Phospho-p38 Mitogen-activated Protein Kinase Research Articles

Off-target effect of high-dose sildenafil on adenosine 5'- diphosphate and collagen-induced platelet activation through mitogen-activated protein kinase pathway in treated BALB/C mice and in vitro experiments: A preliminary study.

Sildenafil, a common over-the-counter pill often self-administered at high doses for erectile dysfunction, has been reported to rarely cause prothrombotic events and sudden cardiac death in a few case reports. Therefore, we investigated the in vitro and in vivo effect of sildenafil treatment and dosage on platelet activation and mitogen-activated protein kinase (MAPK) phosphorylation. BALB/C mice were segregated into four groups, each having four mice each (control, low [3.25 mg/kg], medium [6.5 mg/kg], and high [13 mg/kg] sildenafil), and after the treatment, blood was drawn from each mouse and washed platelets prepared. Washed platelets were incubated with CD41 PE-Cy7 and Phospho-p38 MAPK PE antibodies and analyzed using a flow cytometer for platelet activation and adenosine 5'- diphosphate (ADP)/collagen-induced MAPK phosphorylation. Washed platelets obtained from the venous blood of 18 human volunteers, were incubated with PAC-1 FITC and Phospho-p38 MAPK PE antibodies, and platelet activation (ADP and collagen), followed by flow cytometry analysis. There was a significant increase in both platelet activation as well as MAPK phosphorylation in the presence of collagen in the high-dose (13 mg/kg) sildenafil group (P = 0.000774). Further, increased platelet activation was observed in samples that were treated with high-dose sildenafil as compared to the untreated samples (P < 0.00001). These studies show the risk of prothrombotic episodes in patients on high-dose sildenafil (100 mg), in those with even mild endothelial dysfunction due to ADP, and collagen-induced platelet activation through MAPK phosphorylation, which was not seen in the low-and intermediate-dose cohorts.

TuBG1 promotes hepatocellular carcinoma via ATR/P53-apoptosis and cycling pathways

BackgroundAs reported, γ-tubulin (TuBG1) is related to the occurrence and development of various types of malignant tumors. However, its role in hepatocellular cancer (HCC) is not clear. The present study was to investigate the relationship between TuBG1 and clinical parameters and survival in HCC patients. MethodsThe correlation between TuBG1 and clinical parameters and survival in HCC patients was explored by bioinformatics analysis. Immunohistochemistry was used for the verification. The molecular function of TuBG1 was measured using colony formation, scratch assay, trans-well assay and flow cytometry. Gene set enrichment analysis (GSEA) was used to pick up the enriched pathways, followed by investigating the target pathways using Western blotting. The tumor-immune system interactions and drug bank database (TISIDB) was used to evaluate TuBG1 and immunity. Based on the TuBG1-related immune genes, a prognostic model was constructed and was further validated internally and externally. ResultsThe bioinformatic analysis found high expressed TuBG1 in HCC tissue, which was confirmed using immunohistochemistry and Western blotting. After silencing the TuBG1 in HCC cell lines, more G1 arrested cells were found, cell proliferation and invasion were inhibited, and apoptosis was promoted. Furthermore, the silence of TuBG1 increased the expressions of Ataxia-Telangiectasia and Rad-3 (ATR), phospho-P38 mitogen-activated protein kinase (P-P38MAPK), phospho-P53 (P-P53), B-cell lymphoma-2 associated X protein (Bax), cleaved caspase 3 and P21; decreased the expressions of B-cell lymphoma-2 (Bcl-2), cyclin D1, cyclin E2, cyclin-dependent kinase 2 (CDK2) and CDK4. The correlation analysis of immunohistochemistry and clinical parameters and survival data revealed that TuBG1 was negatively correlated with the overall survival. The constructed immune prognosis model could effectively evaluate the prognosis. ConclusionsThe increased expression of TuBG1 in HCC is associated with poor prognosis, which might be involved in the occurrence and development of HCC.

Activation of p38 MAPK hinders the reactivation of visual cortical plasticity in adult amblyopic mice

ObjectiveTo investigate the impact of p38 mitogen-activated protein kinase (MAPK) signaling on reactivating visual cortical plasticity in adult amblyopic mice. Materials and methodsReverse suture (RS), environment enrichment (EE), and combined with left intracerebroventricular injection of p38 MAPK inhibitor (SB203580, SB) or p38 MAPK agonist (dehydrocorydaline hydrochloride, DHC) were utilized to treat adult amblyopic mice with monocular deprivation (MD). The visual water task, visual cliff test, and Flash visual-evoked potential were used to measure the visual function. Then, Golgi staining and transmission electron microscopy were used to assess the reactivation of structural plasticity in adult amblyopic mice. Western blot and immunohistochemistry detected the expression of ATF2, PSD-95, p38 MAPK, and phospho-p38 MAPK in the left visual cortex. ResultsNo statistically significant difference was observed in the visual function in each pre-intervention group. Compared to pre-intervention, the visual acuity of deprived eyes was improved significantly, the impairment of visual depth perception was alleviated, and the P wave amplitude and C/I ratio were increased in the EE + RS, the EE + RS + SB, and the EE + RS + DMSO groups, but no significant difference was detected in the EE + RS + DHC group. Compared to EE + RS + DHC group, the density of dendritic spines was significantly higher, the synaptic density of the left visual cortex increased significantly, the length of the active synaptic zone increased, and the thickness of post-synaptic density (PSD) thickened in the left visual cortex of EE + RS, EE + RS + SB, and EE + RS + DMSO groups. And that, the protein expression of p-p38 MAPK increased while that of PSD-95 and ATF2 decreased significantly in the left visual cortex of the EE + RS + DHC group mice. ConclusionRS and EE intervention improved the visual function and synaptic plasticity of the visual cortex in adult amblyopic mice. However, activating p38 MAPK hinders the recovery of visual function by upregulating the phosphorylation of p38 MAPK and decreasing the ATF2 protein expression.

Fecal Microbiota Transplantation Alleviated Paclitaxel-Induced Peripheral Neuropathy by Interfering with Astrocytes and TLR4/p38MAPK Pathway in Rats.

Paclitaxel-induced peripheral neuropathy (PIPN) constitutes a refractory and progressive adverse consequence of paclitaxel treatment, causing pain and sensory anomalies in cancer survivors. Although the gut-brain axis is involved in multiple disorders including cancer, its impact on peripheral pain conditions remains elusive. Thus, we assessed the importance of gut microbiota and related mechanisms in PIPN. By implementing fecal microbiota transplantation (FMT) in a rat PIPN model (ie, rats treated with paclitaxel; hereafter as PIPN rats), we explored the effect of gut microbiota on PIPN rats using multiple methods, including different behavioral tests, 16S ribosomal DNA (rDNA) sequencing, and biochemical techniques. Sequencing of 16S rDNA revealed that the abundance of genera Bacteroides and UCG-005 increased, while that of genera Turicibacter, Clostridium sensu stricto 1 and Corynebacterium decreased in the PIPN rats. However, when treated with FMT using fecal from normal rats, the mechanical allodynia and thermal hyperalgesia in PIPN rats were significantly alleviated. In addition, FMT treatment reduced the expression of toll-like receptor 4 (TLR4), phospho-p38 mitogen-activated protein kinase (p-p38MAPK), and the astrocytic marker glial fibrillary acidic protein in the colon and spinal dorsal horn. TAK242 (a TLR4 inhibitor) significantly alleviated the behavioral hypersensitivity of PIPN rats and inhibited the TLR4/p38MAPK pathway in astrocytes in these rats. The gut microbiota played a critical role in PIPN. Future therapies treating PIPN should consider microbe-based treatment as an option.

Efficacy of Buzhong Yiqi decoction on benign prostatic hyperplasia and its possible mechanism.

To explored the mechanism of Buzhong Yigi decoction ( BZYQD) in inhibiting prostatic cell proliferation effect. The compounds of BZYQD consisted with eight herbs were searched in TCMSP databases and the putative targets of BZYQD were collected in Drugbank database. Then, "Benign prostatic hyperplasia" (BPH) was used to find the targets based on the GeneCards, Online Mendelian Inheritance in Man (OMIM) and Therapeutic Target Database (TTD) databases, and they were further used to collect further collect the intersection targets between BZYQD and BPH by counter-selection. Next, Herb-Compound-Target-Disease network was constructed by Cytoscape software and protein interaction network was built by Search tool for recurring instances of neighbouring genes (STRING) database. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were analyzed by Database for Annotation, Visualization and Integrated Discovery (DAVID) database to predict the mechanism of the intersection targets. Mitogen activated protein kinase 8 (MAPK8), interleukin 6 (IL-6) and quercetin were chosen to perform molecular docking. Then 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was to detect the bility of BPH-1 (BPH epithelial cell line) by treated with quercetin at the concentrations of 15, 30, 60, 120 μM for 12, 24, 48, 72 h. The production of IL-6, tumor necrosis factor-α (TNF-α), IL-1β and were mRNA expression detected by enzyme-linked immunosorbent assay kit and quantitative real-time polymerase chain reaction. Western blot was used to detect the expression of phospho-p38 mitogen-activated protein kinase (p-P38) and matrix metalloprotein-9 (MMP-9). A total 151 chemical ingredients of 8 herbs and 1756 targets in BZYQD, 105 common targets of BZYQD and BPH which mainly involving with MAPK8, IL-6, and so on. GO enrichment analysis got 352 GO entries (0.05) which included 208 entries of biological process, 64 entries of cell component and 80 entries of molecular function. KEGG pathway Enrichment analyses got 20 significant pathways which mainly involved with MAPK signaling way. MTT assay indicated quercetin inhibited the viability of BPH-1 cells by time-and dose-dependent manner. Quercetin decreased the IL-6, TNF-α and IL-1β production and mRNA expression, and the expression of p-P38 and MMP-9 were also obviously reduced after treated with quercetin. BZYQD inhibited BPH through suppressing inflammatory response which might involving with regulating the MAPK signaling way.

Exogenous Lipoxin A4 attenuates IL4-induced Mucin Expression in Human Airway Epithelial Cells.

Introduction: The proinflammatory cytokine interleukin-4 (IL-4) induces mucus hypersecretion by human airway epithelial cells and the MAP kinase signalling pathway may be important in terms of IL-4-induced MUC5AC gene expression. Lipoxin A4 (LXA4) is an arachidonic acid-derived mediator that promotes inflammation by binding to the anti-inflammatory receptors (ALXs) or the formyl-peptide receptor like-1 (FPRL1) protein expressed by airway epithelial cells. Here, we explore the effects of LXA4 on IL-4-induced mucin gene expression in, and secretion from, human airway epithelial cells. Methods: We co-treated cells with IL-4 (20 ng/mL) and LXA4 (1 nM) and measured the expression levels of mRNAs encoding MUC5AC and 5B via real-time polymerase chain reaction; protein expression levels were determined by Western blotting and immunocytofluorescence. The ability of IL-4 and LXA4 to suppress protein expression was determined by Western blotting. Results: IL-4 increased MUC5AC and 5B gene and protein expression. LXA4 suppressed IL-4-induced MUC5AC and 5B gene and protein expression by interacting with the IL4 receptor and mitogen-activated protein kinase (MAPK) pathway, including both phospho-p38 MAPK and phospho-extracellular signal-regulated kinase (phospho-ERK). IL-4 and LXA4 increased and decreased, respectively, the number of cells that stained with anti-MUC5AC and 5B antibodies. Conclusions: LXA4 may regulate mucus hypersecretion induced by IL4 in human airway epithelial cells.

Glycolytic Regulation of Intestinal Stem Cell Self-Renewal and Differentiation

The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis. An imbalance in this highly regimented process within the intestinal crypts is associated with several intestinal pathologies. Although metabolic changes are known to play a pivotal role in cell proliferation and differentiation, how glycolysis contributes to intestinal epithelial homeostasis remains to be defined. Small intestines were harvested from mice with specific hexokinase 2 (HK2) deletion in the intestinal epithelium or LGR5+ stem cells. Glycolysis was measured using the Seahorse XFe96 analyzer. Expression of phospho-p38 mitogen-activated protein kinase, the transcription factor atonal homolog 1, and intestinal cell differentiation markers lysozyme, mucin 2, and chromogranin A were determined by Western blot, quantitative real-time reverse transcription polymerase chain reaction, or immunofluorescence, and immunohistochemistry staining. HK2 is a target gene of Wnt signaling in intestinal epithelium. HK2 knockout or inhibition of glycolysis resulted in increased numbers of Paneth, goblet, and enteroendocrine cells and decreased intestinal stem cell self-renewal. Mechanistically, HK2 knockout resulted in activation of p38 mitogen-activated protein kinase and increased expression of ATOH1; inhibition of p38 mitogen-activated protein kinase signaling attenuated the phenotypes induced by HK2 knockout in intestinal organoids. HK2 knockout significantly decreased glycolysis and lactate production in intestinal organoids; supplementation of lactate or pyruvate reversed the phenotypes induced by HK2 knockout. Our results show that HK2 regulates intestinal stem cell self-renewal and differentiation through p38 mitogen-activated protein kinase/atonal homolog 1 signaling pathway. Our findings demonstrate an essential role for glycolysis in maintenance of intestinal stem cell function.

Rab7l1 plays a role in regulating surface expression of toll like receptors and downstream signaling in activated macrophages

Rab GTPases are known for controlling intracellular membrane traffic in a GTP-dependent manner. Rab7l1, belonging to family of Rab GTPases, is important for both endosomal sorting and retrograde transport. In our previous study, we identified a novel role of Rab7l1 in phagosome maturation. However, its role in regulating macrophage innate-effector signaling and cytokine response is not clearly understood. In this study, we have demonstrated that upon treatment of Rab7l1-knocked-down (Rab7l1-KD) THP-1 macrophages with lipopolysaccharide (LPS) and Pam3CSK4 has led to higher induction levels of tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) as compared to the control cells that received scrambled shRNA. Similar results were observed in Rab7l1-KD RAW 264.7 and Balb/c peritoneal macrophages. The phospho-ERK 1/2 (extracellular signal-regulated kinase 1/2) and phospho-p38 MAPK (mitogen-activated protein kinase) levels, known to be responsible for higher induction of TNF-α and IL-10 respectively, were higher in Rab7l1-KD THP-1 macrophages which also displayed higher nuclear translocation of p50/p65 nuclear factor kappa B (NF-κB) upon stimulation with LPS. Surface expression levels of toll-like receptor 2 (TLR2), TLR4 and CD14 receptors were higher in Rab7l1-KD THP-1 macrophages as compared to the control cells. However, intracellular levels of these receptors were lower in Rab7l1-KD THP-1 macrophages as compared to the control group. Together, our study suggests that Rab7l1 has a role in regulating MAPK signaling and cytokine effector responses in macrophages by regulating the surface expression of membrane receptors.

Chinese herbal medicine alleviates the pathogenesis of polycystic ovary syndrome by improving oxidative stress and glucose metabolism via mitochondrial Sirtuin 3 signaling

BackgroundPolycystic ovary syndrome (PCOS) is one of the most common endocrine disorders among women, and the curative effects of its current management are not satisfactory. A formula of Chinese herbal medicine (CHM), called Bu-Shen-Tian-Jing Formula (BSTJF), has clinically shown beneficial effects in treating PCOS. PurposeThis study aimed to investigate the mechanism underlying BSTJF for treatment of PCOS. MethodsWhole blood samples were collected from women with PCOS treated and not treated with BSTJF (n = 5 per group). Whole transcriptome sequencing of leukocytes and untargeted metabonomic analysis of the plasma were performed. Three groups of 18 female Sprague-Dawley rats were randomly selected: control, PCOS, and BSTJF. A PCOS rat model was established using testosterone propionate. The estrous cycle; glucose tolerance; ovarian morphology; serum markers of oxidative stress; and expression of Sirtuin 3 (SIRT3), phospho-p38 mitogen-activated protein kinase, phosphatidylinositol 3-kinase (PI3K), and phospho-protein kinase B in the ovary were measured. Palmitate was initially applied to KGN cells, followed by freeze-dried BSTJF powder. The glucose uptake, reactive oxygen species (ROS) production, and protein levels of SIRT3, PI3K, and glucose transporter type 4 (GLUT4) were detected in KGN cells. ResultsThe transcriptomic and metabolomic profiles showed alterations in 572 genes and 73 metabolites in women with PCOS treated with BSTJF. The enriched pathways in women with PCOS treated with BSTJF were mainly involved in inflammation, insulin resistance, glucose and lipid metabolism, and neuro and associated signaling pathways. In PCOS rat models, BSTJF improved the estrous cycle, glucose tolerance, and ovarian morphology; relieved oxidative stress; increased ovarian SIRT3 expression; inhibited p38 MAPK activation; and promoted the activation of PI3K/AKT signaling in the ovary. In the in-vitro study with KGN cells, BSTJF rescued the palmitate-induced impaired glucose uptake and SIRT3 expression, reduced mitochondrial ROS production mediated by SIRT3, and restored the impaired insulin-induced PI3K/AKT signaling pathway. ConclusionBSTJF effectively alleviated the pathogenesis of PCOS by improving oxidative stress and glucose metabolism via mitochondrial SIRT3 and the following insulin signaling pathway. This study innovatively revealed the action mechanism of CHM in treating PCOS.

Coriolus versicolor and its bioactive molecule are potential immunomodulators against cancer cell metastasis via inactivation of MAPK pathway

Ethnopharmacological relevanceCoriolus versicolor (CV) has been used in traditional Chinese medicine for over 2000 years as a premium medicine for enhancing good health and longevity. The immunomodulatory and anti-cancer effects of polysaccharopeptides (PSP) from cultured CV have been extensively studied; however, the effect and the mechanism of action of other small molecules from CV remain unknown.Aim of the study: we aim to examine the immunomodulatory and anti-cancer effects of the small molecules from CV (SMCV) and identify the active compounds that are responsible for the biological effects against glioblastoma multiforme cells. Materials and methodsThe effects of SMCV/active compound on cytokine and MMP mRNA expressions and productions were assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. An active compound from SMCV was identified with a bioassay-guided fractionation scheme. The potential mode of action of the active compound was further investigated by identifying the cell signaling pathway. The protein expressions of phospho-ERK, phospho-JNK and phospho-p38 MAPKs were measured by Western Blotting. The anti-invasive effect of SMCV/bioactive compound against T98G, lung carcinoma (A549), and breast adenocarcinoma (MDA-MB-231) cells were determined using invasion assay. ResultsOur results showed that SMCV had strong immunomodulatory effect by suppressing LPS-induced TNF-α production, whereas increasing poly I:C-induced IFN-β level in PBMac. SMCV not only possessed indirect anti-cancer effect by suppressing TNF-α-induced MMP-3 production in glioblastoma T98G cells, but also directly reduced the invasion ability of malignant cells including T98G, A549 and MDA-MB-231. Using bioassay-guided fractionation scheme, we isolated 9-KODE methyl ester (compound AM) that was responsible for the bioactivity of SMCV. This compound suppressed TNF-α-induced MMP-3 production in T98G cells and the suppression may be correlated with the inactivation of p38 mitogen-activated protein kinase (MAPK) pathway. Moreover, compound AM also directly reduced T98G cell invasion. ConclusionResults of our present study provides scientific evidence that SMCV possesses immunomodulatory and anti-cancer effects. Its bioactive compound, compound AM, is a potential new drug candidate against the invasion and metastasis of glioblastoma cells.

Chemotherapy-induced nephrotoxicity was improved by crocin in mouse model.

Cisplatin (CDDP) has been widely used in cancer therapy, but it has been linked to side effects such as nephrotoxicity. Crocin is a carotenoid found in crocus and gardenia flowers that has been shown to have anti-oxidant properties, inhibit tumor growth, and provide neuroprotection. The purpose of this study was to investigate the protective effect of crocin against CDDP-induced nephrotoxicity in a mouse model. Kunming mice were administered orally with crocin for 7 days at the dose of 6.25 mg/kg and 12.5 mg/kg per body weight daily and were injected with CDDP via intraperitoneal route at the dose of 10 mg/kg per body weight. Using commercial kits, the oxidative stress markers glutathione, malondialdehyde, catalase, glutathione peroxidase, and superoxide dismutase were measured in the kidneys of mice. Immunohistochemistry was used to assess the levels of p53, cleaved caspase-3, and phospho-p38 mitogen-activated protein kinase in the kidneys. Crocin significantly reduced CDDP-induced changes in serum creatinine and blood urea nitrogen levels, according to the findings. Crocin reduced malondialdehyde levels and increased glutathione, glutathione peroxidase, catalase, and superoxide dismutase levels in CDDP-induced lipid peroxidation. Crocin also significantly inhibited p38 mitogenactivated protein kinase activation, p53 expression, and caspase-3 cleavage. In conclusion, crocin protects against CDDP-induced oxidative stress and nephrotoxicity by attenuating the activation of p38 mitogen-activated protein kinase and caspase-3 cleavage.

Inhibition of P2Y6 receptor expression in Kupffer cells alleviates alcoholic steatohepatitis in mice

Inflammation plays an important role in the progression of alcohol-related liver disease (ALD). UDP-P2Y6 signaling is involved in many human diseases. The purinergic P2Y6 receptor, an important regulator of inflammation and phagocytosis, has attracted attention, but its role in alcoholic steatohepatitis remains unclear. Here, we found that P2Y6 levels were significantly elevated in Kupffer cells in the livers of mice with alcoholic steatohepatitis and ethanol (EtOH)-induced RAW264.7 cells. In this study, mice with alcoholic steatohepatitis were intraperitoneally injected with MRS2578, a specific inhibitor of the P2Y6 receptor, and P2Y6 was silenced in EtOH-induced RAW264.7 cells. We found a marked improvement in steatosis and inflammation in the livers of mice with alcoholic steatohepatitis and EtOH-induced RAW264.7 cells. However, P2Y6 activation in vivo and overexpression in vitro showed contrasting results. In addition, the expression of phospho-p38 mitogen-activated protein kinase (p-p38 MAPK), a phosphorylated protein in the p38 MAPK signaling pathway, was significantly altered after P2Y6 silencing or overexpression in vitro. P2Y6 can induce the activation of the p38 MAPK signaling pathway by mediating the calcium influx, whereas inhibition of the expression of P2Y6 can block the inflammatory process to some extent and thus improve the inflammatory response. The results of this study suggested that targeting P2Y6 signaling may be a potentially effective strategy for the treatment of alcoholic steatohepatitis.

Corydalis decumbens Can Exert Analgesic Effects in a Mouse Neuropathic Pain Model by Modulating MAPK Signaling.

Objectives This study is aimed at investigating the analgesic effect of the administration of Corydalis decumbens (CD) in a mouse model of postherpetic neuralgia (PHN) and at elucidating its mechanism of analgesic action. Methods Adult Kunming (KM) mice were randomly divided into control, CD, and vehicle-treated groups. Neuropathic pain was induced with a single intraperitoneal injection of resiniferatoxin (RTX). Thermal hyperalgesia was assessed with a hot/cold plate test, and mechanical allodynia was evaluated using von Frey filaments. The activation states of astrocytes, microglia, and the mitogen-activated protein kinase (MAPK) pathway in the spinal cord were determined by immunofluorescence staining and Western blot analysis of Iba-1, GFAP, phospho-p38, and phospho-Jun N-terminal kinase (JNK). Results RTX diminished thermal sensitivity and gradually increased sensitivity to tactile stimulation. The expression of Iba-1, GFAP, phospho-p38 MAPK, and phospho-JNK was upregulated in the RTX-induced postherpetic neuralgia mouse model. Systemic treatment with CD significantly ameliorated thermal sensitivity and mechanical hyperalgesia and was accompanied by a reduction in the expression of Iba-1 and GFAP and reduced phosphorylation of p38 and JNK. Conclusions This study suggests that CD is effective at ameliorating mechanical hyperalgesia in PHN mice and that its mechanism of action may involve modulation of MAPK phosphorylation and glial cell activation. Thus, CD may be a promising alternative therapy for PHN.

Regulation of microRNA-126 on the polarization of human macrophages stimulated by Porphyromonas gingivalis lipopolysaccharide

Objective: To study the effect of microRNA-126 (miR-126) on the polarization of human monocyte-derived macrophages stimulated by Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS). Methods: Macrophages derived from human myeloid leukemia mononuclear cells were stimulated by Pg-LPS (5 mg/L) and by Pg-LPS (5 mg/L) after 24 h-transfection of miR-126 mimic or negative control RNA for 48 h, respectively. Real-time quantitative-PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western blotting were conducted to detect the changes in miR-126, pro-inflammatory factor tumor necrosis factor-α (TNF-α), anti-inflammatory factors interleukin-10 (IL-10), inducible nitric oxide synthase (iNOS), arginase-1 (Arg-1) and M1 polarization-related pathways such as nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Results: Compared with non-LPS stimulation group (TNF-α: 1.000±0.020, iNOS: 1.125±0.064, miR-126: 1.004±0.113, IL-10: 1.003±0.053, Arg-1: 1.130±0.061), the mRNA levels of TNF-α (3.105±0.278) and iNOS (4.296±0.003) increased significantly (t=6.53, P=0.003; t=42.63, P<0.001, respectively), while miR-126, IL-10 and Arg-1 expressions (0.451±0.038, 0.545±0.004 and 0.253±0.017) decreased significantly (t=7.95, P=0.001; t=7.36, P=0.002; t=11.94, P<0.001, respectively) after Pg-LPS stimulated by human-derived macrophages for 48 h. The protein expression of iNOS, TNF-α, Arg-1 and IL-10 were consistent at mRNA levels. Meanwhile, the expressions of phospho-NF-κB p65 (p-p65), phospho-extracellular signal-regulated kinase (p-ERK) and phospho-p38 MAPK (p-p38) increased significantly, while the expression of Arg-1 decreased significantly. Compared with the negative controls (scramble RNA) (TNF-α: 1.141±0.197, iNOS: 1.173±0.115, IL-10: 1.032±0.138, Arg-1: 0.933±0.044), the mRNA levels of TNF-α (0.342±0.022) and iNOS (0.588±0.085) expressions significantly decreased (t=5.35, P=0.006; t=5.05, P=0.007), while IL-10 (1.786±0.221) and Arg-1 expressions (2.152±0.229) significantly increased (t=3.71, P=0.021; t=6.21, P=0.003) after Pg-LPS stimulation with miR-126 mimic transfection. The relative protein expressions of iNOS, p-p65, p-ERK and p-p38 significantly decreased (t=13.00, P<0.001; t=6.98, P=0.002; t=10.86, P<0.001; t=8.32, P=0.001), while the protein level of Arg-1 significantly increased (t=12.08, P<0.001). Conclusions: Pg-LPS could promote M1 polarization of macrophages. miR-126 might inhibit the effect of Pg-LPS on the M1 polarization of macrophages through down-regulating NF-κB and MAPK signaling pathways.

Cyclosporin-A reduced the cytotoxicity of propranolol in HUVECs via p38 MAPK signaling.

Propranolol (PROP) is a nonselective β-adrenergic receptor antagonist used to treat hypertension and cardiac arrhythmias. Oral administration of PROP has recently emerged as a new treatment modality for hemangiomas. However, the side effects of PROP at the cellular level have not been adequately described.The present study investigates and highlights the mechanisms of coupling of the drugs cyclosporin-A (CyA) and PROP on cell proliferation and the occurrence of apoptosis. It also relays the antioxidant effect of PROP on human umbilical vein endothelial cells (HUVECs).HUVECs were treated with CyA and PROP. At 24 hours after treatment, the levels of reactive oxygen species (ROS), cell proliferation, and apoptosis were determined using the ROS kit, MTT assay, and Annexin V staining. In addition, the related proteins of phospho-p38 mitogen-activated protein kinase were determined by western blotting. Subsequently, HUVECs pretreated with CyA or PROP were treated with the p38 inhibitor (SB203580). Finally, the ROS level, cell proliferation, and apoptosis were measured again in both active HUVECs and HUVECs, in which the p38 proteins were inhibited.The combination of CyA and PROP reversed the effect of CyA on cell viability, reduced the ROS level and the cell apoptosis induced by PROP. Moreover, inhibition of p38 protein catalase activity immediately stopped the effect of CyA–propranolol in HUVECs.The effect of the CyA–propranolol combination on HUVECs is associated with the p38 pathway changes, which is proven to be a potential chemotherapeutic agent that minimizes the side effects of PROP in hemangioma therapy.

Effects of Pioglitazone Pretreatment on the Expression of NF-κB, ICAM-1 and p38MAPK Pathway in Pancreatic Tissue of Rats with Acute Pancreatitis

To investigate the effects of pioglitazone pretreatment on the expression of nuclear factor kappa B and intercellular adhesion molecule-1 and p38 mitogen activated protein kinase pathway in pancreatic tissue of rats with acute pancreatitis. 72 specific pathogen-free adult female Sprague–Dawley rats and male Sprague–Dawley rats were randomly divided into sham operation group, model group and pioglitazone pretreatment group (pretreatment group), with 24 rats in each group. Compared with sham operation group, the amount of ascites in model group was significantly increased, the levels of serum and ascites amylase, serum tumor necrosis factor-alpha, interleukin-6, pancreatic gross score, pancreatic histological score, nuclear factor kappa B and intercellular adhesion molecule-1 in pancreatic tissue were significantly increased. Compared with the model group, the amount of ascites in the pretreatment group was significantly reduced and the levels of serum and ascites amylase, serum tumor necrosis factor-alpha, interleukin-6, pancreatic gross score and pancreatic histological score, the expression levels of nuclear factor kappa B, intercellular adhesion molecule-1 and phospho-p38 mitogen activated protein kinase in pancreatic tissue were significantly decreased (p<0.05). In the sham operation group, the pancreatic tissue structure was clear with occasional inflammatory cells or mild hyperemia and edema and the acinar lobules were intact; in the model group, the pancreatic stroma showed hyperemia and edema, even necrosis, with obvious inflammatory cell infiltration and acinar lobule structure disorder and the pathological changes were gradually aggravated with the extension of time; in the pretreatment group, the pancreatic tissue inflammatory cell infiltration and other pathological changes were observed compared with the model group, it was significantly improved. Pioglitazone pretreatment can inhibit the inflammatory response, reduce the level of amylase, inhibit the expression of nuclear factor kappa B and intercellular adhesion molecule-1 and inhibit the activity of intercellular adhesion molecule-1 signaling pathway in rats with acute pancreatitis.

Role of genistein on the yield, structure and immunomodulatory activity of Monascus exopolysaccharides.

Manipulating the structures, physicochemical properties, and monosaccharide compositions of exopolysaccharides (EPS) isolated from microorganisms has been reported to enhance their biological activities. Hence, the aim of this work was to examine the effects of genistein addition during fermentation on the amount, physicochemical properties, and immunomodulatory activity of EPS secreted by M. purpureus. Results showed that genistein addition significantly increased M. purpureus biomass and EPS yield to 2.42 g L-1 and 6.08 g L-1, respectively, and affected the physicochemical properties and structures of EPS. Furthermore, EPS produced by genistein-treated M. purpureus (G-EMP) improved the immunomodulatory activity of RAW264.7 macrophages by increasing the secretion of nitric oxide and cytokines. Moreover, phospho-Jun N-terminal kinase (p-JNK), phospho-extracellular regulated protein kinase (p-ERK), phospho-p38 (p-p38) mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) phospho-p65 (p65) proteins were remarkably upregulated by G-EMP stimulation, blocking Toll-like receptor 4 (TLR4) that dramatically reduced the pinocytic and phagocytic capacities. Overall, these findings provide potential rationales for the application of genistein in improving the EPS yield of M. purpureus.

Selenoproteins Protect Against Avian Liver Necrosis by Metabolizing Peroxides and Regulating Receptor Interacting Serine Threonine Kinase 1/Receptor Interacting Serine Threonine Kinase 3/Mixed Lineage Kinase Domain-Like and Mitogen-Activated Protein Kinase Signaling

Liver necroptosis of chicks is induced by selenium (Se)/vitamin E (VE) deficiencies and may be associated with oxidative cell damage. To reveal the underlying mechanisms of liver necrosis, a pool of the corn–soy basal diet (10 μg Se/kg; no VE added), a basal diet plus all-rac-α-tocopheryl acetate (50 mg/kg), Se (sodium selenite at 0.3 mg/kg), or both of these nutrients were provided to day-old broiler chicks (n = 40/group) for 6 weeks. High incidences of liver necrosis (30%) of chicks were induced by –SE–VE, starting at day 16. The Se concentration in liver and glutathione peroxidase (GPX) activity were decreased (P < 0.05) by dietary Se deficiency. Meanwhile, Se deficiency elevated malondialdehyde content and decreased superoxide dismutase (SOD) activity in the liver at weeks 2 and 4. Chicks fed with the two Se-deficient diets showed lower (P < 0.05) hepatic mRNA expression of Gpx1, Gpx3, Gpx4, Selenof, Selenoh, Selenok, Selenom, Selenon, Selenoo, Selenop, Selenot, Selenou, Selenow, and Dio1 than those fed with the two Se-supplemented diets. Dietary Se deficiency had elevated (P < 0.05) the expression of SELENOP, but decreased the downregulation (P < 0.05) of GPX1, GPX4, SELENON, and SELENOW in the liver of chicks at two time points. Meanwhile, dietary Se deficiency upregulated (P < 0.05) the abundance of hepatic proteins of p38 mitogen-activated protein kinase, phospho-p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, phospho-c-Jun N-terminal kinase, extracellular signal-regulated kinase, phospho-mitogen-activated protein kinase, receptor-interacting serine-threonine kinase 1 (RIPK1), receptor-interacting serine-threonine kinase 3 (RIPK3), and mixed lineage kinase domain-like (MLKL) at two time points. In conclusion, our data confirmed the differential regulation of dietary Se deficiency on several key selenoproteins, the RIPK1/RIPK3/MLKL, and mitogen-activated protein kinase signaling pathway in chicks and identified new molecular clues for understanding the etiology of nutritional liver necrosis.

Protective effects of curcumin against methotrexate-induced testicular damage in rats by suppression of the p38-MAPK and nuclear factor-kappa B pathways.

ObjectiveThe present study aimed to investigate the possibility that curcumin (CMN) protects against methotrexate (MTX)-induced testicular damage by affecting the phospho-p38 (p-p38) mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways.MethodsEighteen male Wistar albino rats were randomly divided into three groups. The control group was given an intragastric administration of dimethyl sulfoxide (DMSO) daily for 14 days, the MTX group was given a single intraperitoneal dose of MTX (20 mg/kg) on the 11th day, and the MTX+CMN group was given intragastric CMN (100 mg/kg/day, dissolved in DMSO) for 14 days and a single intraperitoneal dose of MTX (20 mg/kg) on the 11th day. At the end of the experiment, all animals were sacrificed and the testicular tissues were removed for morphometry, histology, and immunohistochemistry. Body and testicular weights were measured.ResultsBody weights, seminiferous tubule diameter, and germinal epithelium height significantly decreased in the MTX group compared to the control group. Whereas, the number of histologically damaged seminiferous tubules and interstitial space width significantly increased in the MTX group. In addition, the number of p-p38 MAPK immunopositive cells and the immunoreactivity of NF-κB also increased in the MTX group compared to the control group. CMN improved loss of body weight, morphometric values, and histological damage due to MTX. CMN also reduced the number of p-p38 MAPK immunopositive cells and the NF-κB immunoreactivity.ConclusionCMN may reduce MTX-induced testicular damage by suppressing the p38 MAPK and NF-κB signaling pathways.

Angelica sinensis extract promotes neuronal survival by enhancing p38 MAPK–mediated hippocampal neurogenesis and dendritic growth in the chronic phase of transient global cerebral ischemia in rats

Ethnopharmacological relevanceAngelica sinensis (Oliv.) Diels (ASD), commonly known as Dang Gui, is a popular Chinese herb that has long been used to treat ischemic stroke. However, the effects of ASD in chronic cerebral ischemia and its underlying mechanisms still remain unclear. Aim of the studyThis study aimed to determine the effects of the ASD extract on hippocampal neuronal survival at 28 d after transient global cerebral ischemia (GCI) and to investigate the precise mechanisms underlying the p38 mitogen-activated protein kinase (MAPK)-related signaling pathway's involvement in hippocampal neurogenesis. Materials and methodsRats underwent 25 min of four-vessel occlusion. The ASD extract was intragastrically administered at doses of 0.25 g/kg (ASD-0.25 g), 0.5 g/kg (ASD-0.5 g), 1 g/kg (ASD-1 g), 1 g/kg after dimethyl sulfoxide administration (D + ASD-1 g), or 1 g/kg after SB203580 (a p38 MAPK inhibitor) administration (SB + ASD-1 g) at 1, 3, 7, 10, 14, 17, 21, and 24 d after transient GCI. ResultsASD-0.5 g, ASD-1 g, and D + ASD-1 g treatments had the following effects: upregulation of bromodeoxyuridine (BrdU) and Ki67 expression, and BrdU/neuronal nuclei (NeuN) and Ki67/nestin co-expression in the hippocampal dentate gyrus (DG); upregulation of microtubule-associated protein 2/NeuN co-expression, and NeuN and glial fibrillary acidic protein (GFAP) expression, and downregulation of tumor necrosis factor-α/GFAP co-expression in the hippocampal CA1 region; upregulation of phospho-p38 MAPK (p-p38 MAPK), phospho-cAMP response element-binding protein (p-CREB), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and vascular endothelial growth factor A (VEGF-A) expression in the hippocampus. SB + ASD-1 g treatment abrogated the effects of ASD-1 g on the expression of these proteins. ConclusionsASD-0.5 g and ASD-1 g treatments promotes neuronal survival by enhancing hippocampal neurogenesis. The effects of the ASD extract on astrocyte–associated hippocampal neurogenesis and dendritic growth are caused by the activation of p38 MAPK–mediated CREB/BDNF, GDNF, and VEGF-A signaling pathways in the hippocampus at 28 d after transient GCI.