Animals ; Drug Tolerance ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Morphine ; Phosphorylation ; Rats ; Signal Transduction

OBJECTIVETo investigate the role of the key intracellular signaling molecule glycogen synthase kinase-3 beta in the mechanism of liver ischemia reperfusion (IR).

METHODSC57BL/6 mice were subjected to 90 min warm liver cephalad lobe ischemia, followed by various length of reperfusion. Experiment groups included sham control group, liver IRI model group and glycogen synthase kinase-3 beta inhibitor-treated group (SB216763 in DMSO, 25 g/kg, i.p, 2 hour prior to the onset of liver ischemia). The expression of glycogen synthase kinase-3 beta protein was analysed by Western blotting. The serum ALT levels were determined to reflect the function of liver. The affected liver lobes were harvested for histology analysis. The inflammatory gene expression was detected by Quantitative PCR.

RESULTSBy western blot analysis, we found that ischemia itself activated glycogen synthase kinase-3 beta by a significant decrease of its phosphorylation. Glycogen synthase kinase-3 beta inhibitor SB216763-pretreatment ameliorated the liver damages significantly as compared to the controls (sALT: 2046+/-513 U/L vs 5809+/-1689 U/L, P = 0.0153), and suppressed the gene expressions of IL-12, TNFa, IL-1b and IL-6.

CONCLUSIONSThis study demonstrated that the ischemia process modulated liver innate immune activation via a GSK-3-dependent mechanism which favored the development of a pro-inflammation response and lead to liver tissue damages. GSK-3b may be a new therapeutic target to ameliorate liver IRI in transplant patients.

Animals ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Inflammation ; metabolism ; Liver ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Reperfusion Injury ; metabolism ; pathology

Animals ; Apoptosis ; Cardiomegaly ; metabolism ; pathology ; Glycogen Synthase Kinase 3 ; metabolism ; Mice ; Mice, Transgenic ; Myocytes, Cardiac ; cytology

OBJECTIVETo research the effects of glycogen synthase kinase (GSK3β) overexpression and GSK3β inhibitor SB-216763 on the proliferation of hepatic oval cells in rats and its regulatory mechanisms by Wnt signaling pathway.

METHODSThe hepatic oval cells WBF-344 were divided into the blank control group, GSK3β over-expression group, DMSO control group and GSK3β inhibitor groups, while the inhibitor groups set up three concentration gradients, that was 1, 5, 10 µmol/L. Using the GSK3β over-expression lentivirus, which had been identified correctly, and SB-216763 dealt with the cells WBF-344. The cells morphology of each group was observed under the phase contrast inverted microscope, and the expression of fluorescence in the lentivirus-transfected group was observed under the fluorescent microscope. The proliferation of each group cells was tested by CCK8 kits. The cells' apoptosis was detected by AnnexinV-FITC/PI kits. The expression of GSK3β, β-catenin and cyclin D1 were detected by Western blot.

RESULTSThe cells of GSK3β over-expression group were fewer and obvious aging. However, in each inhibitor added group, the cells' division and proliferation was vigorous, and the condition was good. Moreover, the cells' proliferation was getting stronger with the concentration of SB-216763 increasing. A large number of green fluorescence was expressed in the lentivirus-transfected cells. The cells' proliferation in GSK3β over-expression group restrained (t = 7.178, P < 0.01, as compared with control), while the cells' proliferation was vigorous in inhibitor groups (F = 45.030, P < 0.01, as compared with control). Flow Cytometry showed that the cells apoptosis was significant in GSK3β over-expression group. Western blot showed that the expression of GSK3β was increased, while the expression of β-catenin and cyclin D1 was decreased in the over-expression group. The expression of GSK3β had no significant difference among the control group and inhibitor groups. However, the expression of β-catenin and cyclin D1 was significantly increased with the concentration of SB-216763 increasing.

CONCLUSIONSThe overexpression of GSK3β can inhibit the Wnt signaling pathway, thus restrain the cells' proliferation and promotes apoptosis. SB-216763 can activate the Wnt pathway, thus promotes cells' proliferation.

Animals ; Cell Line ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Glycogen Synthase Kinases ; metabolism ; Hepatocytes ; drug effects ; Indoles ; pharmacology ; Male ; Maleimides ; pharmacology ; Rats ; Transfection ; Wnt Signaling Pathway ; beta Catenin ; metabolism

OBJECTIVETo study the molecular mechanism of curcumin in human esophageal carcinoma cell line (EC109).

METHODSEC109 cells were cultivated in vitro. When 80%-90% confluence was reached, they were treated with curcumin in different concentrations (15-120 µmol/L). The effects on cell proliferation were examined by CCK-8 colorimetry. The ultrastructure of EC109 cells were detected with transmission electron microscope(TEM). The cells apoptosis was observed with laser confocal microscope(LCM) by AnnexinV-FITC/PI double staining. The proteins level of PTEN, AKT, GSK3β and Caspase 3 were tested by flow cytometry(FCM) .

RESULTSCCK-8 test showed that curcumin could inhibit the proliferation of EC109 cells in a time- and concentration-dependent manner. TEM and LCM examinations indicated that curcumin could make EC109 cells apoptosis. The data of FCM showed that curcumin could increase the expression of PTEN, GSK3β and Caspase 3, decreased the expression of AKT.

CONCLUSIONThe effects of curcumin on inhibiting proliferation and promoting apoptosis of EC109 cells were related with increased expression of PTEN and inhibition of PI3K/AKT signaling pathway.

Apoptosis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Curcumin ; pharmacology ; Esophageal Neoplasms ; metabolism ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Humans ; PTEN Phosphohydrolase ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction

Proto-Oncogene Proteins c-akt/metabolism* ; Butyrates ; Ligands ; Glucose ; Receptors, G-Protein-Coupled/metabolism* ; Glycogen Synthase Kinases/metabolism* ; Glycogen Synthase Kinase 3 beta ; Phosphorylation

OBJECTIVETo explore the effects of ischemic postconditioning on ischemia/reperfusion injury in isolated hypertrophied rat heart and investigate the signal transduction pathway changes induced by ischemia postconditioning.

METHODSCardiac hypertrophy was induced in rats by abdominal aortic banding, and isolated hypertrophied rat heart ischemia/reperfusion model was made by Langendorff technique to evaluate the effects of ischemia postconditioning on left ventricular systole pressure, coronary artery flow, creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) release, myocardial infarction size, and the level of myocardial phospho-protein kinase B/Akt (Ser473), phospho-glycogen synthase kinase-3beta (Ser9). Following groups were studied (n = 12 each group): IR, 30 min ischemia (I)/60 min Reperfusion (R); Post: 30 min ischemia, 6 circles of 10 s I/10 s R followed by 60 min R; Post Wort: 30 min ischemia, 6 circles of 10 s I/10 s R, wortmannin (10(-7) mol/L) followed by 60 min R; Wort: 30 min ischemia, wortmannin (10(-7) mol/L) followed by 60 min R.

RESULTSLeft ventricular systolic pressure and coronary artery flow were significantly increased, myocardial infarction size and the release of CPK, LDH significantly reduced in Post group compared to that in IR group. Phospho-protein kinase B/Akt (Ser473) and phospho-glycogen synthase kinase-3beta (Ser9) levels were also significantly higher in Post group than that in IR group. Phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin prevented the increase of phospho-protein kinase B/Akt (Ser473) and phospho-glycogen synthase kinase-3beta (Ser9) induced by ischemic postconditioning, but only partly abolished the cardioprotection of ischemic postconditioning.

CONCLUSIONIschemic postconditioning attenuates ischemia/reperfusion injury in isolated hypertrophied rat heart. The cardioprotective effects of ischemic postconditioning were partly mediated through PI3K/Akt/GSK-3beta signaling pathway.

Animals ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Ischemic Preconditioning, Myocardial ; Male ; Myocardial Reperfusion Injury ; metabolism ; therapy ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Signal Transduction

Glycogen synthase kinase3 (GSK3α and GSK3β) are serine/threonine protein kinases acting on numerous substrates and involved in the regulation of various cellular functions such as their proliferation, survival, glycogen metabolism, and autophagy. Accumulating evidence indicates that the expression of GSK3α is increased mainly in androgendependent while that of GSK3β in androgenindependent prostate cancer, and that GSK3β is also involved in the regulation of the transactivation of the androgen receptor (AR) and growth of prostate cancer. Animal experiments have proved that some GSK3 inhibitors, such as lithium, can significantly suppress tumor growth in different animal models of prostate cancer. The GSK3 inhibitor is promising to be an important agent for the clinical management of prostate cancer.
Androgens ; Animals ; Cell Line, Tumor ; Glycogen Synthase Kinase 3 ; antagonists & inhibitors ; metabolism ; Glycogen Synthase Kinase 3 beta ; antagonists & inhibitors ; metabolism ; Humans ; Male ; Neoplasm Proteins ; metabolism ; Neoplasms, Hormone-Dependent ; enzymology ; metabolism ; Prostatic Neoplasms ; drug therapy ; enzymology ; pathology ; Receptors, Androgen ; metabolism

OBJECTIVETo determine the mechanism underlying the therapeutic activities of glycogen synthase kinase 3b (GSK3b) against hepatic ischemia-reperfusion (H-IR) injury by investigating the inhibitive effects of GSK3b on inflammation mediated by Toll-like receptor 4 (TLR4).

METHODSC57BL/6 male mice were subjected to 90 min of warm liver cephalad lobe ischemia, followed by reperfusion for various lengths of time. The mice were divided into three groups: the H-IR untreated model (control group), and the H-IR inflammation-induced models that received an intraperitoneal injection of purified lipopolysaccharide (LPS) endotoxin alone (inflammation group) or with pretreatment of the SB216763 GSK3b-specific inhibitor (intervention group). To create a parallel isolated cell system for detailed investigations of macrophages, marrow-derived stem cells were isolated from femurs of the H-IR control group of mice and used to derive primary macrophages. The cells were then divided into the same three groups as the whole mouse system: control, LPS-induced inflammation model, and inflammation model with SB216763 intervention. Differential expressions of inflammation-related proteins and genes were detected by Western blotting and real-time quantitative PCR, respectively.

RESULTSThe phosphorylation levels of ERK, JNK and p38 MAPK were induced in liver at 1 h after reperfusion, but then steadily decreased and returned to baseline levels by 4 h after reperfusion. In addition, the phosphorylation levels of ERK and JNK were induced in macrophages at 15 min after LPS stimulation, while the phosphorylation level of p38 MAPK was induced at 1 h; SB216763 pretreatment suppressed the LPS-stimulated ERK, JNK and p38 phosphorylation in macrophages. In the mouse model, GSK3b activity was found to promote the gene expression of anti-inflammatory cytokine IL-10 (control: 0.21 ± 0.08, inflammation: 0.83 ± 0.21, intervention: 1.76 ± 0.67; F = 3.16, P = 0.027) but to significantly inhibit the gene expression of pro-inflammatory cytokines IL-12 (control: 0.11 ± 0.05, inflammation: 0.85 ± 0.11, intervention: 0.43 ± 0.10; F = 2.67, P = 0.038), TNF-a, (control: 0.052 ± 0.012, inflammation: 8.11 ± 0.98, intervention: 3.9 ± 0.82; F = 4.13, P = 0.016), IL-6 (control: 0.22 ± 0.08, inflammation: 6.37 ± 0.81, intervention: 2.11 ± 0.63; F = 3.21, P = 0.024), and IL-1b (control: 0.12 ± 0.07, inflammation: 2.51 ± 0.62, and intervention: 1.28 ± 0.33; F = 2.22, P = 0.030).

CONCLUSIONInhibition of GSK3b selectively regulates the expression of anti-inflammatory and pro-inflammatory cytokines in liver Kupffer cells (liver macrophages). This process may be one of the mechanisms underlying the ability of GSK3b to ameliorate hepatic ischemia-reperfusion injury, possibly because inhibition of pro-inflammatory cytokines may indirectly mediate liver cell apoptosis.

Animals ; Cells, Cultured ; Cytokines ; metabolism ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Inflammation ; metabolism ; pathology ; Lipopolysaccharides ; adverse effects ; Liver ; pathology ; Macrophages ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Reperfusion Injury ; Toll-Like Receptor 4 ; metabolism

This study investigated the role of glycogen synthase kinase-3β (GSK-3β) in isoflurane-induced neuroinflammation and cognitive dysfunction in aged rats. The hippocampi were dissected from aged rats which had been intraperitoneally administered lithium chloride (LiCl, 100 mg/kg) and then exposed to 1.4% isoflurane for 6 h. The expression of GSK-3β was detected by Western blotting. The mRNA and protein expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 were measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Morris water maze was employed to detect spatial memory ability of rats. The results revealed that the level of GSK-3β was upregulated after isofurane exposure. Real-time PCR analysis demonstrated that isoflurane anesthesia increased mRNA levels of TNF-α, IL-1β and IL-6, which was consistent with the ELISA results. However, these changes were reversed by prophylactic LiCl, a non-selective inhibitor of GSK-3β. Additionally, we discovered that LiCl alleviated isoflurane-induced cognitive impairment in aged rats. Furthermore, the role of GSK-3β in isoflurae-induced neuroinflammation and cognitive dysfunction was associated with acetylation of NF-κB p65 (Lys310). In conclusion, these results suggested that GSK-3β is associated with isoflurane-induced upregulation of proinflammatory cytokines and cognitive disorder in aged rats.
Animals ; Cognition Disorders ; chemically induced ; metabolism ; pathology ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Inflammation ; chemically induced ; metabolism ; pathology ; Isoflurane ; adverse effects ; Male ; Neurons ; drug effects ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley