Expression Of p-p38MAPK Research Articles

Novel role of silent information regulator 1 in acute endothelial cell oxidative stress injury

Silent information regulator 1 (SIRT1), a class III histone deacetylase, retards aging and plays roles in cellular oxidative stress injury (OSI). However, the biological context in which SIRT1 promotes oxidative injury is not fully understood. Here, we show that SIRT1 essentially mediates hydrogen peroxide (H2O2)-induced cytotoxicity in human umbilical vein endothelial cell (HUVEC). In HUVECs, SIRT1 protein expression was significantly increased in a dose-dependent manner after H2O2 treatment, whereas the acetylation levels of the NF-κB p65 subunit and p53 were decreased. EX527 (a specific SIRT1 inhibitor) conferred protection to the HUVECs against H2O2, as indicated by an improved cell viability, adhesion, an enhanced migratory ability, a decreased apoptotic index, decreased reactive oxygen species (ROS) production and reductions in several biochemical parameters. Immunofluorescence and Western blot analyses demonstrated that H2O2 treatment up-regulated SIRT1, phosphorylated-JNK (p-JNK), p-p38MAPK, and p-ERK expression. EX527 pretreatment reversed these effects on SIRT1, p-JNK, and p-p38MAPK but further increased the p-ERK levels. Similar results were confirmed in SIRT1 siRNA experiments. In summary, SIRT1 signaling pathway inhibition imparts protection against acute endothelial OSI, and modulation of MAPKs (JNK, p38MAPK, and ERK) may be involved in the protective effect of SIRT1 inhibition.

Effects of UII/UT system on the expression of inflammatory signal molecules p38 MAPK and NF-κB in LPS-stimulated Kupffer cells

Objective To investigate the effects of urotensin Ⅱ/urotensin Ⅱreceptor （ UⅡ/UT） system on the expression of inflammatory signal molecules p 38 mitogen-activated protein kinase （ p38 MAPK） and nuclear factor-κB （ NF-κB ） in lipopolysaccharide （ LPS ）-stimulated Kupffer cells （ KCs ） . Methods Rat KCs were isolated and purified by means of in situ perfusion and density gradient centrifuga-tion.The isolated cells were randomly divided into six treatment groups including group 1：UⅡ（-） urantide （-）LPS（-）, group 2：UⅡ（＋）urantide（-）LPS（-）, group 3： UⅡ（-）urantide（＋）LPS（-）, group 4：UⅡ（-）urantide（-）LPS（＋）, group 5：UⅡ（＋） urantide（-） LPS（＋） and group 6：UⅡ（-）urantide（＋） LPS（＋） .Western blot assay was performed to detect p 38 MAPK/p-p38 MAPK protein and NF-κB p65 sub-unit.The DNA-binding activity of NF-κB was tested by electrophoretic mobility shift assay （EMSA）.Re-sults There was no significant difference with the expression of p 38 MAPK protein in KCs among the six groups （P〉0.05）.The expression of p65 protein and p-p38 MAPK and the DNA-binding activity of NF-κB were significantly enhanced in LPS-stimulated KCs from groups 4, 5 and 6 in comparison with those in group 1 （P〈0.01）.No significant differences with the levels of p65 protein and phosphor-p38 MAPK and the DNA-binding activity of NF-κB were observed between UⅡ/urantide-treated cells （ group 2 or group 3） and untreated cells （group 1） （all P〉0.05）, but that were decreased in group 6 than those in group 4 （all P〈0.01）.Conclusion UⅡ/UT system participated in the activation of p38 MAPK and NF-κB signaling pathways in LPS-stimulated primary Kupffer cells . Key words: UrotensinⅡ; UrotensinⅡreceptor; Urantide; Kupffer cell; p38 MAPK; NF-κB

Effects of Xin-Ji-Er-Kang formula on 2K1C-induced hypertension and cardiovascular remodeling in rats

Ethnopharmacological relevanceXin-Ji-Er-Kang (XJEK), a Chinese herbal formula, is effective against hypertension induced coronary heart disease, viral myocarditis and toxic myocarditis. In this study, the effect of XJEK on cardiovascular system was investigated. To test the hypothesis that Xin-Ji-Er-Kang (XJEK) has an anti-hypertensive effect mediated through attenuation of cardiac remodeling, and amelioration of vascular endothelial dysfunction and oxidative stress. Materials and methodsHypertension was induced in Wistar rats by 2 kidney 1 clip (2K1C) treatment. The hypertensive rats were then randomly assigned into four groups and treated as follows: group 1 (Sham-operated [Sh-Op] group received only drinking water), group 2 (induced hypertensive model+no treatment), and group 3 (induced hypertensive+a single daily oral dose of 24gkg−1 XJEK treatment) and group 4 (induced hypertensive+a single oral dose of 15mgkg−1 Fosinopril treatment). The rats in all the defined groups were respectively treated for a period of 4 weeks. Cardiovascular parameter such as systolic blood pressure (SBP) was measured weekly by using tail-cuff apparatus; left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and the rate of the rise in left ventricular pressure (±dp/dt max) were measured by using a PowerLab 8/30 apparatus (AD Instruments, Australia) at the end of the 8th week; heart weight/body weight (HW/BW) was determined as an index of myocardial hypertrophy (MH). Hematoxylin and eosin (H&E) and Van Gieson (VG) stain were used to assess the cardio-histological changes. Colorimetric analysis was used to assay serum superoxide dismutase (SOD) activity, malondialdehyde (MDA), nitric oxide (NO), and hydroxyproline (Hyp) contents in cardiac tissue. Angiotensin II (Ang II) content in serum was assessed by radioimmunoassay; tetrahydrobiopterin (BH4) content in cardiac tissue, BNP and endothelial NOS (eNOS) in serum were determined by using ELISA, and the protein expressions of c-Jun NH2-terminal kinase (JNK), P-JNK, p38, P-p38, and NADPH oxidases-2 (Nox-2) were measured by western blot. ResultsXJEK therapy could impair the heart systolic and diastolic function, potently improve the heart weight index, inhibit the elevation of HW/BW ratio, and markedly ameliorate hemodynamic indices and vascular remodeling index. It has blunted the decrease of SOD, NO and the increase in MDA and Ang II serum contents, myocardial cross-section area (CSA), collagen volume fraction (CVF) and perivascular circumferential collagen area (PVCA) compared to the hypertensive model group. It also reduced the serum content of Hyp while increased BH4 levels in cardiac tissue. In addition, the expressions of Nox-2, P-JNK and P-p38MAPK were all suppressed compared to the hypertensive model group. Moreover, treatment with XJEK improved endothelial dysfunction (ED) manifested by promoting eNOS activities and enhancing the NO activity in serum. ConclusionThe results of the present study show that XJEK attenuates 2K1C-induced hypertension in rats, which confirms our hypothesis that XJEK has an anti-hypertensive and cardiovascular remodeling effect via attenuation of cardiac remodeling and improvement of endothelial dysfunction and oxidative stress.

The mechanism of hepatoprotective effect of sesquiterpene rich fraction from Cichorum glandulosum Boiss. et Huet on immune reaction-induced liver injury in mice

Ethnopharmacological relevanceCichorum glandulosum Boiss. et Huet is a traditional Uygur herbal medicine that has been used as a cholagogic and diuretic agent to improve liver function. However, the mechanism is not known for the liver-protective function. We investigated the antioxidant effects of plant extraction (CGE60) in vitro and in vivo, and find the mechanism of liver protection in Bacille Calmette-Guerin vaccine (BCG)+Lipopolysaccharides (LPS) induced liver injury in mice. Materials and methodsCGE60 was made, and the antioxidant activity was investigated by comparing the ability of scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH), and 2,2-azinobis(3-ehtylbenzothiazolin-6-sulfnicAcid) diammonium salt (ABTS) free radicals in vitro. Then, CGE60 was administrated in mice of liver damage model which was induced in mice using the BCG+LPS protocol. The CGE 60 extract was tested at three dosages: 50mg/kg, 100mg/kg, and 200mg/kg. Product of lipid peroxidation (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX,), nitric oxide (NO), nitric oxide synthetase (NOS), hydroxyproline and alkaline phosphatase (ALP) contents were evaluated in liver to determine the CGE60 activity in the hepatic injury model. Tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and transforming growth factor-β (TGF-β) proteins were determined in the liver tissues using ELSIA. The signaling activities were evaluated in Western blot. ResultsCGE60 exhibited strong antioxidant ability in vitro. With oral administration, CGE60 significantly increased the activity of CAT, SOD, GSH-PX, and decreased the level of NO, NO synthase, hydroxyproline, ALP and lipid peroxidation liver of in the BDG+ LPS model. CGE60 attenuated hepatic inflammation via down- regulation of TNF-α, IL-6 and TGF-β. CGE60 blocked protein expression of cytochrome P450 2E1 (CYP2E1), nuclear factor kappa-B (NF-κB), phosphorylation of extracellular signal-regulated kinase (p-ERK1/2), and cyclooxygenase-2 (COX-2),but activated the expression of p-P38 MAPK. ConclusionThis study suggests that CGE60 possesses antioxidant activity and this activity associates with hepatoprotective effect in the mice of BCG +LPS model, and the mechanisms underlying these effects may involve antioxidant actions and anti-inflammation activities.

Effects of butylphthalide on cognitive function, apoptosis and p-p38MAPK expression in hippocampus of rat model of vascular dementia

Objective To study the effects of butylphthalide on cognitive function, apoptosis and p-p38MAPK in hippocampus of rat model of vascular dementia. Methods The vascular dementia (VD) model was established by two vascular (2VO) method, and then sixty male Wistar rats were randomly divided into VD group, sham operation group and NBP (butylphthalide) group. Rats in NBP group were given 120 mg·kg-1·day-1 dose butylphthalide by gavage, and rats in VD group and sham operation group were given the same dose vegetable oil .The cognitive function of each rat was tested by Morris water maze.The expression of p-p38MAPK in the hippocampus was observed by Western blot; and the apoptosis was observed in hippocampal CAl region by TUNEL staining. Results The hidden platform escape latency of NBP group((48.72±7.01)s, (42.41±4.06)s, (40.34±2.46)s) was significantly shortened compared with those of VD group((82.71±8.27)s, (80.36±9.65)s, (77.74±6.33)s)(P< 0.01); and the former platform quadrant time and the number of passing through the platform of NBP group((26.45±4.66)s , (1.84±0.82)times)were significantly prolonged (P<0.01) compared with those of VD group ((18.67±5.39)s, (1.32±0.61)times); the apoptosis and the expression of p38MAPK phosphorylation in hippocampus in NBP group ((153.65±9.85), (0.42±0.04)) significantly reduced (P<0.01) compared with those of VD group ((209.46±11.49), (0.88±0.10)). Conclusion Butylphthalide can improve the learning and memory ability of VD rats, the reduce apoptosis in the hippocampus by the inhibition of the P38MAPK pathway.This may be one of the ways by which butylphthalide can treat vascular dementia. Key words: Butylphthalide; Vascular dementia; Apoptosis; p38MAPK; Cognitive function

Knockdown of VEGFR2 inhibits proliferation and induces apoptosis in hemangioma-derived endothelial cells

Angiogenesis is a process of development and growth of new capillary blood vessels from pre-existing vessels. Angiogenic growth factors play important roles in the development and maintenance of some malignancies, of which vascular endothelial growth factor (VEGF)/VEGFR2 interactions are involved in proliferation, migration, and survival of many cancer cells. The aim of this study was to investigate the function of VEGFR2 in human hemangiomas (HAs). Using immunohistochemistry assay, we examined the expression levels of VEGF, VEGFR2, Ki-67, glucose transporter-1 (Glut-1), phosphorylated protein kinase B (p-AKT) and p-ERK in different phases of human HAs. Positive expression of VEGF, VEGFR2, Ki-67, Glut-1, p-AKT and p-ERK was significantly increased in proliferating phase HAs, while decreased in involuting phase HAs (P=0.001; P=0.003). In contrast, cell apoptotic indexes were decreased in proliferating phase HAs, but increased in involuting phase HAs (P<0.01). Furthermore, we used small hairpin RNA (shRNA)-mediated VEGFR2 knockdown in primary HA-derived endothelial cells (HemECs) to understand the role of VEGF/VEGFR2 signaling. Knockdown of VEGFR2 by Lv-shVEGFR2 inhibited cell viability and induced apoptosis in primary HemECs companied with decreased expression of p-AKT, p-ERK, p-p38MAPK and Ki-67 and increased expression of caspase-3 (CAS-3); Overexpression of VEGFR2 promoted cell viability and blocked apoptosis in Lv-VEGFR2-transfected HemECs. Taken together, our findings demonstrate that, increased expression of VEGFR2 is involved in the development of primary HemECs possibly through regulation of the AKT and ERK pathways, suggesting that VEGFR2 may be a potential therapeutic target for HAs.

Dry eye management in a Sjögren’s syndrome mouse model by inhibition of p38-MAPK pathway

BackgroundTo investigate the therapeutic effect of p38-MAPK inhibitor, SB203580, on dry eye in a mouse model of Sjögren’s syndrome (MRL/lpr mice).Methods18 female BALB/c mice and 44 female MRL/lpr mice were included. Mice were randomly assigned to the control or treatment group. The expression of phospho-p38 MAPK in lacrimal glands of BALB/c mice was determined by Western blot analysis following IL-1β treatment at various time points. Different doses of SB203580 were injected into lacrimal glands of MRL/lpr mice and phenol red thread test was performed seven days post-injection. Moreover, the levels of acetylcholine and norepinephrine expression in lacrimal glands of MRL/lpr mice were measured using spectrofluoremetric assay and the histopathology of lacrimal glands was also evaluated.ResultsThe expression of p-p38 MAPK in lacrimal glands of BALB/c mice gradually increased following incubation with IL-1β ex vivo. Injection of SB203580 into lacrimal glands significantly improved the results of phenol red thread test in MRL/lpr mice. In addition, the secretions of acetylcholine and norepinephrine increased significantly compared to the control group. Less lymphocytes infiltration was observed in pathological section of lacrimal glands following SB203580 injection.ConclusionsOur results indicate that the activation of p38-MAPK pathway plays an important role in dry eye of a Sjögren’s syndrome mouse model. Inhibition of p38-MAPK pathway by SB203580 might have potential therapeutic effect on Sjögren’s syndrome associated dry eye.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1256849631103092.

Effects of Chaihu-Shugan-San and Shen-Ling-Bai-Zhu-San on p38 MAPK Pathway in Kupffer Cells of Nonalcoholic Steatohepatitis.

This study aimed to investigate the effects of Chaihu-Shugan-San (CSS), Shen-Ling-Bai-Zhu-San (SLBZS), and integrated recipe of the above two recipes on inflammatory markers and proteins involved in p38 MAPK pathway in Kupffer cells of NASH rats induced by high fat diet (HFD). Rats were administered at low or high dose of CSS, SLBZS, and integrated recipe except normal group and model group for 16 weeks. The levels of hepatic lipid, TNF-α, IL-1, and IL-6 in liver tissues were measured. Kupffer cells were isolated from livers to evaluate expressions of TLR4, p-p38 MAPK, and p38 MAPK by Western blotting. The results showed that the NASH model rats successfully reproduced typical pathogenetic and histopathological features. Levels of hepatic lipid and liver tissues inflammatory factors in high-dose SLBZS group and integrated recipe group were all lower than that of model group decreased observably. Expressions of TLR4, p-p38 MAPK, and p38 MAPK in Kupffer cells were decreased in all treatment groups, but there was no significant difference between treatment groups. The high-dose SLBZS group had the lowest expression levels of TLR4, and the most visible downtrend in the expression levels of p-p38 MAPK and p38 MAPK was found in the high-dose integrated recipe group. The ratio of p-p38 MAPK to total p38 MAPK protein was obviously increased in all treatment groups. Therefore, our study showed that the activation of p38 MAPK pathway in Kupffer cells might be related to the release of inflammatory factors such as TNF-α, IL-1, and IL-6 in NASH rats. High dose of SLBZS and integrated recipe might work as a significant anti-inflammatory effect in Kupffer cells of NASH rats induced by HFD through suppression of p38 MAPK pathway. It indicated that p38 MAPK pathway may be the possible effective target for the recipes.

Effects of Poly (ADP-ribose) Polymerase Inhibitor 3-Aminobenzamide on Blood–Brain Barrier and Dopaminergic Neurons of Rats with Lipopolysaccharide-Induced Parkinson’s Disease

Neuro-inflammation and dysfunction of blood-brain barrier play an important role in the occurrence, development, and neuronal degeneration of Parkinson's disease (PD). Studies have demonstrated that a variety of cytokines such as TNF-α and IL-1β destroy the structure and function of blood-brain barrier. The damage to blood-brain barrier results in death of dopaminergic neurons, while protection of blood-brain barrier slows down the progression of PD. Also, it has been shown that activation of poly (ADP-ribose) polymerase (PARP) plays an important role in causing damage to blood-brain barrier. In addition, the PARP inhibitor 3-AB has been shown to protect blood-brain barrier from damage and has neuroprotective effects. In this study, using a lipopolysaccharide (LPS)-induced PD rat model, we investigated whether 3-AB protects blood-brain barrier and dopaminergic neurons from functional damage. LPS significantly increased Evans blue content in the substantia nigra which peaked at 12 h, while administration of 3-AB significantly inhibited the LPS-induced increase in Evans blue content and also significantly increased the expression of the tight junction-associated proteins claudin-5, occludin and ZO-1. 3-AB also increased the number of tyrosine hydroxylase positive cells and reduced the IL-1β and TNF-α content significantly. According to western blot analysis, 3-AB significantly reduced the p-ERK1/2 expression, while the expression of p-p38MAPK increased. These results suggest that 3-AB protects the blood-brain barrier from functional damage in an LPS-induced PD rat model and dopaminergic neurons are protected from degeneration by upregulation of tight junction-associated proteins. These protective effects of 3-AB may be related to modulation of the ERK1/2 pathway.

Protective Effect of Shikonin in Experimental Ischemic Stroke: Attenuated TLR4, p-p38MAPK, NF-κB, TNF-α and MMP-9 Expression, Up-Regulated Claudin-5 Expression, Ameliorated BBB Permeability

Inflammatory damage plays an important role in cerebral ischemic pathogenesis and represents a new target for treatment of stroke. Shikonin has gained attention for its prominent anti-inflammatory property, but up to now little is known about shikonin treatment in acute ischemic stroke. The aim of this study was to evaluate the potential neuroprotective role of shikonin in cerebral ischemic injury, and investigate whether shikonin modulated inflammatory responses after stroke. Focal cerebral ischemia in male ICR mice was induced by transient middle cerebral artery occlusion. Shikonin (10 and 25 mg/kg) was administered by gavage once a day for 3 days before surgery and another dosage after operation. Neurological deficit, infarct volume, brain edema, blood-brain barrier (BBB) dysfunction, and inflammatory mediators were evaluated at 24 and 72 h after stroke. Compared with vehicle group, 25 mg/kg shikonin significantly improved neurological deficit, decreased infarct volume and edema both at 24 and 72 h after transient ischemic stroke, our data also showed that shikonin inhibited the pro-inflammatory mediators, including TLR4, TNF-α, NF-κB, and phosphorylation of p38MAPK in ischemic cortex. In addition, shikonin effectively alleviated brain leakage of Evans blue, up-regulated claudin-5 expression, and inhibited the over-expressed MMP-9 in ischemic brain. These results suggested that shikonin effectively protected brain against ischemic damage by regulating inflammatory responses and ameliorating BBB permeability.

Effects of p38MAPK inhibitor on the occurrence of acute GVHD and intestine damage after allogeneic hematopoietic stem cell transplantation in mice

To explore the effects of p38MAPK inhibitor SB203580 (SB) on the occurrence of acute GVHD and intestine damage after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in mice. Sixty BALB/c mice, as recipients, were randomized to control group, irradiation group, model group and intervention group. C57BL/6 mice, as donors, were raised to prepare the bone marrow cells (BMCs) and spleen cells (SCs), which were injected into irradiated recipients mice by tail vein. Except control group, other groups accepted 7.5Gy total body irradiation. Model group and intervention group were infused with BMCs 5×10⁶ and SCs 5×10⁵ by less than 4 h after irradiation. SB was injected into intervention group by intraperitoneally, but only DMSO for model group. The general status and survival rate of each group were evaluated. The expression of p-p38MAPK, Fas and FasL in intestine were determined by RT-PCR, Western blot and immunohistochemistry (IHC). The weight changes of intervention group (13.00±0.50)% was significantly lighter than that of model group (25.00±0.75)% (P<0.05). The clinical score of acute GVHD in the intervention group (3.33±0.82) was significantly lower than that of model group (6.33±1.36) (P<0.05). The expression levels of p-p38MAPK, Fas and FasL in small intestine of intervention group (1.43±0.02, 0.81±0.03, 0.97±0.03) were lower than those of model group (1.76±0.05, 1.52±0.04, 1.48±0.04). SB inhibited the activation of p38MAPK and Fas/ FasL signal pathway and alleviated the apoptosis of small intestine. And SB could relieve small intestine damages induced by allogeneic T lymphocytes.

GW24-e1393 The effect of Bim silencing by siRNA on apoptosis induced by hypoxia in rat cardiomyocyte

ObjectivesTo investigate the effect of BH3 only protein Bim (Bcl-2 interacting the mediator of the cell death) on apoptosis induced by hypoxia in rat cardiomyocyte.MethodsThe Rat cardiomyocytes, 1-3days after birth,...

Expression and activation of P38 MAP kinase in invasive ductal breast cancers: Correlation with expression of the estrogen receptor, HER2 and downstream signaling phosphorylated proteins

MAP kinase signaling proteins have major implications in the molecular oncogenesis of breast cancers and have been extensively investigated as putative targets for therapy. This study reports the investigation of the expression of P38 MAPK and its phosphorylated form (p-P38 MAPK) in clinical specimens of invasive breast carcinomas and their correlation with estrogen receptor (ER) and HER2 expression, as well as MAPK and PI3 kinase-AKT pathway signaling phosphorylated proteins. Expression levels of P38 MAPK and p-P38 MAPK as well as p-AKT, p-GSK3β, p-S6 kinase, p-MEK1 and p-ERK1/2 were quantitatively assessed using multiplex bead immunoassay in frozen specimens from 45 invasive ductal breast cancers. Twenty-nine specimens were ER+, 15 were HER2+ and 10 were triple‑negative breast cancers (TNBCs). P38 MAPK was found to be expressed in all tumor specimens and was significantly (P=0.002) overexpressed in ER+ tumors. P38 MAPK expression was lower in TNBCs than in all of the other tumors. The median expression of p-P38 MAPK was also higher in ER+ tumors while lower in the TNBCs. HER2 status had no effect on P38 MAPK and p-P38 MAPK expression. No variation in the phosphorylation rate of P38 MAPK was observed in relation with ER, HER2 or TNBC status. Significantly higher (P=0.0048) expression of p-AKT was observed in HER2+ tumors. No significant difference in p-MEK1, p-GSK3β and p-S6K expression was found in any other comparisons based on ER and HER2 expression subtypes. Investigation of the expression of multiple phosphorylated signaling proteins can be used for personalized targeted therapy. In invasive breast cancer, the overexpression of P38 MAPK may serve as a biomarker for the evaluation of P38 MAPK inhibitors.

Protective effects of Sapindus saponins in spontaneously hypertensive rats

To investigate the protective effects of Sapindus saponins in spontaneously hypertensive rats, and the possible cellular and molecular mechanisms. Thirty-two 16-week-old spontaneously hypertensive rats were randomly divided into four groups (8 in each group): model group (placebo), positive control group (27 mg/kg of Captopril Tablets), Sapindus saponins groups (27 mg/kg and 108 mg/kg, respectively). Another 8 healthy Wistar-Kyoto strain (WKY) rats were used as the normal group. The animals were treated for 8 weeks. Blood pressure of rats was determined by non-invasive blood pressure meter (BP-6). Furthermore, the contents of angiotensin II (Ang II) in plasma and myocardial tissue were determined by enzyme-linked immunosorbent assay (ELISA), the gene expression of receptor angiotensin type 1 (AT1R) in aorta was determined by quantitative realtime polymerase chain reaction (qRT-PCR). The protein expression of transforming growth factor-β1 (TGF-β1) and AT1R in heart was determined by immunohistochemical staining. The protein expression of p-phosphorylation of p38 mitogen-activated protein kinase (p-p38MAPK) was determined by Western blotting. The contents of interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF) in serum were determined by radioimmunoassay. And the histopathological and morphological changes of aorta and heart tissue samples were assessed semi-quantitatively by hematoxylin-eosin (HE) or Masson staining. Thirty minutes after single or continuous treatment, systolic blood pressure (SBP) was reduced significantly in Sapindus saponins groups. And the contents of AngII, IL-1, IL-6 and TNF-α in serum, the expression of AT1R mRNA, p-p38MAPK and TGF-β1 were significantly suppressed dose-dependently (P<0.05 or P<0.01). With the Sapindus saponins treatment, compared with those of the model group, the cardiac and aortic pathological changes were ameliorated significantly. Our findings suggest that Sapindus saponins might have protective effects in spontaneously hypertensive rats, the cellular and molecular mechanisms of which might be relevant to the regulation of inflammatory responses mediated by p-p38MAPK signal pathway based on activated Ang II and AT1R.

Evodiamine induces apoptosis and inhibits metastasis in MDA-MB-231 human breast cancer cells in vitro and in vivo

Breast cancer remains the leading cause of cancer-related deaths among women. Owing to high efficiency and low toxic effects, further exploration of natural compounds from Chinese herbal medicine may be an efficient approach for breast cancer drug discovery. In this study, we investigated the effects of evodiamine on the growth and metastasis of MDA-MB-231 human breast cancer cells invitro and invivo. Invitro, evodiamine inhibited cell migration and invasion abilities through downregulation of MMP-9, urokinase-type plasminogen activator (uPA) and uPAR expression. Evodiamine-induced G0/G1 arrest and apoptosis were associated with a decrease in Bcl-2, cyclinD1 and cyclin-dependent kinase6 (CDK6) expression and an increase in Bax and p27Kip1 expression. Moreover, evodiamine regulated p-ERK and p-p38 MAPK expression. Evodiamine-induced apoptosis was enhanced by its combination with the extracellular signal-regulated kinase (ERK) inhibitor PD98059 or the p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580. Evodiamine-inhibited metastasis was partly blocked by combination with PD98059 or SB203580. Invivo, the administration of evodiamine (10mg/kg) significantly reduced tumor growth and pulmonary metastasis. These results demonstrate that evodiamine possesses antitumor activities via inhibition of cell migration and invasion, arrest of the cell cycle and induction of cell apoptosis in MDA-MB-231 cells.

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.

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

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

Effect of Emodin on Endoplasmic Reticulum Stress in Rats with Severe Acute Pancreatitis

This study aimed to investigate the protective effect of emodin on endoplasmic reticulum (ER) stress in rats with severe acute pancreatitis (SAP) and the underlying molecular mechanism. Sprague-Dawley male rats were randomly divided into sham operation group, SAP model group, and emodin treatment group. SAP was constructed through injecting sodium taurocholate into pancreatic and biliary duct in rats. Half an hour before establishing the animal model, emodin or sodium carboxymethylcellulose was intragastrically administrated to the rats in respective group. Rats were killed at 3, 6, and 12 h postdisease induction. The amylase, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) levels in serum, pancreatic histopathology, acinar ER ultrastructure, protein expression of Bip, IRE1α,TRAF2, ASK1, p-JNK, and p-p38 MAPK in pancreas were examined. Sodium taurocholate induced pancreatic injury and ER lumen dilated in exocrine pancreas in rats at 3-, 6-, and 12-h time points. ER stress transducers Bip, IRE1α, and their downstream molecules TRAF2, ASK1 in pancreatitis were upregulated. Furthermore, phosphorylation of JNK and p38MAPK in pancreas was increased, which induced high expression level of inflammatory cytokines such as TNF-α and IL-6. Treatment with emodin obviously ameliorated pancreatic injury and decreased the release of amylase and inflammatory cytokines. Further studies showed that emodin significantly decreased the expression of Bip, IRE1α, TRAF2, and ASK1, inhibited phosphorylation of JNK and p38 MAPK in pancreas in rats at all time points. Emodin could reduce pancreatic injury and restrain inflammatory reaction in SAP rats partly via inhibiting ER stress transducers IRE1α and its downstream molecules.

Effects of sapindus saponins on inflammatory response mediated by AngⅡ/p38MAPK pathway and cardiac hypertrophy in spontaneously hypertensive rats

To investigate the effects of sapindus saponins on myocardial inflammation mediated by Ang II/ p38MAPK signal pathway and cardiac hypertrophy in spontaneously hypertensive rats. And also to explore the correlation of cardiac hypertrophy and inflammation.Thirty-two 16-week-old spontaneously hypertensive rats (SHR) were randomly divided into four groups, one with placebo as model group, one with captopril tablets (27 mg x kg(-1)) as positive control, one with low-dose sapindus saponins (27 mg x kg(-1)), one with high-dose (108 mg x kg(-1)). And another eight healthy Wistar-Kyoto strain (WKY) rats were used as the normal group. The animals were treated for eight weeks, and the indicators detected were as follows: (1) left ventricular mass index (LVMI); (2) the content of Ang II and hs-CRP in plasma were determined by ELISA; (3) the protein expression of AT1R and VEGF were determined by immunohistochemical method; (4) the protein expression of p-p38MAPK in myocardial cells was determined by Western blot.Sapindus saponins reduced LVMI, and blocked the expression level of Ang II, AT1R, p-p38MAPK, VEGF and hs-CRP in myocardial tissue. Vs the SHR model group, there were significant differences (P < 0.05 or P < 0.01).Our findings suggested that sapindus saponins could inhibited cardiac hypertrophy, the possible mechanisms may be related to the inhibition on inflammatory response mediated by Ang II/p38MAPK pathway.

Role of mitochondrial Bax, caspases, and MAPKs for ceramide-induced apoptosis in renal proximal tubular cells

It remains elusive whether crosstalk exists among mitochondrial Bax, caspases, and mitogen-activated protein kinases (MAPKs), and whether epidermal growth factor (EGF), which may activate MAPKs, affects ceramide-induced apoptosis through the crosstalk in renal proximal tubular cells (RPTCs). Effect of ceramide on expression of mitochondrial Bax and phosphorylated (p)-ERK, p38MAPK and JNK, that of MAPKs inhibition, and of EGF in the presence or absence of MAPKs inhibition on ceramide-induced apoptosis were examined in HK-2 cells. Apoptosis and expression of mitochondrial Bax and p-MAPKs were measured by Hoechst 33258 staining and Western blotting. C2-ceramide, but not dihydroC2-ceramide, inactive C2-ceramide, induced apoptosis at 24 h. C2-ceramide enhanced the mitochondrial Bax expression at 1 h, which was peaked at 3-6 h and decreased at 24 h, but remained increased, compared to control. An inhibitor of caspases, zVAD-fmk, ameliorated ceramide-induced apoptosis, suggesting a role of caspases for ceramide-induced apoptosis. C2-ceramide enhanced the expression of p-ERK and p-p38MAPK, but not p-JNK, at 1 h, which was increased till 24 h. An inhibitor of ERK, PD98059, or of p38MAPK, SB202190, failed to affect C2-ceramide-induced apoptosis. EGF, which enhanced the expression of p-ERK and p-p38MAPK but not p-JNK, ameliorated C2-ceramide-induced apoptosis without affecting mitochondrial Bax. Inhibition of ERK or p38MAPK failed to abolish the protective effect of EGF on C2-ceramide-induced apoptosis. Mitochondrial Bax and caspases, but not MAPKs, play a role for ceramide-induced apoptosis in RPTCs. EGF ameliorates ceramide-induced apoptosis in Bax- and MAPKs-independent pathways. The mechanism of ceramide-induced apoptosis and anti-apoptotic effect of EGF deserves further investigations.