NEAT1 served as a ceRNA to activate p38 MAPK signaling by targeting miR-495 in OME, which may offer a new target for OME treatment.

MiR-195 alleviates ulcerative colitis in rats via MAPK signaling pathway.

To study the effect of micro ribonucleic acid (miR)-195 on the inflammatory response of ulcerative colitis (UC) model rats and to explore its regulatory mechanism, thus providing a new scheme for the clinical treatment of UC. A rat model of UC was prepared by 2, 4, 6-trinitrobenzenesulfonic acid (TNBS)/ethanol assay, and the rats were randomly divided into Control group, Model group, and miR-195 mimic (miR-195 agomir) group. The disease activity index (DAI) in each group was observed. Hematoxylin and eosin (H&E) staining was utilized to detect the pathological changes in the rat colon tissues in each group. The levels of interleukin-6 (IL-6) and IL-1β in the colon tissues of the rats in each group were detected by enzyme-linked immunosorbent assay (ELISA). In addition, the messenger RNA (mRNA) and protein levels of p38 mitogen-activated protein kinase (p38 MAPK) and tumor necrosis factor-alpha (TNF-α) in the colon tissues of each group of rats were examined via Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western blotting, respectively. Compared with those in Control group, the rats in Model group had an increased DAI score, severely pathologically damaged colon tissues, raised levels of IL-6 and IL-1β in the colon tissues and significantly elevated mRNA and protein levels of p38 MAPK and TNF-α. In comparison with those in Model group, the DAI score was decreased, the pathological damage to the rat colon tissues was improved, the levels of IL-6 and IL-1β in the rat colon tissues were reduced, and the mRNA and protein levels of p38 MAPK and TNF-α were notably lowered in miR-195 agomir group. MiR-195 mimics can alleviate the pathological damage to the colon and inflammatory responses in UC model rats, and its mechanism may be related to the inhibition on the p38 MAPK signaling pathway.

Role and mechanism of cardiac insulin resistance in occurrence of heart failure caused by myocardial hypertrophy.

Cardiac insulin resistance plays an important role in the development of heart failure, but the underlying mechanisms remain unclear. Here, we found that hypertrophic hearts exhibit normal cardiac glucose oxidation rates, but reduced fatty acid oxidation rates, compared to Sham controls under basal (no insulin) conditions. Furthermore, insulin stimulation attenuated insulin’s effects on cardiac substrate utilization, suggesting the development of cardiac insulin resistance. Consistent with insulin resistance, p38-MAPK protein levels were reduced in hypertrophic hearts. By contrast, systemic hyperinsulin-euglycemic clamp indicated normal insulin sensitivity. Finally, electron microscopy revealed severe mitochondrial damage in the hypertrophic myocardium. Our results indicate that that cardiac insulin resistance caused by cardiac hypertrophy is associated with mitochondrial damage and cardiac dysfunction. Moreover, our findings suggest that cardiac insulin resistance is independent of systemic insulin resistance, which is also a risk factor for heart failure.

Celastrol ameliorates Aspergillus fumigatus keratitis via inhibiting LOX-1

PurposeTo investigate the effect of Celastrol (CLT) on Aspergillus fumigatus (A. fumigatus) keratitis. MethodsPrimary peritoneal macrophages of C57BL/6 mice were pretreated with CLT before A. fumigatus hyphae stimulation. C57BL/6 mice were infected with A. fumigatus. Mice corneas were treated with CLT from 1 day post infection. Clinical score, PCR, ELISA and Western blot were used to test expression of anti-inflammatory mediators, proinflammatory mediators and Lectin-like oxidized low-density lipoprotein receptor 1(LOX-1). The protein levels of p38MAPK after pretreated with CLT in macrophages of C57BL/6 mice challenged with A. fumigatus were tested by Western blot. ResultsC57BL/6 mice treated with CLT from 1 day post infection showed decreased disease, IL-1β, TNF-α, IL-10, TGF-β, MIP-2 and LOX-1 levels. CLT treatment markedly inhibiting mRNA and proteins levels of anti-inflammatory mediators, proinflammatory mediators and LOX-1 in macrophages of C57BL/6 mice compared with control group. CLT pretreatment before A. fumigatus stimulation obviously inhibiting protein levels of p38MAPK versus DMSO pretreated group in macrophages of C57BL/6 mice challenged with A. fumigatus. ConclusionThese data provide evidences that CLT ameliorates A. fumigatus keratitis of C57BL/6 mice via inhibiting LOX-1. CLT pretreatment before A. fumigatus stimulation decreased levels of inflammation in macrophages of C57BL/6 mice, which may be regulated by p-p38MAPK.

GABA, γ-Aminobutyric Acid, Protects Against Severe Liver Injury

BackgroundAcute liver failure (ALF) from severe acute liver injury is a critical condition associated with high mortality. The purpose of this study was to investigate the impact of preemptive administration of γ-aminobutyric acid (GABA) on hepatic injury and survival outcomes in mice with experimentally induced ALF. Materials and methodsTo induce ALF, C57BL/6NHsd mice were administered GABA, saline, or nothing for 7 d, followed by intraperitoneal administration of 500 μg of tumor necrosis factor α and 20 mg of D-galactosamine. The study mice were humanely euthanized 4-5 h after ALF was induced or observed for survival. Proteins present in the blood samples and liver tissue from the euthanized mice were analyzed using Western blot and immunohistochemical and histopathologic analyses. For inhibition studies, we administered the STAT3-specific inhibitor, NSC74859, 90 min before ALF induction. ResultsWe found that GABA-treated mice had substantial attenuation of terminal deoxynucleotidyl transferase dUTP nick end labeling–positive hepatocytes and hepatocellular necrosis, decreased caspase-3, H2AX, and p38 MAPK protein levels and increased expressions of Jak2, STAT3, Bcl-2, and Mn-SOD, with improved mitochondrial integrity. The reduced apoptotic proteins led to a significantly prolonged survival after ALF induction in GABA-treated mice. The STAT3-specific inhibitor NSC74859 eliminated the survival advantage in GABA-treated mice with ALF, indicating the involvement of the STAT3 pathway in GABA-induced reduction in apoptosis. ConclusionsOur results showed that preemptive treatment with GABA protected against severe acute liver injury in mice via GABA-mediated STAT3 signaling. Preemptive administration of GABA may be a useful approach to optimize marginal donor livers before transplantation.

Protective Effects of Lactic Acid Bacteria Against TLR4 Induced Inflammatory Response in Hepatoma HepG2 Cells Through Modulation of Toll-Like Receptor Negative Regulators of Mitogen-Activated Protein Kinase and NF-κB Signaling.

The beneficial effects of probiotics in several liver diseases have been investigated in both animal and clinical models; however, the precise mechanisms responsible for their effects have not yet been elucidated. Gut transmitted endotoxins such as LPS have been shown to play critical roles in hepatic inflammation and injury. Therefore, in this study, we investigated the beneficial role of selected lactic acid bacteria (LABs) on reduction of hepatic steatosis (HS) and attenuation of LPS induced inflammatory response in vitro. Total cellular fluid (TCF) of LABs treatment reduced HS by decreasing the amount of lipid accumulation in vitro. Additionally, HepG2 cells exposed to LPS showed increased expression of exacerbated inflammatory cytokines, such as IL-6, CXCL8, CCL2, and TNF-α, but these effects were counteracted when cells were treated with TCF of LABs prior to LPS challenge. Moreover, TCF of LABs was able to modulate mRNA levels of TLR negative regulators and protein levels of p38 MAPK and p65 NF-κB transcription factors. However, these modulations were differed remarkably between both free fatty acid treated and untreated HepG2 cells. Heat-killed LABs were also indirectly suppressed THP-1 cells to produce higher level of IL-10, TLR4, and lower at genes level of TGF-β, IL-1β, and IL-6, and at protein level of TNF-α in response to LPS. Taken together, our findings indicate that selected LABs exhibit profound immunoregulatory effects on liver cells via modulation of TLR negative regulators of the MAPK and NF-κB pathways.

2-Chloroethanol Induced Upregulation of Matrix Metalloproteinase-2 in Primary Cultured Rat Astrocytes Via MAPK Signal Pathways.

This study was to explore the mechanisms underlying 1,2-dichloroethane (1,2-DCE) induced brain edema by focusing on alteration of matrix metalloproteinase-2 (MMP-2) in rat astrocytes induced by 2-chloroethanol (2-CE), an intermediate metabolite of 1,2-DCE in vivo. Protein and mRNA levels of MMP-2, and the phosphorylated protein levels of p38 MAPK (p-p38), extracellular signal regulated protein kinase (p-ERK1/2) and c-Jun N-terminal kinase (p-JNK1/2) in astrocytes were examined by immunostaining, western blot or real-time RT-PCR analysis. Findings from this study disclosed that protein levels of MMP-2 were upregulated by 2-CE in astrocytes. Meanwhile, protein levels of p-p38, p-ERK1/2 and p-JNK1/2 were also increased apparently in the cells treated with 2-CE. Moreover, pretreatment of astrocytes with SB202190 (inhibitor of p38 MAPK), U0126 (inhibitor of ERK1/2) or SP600125 (inhibitor of JNK1/2) could suppress the upregulated expression of p-p38, p-ERK1/2, and p-JNK1/2. In response to suppressed protein levels of p-p38 and p-JNK1/2, the protein levels of MMP-2 also decreased significantly, indicating that activation of MAPK signal pathways were involved in the mechanisms underlying 2-CE-induced upregulation of MMP-2 expression.

Effect of Gallic Acid on Regression of Murine CCL4-Induced Hepatic Cirrhosis

Hepatic cirrhosis can be induced by chronic exposure to reactive oxygen species (ROS). However, phenolic compounds, such as gallic acid (GA), appear to inhibit ROS-induced oxidative stress. We aimed to investigate the effects of GA on murine hepatic fibrogenesis. The effects of GA were evaluated on the regression of liver cirrhosis that was induced by chronic intraperitoneal CCl4 administration (20% v/v) in C57 mice for a period of 10 weeks. The animals were treated intraperitoneally and distributed in three groups, as follows: SHAM group – 8 weeks with Olive oil (CCL4 solution vehicle); C group – 8 weeks with CCL4 solution, and then 2 more weeks with deionized water (Gallic Acid solution vehicle); and C + GA group – 8 weeks with CCL4 solution, and then 2 more weeks with Gallic Acid solution (100 mg/Kg/day) on alternated days. The cirrhosis and inflammation-related factors were estimated using histological, western blotting and PCR-RT analysis. There was a significant decrease in the collagen deposition, as indicated by Sirius red staining, in the C + GA group compared to the C group (p<0.05). This improvement was accompanied by a reduction in the number of α-SMA-positive cells observed in these animals (p<0.05). The expression of the Procollagen α1(I), TGFβ1 and TIMP1 genes was also diminished by GA administration compared to the control (p<0.001). Additionally, proteomic studies revealed reduced p65 NFκB and p38 MAPK protein levels in the C + GA group. These findings reveal the effect of GA on the regression of cirrhosis. The mechanisms of this process might involve the anti-inflammatory activity of GA, which represses the TGFβ1, p65 NFκB, and p38 MAPK-mediated signaling pathways.

Involvement of AP-1 in p38MAPK signaling pathway in osteoblast apoptosis induced by high glucose.

We investigated the effect of p38MAPK/AP-1 (activator protein-1) signaling on the apoptosis of osteoblasts induced by high glucose. A lentivirus vector of small hairpin RNA (shRNA) targeting p38MAPK was constructed in vitro. Osteoblasts MC3T3-E1 cultured in vitro were treated with vehicle, high glucose, p38MAPK-shRNA transfection, p38MAPK inhibitor, and unrelated shRNA transfection. Apoptosis, protein levels of p38MAPK, and activities of AP-1 in MC3T3-E1 osteoblasts were measured using TUNEL and flow cytometry, Western blot analysis, and an electrophoretic mobility shift assay. Compared with the vehicle group, high glucose induced apoptosis of MC3T3-E1 osteoblasts and activated p38MAPK and AP-1. p38MAPK-shRNA transfection blocked the effect of high glucose stimulation, and the p38MAPK inhibitor showed similar effects as those observed in p38MAPK transfection. Unrelated shRNA had no effect on these changes in MC3T3-E1 osteoblasts induced by high glucose. Therefore, our results suggest that p38MAPK-shRNA reduce apoptosis of MC3T3-E1 osteoblasts induced by high glucose by inhibiting the p38MAPK-AP-1 signaling pathway.

Octaphlorethol A isolated from Ishige foliacea inhibits α-MSH-stimulated induced melanogenesis via ERK pathway in B16F10 melanoma cells.

In this study, the potent skin-whitening effects of Octaphlorethol A (OPA) isolated from Ishige foliacea was investigated through inhibitory effect of melanin synthesis and tyrosinase activity in alpha-melanocyte stimulating hormone (α-MSH) induced B16F10 melanoma cells. OPA markedly inhibited melanin synthesis and tyrosinase activity in a concentration-dependent manner. We also found that OPA decreased microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 and -2 (TRP-1 and TRP-2) protein expressions. Moreover, OPA reduces p38 MAPK protein levels and activates extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinases (JNKs) protein expressions in B16F10 cells. A specific ERK inhibitor PD98059 significantly blocks OPA-inhibited melanin synthesis and tyrosinase activity, whereas a p38MAP and JNK inhibitor had no effect. These findings provide evidence demonstrating that the anti-melanogenic effect of OPA is mediated through the activation of ERK signal pathway in B16F10 cells. These results indicate that OPA has the potential to be used as a melanogenesis inhibitor in the food and cosmetics industry.

Effect of pioglitazone on inflammatory signaling pathways induced by pancreatitis-associated ascitic fluid in human monocytic cell line THP-1

Objective To investigate the inhibitory effect of pioglitazone on inflammatory signaling pathways induced by human pancreatitis-associated ascitic fluid(PAAF) in human monocytic cell line THP-1.Methods PAAF was collected from patients with severe acute pancreatitis.The cultured THP-1 cells were divided into 4 groups: control group(C),PAAF group(A),PPARγ agonist pioglitazone group(P),and PPARγ antagonist GW9662 group(G).Cells in group A were stimulated with PAAF,those in group P were treated with 20 μmol/L PPARγ agonist pioglitazone 2 h after stimulation with PAAF,and those in group G were treated with PPARγ antagonist GW9662,pioglitazone(30 min later) and PAAF(2 h later) in turn.After incubation for 12 h,TNF-α and IL-6 mRNA expression was measured by real-time PCR,and Western blotting analysis was used to examine the expression of the phospho-p38-mitogen-activated protein kinase(MAPK),nuclear transcription factor(NF-κB) p65 levels,and IκB. Results Compared with group C,TNF-α and IL-6 mRNA expression in group A was increased,NF-κB and p38MAPK protein levels were increased,and IκB protein level was decreased(P0.05).Compared with group A,TNF-α and IL-6 mRNA expression was decreased in group P,NF-κB and p38MAPK protein levels were down-regulated,and IκB protein level was increased(P0.05).Compared with group P,TNF-α and IL-6 mRNA expression was increased in group G,NF-κB and p38MAPK protein levels were increased,and IκB protein level was decreased(P0.05).Conclusion The results show that pioglitazone can inhibit PAAF-stimulated THP-1 inflammation by blocking p38MAPK and NF-κB signaling pathway.

P38MAPK/p53-Mediated Bax induction contributes to neurons degeneration in rotenone-induced cellular and rat models of Parkinson’s disease

Rotenone is an environmental neurotoxin that induces degeneration of dopaminergic (DA) neurons in substantia nigra pars compacta (SNpc), which ultimately results in parkinsonism, but the molecular mechanisms of selective degeneration of nigral DA neurons are not fully understood. In the present study, we investigated the induction of p38MAPK/p53 and Bax in SNpc of Lewis rats after chronic treatment with rotenone and the contribution of Bax to rotenone-induced apoptotic commitment of differentiated PC12 cells. Lewis rats were subcutaneously treated with rotenone (1.5mg/kg) twice a day for 50days and the loss of tyrosine hydroxylase (THase), motor function impairment, and expression of p38MAPK, P-p38MAPK, p53, and Bax were assessed. After differentiated PC cells were treated with rotenone (500nM) for 6–36h, protein levels of p38MAPK and P-p38MAPK, p53 nuclear translocation, Bax induction and cell death were measured. The results showed that rotenone administration significantly reduced motor activity and caused a loss of THase immunoreactivity in SNpc of Lewis rats. The degeneration of nigral DA neurons was accompanied by the increases in p38MAPK, P-p38MAPK, p53, and Bax protein levels. In cultured PC12 cells, rotenone also induced an upregulation of p38MAPK, P-p38MAPK, p53 and Bax. Pharmacological inhibition of p38MAPK with SB203580 (25μM) blunted rotenone-induced cell apoptosis. Treatment with SB203580 prevented the p53 nuclear translocation and upregulation of Bax. Inhibition of p53 with pifthrin-alpha or Bax with siRNAs significantly reduced rotenone-induced Bax induction and apoptotic cell death. These results suggest that the p38MAPK/p53-dependent induction of Bax contributes to rotenone’s neurotoxicity in PD models.

Hypoxic Preconditioning Enhances Survival of Human Adipose-Derived Stem Cells and Conditions Endothelial Cells In Vitro

To grow more robust cardiac tissue for implantation in vivo, strategies to improve survival of implanted stem cells are required. Here we report the protective effects of hypoxic preconditioning (HPC) and identify mechanisms for improving survival of adipose-derived stem cells (ASC) in vitro. Human ASC were preconditioned for 24 h with hypoxia and then exposed to simulated ischemia for a further 24 h. HPC significantly increased ASC viability, and reduced cell injury and apoptosis compared with non-preconditioned cells under ischemic conditions, as shown by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), lactate dehydrogenase-release, and caspase activity assays. Preconditioned ASC increased levels of hypoxia-inducible factor-1 alpha and secreted significantly more of the downstream target vascular endothelial growth factor (VEGF-A; 13-fold) compared with control during the 24 h. Exogenous VEGF (50 ng/mL) increased phosphorylation of Akt without affecting ERK1/2, JNK, or p38 MAPK protein levels. Phospho-Akt was also increased in preconditioned ASC compared with non-preconditioned ASC, an effect that may be mediated via VEGF-A. Importantly, the protective effects of HPC were abolished by a neutralizing antibody against VEGF-A and the phosphoinositol 3-kinase inhibitor LY294002, demonstrating the importance of VEGF-A and Akt in hypoxia-induced ASC survival. Importantly, we showed that media derived from hypoxic preconditioned ASC support endothelial cell survival and endothelial tube formation in vitro. Our in vitro findings indicate that HPC may be a promising strategy to improve survival of ASC and promote angiogenesis in ischemic environments.

Expression of growth arrest and DNA damage-inducible 45α and p38 mitogen-activated protein kinase in placentas of women complicated with preeclampsia

Objective To evaluate the expression of Gadd45α and p38 MAPK in placentas and the correlations of Gadd45α protein and serum soluble vascular endothelial growth factor receptor-1 (sFlt-1) and soluble endoglin (sEng) in preeclampsia(PE). Methods Fifty-four pregnant women who delivered from September 2009 to March 2010 in the First Affiliated Hospital of Chongqing Medical University were chosen as the subjects. They were classified into mild preeclampsia group (n=20),severe preeclampsia group (n=16) and the control group (normal pregnant women underwent elective cesarean sections at term without labor and perinatal complications, n=18). Western blot and immunohistochemistry were employed to determine the expression and localization of Gadd45α and p-p38 MAPK protein respectively. Gadd45α mRNA level was determined by quantitative real-time PCR. The levels of seum sFlt-1 and sEng were measured by enzyme-linked immunosorbent assay (ELISA). One-way ANOVA and LSD-t test were applied for statistical analysis. Results (1)Immunohistochemistry identified that the positive stained cells were mostly located in trophoblast cells in normotensive placentas, whereas in preeclamptic placentas Gadd45α protein and p-p38 MAPK protein were detected in trophoblast and endothelial cells, as well as a few stromal cells at increased levels.(2)The mRNA levels of Gadd45α was significantly elevated in mild and severe preeclampsia groups compared to the control group (2.10±0.11 and 3.33±0.13 vs 1.01±0.18, P＜0.05), and Gadd45α mRNA level in severe group was significantly higher than in mild group (P＜0.05).(3)The data of Western blot revealed that the Gadd45α protein levels in each group were 0.22±0.11, 0.65±0.15 and 1.34±0.17, respectively, with significant differences between each group(P＜0.05). The p-p38 MAPK protein levels in each group were 0.32±0.08, 0.72±0.12 and 1.45±0.21, respectively, with significant differences between each group (P＜0.05). p38 MAPK protein levels in the total groups showed no difference(P＞0.05).(4)Compared with the control group, sFlt-1 and sEng concentrations in maternal circulation were significantly increasing in mild and severe preeclampsia groups, and concentrations in severe group were significantly higher than those in mild group (P＜0.05).(5) There were positive correlations between Gadd45α protein levels and the concentrations of serum sFlt-1 and sEng in each group( r=0.88 and 0.87, respectively all P＜0.05). Conclusions Upregulation of Gadd45α in preeclampsia placentas may play an important role in the pathogenesis of preeclampsia. It may induce the increased maternal serum levels of sFlt-l and sEng by activating p38 MAPK signaling pathway, leading to deficient cytotrophoblastic invasion and abnormal placental vascular reconstruction during pregnancy. Key words: Pre-eclampsia; Nuclear proteins; Cell cycle proteins; p38 Mitogen-activated protein kinases; Vascular endothelial growth factor receptor-1; Receptors, cell surface; Antigens, CD

Effects of human leukocyte antigen-G on p38 mitogen-activated protein kinase signaling pathway in HTR-8/SVneo cell line

To investigate the role of human leukocyte antigen-G (HLA-G) on the invasion and the molecular mechanism involved in this cellular progress in HTR-8/SVneo cell line. There were three groups: groups of transfection, negative control and blank control, which corresponding to treatment by HLA-G specific siRNA, negative siRNA and only lipofectamine 2000 using lipofection technology in HTR-8/SVneo cell line. The efficiency of down-regulated of HLA-G was detected by reverse transcription-polymerase chain reaction and western blot analysis in mRNA and protein level, respectively. Changes of p38 mitogen-activated protein kinases (p-p38MAPK)/p38MAPK protein levels and the cell invasion were respectively detected by western blot analysis and transwell test. (1) The mRNA levels of HLA-G transfection group, negative control group and blank control group were 0.26±0.08, 0.71±0.11, 0.79±0.07, respectively. There was significant difference between transfection group and negative control group (P<0.01), while there was no significant difference between negative control group and blank control group (P>0.05). The efficiencies of down-regulated of HLA-G were (69.8±6.3)%, (14.9±2.2)%, 0 in transfection group, negative control group and blank control group respectively in mRNA level. (2) In protein levels, HLA-G were 0.20±0.15, 0.75±0.12, 0.76±0.21 in transfection group, negative control group and blank control group, respectively. There was significant difference between transfection group and negative control group (P<0.01), whereas there was no significant difference between negative control group and blank control group (P>0.05). The efficiencies of down-regulated of HLA-G were (81.1±14.4)%, (18.0±7.7)%, 0 in transfection group, negative control group and blank control group respectively. (3) The invasive number of transfection group, negative control group and blank control group were 57±38, 364±79 and 260±84, respectively, with a significant difference between transfection group and negative control group (P<0.01). There was no significant difference between negative control group and blank control group (P>0.05). (4) The p-p38MAPK/p38MAPK values of the HLA-G transfection group, negative control group and blank control group were 0.74±0.04, 0.47±0.09 and 0.36±0.21, respectively. HLA-G transfection group was significantly different compared with the other two groups (P<0.01). (5) Without or with SB203580, the p-p38MAPK/p38MAPK values of the HLA-G transfection group were 0.89±0.09 and 0.16±0.04, the values of negative control group and blank control group were 0.76±0.08, 0.14±0.03 and 0.51±0.05, 0.03±0.01, respectively. There was significant difference between without SB203580 and with SB203580 (P<0.01). (6) Without or with SB203580, the invasive number of transfection group were 51±13 and 90±21, respectively, which was significantly different (P<0.01). The invasive number of negative control group and blank control group were 290±52, 298±33 and 290±73, 264±64, respectively, which was no significant difference between without SB203580 and with SB203580 (P>0.05). HLA-G gene may regulate invasion of trophoblast-derived cell line HTR-8/SVneo via p38MAPK signaling pathway. The lower expression of HLA-G in trophoblast cells may lead to the occurrence of pathologic pregnancy.