OBJECTIVE: To study the changes and significance of apoptosis signal-regulating kinase 1 (ASK1) in left ventricular remodeling in FVB/N mice. METHODS: A total of 54 FVB/N mice were randomly divided into 4 groups: 0 d group with 8 mice, 7 d group with 10 mice, 14 d group with 16 mice, and 21 d group with 20 mice. A model of cardiac remodeling was established by intraperitoneal injection of isoproterenol (ISO) at a daily dose of 30 mg/kg, and the 7 d, 14 d, and 21 d groups were injected for 7, 14, and 21 consecutive days respectively. The 0 d group was given intraperitoneal injection of an equal volume of normal saline. Echocardiography was used to measure left ventricular posterior wall thickness at end diastole (dLVPW) and the ratio of heart weight to tibia length (HW/TL) was measured. Hematoxylin-eosin staining was used to measure left ventricular myocardial fiber diameter. Picric-Sirius red staining was used to measure myocardial collagen deposition area in the left ventricle. Quantitative real-time PCR was used to measure the mRNA expression of ASK1, type I collagen (collagen I), and B-type natriuretic peptide (BNP). The mortality rate was observed for each group. RESULTS: There were gradual increases in HW/TL, myocardial fiber diameter, and dLVPW after 0, 7, and 14 days of ISO injection (P<0.05). There were no significant changes in HW/TL ratio and dLVPW from days 14 to 21 of ISO injection (P>0.05), while there was a significant reduction in myocardial fiber diameter (P<0.05), which was similar to the value on day 7 (P>0.05). There were significant increases in myocardial collagen deposition area and the mRNA expression of collagen I, ASK1, and BNP after 0, 7, 14, and 21 days of ISO injection, which reached the peaks on day 21 (P<0.01). The mRNA expression of ASK1 was positively correlated with myocardial collagen deposition area and the mRNA expression of collagen I and BNP and had a weak correlation with HW/TL, myocardial fiber diameter, and dLVPW. There was a significant increase in the mortality rate of the mice over the time of ISO injection. CONCLUSIONS The expression of ASK1 in the myocardium is closely associated with left ventricular remodeling. The increase of ASK1 expression may lead to the aggravation of left ventricular remodeling, and the mechanism of which needs further study.
Animals ; Isoproterenol ; MAP Kinase Kinase Kinase 5 ; Mice ; Myocardium ; Myocytes, Cardiac ; Ventricular Remodeling

ObjectiveTo explore the inhibitory effect of polyphyllin Ⅰ (PPⅠ) on the proliferation of castration-resistant prostate cancer PC3 cells and its molecular mechanism.

METHODSWe cultured human prostate cancer PC3 cells in vitro and treated them with PPⅠ at the concentrations of 0 (blank group), 0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 μmol/L for 24, 48, and 72 hours, respectively. Then we detected the proliferation of the cells by MTT assay, measured their apoptosis by flow cytometry, and determined the expressions of p-ERK1/2, ERK1/2, NF-κB/p65 and DNMT1 proteins as well as the level of NF-κB/p65 in the cells additionally treated with the ERK1/2 inhibitor SP600125 by Western blot.

RESULTSCompared with the blank control group, the PPⅠ-treated PC3 cells showed a concentration- and time-dependent reduction of the survival rate (1.00 ± 0.00 vs 0.85 ± 0.05, P < 0.01) at 0.4 μmol/L after 48 hours of intervention, concentration-dependent early apoptosis at 0.8 μmol/L (4.83 ± 0.95 vs 13.83 ± 2.97, P < 0.01), time-dependent increase of the expressions of p-ERK1/2 (1.00 ± 0.00 vs 1.73 ± 0.17, P < 0.01) and ERK1/2 (1.00 ± 0.00 vs 1.36 ± 0.12, P < 0.01) at 2 hours, and concentration-dependent decrease of the expressions of NF-κB/p65 and DNMT1 at 1.2 μmol/L (1.00 ± 0.00 vs 0.78 ± 0.10 and 0.63 ± 0.06, P < 0.01) and 1.6 μmol/L (1.00 ± 0.00 vs 0.67 ± 0.11 and 0.52 ± 0.09, P<0.01). Inhibition of ERK1/2 phosphorylation with PD98059 markedly reversed PPⅠ-induced decrease of the NF-κB/p65 expression as compared with that in the PPⅠ group (0.86 ± 0.18 vs 0.43 ± 0.09, P < 0.05).

CONCLUSIONSPPⅠ induces the early apoptosis and suppresses the proliferation of PC3 cells, probably by activating the ERK1/2 pathway and inhibiting the expressions of the NF-κB/p65 and DNMT1 proteins.

Apoptosis ; Cell Proliferation ; drug effects ; DNA (Cytosine-5-)-Methyltransferase 1 ; metabolism ; Diosgenin ; analogs & derivatives ; pharmacology ; Flavonoids ; metabolism ; Humans ; MAP Kinase Signaling System ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; NF-kappa B ; metabolism ; PC-3 Cells ; Phosphorylation ; Prostatic Neoplasms, Castration-Resistant ; drug therapy ; metabolism ; pathology ; Signal Transduction ; Transcription Factor RelA ; metabolism

Escherichia coli ; chemistry ; cytology ; Fluorine Radioisotopes ; MAP Kinase Kinase 5 ; chemistry ; metabolism ; MAP Kinase Kinase Kinase 3 ; chemistry ; metabolism ; Nuclear Magnetic Resonance, Biomolecular ; Protein Binding ; Protein Interaction Mapping ; methods ; Protein Interaction Maps

OBJECTIVETo examine whether sodium tanshinone II A sulfonate (STS), the main effective component of Salvia miltiorrhiza is effective in relieving the microcirculatory disturbance of small intestine by suppressing the production of reactive oxygen species (ROS) in rats with sepsis.

METHODSA rat model of sepsis was induced by cecal ligation and puncture (CLP). Rats (n =40) were randomly divided into 4 groups: sham-operated group (sham, n =10), sepsis group (CLP, n =10), STS treatment group (STS, n =10) and ROS scavenger dimethylthiourea (DMTU, n =10) group. Animals in the STS group were injected with STS (1 mg/kg) for 10 min through the right external jugular vein after the CLP operation, and animals in the CLP group were given the same volume of normal saline after the CLP operation. Animals in the DMTU group were intraperitoneally injected with 5 mL/kg of 20% DMTU 1 h before CLP. The histopathologic changes in the intestinal tissues and changes of mesenteric microcirculation were observed. The levels of ROS in intestinal tissues from each group were qualitatively evaluated using a fluorescent microscope. The expressions of apoptosis signal-regulating kinase (ASK1), phosphorylated ASK1 (phospho-ASK1), p38 mitogen-activated protein kinases (p38 MAPK), phosphorylated p38 MAPK (phospho-p38 MAPK) and tissue factor (TF) were determined by Western blotting.

RESULTSIt was shown that there were obvious microcirculatory disturbance (P <0.05) and tissue injuries in intestinal tissues after CLP operation. The levels of ROS production, phospho-ASK1, phospho-p38 MAPK and TF were increased. Both STS and DMTU suppressed ROS, phospho-ASK1, phospho-p38 MAPK and TF production, and ameliorated the microcirculatory disturbance and tissues injury (P <0.01).

CONCLUSIONSTS can ameliorate the microcirculatory disturbance of the small intestine by attenuating the production of ROS in rats with sepsis.

Animals ; Intestine, Small ; blood supply ; drug effects ; pathology ; MAP Kinase Kinase Kinase 5 ; metabolism ; Male ; Microcirculation ; drug effects ; Phenanthrenes ; chemistry ; pharmacology ; therapeutic use ; Phosphorylation ; drug effects ; Rats, Wistar ; Reactive Oxygen Species ; metabolism ; Sepsis ; drug therapy ; enzymology ; pathology ; physiopathology ; Thromboplastin ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

BACKGROUND/OBJECTIVES: Resveratrol, a natural polyphenol, has multiple functions in cellular responses including apoptosis, survival, and differentiation. It also participates in the regulation of inflammatory response and oxidative stress. MicroRNA-Let-7A (miR-Let7A), known as a tumor suppressor miRNA, was recently reported to play a crucial role in both inflammation and apoptosis. Therefore, we examined involvement of miR-Let7A in the modulation of inflammation and cell survival/apoptosis regulated by resveratrol. MATERIALS/METHODS: mRNA expression of pro-/anti-inflammatory cytokines and sirtuin 1 (SIRT1), and protein expression of apoptosis signal-regulating kinase 1 (ASK1), p-ASK1, and caspase-3 and cleaved caspase-3 were measured, and cell viability and Hoechst/PI staining for apoptosis were observed in Lipopolysaccharide (LPS)-stimulated human THP-1 macrophages with the treatment of resveratrol and/or miR-Let7A overexpression. RESULTS: Pre-treatment with resveratrol (25-200 µM) resulted in significant recovery of the reduced cell viabilities under LPS-induced inflammatory condition and in markedly increased expression of miR-Let7A in non-stimulated or LPS-stimulated cells. Increased mRNA levels of tumor necrosis factor-α and interleukin (IL)-6 induced by LPS were significantly attenuated, and decreased levels of IL-10 and brain-derived neurotrophic factor were significantly restored by resveratrol and miR-Let7A overexpression, respectively, or in combination. Decreased expression of IL-4 mRNA by LPS stimulation was also significantly increased by miR-Let7A overexpression co-treated with resveratrol. In addition, decreased SIRT1 mRNA levels, and increased p-ASK1 levels and PI-positive cells by LPS stimulation were significantly restored by resveratrol and miR-Let7A overexpression, respectively, or in combination. CONCLUSIONS: miR-Let7A may be involved in the inflammatory response and cell survival/apoptosis modulated by resveratrol in human THP-1 macrophages.
Apoptosis ; Brain-Derived Neurotrophic Factor ; Caspase 3 ; Cell Survival ; Cytokines ; Humans ; Inflammation ; Interleukin-10 ; Interleukin-4 ; Interleukins ; Macrophages* ; MAP Kinase Kinase Kinase 5 ; MicroRNAs ; Necrosis ; Oxidative Stress ; RNA, Messenger ; Sirtuin 1

OBJECTIVETo investigate the effect of suppressing apoptosis signal regulating kinase 1 (ASK1) on glial fibrillary acidic protein (GFAP) and vimentin expressions at the injury site and on hindlimb mobility in rats after spinal cord injury (SCI).

METHODSThe rat models of SCI were established by extradural compression of the spinal cord using an aneurysm clip. The injured rats were treated with normal saline (model group), ASK1 specific inhibitor thioredoxin (Trx group), or ASK1 monoclonal antibody (Anti-ASK1 group), and the rats receiving a sham operation underwent laminectomy without SCI. The expression of GFAP and vimentin were detected by Western blotting and immunofluorescence assay at 1, 7, 14 and 28 days after SCI. The motion function of the hindlimbs of the injured rats was assessed with Basso Beattie Bresnahan (BBB) scores, and somatosensory-evoked potentials (SEP) and motor-evoked potentials (MEP) were determined to examine the electrophysiological changes.

RESULTSAt 1 day after SCI, the expressions of GFAP and vimentin showed no significant differences among the groups; at 7, 14 and 28 days after SCI, GFAP and vimentin expressions significantly increased in Trx and Anti-ASK1 groups compared with those in the model group (P<0.01). The BBB scores showed no significant differences among the groups at 1, 7 and 14 days after SCI, while at 28 days, the BBB scores in Trx and Anti-ASK1 groups were significantly higher than those in the model group (P<0.01). At 28 days after SCI, the latent period of SEP and MEP decreased and the amplitude increased significantly in Trx and Anti-ASK1 groups compared with that in the model group (P<0.01).

CONCLUSIONBlocking ASK1 can inhibit the expression of GFAP and vimentin in glial scars and improve the outcomes of hindlimb mobility in rats after SCI.

Animals ; Disease Models, Animal ; Evoked Potentials, Motor ; Evoked Potentials, Somatosensory ; Glial Fibrillary Acidic Protein ; metabolism ; Hindlimb ; physiopathology ; MAP Kinase Kinase Kinase 5 ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; metabolism ; Vimentin ; metabolism

OBJECTIVETo investigate the expression of ASK1 and PRKCD in the process of monocyte differentiation, and explore their role in functional changes of hypersplenism spleen macrophages (Mφ) in portal hypertension (PH).

METHODSU937 cells were stimulated to differentiate into monocyte/macrophage-like cells by cultivation in PMA and the mRNA expressions of ASK1 and PRKCD were detected by q-PCR and the changes of protein expression were identified by western blot analysis. The secretion of phagocytose related cytokines such as IL-10 and TNF-α were tested by ELISA, and the function of the macrophage-like cells were studied by chicken red blood cell phagocytose test.

RESULTSThe expressions of PRKCD and ASK1 mRNA were gradually decreased along with the cell differentiation, while the secretion of TNF-α was increased, IL-10 secretion reached a maximum at 24 h after PAM stimulation, and then gradually fell. The expression of ASK1 and p-ASK1 were rapidly increased compared with the non-stimulated U937 cells, while the expression of PRKCD and p-PRKCD were sightly declined. The phagocytose test show that U937 cells induced with PMA were able to swallow the chicken red blood cell.

CONCLUSIONUp-regulated protein expression of ASK1 and p-ASK1 and down-regulated protein expression of PRKCD and p-PRKCD in the process of PMA induced monocyte differentiation, are consist with the expression changes of splenic macrophage phagocytosis in hypersplenism, which leads to increased activity of Mφ.

Cell Differentiation ; Down-Regulation ; Humans ; Hypersplenism ; Hypertension, Portal ; Interleukin-10 ; secretion ; MAP Kinase Kinase Kinase 5 ; physiology ; Macrophages ; cytology ; Phagocytosis ; Protein Kinase C-delta ; physiology ; RNA, Messenger ; Tumor Necrosis Factor-alpha ; secretion ; U937 Cells

Neural stem cells (NSCs) have been suggested as a groundbreaking solution for stroke patients because they have the potential for self-renewal and differentiation into neurons. The differentiation of NSCs into neurons is integral for increasing the therapeutic efficiency of NSCs during inflammation. Apoptosis signal-regulating kinase 1 (ASK1) is preferentially activated by oxidative stress and inflammation, which is the fundamental pathology of brain damage in stroke. ASK1 may be involved in the early inflammation response after stroke and may be related to the differentiation of NSCs because of the relationship between ASK1 and the p38 mitogen-activated protein kinase pathway. Therefore, we investigated whether ASK1 is linked to the differentiation of NSCs under the context of inflammation. On the basis of the results of a microarray analysis, we performed the following experiments: western blot analysis to confirm ASK1, DCX, MAP2, phospho-p38 expression; fluorescence-activated cell sorting assay to estimate cell death; and immunocytochemistry to visualize and confirm the differentiation of cells in brain tissue. Neurosphere size and cell survival were highly maintained in ASK1-suppressed, lipopolysaccharide (LPS)-treated brains compared with only LPS-treated brains. The number of positive cells for MAP2, a neuronal marker, was lower in the ASK1-suppressed group than in the control group. According to our microarray data, phospho-p38 expression was inversely linked to ASK1 suppression, and our immunohistochemistry data showed that slight upregulation of ASK1 by LPS promoted the differentiation of endogenous, neuronal stem cells into neurons, but highly increased ASK1 levels after cerebral ischemic damage led to high levels of cell death. We conclude that ASK1 is regulated in response to the early inflammation phase and regulates the differentiation of NSCs after inflammatory-inducing events, such as ischemic stroke.
Animals ; Cell Death ; Infarction, Middle Cerebral Artery/*metabolism ; Lipopolysaccharides/pharmacology ; MAP Kinase Kinase Kinase 5/genetics/*metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Microtubule-Associated Proteins/genetics/metabolism ; Neural Stem Cells/cytology/drug effects/*metabolism ; *Neurogenesis ; Neuropeptides/genetics/metabolism ; p38 Mitogen-Activated Protein Kinases/genetics/metabolism

Epstein-Barr virus (EBV) latent infection transforms B lymphocytes into proliferating lymphoblastoid cell lines (LCLs). EBV latent infection membrane protein 1 (LMP1) is required for EBV-mediated B lymphocyte transformation, and LMP1-induced NF-kappaB activation is essential for LCL survival. Previously, it was reported that the level of reactive oxygen species (ROS) and the expression of apoptosis signal-regulating kinase 1 (ASK1) are elevated in EBV-positive Burkitt's lymphoma (BL) cells, the potential role of ASK1 in LMP1-induced NF-kappaB activation was thus investigated in this study. In EBV-positive BL cells, ASK1 was highly expressed and activated. In addition, TRAF6-ASK1 interaction was significantly increased in EBV-positive BL cells. Interestingly, the expression of LMP1 alone facilitated ASK1 activation. The expression of a dominant negative ASK1 mutant (ASK1KM) strongly blocked LMP1-induced NF-kappaB activation. Furthermore, LMP1-induced NF-kappaB activation was significantly reduced in ASK1 knock out (ASK1-/-) mouse embryonic fibroblasts (MEFs). Taken together, these results demonstrate that ASK1 is activated by LMP1 and is critical for LMP1-induced NF-kappaB activation.
Animals ; B-Lymphocytes ; Burkitt Lymphoma ; Cell Line ; Fibroblasts ; Herpesvirus 4, Human ; Lymphocyte Activation ; MAP Kinase Kinase Kinase 5 ; Membrane Proteins ; Mice ; NF-kappa B ; Reactive Oxygen Species

In this study, we focused to identify whether eupatilin (5,7-dihydroxy-3',4',6-trimethoxyflavone), an extract from Artemisia argyi folium, prevents H2O2-induced injury of cultured feline esophageal epithelial cells. Cell viability was measured by the conventional MTT reduction assay. Western blot analysis was performed to investigate the expression of 5-lipoxygenase by H2O2 treatment in the absence and presence of inhibitors. When cells were exposed to 600 microM H2O2 for 24 hours, cell viability was decreased to 40%. However, when cells were pretreated with 25~150 microM eupatilin for 12 hours, viability was significantly restored in a concentration-dependent manner. H2O2-treated cells were shown to express 5-lipoxygenase, whereas the cells pretreated with eupatilin exhibited reduction in the expression of 5-lipoxygenase. The H2O2-induced increase of 5-lipoxygenase expression was prevented by SB202190, SP600125, or NAC. We further demonstrated that the level of leukotriene B4 (LTB4) was also reduced by eupatilin, SB202190, SP600125, NAC, or nordihydroguaiaretic acid (a lipoxygenase inhibitor) pretreatment. H2O2 induced the activation of p38MAPK and JNK, this activation was inhibited by eupatilin. These results indicate that eupatilin may reduce H2O2-induced cytotoxicity, and 5-lipoxygenase expression and LTB4 production by controlling the p38 MAPK and JNK signaling pathways through antioxidative action in feline esophageal epithelial cells.
Anthracenes ; Arachidonate 5-Lipoxygenase ; Artemisia ; Blotting, Western ; Cell Survival ; Epithelial Cells ; Flavonoids ; Hydrogen ; Hydrogen Peroxide ; Imidazoles ; Leukotriene B4 ; Lipoxygenase ; MAP Kinase Signaling System ; Nordihydroguaiaretic Acid ; p38 Mitogen-Activated Protein Kinases ; Pyridines