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 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

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

OBJECTIVETo investigate the role of integrin-linked kinase (ILK) signaling pathway in the skin lesions and wound healing in diabetic rats.

METHODSThirty-six SD rats were divided into diabetic wound group (D) and non-diabetic wound group (N) according to the random number table, with 18 rats in each group. 10 g/L streptozocin (60 mg/kg) was intraperitoneally injected in rats in group D, while the rats in group N were given same quantity of sodium citrate buffer. Two weeks after successful reproduction of diabetic model of rats in group D, two full-thickness skin of an area of 2 cm × 2 cm was resected on both sides of back of rats in the two groups. Wounds of three rats of each group were photographed and examined on post injury day (PID) 1, 3, 7, 10, 14, and 21, and the wound healing rates were calculated. The non-injured skin and wound tissue (central part) on back of three rats of the rest 15 rats in the two groups were harvested on PID 3, 7, 10, 14, and 21, respectively. Morphology of the non-injured skin tissue was observed with HE staining, and the thickness of full-thickness skin and epidermis were measured. The mRNA expression levels of ILK, protein kinase B (Akt), and glycogen synthase kinase-3β (GSK-3β) in non-injured skin tissue were determined with real-time fluorescent quantitative RT-PCR. The protein expression levels of ILK, Akt, phosphorylated Akt, GSK-3β, and phosphorylated GSK-3β in non-injured skin tissue, and ILK, phosphorylated Akt in wound tissue were assessed with Western blotting. Data were processed with two independent-sample t test, one-way analysis of variance, SNK test and analysis of variance of factorial design.

RESULTS(1) After injury, the wound scabs of rats in group N were dry, and red granulation tissue with no excretion were seen when the scabs fell off, and the wound healed fast. After injury, excretion under the wound scabs of rats in group D was seen, and the scabs easily fell off with exposure of pink granulation tissue with much excretion, and the wounds healed slowly. Except for PID 3, the wound healing rate of rats in group D was significantly lower than that in group N on other PIDs (with t values from 3.858 to 13.738, P<0.05 or P<0.01). (2) On PID 3, the hair follicles and blood vessels in the non-injured skin tissue of rats in group N were rich, and the epidermis was composed of stratified cells in form of basal cells and keratinocyte, and the hair follicles and blood vessels in the non-injured skin tissue of rats in group D were scarce, and the epidermis was nearly composed of one-layer of cells. The thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group N was similar from PID 3 to 21, and the thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group D on PID 3 was respectively (1 074 ± 66) and (15.1 ± 3.8) μm, and they gradually thinned out to (785 ± 122) and (9.7 ± 2.1) μm on PID 21, respectively. The thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group N were significantly thicker than those in group D on each PID (with t values from 4.620 to 23.549, P values below 0.001). (3) From PID 3 to 21, the mRNA expression levels of ILK and Akt in non-injured skin tissue of rats in group D were significantly lower than those in group N (with t values respectively 4.779 and 3.440, P values below 0.05), the mRNA expression levels of GSK-3β in non-injured skin tissue of rats were similar in two groups (t=0.363, P>0.05). (4) From PID 3 to 21, the protein expression levels of ILK, Akt and phosphorylated Akt in non-injured skin tissue of rats in group D were significantly lower than those in group N (with t values from 2.630 to 6.209, P<0.05 or P<0.01); the protein expression levels of GSK-3β in non-injured skin tissue of rats in two groups were similar (t=0.652, P>0.05); the protein expression level of phosphorylated GSK-3β in non-injured skin tissue of rats in group D was significantly higher than that in group N (t=4.131, P<0.001). The protein expression levels of ILK in wound tissue of rats in two groups were similar on each PID (with t values from 0.381 to 2.440, P values above 0.05). Except for PID 3, the protein expression levels of phosphorylated Akt in wound tissue of rats in group N were significantly higher than that in group D on other PIDs (with t values from 4.091 to 20.555, P<0.05 or P<0.01). From PID 3 to 21, the protein expression levels of ILK in wound tissue and non-injured skin tissue of rats in group N were similar (F=2.522, P>0.05), and the protein expression level of phosphorylated Akt in wound tissue was significantly higher than that in non-injured skin tissue (F=117.329, P<0.001); the protein expression levels of ILK in wound tissue and non-injured skin tissue of rats in group D were similar (F=1.337, P>0.05), and the protein expression level of phosphorylated Akt in wound tissue was significantly higher than that in non-injured skin tissue (F=184.120, P<0.001).

CONCLUSIONSThe skin lesion of diabetic rats may be related to the declined expression levels of ILK, Akt and phosphorylated Akt in the ILK signaling pathway. The refractory healing of wound in diabetic rats may be related to the declined expression level of phosphorylated Akt.

Animals ; Diabetes Mellitus, Experimental ; enzymology ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Phosphorylation ; Protein-Serine-Threonine Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Skin ; injuries ; Wound Healing

OBJECTIVETo explore the association between the abnormal phosphorylation sites found in Alzheimer disease (AD) tau and the inhibition of its biological activity.

METHODSUltracentrifugation, chromatography, manual Edman degradation and autosequence techniques were used to prepare and phosphorylate human recombinant tau, isolate and purify 32P tau peptides and determine phosphorylation sites.

RESULTSPhosphorylation of tau by casein kinase 1 (CK-1), cyclic AMP-dependent protein kinase (PKA) and glycogen synthetase kinase 3 (GSK-3) separately inhibited its biological activity and the inhibition of this activity by (CSK-3 was significantly increased if tau was prephosphorylated by CK-1 or PKA. The most potent inhibition was seen by a combined phosphorylation of tau with PKA and GSK-3. The treatment of tau by PKA and GSK-3 combination induced phosphorylation of tau at Ser-195, Ser-198, Ser-199, Ser-202, Thr-205, Thr-231, Ser-235, Ser-262, Ser-356, Ser-404, whereas Thr-181, Ser-184, Ser-262, Ser-356 and Ser-400 were phosphorylated by GSK-3 alone under the same condition.

CONCLUSIONPhosphorylation of tau by PKA plus GSK-3 at Thr-205 might play a key role in tau pathology in AD.

Alzheimer Disease ; metabolism ; Binding Sites ; Casein Kinases ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; Glycogen Synthase Kinase 3 ; metabolism ; Humans ; In Vitro Techniques ; Microtubules ; metabolism ; Phosphorylation ; Protein Kinases ; metabolism ; tau Proteins ; metabolism

OBJECTIVE: This study investigated the effects of bis (2-butoxyethyl) phthalate (BBOP) on the onset of male puberty by affecting Leydig cell development in rats. METHODS: Thirty 35-day-old male Sprague-Dawley rats were randomly allocated to five groups mg/kg bw per day that were gavaged for 21 days with BBOP at 0, 10, 100, 250, or 500 mg/kg bw per day. The hormone profiles; Leydig cell morphological metrics; mRNA and protein levels; oxidative stress; and AKT, mTOR, ERK1/2, and GSK3β pathways were assessed. RESULTS: BBOP at 250 and/or 500 mg/kg bw per day decreased serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels mg/kg bw per day (P < 0.05). BBOP at 500 mg/kg bw per day decreased Leydig cell number mg/kg bw per day and downregulated Cyp11a1, Insl3, Hsd11b1, and Dhh in the testes, and Lhb and Fshb mRNAs in the pituitary gland (P < 0.05). The malondialdehyde content in the testis significantly increased, while Sod1 and Sod2 mRNAs were markedly down-regulated, by BBOP treatment at 250-500 mg/kg bw per day (P < 0.05). Furthermore, BBOP at 500 mg/kg bw per day decreased AKT1/AKT2, mTOR, and ERK1/2 phosphorylation, and GSK3β and SIRT1 levels mg/kg bw per day (P < 0.05). Finally, BBOP at 100 or 500 μmol/L induced ROS and apoptosis in Leydig cells after 24 h of treatment in vitro (P < 0.05). CONCLUSION: BBOP delays puberty onset by increasing oxidative stress and apoptosis in Leydig cells in rats. UNLABELLED The graphical abstract is available on the website www.besjournal.com.
Rats ; Male ; Animals ; Leydig Cells/metabolism* ; Testosterone ; Glycogen Synthase Kinase 3 beta/pharmacology* ; Rats, Sprague-Dawley ; Sexual Maturation ; Testis ; Oxidative Stress ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis

Type 1 diabetes mellitus (T1DM)-induced cognitive dysfunction is common, but its underlying mechanisms are still poorly understood. In this study, we found that knockout of conventional protein kinase C (cPKC)γ significantly increased the phosphorylation of Tau at Ser214 and neurofibrillary tangles, but did not affect the activities of GSK-3β and PP2A in the hippocampal neurons of T1DM mice. cPKCγ deficiency significantly decreased the level of autophagy in the hippocampal neurons of T1DM mice. Activation of autophagy greatly alleviated the cognitive impairment induced by cPKCγ deficiency in T1DM mice. Moreover, cPKCγ deficiency reduced the AMPK phosphorylation levels and increased the phosphorylation levels of mTOR in vivo and in vitro. The high glucose-induced Tau phosphorylation at Ser214 was further increased by the autophagy inhibitor and was significantly decreased by an mTOR inhibitor. In conclusion, these results indicated that cPKCγ promotes autophagy through the AMPK/mTOR signaling pathway, thus reducing the level of phosphorylated Tau at Ser214 and neurofibrillary tangles.
AMP-Activated Protein Kinases/metabolism* ; Animals ; Autophagy ; Diabetes Mellitus, Type 1 ; Glucose ; Glycogen Synthase Kinase 3 beta/metabolism* ; Mice ; Phosphorylation ; Protein Kinase C/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; tau Proteins/metabolism*

One of the pathological feathers of Alzheimer's disease (AD) is neurofibrillary tangles (NFTs), which consist of paired helical filaments (PHFs) formed by hyperphosphorylated microtubule-associated protein tau. To study the role of mitogen-activated protein kinase (MAPK) in tau hyperphosphorylation and the underlying mechanism, wild type mouse neuroblastoma cells (N2a) were dealt with different concentrations (0.1 microg/mL, 0.2 microg/mL and 0.4 microg/mL) of anisomycin (an activator of MAPK) for 6 h. The relationship between MAPK activity and tau phosphorylation at some Alzheimer-sites was analyzed, and the activities of protein kinase A (PKA) and glycogen synthase kinase-3 (GSK-3) were detected. The results showed that anisomycin activated MAPK in a dose-dependent manner, but tau hyperphosphorylation at Ser-198/199/202 and Ser-396/404 sites was only observed when the concentration of anisomycin was at the level of 0.4 microg/mL, and the alteration of tau phosphorylation at Ser-214 showed no significant difference in different groups. 0.2 microg/mL and 0.4 microg/mL of anisomycin led to an increase in the activity of GSK-3, respectively, but had no effect on the activity of PKA. Lithium chloride, a specific inhibitor of GSK-3, completely abolished the anisomycin-induced elevation of tau phosphorylation without any effect on the activity of MAPK. In conclusion, overactivation of MAPK up to a certain degree induces tau hyperphosphorylation at Ser-198/199/202 and Ser-396/404 sites, and this is probably related to the effect of activated GSK-3 by MAPK.
Alzheimer Disease ; pathology ; Animals ; Anisomycin ; pharmacology ; Cell Line, Tumor ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; Glycogen Synthase Kinase 3 ; metabolism ; Mice ; Mitogen-Activated Protein Kinases ; metabolism ; Neurofibrillary Tangles ; pathology ; Phosphorylation ; tau Proteins ; metabolism

The apoptosis of nucleus pulposus cells (NPCs) is the main cellular process of intervertebral disc degeneration (IVDD). Our previous studies showed that 17β-estradiol (E
Animals ; Apoptosis ; Estradiol/pharmacology* ; Glycogen Synthase Kinase 3 beta ; Interleukin-1beta ; Nucleus Pulposus/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats ; Signal Transduction ; TOR Serine-Threonine Kinases