p-p38MAPK Protein Research Articles

Role of P38 mitogen-activated protein kinase on Cx43 phosphorylation in cerebral vasospasm after subarachnoid hemorrhage

Objectives: Cx43 phosphorylation is involved in the pathogenesis of cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH). However, the exact phosphorylation mechanism of Cx43 in CVS is not fully elucidated. Thus, we examined the role of P38MAPK in CVS and Cx43 phosphorylation, using a double hemorrhage rat model.Methods: Sprague–Dawley rats weighing 300–350 g were grouped into sham, SAH, vehicle, and SAH + SB203580. SAH was induced by double injecting blood into the prechiasmatic cisterns. Neurological score was measured with the Garcia scoring system, and the diameters of basilar arteries and the expression of pCx43, pP38MAPK, and P38MAPK proteins were measured through pressure myograph measurement and Western blot analysis, respectively.Results: The neurological scores remarkably decreased after SAH but remarkably improved after SB203580 was used. The results of pressure myograph analysis on the SAH and vehicle groups showed the considerable narrowing of the basilar arteries in comparison with that of the sham group. By contrast, the arterial diameters in the SAH + SB203580 group were much larger than those observed in the SAH and vehicle groups. Moreover, the P38MAPK expression in the sham group had no substantial change in contrast to the SAH and vehicle groups, and pCx43 and pP38MAPK increased in the SAH and vehicle groups. Meanwhile, the SAH + SB203580 group showed marked decrease in Cx43 and P38MAPK phosphorylation levels relative to the SAH and vehicle groups.Conclusions: P38MAPK pathway facilitates the development of CVS through the upregulation of Cx43 phosphorylation.

AOS ameliorates monocrotaline-induced pulmonary hypertension by restraining the activation of P-selectin/p38MAPK/NF-κB pathway in rats

Perivascular inflammation, vascular luminal area reduction and hemodynamics changes are important pathophysiologic bases of pulmonary hypertension (PH). In this study, PH was induced by an intraperitoneal single injection of monocrotaline (MCT, 60 mg/kg). Alginate oligosaccharides (AOS), one of the most famous marine drugs, provided protections in the perivascular inflammation, vascular luminal area reduction and hemodynamics changes of the PH rat induced by MCT. The downregulation of P-selectin plays an important role in the protective effects of AOS against MCT induced PH. The results showed that the treatment with AOS (5, 10, or 20 mg/kg) dose-dependently decreased the expression of P-selectin in serum, pulmonary tissue and pulmonary artery of MCT-induced pulmonary arterial hypertension rats. What’s more, the study showed that the protective effects were mediated by the inhibition of p38MAPK/NF-κB pathway, which was caused by reducing the p-p38MAPK protein expression, IκBα degradation and nuclear transcription of NF-κB protein in the pulmonary artery of MCT-induced PH rats. These findings provided an alternative potent medicine for the prevention and therapy of PH.

Metformin Regulates the Expression of SK2 and SK3 in the Atria of Rats With Type 2 Diabetes Mellitus Through the NOX4/p38MAPK Signaling Pathway

We previously found that metformin regulates the ion current conducted by the small conductance calcium-activated potassium channels (SK channels) in the atria of rats with type 2 diabetes mellitus (T2DM) as well as the mRNA and protein expression of the SK2 and SK3 subtypes of SK channels. In this study, we hypothesized that the nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4)/p38 mitogen-activated protein kinase (p38MAPK) signaling pathway was involved in the metformin-mediated regulation of SK2 and SK3 expression in the atria of rats with T2DM. We randomly divided Wistar rats into the control group, the untreated T2DM group, the metformin-treated group, the group receiving subcutaneous injections of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor diphenyleneiodonium (DPI), and the group receiving tail vein injections of the p38MAPK agonist anisomycin. Real-time polymerase chain reaction, Western blot, and immunohistochemistry were applied to examine the expression levels of SK2, SK3, NOX4, and phospho-p38MAPK (p-p38MAPK) mRNAs and proteins in the atrial tissue of relevant groups. We observed that the expression levels of NOX4 mRNA and protein and p-p38MAPK protein were significantly elevated in the atria of rats with T2DM compared with the control group. In addition, SK2 protein expression was reduced, whereas SK3 protein expression was increased. The 8-week treatment with metformin markedly reduced the expression levels of NOX4 mRNA and protein and p-p38MAPK protein, upregulated the SK2 expression, and downregulated the SK3 expression. Tail vein injection with anisomycin significantly increased the p-p38MAPK expression while further inhibiting the expression of SK2 and enhancing the expression of SK3. Subcutaneous injection with DPI considerably inhibited the expression of NOX4, further enhanced the expression of SK2 and suppressed the expression of SK3. In addition, subcutaneous injection with DPI significantly suppressed the phosphorylation of p38MAPK. In conclusion, the NOX4/p38MAPK signaling pathway mediates the downregulation of SK2 and the upregulation of SK3 in the atria of rats with T2DM. Long-term metformin treatment upregulates SK2 protein expression and downregulates SK3 protein expression by inhibiting the NOX4/p38MAPK signaling pathway.

Protective Effects of Calcitonin Gene-Related Peptide-Mediated p38 Mitogen-Activated Protein Kinase Pathway on Severe Acute Pancreatitis in Rats.

Calcitonin gene-related peptide (CGRP) has antioxidant and anti-inflammatory activities on the pathological damage of acute pancreatitis. However, its molecular mechanism on severe acute pancreatitis (SAP) remains unknown. To evaluate the influence of CGRP-mediated p38MAPK signaling pathway in rats with SAP. SD rats were randomly divided into Sham group, SAP group, CGRP group (SAP rats injected with CGRP), SB203580 group (rats injected with p38MAPK pathway inhibitor SB203580), and CGRP8-37 group (SAP rats injected with CGRP8-37). Serum amylase and lipase activities were determined. Histopathological observations were evaluated, and the expression of inflammatory cytokines and oxidative stress-related indexes were measured. Compared with Sham group, SAP rats were increased in the activities of serum amylase and lipase, the pathologic assessment of pancreatic tissue, the levels of TNF-α, IL-1β, IL-6, and IL-8, the content of MDA and MPO, and the expressions of CGRP, and p-p38MAPK protein, but they were decreased in SOD activity and GSH content. The above alterations were aggravated in the CGRP8-37 group when compared with SAP group. Besides, in comparison with SAP group, rats in the CGRP and SB203580 groups presented a reduction in the activities of serum amylase and lipase, the levels of inflammatory cytokines, the content of MDA and MPO, and the expressions of p-p38MAPK protein, while showed an elevation in SOD activity and GSH content. Pretreatment with CGRP alleviated oxidative stress and inflammatory response of SAP rats possibly by suppressing the activity of p38MAPK pathway, and thereby postponing the disease progression.

Epigallocatechin-3-gallate protected vanadium-induced eggshell depigmentation via P38MAPK-Nrf2/HO-1 signaling pathway in laying hens.

It has been demonstrated that tea polyphenol (TP) epigallocatechin-3-gallate (EGCG) can confer protection against vanadium (V) toxicity in laying hens; however, our understanding of the molecular mechanisms beyond this effect are still limited. In this study, 360 hens were randomly assigned to the 3 groups to study whether the potential mechanism P38MAPK-Nrf2/HO-1 signaling pathway is involved in the protective effect of EGCG on eggshell pigmentation in vanadium challenged laying hens. Treatments included a control group, a 10mg/kg V (V10), and a V10 plus 130mg/kg of EGCG group (V10+EGCG130). Both eggshell color and protoporphyrin IX were decreased in the V10 group compared with the control diet, while EGCG130 treatment partially improved shell color and protoporphyrin IX (P < 0.05). The V10 exposure induced higher cell apoptosis rate and oxidative stress in birds as evidenced by the histological apoptosis status, decreased uterine glutathione-S transferase (GST) and high abundance of malondialdehyde (MDA) compared with the control group, whereas EGCG130 markedly alleviated oxidative stress via reducing MDA generation (P < 0.05). Dietary vanadium reduced ferrochelatase, NF-E2-related factor 2 (Nrf2), and heme oxygenase (HO-1) mRNA expression, while EGCG up-regulated Nrf2 and HO-1 expression (P < 0.05). Protein levels of Nrf2, HO-1 and phospho-p38 (P-P38) MAPK were reduced in V10 group, while dietary supplementation with 130mg/kg EGCG markedly increased Nrf2, HO-1 and P-P38 MAPK protein levels in the uterus compared with the V10 group (P < 0.01). In conclusion, EGCG improved eggshell color and antioxidant system in V10-challenged hens, which seems to be associated with P38MAPK-Nrf2/HO-1 signaling pathway.

Muscle-derived IL-6 improved insulin resistance of C2C12 cells through activating AMPK and inhibiting p38MAPK signal pathway in vitro

This paper was to probe into whether IL-6 improved insulin resistance by AMPK and p38MAPK signal pathway. The C2C12 cell lines were purchased from ATCC. The cells of control group were cultured in high-glucose DMEM culture medium. CaCl2 with different concentrations was respectively used to culture C2C12 cells; then, glucose concentrations in the nutrient solution following 24 h were measured in the preliminary experiment. Firstly, cells of A group (control group) were normally cultured C2C12 cells, cells of B, C, and D group were respectively cultured with palmitic acid 48 h, palmitic acid 24 h, and IL-6 24 h, and IL-6-shRNA 48 h, glucose concentrations, IL-6 mRNA and GLUT4 mRNA levels, p-p38MAPK, p-AMPK, p-IRS-1, and p-PI-3K protein contents of each group in the nutrient solution following 48 h were measured. Secondly, cells of A group (control group) were palmitic acid-cultured C2C12 cells, cells of B and C group were respectively cultured with palmitic acid 24 h and CaCl2 24 h, and palmitic acid 24 h CaCl2 24 h, and IL-6-shRNA 24 h, and the same indexes were measured with the first experiment. The preliminary experimental result showed that CaCl2 feed can induce C2C12 cell contraction, and CaCl2 with different concentrations was able to decrease significantly glucose concentrations in the nutrient solution following 24 h (p < 0.05). The result of the first experiment showed that, compared with B group, p-AMPK, p-IRS-1, and p-PI-3K protein contents, GLUT4 mRNA level, and glucose ingestion capacity of C group significantly increased (p < 0.05), and p-p38MAPK protein content of it significantly decreased (p < 0.05). Compared with A group, those of D group significantly decreased (p < 0.05), and p-p38MAPK protein contents of it significantly increased (p < 0.05). The result of the second experiment showed that, compared with A group, those of B group significantly increased (p < 0.05), and p-p38MAPK protein content significantly decreased (p < 0.05). Compared with B group, those of C group significantly decreased (p < 0.05), and p-p38MAPK protein contents significantly increased (p < 0.05). This experiment firstly discovered that exogenous calcium-cultured can induce contraction of C2C12 cells. Furthermore, muscle-derived IL-6 improved insulin resistance of C2C12 cells through activating AMPK and inhibiting p38MAPK signal pathway.

Angiotensin-converting enzyme 2-angiotensin (1-7)-Mas axis prevents pancreatic acinar cell inflammatory response via inhibition of the p38 mitogen-activated protein kinase/nuclear factor-κB pathway.

The aim of the present study was to investigate the role of the angiotensin-converting enzyme (ACE)2-angiotensin‑(Ang)-(1-7)-Mas axis in the pathogenesis of pancreatitis and the association between this axis and the p38mitogen-activated protein kinase (p38MAPK)/nuclear factor(NF-κB) signaling pathway in pancreatic acinar cells. Mouse pancreatic acinar cancer (MPC-83) cells were stimulated with 10nM caerulein (CAE) to create an invitro model of acute pancreatitis, and collected for analysis at 2, 6, 12, 24 and 48h post stimulation. In addition, cells were pretreated with different concentrations of Ang‑(1‑7), Ang‑(1‑7) antagonist A779, p38MAPK inhibitor SB203580 or ACE2 inhibitor DX600 for 30min, and then stimulated with CAE for 24h. The ACE2, Mas receptor, p38MAPK, phosphorylated (p)-p38MAPK and NF-κB expression levels were evaluated using western blotting and immunofluorescence. p38MAPK, NF-κB, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-8 and IL-10 mRNA expression levels were assessed using reverse transcription-quantitative polymerase chain reaction. The results of the immunofluorescence assay demonstrated that ACE2 and p38MAPK were present mainly in the cytoplasm, while the Mas receptor was located mainly in the cell membrane. ACE2, p38MAPK and p-p38MAPK protein levels were significantly increased (P<0.05) following stimulation with CAE compared with those in the control group and peaked at 24h. Mas receptor protein levels were significantly upregulated (P<0.05) between 6 and 24h, peaking at 12h. Ang‑(1‑7) and SB203580 downregulated p-p38MAPK and NF-κB expression and the mRNA levels of inflammatory factors IL-6, TNF-α and IL-8, but upregulated the mRNA level of inflammatory factor IL-10 compared with those treated with CAE alone. These results were supported by the opposite outcomes observed for cells treated with A779 or DX600. Therefore, it was concluded that the ACE2-Ang‑(1‑7)-Mas axis significantly inhibits pancreatitis by inhibition of the p38MAPK/NF-κB signaling pathway.

Crosstalk Influence between P38MAPK and Autophagy on Mitochondria-Mediated Apoptosis Induced by Anti-Fas Antibody/Actinomycin D in Human Hepatoma Bel-7402 Cells.

Our previous study indicated that anti-Fas antibody/actinomycin D (AF/AD) induced apoptosis of human hepatocellular carcinoma Bel-7402 cells; however, crosstalk influence between P38MAPK and autophagy on mitochondria-mediated apoptosis induced by AF/AD in Bel-7402 cells remains unclear. Therefore, effect of AF/AD on apoptosis, autophagy, phosphorylated-P38MAPK (p-P38MAPK), and membrane potential (ΔΨm) with or without the P38MAPK inhibitor SB203580 or the autophagy inhibitor 3-methyladenine (3-MA) in Bel-7402 cells was investigated in the present study. The results showed that AF/AD resulted in induction of apoptosis concomitant with autophagy, upregulation of p-P38MAPK and autophagy-associated gene proteins (Atg5-Atg12 protein complex, Atg7, Atg10, Beclin-1, LC3 I, and LC3 II), and downregulation of ΔΨm in Bel-7402 cells. In contrast, SB203580 attenuated the effects of AF/AD in Bel-7402 cells. Furthermore, the findings also demonstrated that 3-MA inhibited the impact of AF/AD on autophagy, Atg5-Atg12 protein complex, Atg7, Atg10, Beclin-1, LC3 I, LC3 II, and ΔΨm, and promoted the influence of AF/AD on apoptosis and p-P38MAPK in Bel-7402 cells. Taken together, we conclude that crosstalk between P38MAPK and autophagy regulates mitochondria-mediated apoptosis induced by AF/AD in Bel-7402 cells.

Protective effects of bellidifolin in hypoxia-induced in pheochromocytoma cells (PC12) and underlying mechanisms

ABSTRACTBellidifolin, a xanthone compound derived from plants of Gentiana species, is known to exert a variety of pharmacological activities including anti-oxidation, anti-inflammatory and antitumor actions as well as a protective effect on cerebral ischemic nerve injury. The aim of this study was to examine the protective effects of bellidifolin on nerve injury produced by hypoxia and possible underlying mechanisms using pheochromocytoma cells (PC12). Data showed that the viability of PC12 cells subjected to hypoxia resulted in a significant decrease; however; pretreatment with certain concentrations of bellidifolin (20 or 40 μmol/L) prior to hypoxia significantly increased the survival rate. The results of immunohistochemical staining analysis revealed that there were no marked alterations in the expression of pERK protein between all bellidifolin groups while the expression of p-p38MAPK protein was significantly enhanced by hypoxia. Pretreatment with different concentrations of bellidifolin followed by hypoxia significantly decreased the expression of p-p38MAPK protein. The results of western blot analysis showed that hypoxia induced the expression of the MAPK signaling pathway downstream of the key apoptosis factor caspase-3. Compared to hypoxia, the expression of caspase-3 in the presence of belliidifolin was significantly lower. Data suggest that bellidifolin may contribute to the protective effects associated with nerve injury initiated by hypoxia by mechanisms related to inhibition of cell apoptosis independent of the ERK pathway, but may involve blockade of p38MAPK signaling pathway activation and downstream caspase-3 expression.

Effect of macrophages on podocytes apoptosis in diabetic nephropathy

Objective To investigate the effect of macrophages on podocytes apoptosis in diabetic nephropathy. Methods Differentiated mouse macrophages (RAW264.7) were exposed to normal glucose, high glucose, then the conditioned media (CM) was collected and considered as NC-CM or HG-CM, respectively. Western blotting and immunofluorescent staining were used to detect the specific markers for M1 macrophages (iNOS) and M2 macrophages (MR). ELISA was used to detect the concentration of TNF-α in the CM. Then normal PRMI 1640 media (control), NC-CM or HG-CM was added to podocytes. In some experiments, ROS inhibitors (Tempo), p38 MAPK inhibitor (SB203580), anti-TNF-α neutralizing antibody, and IgG1 isotype control were respectively added to cells with HG-CM. Besides, recombinant mouse TNF-α alone was applied to incubate podocytes. Podocytes apoptosis was accessed by Annexin V-FITC/PI and Hoechst33342 staining. DCFH-DA staining was used to analyse ROS level. Western blotting was used to detect cleaved casepase-3, p38MAPK, and p-p38MAPK protein. Results Macrophages were activated when exposed to high glucose, displaying pro-inflammatory M1 polarization with higher iNOS and lower MR expression. HG-CM but not NC-CM trigged podocytes apoptosis, up-regulated ROS, cleaved casepase-3 and p-p38MAPK. However, the podocytes apoptosis trigged by HG-CM was abolished by either a ROS inhibitor (Tempo) or a p38 MAPK inhibitor (SB203580). Additionally, TNF-α was increased in the HG-CM. TNF-α protein in macrophage was aslo increased when exposed to high glucose. Anti-TNF-α neutralizing antibody blunted the apoptotic response, excess ROS generation and p-p38 MPAK expression in podocytes induced by HG-CM. Moreover, addition of recombinant TNF-α similarly led to podocytes apoptosis, increased ROS and p38 MPAK expression. Conclusion M1 macrophages activated by high glucose released TNF-α to promote podocytes apoptosis via ROS-p38 MAPK pathway. (Chin J Endocrinol Metab, 2017, 33: 680-686) Key words: Macrophage; Podocytes; Apoptosis; Diabetic nephropathy

Effect of plumbagin on epithelial-mesenchymal transition and underlying mechanisms in human tongue squamous cell carcinoma cells

Objective: To study the effect of plumbagin on epithelial-mesenchymal transition (EMT) and underlying mechanisms in human tongue squamous cell carcinoma (TSCC) cells. Methods: Methyl thiazolyl tetrazolium assay was apllied to examine the proliferation inhibition effect and half maximal inhibitory concentration (IC(50)) of plumbagin (0.1, 1.0, 5.0, 10.0, 20.0 μmol/L) in 12, 24, 48 h in TSCC cells. Transwell assay was used to count the number of transmembrane cells and scratch test was performed to examine cells mobility. The flow cytometry was applied to measure intracellular reactive oxygen species (ROS) level in control group, plumbagin group (1.0 μmol/L, 24 h) and glutathione (GSH)+plumbagin group. The expression of E-cadherin, vimentin, Slug, p38 mitogen activated protein kinases (p38MAPK) and phospho-p38MAPK (p-p38MAPK) proteins were determined by Western blotting. The expression of E-cadherin, vimentin and Slug were detected by Western blotting in control group, plumbagin group, activator combined group (p38MAPK activator+plumbagin) and inhibitor combined group (p38MAPK inhibitor+plumbagin). Results: After the treatment of plumbagin for 12, 24, and 48 h, the IC(50) of TSCC cells were 10.3, 3.1, 1.5 μmol/L. After treated by 1.0 μmol/L plumbagin for 24 h, the number of transmembrane cells were significantly reduced ([50±13], P<0.05) in comparison to control group (204±6), as well as the cells mobility ([18.2±2.3]%, P<0.05) in comparison to control group ([49.3±1.2]%). Compared to control group (2.32±0.52), the ROS level was increased in plumbagin group (902.20±10.69), while compared to plumbagin group, the ROS level was reduced in GSH combined group (2.18±0.15). In comparison to control group, the expression of E-cadherin was up-regulated (P<0.05), and vimentin, Slug, p-p38MAPK/p38MAPK were down-regulated in plumbagin group (P<0.05). In comparison to plumbagin group, the expression of E-cadherin was down-regulated (P<0.05), and vimentin, Slug, p-p38MAPK/p38MAPK were up-regulated in GSH combined group (P<0.05). Treatment of cells with p38MAPK activator could decrease the expression of E-cadherin significantly (P<0.05) and increase the expression of vimentin (P<0.05) and Slug (P<0.05) in comparison to plumbagin group. Treatment of cells with p38MAPK inhibitor could increase the expression of E-cadherin significantly (P<0.05) and decrease the expression of vimentin (P<0.05) and Slug (P<0.05) in comparison to plumbagin group. Conclusions: Plumbagin inhibits EMT via ROS/p38MAPK-mediated pathway in human TSCC cells.

α7 nicotinic acetylcholine receptor agonist reduced myocardial fibrosis possibly through inhibiting p38 mitogen activated protein kinase signaling pathway

Objective To investigate the role of α7 nicotinic acetylcholine receptor (α7nAchR) agonist in myocardial fibrosis and its possible mechanism. Methods The cardiac fibroblasts were isolated and cultured from neonatal rats. The cardiac fibroblasts were divided into the following four groups: blank control group (cardiac fibroblasts without any intervention); model group [cardiac fibroblasts treated with 10-6 mol/L angiotensin Ⅱ (Ang Ⅱ) only]; α7nAchR agonist group (cardiac fibroblasts treated with 5×10-6 mol/L PNU-282987, and 1 h later treated with 10-6 mol/L Ang Ⅱ); α7nAchR antagonist group (cardiac fibroblasts treated with 10-6 mol/L MLA, and 1 h later treated with 10-6 mol/L Ang Ⅱ). The proliferation abilities of cardiac fibroblasts in different treatment groups were measured using WST-1 method. The expression levels of α7nAchR genes in cardiac fibroblasts in different treatment groups were examined by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR). The expression levels of α7nAchR, type Ⅰ and type Ⅲ collagen, α-smooth muscle actin (α-SMA), p38 mitogen activated protein kinase (p38MAPK) and phosphorylated p38MAPK (p-p38MAPK) proteins in cardiac fibroblasts were detected by Western blotting. Results (1) The final absorbance in the α7nAchR agonist group (0.50±0.13) was significantly lower than α7nAchR antagonist group and model group (0.75±0.10, 0.63±0.11, F=10.567, P=0.000). It suggested that activation of α7nAchR could inhibit the proliferation of cardiac fibroblasts. (2) The relative expression levels of α7nAchR mRNA and protein in α7nAchR agonist group (0.87±0.15, 0.76±0.08) were significantly higher than the α7nAchR antagonist group (0.45±0.09, 0.40±0.14), model group (0.62±0.11, 0.59±0.10) and blank control group (0.32±0.13, 0.30±0.07, F=25.402, P=0.000). It suggested that α7nAchR agonists can upregulate the expression of α7nAchR gene in cardiac fibroblasts. (3) The relative expression levels of type Ⅰ and type Ⅲ collagen proteins, α-SMA protein and p-p38MAPK protein in α7nAchR agonist group (0.53±0.09, 0.50±0.12, 0.38±0.08, 0.27±0.09) were lower than the model group (0.65±0.12, 0.62±0.10, 0.57±0.11, 0.45±0.11) and α7nAchR antagonist group (0.81±0.13, 0.71±0.11, 0.70±0.10, 0.68±0.08), while were higher than the blank control group (0.41±0.08, 0.35±0.06, 0.19±0.07, 0.16±0.07, F=29.647, 32.962, 30.549, 53.665, P=0.000, 0.000, 0.000, 0.000). The relative expression of p38MAPK protein in the α7nAchR agonist group (0.71±0.12) was higher than the model group (0.52±0.10) and α7nAchR antagonist group (0.35 ± 0.09), but was lower than the blank control group (0.85 ± 0.14, F=44.347, P=0.000). Conclusion α7nAchR agonists could inhibit the proliferation of cardiac fibroblasts and reduce the synthesis of collagen between cells. The mechanism might be related to inhibition of p38MAPK signaling pathway activation. Key words: Cardiac fibroblasts; α 7 nicotinic acetylcholine receptor agonist; Fibrosis; p38 mitogen-activated protein kinase signaling pathway

Effects of AQP5 gene silencing on proliferation, migration and apoptosis of human glioma cells through regulating EGFR/ERK/ p38 MAPK signaling pathway.

We investigated the effects of aquaporin 5 (AQP5) gene silencing on the proliferation, migration and apoptosis of human glioma cells through regulating the EGFR/ERK/p38MAPK signaling pathway. qRT-PCR was applied to examine the mRNA expressions of AQP5 in five human glioma cell lines. U87-MG, U251 and LN229 cells were selected and assigned into blank, vector, AQP5 siRNA and FlagAQP5 groups. MTT assay was used to measure cell proliferation. Flow cytometry (FCM) with AnnexinV-FITC/PI double staining and PI staining were employed to analyze cell apoptosis and cell cycle respectively. Scratch test was used to detect cell migration. Western blotting was performed to determine the EGFR/ERK/p38 MAPK signaling pathway-related proteins. Results showed that the positive expression of AQP5 in primary glioblastoma was associated with the tumor size and whether complete excision was performed. The mRNA expressions of AQP5 in cell lines of U87-MG, U251 and LN229 were significantly higher than in U373 and T98G. The proliferation rates of U87-MG, U251 and LN229 cells in the AQP5 siRNA group were lower than in the vector and blank groups. The apoptosis rate increased in the AQP5 siRNA group compared with the vector group. Scratch test demonstrated that AQP5 gene silencing could suppress cell migration. Compared with the vector and blank groups, the AQP5 siRNA group showed decreased expressions of the ERK1/2, p38 MAPK, p-ERK1/2 and p-p38 MAPK proteins. AQP5 gene silencing could inhibit the cell proliferation, reduce cell migration and promote the cell apoptosis of U87-MG, U251 and LN229 by suppressing EGFR/ERK/p38 MAPK signaling pathway.

Amino-modified polystyrene nanoparticles affect signalling pathways of the sea urchin (Paracentrotus lividus) embryos

Polystyrene nanoparticles have been shown to pose serious risk to marine organisms including sea urchin embryos based on their surface properties and consequently behaviour in natural sea water. The aim of this study is to investigate the toxicity pathways of amino polystyrene nanoparticles (PS-NH2, 50 nm) in Paracentrotus lividus embryos in terms of development and signalling at both protein and gene levels. Two sub-lethal concentrations of 3 and 4 μg/mL of PS-NH2 were used to expose sea urchin embryos in natural sea water (PS-NH2 as aggregates of 143 ± 5 nm). At 24 and 48 h post-fertilisation (hpf) embryonic development was monitored and variations in the levels of key proteins involved in stress response and development (Hsp70, Hsp60, MnSOD, Phospho-p38 Mapk) as well as the modulation of target genes (Pl-Hsp70, Pl-Hsp60, Pl-Cytochrome b, Pl-p38 Mapk, Pl-Caspase 8, Pl-Univin) were measured. At 48 hpf various striking teratogenic effects were observed such as the occurrence of cells/masses randomly distributed, severe skeletal defects and delayed development. At 24 hpf a significant up-regulation of Pl-Hsp70, Pl-p38 Mapk, Pl-Univin and Pl-Cas8 genes was found, while at 48 hpf only for Pl-Univin was observed. Protein profile showed different patterns as a significant increase of Hsp70 and Hsp60 only after 48 hpf compared to controls. Conversely, P-p38 Mapk protein significantly increased at 24 hpf and decreased at 48 hpf. Our findings highlight that PS-NH2 are able to disrupt sea urchin embryos development by modulating protein and gene profile providing new understandings into the signalling pathways involved.

MicroRNA-130a Promotes Human Keratinocyte Viability and Migration and Inhibits Apoptosis Through Direct Regulation of STK40-Mediated NF-κB Pathway and Indirect Regulation of SOX9-Meditated JNK/MAPK Pathway: A Potential Role in Psoriasis.

Psoriasis is a chronic inflammatory skin disorder. The aim of this study was to determine a potential role of microRNA (miR)-130a in psoriasis, and underlying mechanism. Expression levels of miR-130a in psoriasis specimens and normal skin tissues were analyzed. MiR-130a mimic, inhibitor, miR-control, small interfering RNA (siRNA) specific serine/threonine kinase 40 (STK40), or sex-determining region Y chromosome-box 9 (SOX9) were transfected to human keratinocyte HaCaT cells, respectively. After transfection, the cell viability, apoptosis, and migration were determined. Luciferase reporter assay, quantitative reverse transcription-polymerase chain reaction, and western blot were performed to explore whether STK40 was a target of miR-130a. The effects of aberrant expressions of miR-130a, STK40, or SOX9 on key proteins of NF-κB and c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase (MAPK) pathway were assessed. The miR-130a levels were significantly higher in patients with psoriasis compared to the healthy controls (p < 0.01). Overexpressing miR-130a strikingly promoted HaCaT cell viability and migration and inhibited apoptosis (p < 0.01 or p < 0.05). We confirmed that STK40 was a direct target of miR-130a, and STK40 was involved in miR-130a-induced cell functions. Overexpressing miR-130a significantly upregulated NF-κB p65, SOX9, p-c-Jun, p-JNK, and p-p38MAPK proteins and silencing miR-130a downregulated them. In addition, silencing STK40 alleviated the effects of anti-miR-130a on SOX9 expression. Furthermore, silencing SOX9 also decreased levels of p-c-Jun, p-JNK, and p-p38MAPK proteins. MiR-130a regulates human keratinocyte HaCaT viability, migration and apoptosis might be by direct regulation of STK40-mediated NF-κB pathway and by indirect regulation of SOX9-mediated downstream JNK/MAPK signaling pathway.

Notoginsenoside R&amp;lt;sub&amp;gt;1&amp;lt;/sub&amp;gt; Attenuates Hypoxia and Hypercapnia-Induced Vasoconstriction &amp;lt;i&amp;gt;In Vitro&amp;lt;/i&amp;gt; by Reducing the Expression of p38

Notoginsenoside R1, the main active ingredient of Panax notoginseng saponins (PNS), has been proposed to play fatal roles in the development of hypoxic hypercapnia-induced pulmonary vasoconstriction (HHPV). Subsequently, pulmonary arterial smooth muscle cells (PASMCs) lead to pulmonary vascular system remodeling and chronic pulmonary disease in the development of HHPV. Despite considerable studies have contributed to pulmonary disease, the mechanism of how Notoginsenoside R1 affects HHPV remains unclear. In this view, we will discuss the effect of notoginsenoside R1 by investigating the expression of p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway in PASMCs under hypoxia and hypercapnia condition. The third order PASMCs of Sprague Dawley (SD) rats were cultured with various concentrations (8, 40, 100 mg/L, respectively) of Notoginsenoside R1. Our data showed that the protein and mRNA expression levels of p-38 MAPK were higher in hypoxic hypercapnia group compared with hypoxic DMSO and normoxia control groups (p 1 treatment groups, the level of p-p38 MAPK protein and p38 MAPK mRNA were significantly decreased with different degrees (p 1 treatment may contribute to attenuate HHPV via decreasing the protein and mRNA expression levels of p-38 MAPK.

Glabridin reduces lipopolysaccharide-induced lung injury in rats by inhibiting p38 mitogen activated protein kinase/extracellular regulated protein kinases signaling pathway

Objective: To investigate whether glabridin has a beneficial effect on lipopolysaccharide (LPS) induced acute respiratory distress syndrome (ARDS) in rats, and to explore the possible underlying mechanisms. Methods: Thirty-two Wistar rats were randomly assigned into control group, model group (LPS group), glabridin group (GLA group), and ulinastatin group (UTI group), with 8 rats in each group. ARDS rat model was reproduced by intraperitoneal injection of LPS (10 mg/kg). The rats in the control group received an equal volume of normal saline at the same times. The rats in GLA group were gavaged by glabridin (30 mg/kg). The rats in UTI group were injected ulinastatin (20 000 U/kg). Animals were sacrificed 12 hours after LPS challenge. Plasma and lung tissue samples were collected. Histopathological evaluation, lung wet/dry (W/D)ratio, tumor necrosis factor-α (TNF-α), interleukin-18 (IL-18), malondialdehyde (MDA), nitric oxide (NO) and superoxide dismutase(SOD)were analyzed. Immunohistochemical method was used to detect the protein expression of p38MAPK and ERK. Western blot method was used to detect the changes of p38 mitogen activated protein kinase (p-p38MAPK) and phosphorylated extracellular regulated protein kinases (pERK) protein expression in lung tissues. Result: In the control groups, lung tissue showed a normal structure and clear pulmonary alveoli under a light microscope. In the model group, ARDS characters such as extensive thickening of the alveolar wall, significant infiltration of inflammatory cells, demolished structure of pulmonary alveoli, and hemorrhage were found. In the GLA and UTI treatment group, these pathological changes in lung were markedly alleviated compare with LPS-induced ARDS group. Compared with control groups, lung W/D ratio, TNF-α and IL-18 in plasma, and lung MDA, NO levels in lung homogenates of the LPS group were increased significantly, while the lung SOD levels of the LPS group were decreased. Compared with the LPS group, lung W/D ratio, TNF-α and IL-18 in plasma , and lung MDA, NO levels in lung homogenates of the GLA group and UTI group were decreased significantly, while the lung SOD levels of the GLA and ulinastatin groups were increased [TNF-α(μg/L): 51.7±10.3 vs 105.7±30.5, IL-18(μg/L): 37.9±13.9 vs 49.2±14.5, MDA (nmol/mgprot): 2.87±0.62 vs 3.81±0.42, NO(μmol/L): 18.96±0.79 vs 28.58±2.51, SOD(U/mgprot): 115.5±15.2 vs 75.9±14.0, all P<0.05]. Immunohistochemistry showed that the positive expressions of p38MAPK and ERK in cytoplasm and nucleus of the glabridin and ulinastatin treatment group were significantly lower than those of the model group. Western blot showed that compared with the control group, the p-p38MAPK and pERK protein expression in LPS group were significantly increased. And the glabridin and ulinastatin inhibited the protein expressions compared with model group. Conclusion: Traditional Chinese medicine glabridin significantly ameliorated the lung injury induced by LPS in rats via reducing inflammation which caused by the inhibition of p38MAPK and ERK signaling pathway and antioxidant effect.

Iridoid glycoside from Cornus officinalis ameliorated diabetes mellitus-induced testicular damage in male rats: Involvement of suppression of the AGEs/RAGE/p38 MAPK signaling pathway

Ethnopharmacological relevanceCornus officinalis (CO) has been widely used as a traditional Chinese medicine for treating diabetes mellitus (DM) and its complications. Iridoid glycoside from C. officinalis (IGCO) can resist apoptosis, hyperglycemia, oxidation and so on. The aim of this study was to investigate the therapeutic effects of IGCO on DM-induced testicular damage through inhibition of the AGEs/RAGE/p38 MAPK signaling pathway. Materials and methodsA DM model of male Wistar rats was induced with streptozotocin injection (30mg/kg, i.p.) and high-fat diet. The DM rats were administrated with IGCO at low and high doses (15 and 30mg/kg, p.o.) for 12 weeks. Testicular damage was evaluated by estimating relative testicular weights, testicular pathohistology, sperm count, live sperm rate, endogenous sex hormone level and activity of testicular marker enzymes. Besides, general diabetic symptoms, renal function, oxidative stress parameters and testicular apoptosis marker were also determined. Finally, the mechanism was explored based on the AGEs/RAGE/p38 MAPK pathway. ResultsIGCO effectively mitigated the general symptoms of DM rats including weight loss, polydipsia, polyphagia, polyuria, elevated blood glucose level and low serum insulin level. Nourishing the kidney evidently, IGCO reduced serum creatinine, urea nitrogen and urine protein excretion, and also markedly protected against DM-induced testicular damage by increasing testis/body weight ratio and live sperm rate, improving the histomorphology of testes, upregulating testosterone, LH, FSH and GnRH levels and preventing the decrease of testicular marker enzymes LDH, ACP and γ-GT. Moreover, IGCO showed considerable anti-oxidative and anti-apoptotic effects, which downregulated the increase of ROS and MDA levels, restored SOD and CAT activities, and decreased spermatogenic cell apoptosis and Bax/Bcl-2 ratio. In the end, the increased AGEs, RAGE and p-p38 MAPK protein levels in DM rats were also reversed by IGCO significantly. ConclusionsThe kidney tonic IGCO well protected DM rats from testicular damage, which may be related to suppression of the AGEs-RAGE-p38 MAPK pathway.

Chi-Ju-Di-Huang-Wan protects rats against retinal ischemia by downregulating matrix metalloproteinase-9 and inhibiting p38 mitogen-activated protein kinase.

BackgroundRetinal ischemia is a retinal disorder related to retinal vascular occlusion, glaucoma, diabetic retinopathy and age-related macular degeneration. The study aimed to evaluate the protective effects and underlying mechanisms of Chi-Ju-Di-Huang-Wan (CJDHW) against retinal ischemia in rats.MethodsHigh intraocular pressure (HIOP)-induced retinal ischemia was established in Wistar rats by raising their intraocular pressure to 120 mmHg for 60 min with in an eye whose anterior chamber was cannulated with a 30-guage needle adapted to a normal saline bottle through an intravenous line. This ischemic insult was followed by 1 or 7 days of reperfusion. The effects of CJDHW were studied by (i) electroretinogram (ERG); (ii) real-time polymerase chain reaction to determine the retinal mRNA levels of Thy-1 and matrix metalloproteinase-9 (MMP-9); (iii) Western blot analysis to determine the retinal protein levels of B cell lymphoma 2 (Bcl-2), heme oxygenase-1 (HO-1), phosphorylated-p38 mitogen-activated protein kinase (P-p38 MAPK) and MMP-9; (iv) hematoxylin and eosin (HE) staining; (v) fluorogold retrograde labeling; and (vi) terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) apoptosis assay. Moreover, after fixation with 4 % paraformaldehyde and 30 % sucrose, the isolated retinas were sectioned and immunolabeled with goat anti-choline acetyltransferase (ChAT) polyclonal antibody, mouse anti-vimentin monoclonal antibody and rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody. The retinal sections were then incubated with rhodamine-conjugated rabbit anti-goat antibody, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-rabbit IgG. A daily oral intake of 3 mL of water (vehicle; Group 2) or CJDHW (2.8 or 4.2 g/kg/day; CJDHW2.8 or CJDHW4.2; Group 3 or 4) was given for 7 consecutive days either before (preischemic drug administration) or after HIOP-induced retinal ischemic injury (postischemic drug administration). In Group 5, an intravitreal injection of 4 μL of 0.5 mM SB203580 (p38 MAPK inhibitor) was performed on the ischemic eye 15 min before retinal ischemia. The control rats received a sham procedure (Group 1) where the saline reservoir was not raised.ResultsThe ischemia-induced changes (Group 2) were significantly modulated by pretreating the rats with 4.2 g/kg/day of CJDHW (Group 4; ERG: P < 0.001 on I/R day 7; HE stain: P < 0.001 on I/R day 7; TUNEL: P = 0.05 on I/R day 7; retrograde labeling: P = 0.007 on I/R day 7; Thy-1 mRNA: P = 0.02; MMP-9 mRNA: P < 0.001; Bcl-2 protein: P = 0.02; HO-1 protein: P = 0.03; P-p38 MAPK protein: P < 0.001; MMP-9 protein: P = 0.02). These modulations included the following features (Group 2 vs. 4), increased ERG b-wave amplitudes (0.38 ± 0.04 vs. 0.81 ± 0.03), increased inner retinal thickness (45.08 ± 2.85 vs. 67.98 ± 5.48 μm), increased ChAT immunolabeling, decreased vimentin/GFAP immunoreactivity, less numerous apoptotic cells in the ganglion cell layer (1.40 ± 0.55 vs. 0.60 ± 0.55), and more numerous retinal ganglion cells (887.73 ± 158.18 vs. 1389.02 ± 53.20). Moreover, increased Thy-1 (0.31 ± 0.15 vs. 0.78 ± 0.32) and decreased MMP-9 mRNA levels were found (4.44 ± 0.84 vs. 1.13 ± 0.34), respectively. Furthermore, the Bcl-2 protein level (0.78 ± 0.08 vs. 1.80 ± 0.34) was increased while the HO-1 (0.99 ± 0.20 vs. 4.15 ± 2.08), P-p38 MAPK (1.12 ± 0.18 vs. 0.57 ± 0.18) and MMP-9 levels were decreased (0.70 ± 0.23 vs. 0.39 ± 0.10). The ischemia-associated increases in P-p38 and MMP-9 protein levels were also attenuated by 0.5 mM SB203580 (P-p38 MAPK: 1.12 ± 0.18 vs. 0.18 ± 0.07, P < 0.001; MMP-9: 0.70 ± 0.23 vs. 0.21 ± 0.07, P = 0.002). This was also the case to the MMP_enzyme activity (Group 2 vs. 4: 5.03 ± 1.57 vs. 1.59 ± 0.47, P = 0.002; Group 2 vs. 5: 5.03 ± 1.57 vs. 1.35 ± 0.41, P = 0.001).ConclusionTreatment of the rats suffering from retinal ischemia with CJDHW inhibited apoptosis, increased antioxidative activity, downregulated MMP-9 and inhibited p38 MAPK.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-016-0109-6) contains supplementary material, which is available to authorized users.

Effects of astragaloside IV on lung injury and inflammation after hemorrhagic shock and resuscitation in rats

Objective To investigate the effects of astragaloside Ⅳ on lung injury and inflammation after hemorrhagic shock and resuscitation in rats. Methods Twenty-four male Sprague Dawley rats were randomly divided in 3 groups, including Sham group, hemorrhagic shock-resuscitation group (HS-R group) and astragaloside Ⅳ+ hemorrhagic shock-resuscitation group (AGS+ HS-R group). Hemorrhagic shock was induced by blood drawing from the femoral artery, and the resuscitation was performed by infusion of shed blood and two-fold volume saline. The operations for Sham group were the same with HS-R group, except that the blood drawing and fluid resuscitation were not performed. The rats in AGS+ HS-R group were subjected to hemorrhagic shock, and 20 mg/kg of astragaloside Ⅳ was intraperitoneally injected at the onset of resuscitation. Two h after resuscitation, histological changes were observed under light microscope, and myeloperoxidase (MPO) activities were determined with a commercial kit. The protein levels of cytoplasmic high mobility group protein B1 (HMGB1) as well as Toll-like receptor 4 (TLR4) and phospho-p38 mitogen activated protein kinase (p-p38MAPK) protein expression levels were detected by Western blotting. The protein levels of cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β were examined by enzyme linked immunosorbent assay (ELISA). Results Histological results showed that the lung injury after hemorrhagic shock and resuscitation was attenuated by the treatment of astragaloside Ⅳ. As compared with HS-R group [(8.2±1.6) U/g], the treatment of astragaloside Ⅳ [(5.2±0.9) U/g] decreased MPO activities by 36.6% (P<0.01). In addition, the astragaloside Ⅳ treatment also down-regulated the expression levels of cytoplasmic HMGB1 as well as TLR4 and p-p38MAPK by 24.9%, 27.5%, and 23.7%, respectively (P<0.01 or P<0.05). The results of ELISA suggested that the lung protein levels of TNF-α, and IL-1β after hemorrhagic shock and resuscitation were decreased by 66.3%, and 44.3% respectively (P<0.01). Conclusion Astragaloside Ⅳ might attenuate the lung injury after hemorrhagic shock and resuscitation via inhibition of HMGB1/TLR4 signaling. Key words: Astragaloside Ⅳ; Hemorrhagic shock; Lung injury; Inflammatory response