Glycogen synthase kinase-3β positively regulates the proliferation of human ovarian cancer cells

Although glycogen synthase kinase-3 （GSK-3） might act as a tumor suppressor since its inhibition is expected to mimic the activation of Wnt-signaling pathway, GSK-3β may contribute to NF-κB activation in cancer cells leading to increased cancer cell proliferation and survival. Here we report that GSK-3β activity was involved in the proliferation of human ovarian cancer cell both in vitro and in vivo. Inhibition of GSK-3 activity by pharmacological inhibitors suppressed proliferation of the ovarian cancer cells. Overexpressing constitutively active form of GSK-3β induced entry into the S phase, increased cyclin D1 expression and facilitated the proliferation of ovarian cancer cells. Furthermore, GSK-3 inhibition prevented the formation of the tumor in nude mice generated by the inoculation of human ovarian cancer cells. Our findings thus suggest that GSK-3β activity is important for the proliferation of ovarian cancer cells, implicating this kinase as a potential therapeutic target in ovarian cancer.

Lithium chloride ameliorates learning and memory ability and inhibits glycogen synthase kinase-3 beta activity in a mouse model of fragile X syndrome

In the present study,Fmr1 knockout mice （KO mice） were used as the model for fragile X syndrome.The results of step-through and step-down tests demonstrated that Fmr1 KO mice had shorter latencies and more error counts,indicating a learning and memory disorder.After treatment with 30,60,90,120,or 200 mg/kg lithium chloride,the learning and memory abilities of the Fmr1 KO mice were significantly ameliorated,in particular,the 200 mg/kg lithium chloride treatment had the most significant effect.Western blot analysis showed that lithium chloride significantly enhanced the expression of phosphorylated glycogen synthase kinase 3 beta,an inactive form of glycogen synthase kinase 3 beta,in the cerebral cortex and hippocampus of the Fmr1 KO mice.These results indicated that lithium chloride improved learning and memory in the Fmr1 KO mice,possibly by inhibiting glycogen synthase kinase 3 beta activity.

The role of glycogen synthase kinase 3 beta in brain injury induced by myocardial ischemia/reperfusion injury in a rat model of diabetes mellitus

Myocardial ischemia/reperfusion injury can lead to severe brain injury.Glycogen synthase kinase 3 beta is known to be involved in myocardial ischemia/reperfusion injury and diabetes mellitus.However,the precise role of glycogen synthase kinase 3 beta in myocardial ischemia/reperfusion injury-induced brain injury is unclear.In this study,we observed the effects of glycogen synthase kinase 3 beta on brain injury induced by myocardial ischemia/reperfusion injury in diabetic rats.Rat models of diabetes mellitus were generated via intraperitoneal injection of streptozotocin.Models of myocardial ischemia/reperfusion injury were generated by occluding the anterior descending branch of the left coronary artery.Post-conditioning comprised three cycles of ischemia/reperfusion.Immunohistochemical staining and western blot assays demonstrated that after 48 hours of reperfusion,the structure of the brain was seriously damaged in the experimental rats compared with normal controls.Expression of Bax,interleukin-6,interleukin-8,terminal deoxynucleotidyl transferase d UTP nick end labeling,and cleaved caspase-3 in the brain was significantly increased,while expression of Bcl-2,interleukin-10,and phospho-glycogen synthase kinase 3 beta was decreased.Diabetes mellitus can aggravate inflammatory reactions and apoptosis.Ischemic post-conditioning with glycogen synthase kinase 3 beta inhibitor lithium chloride can effectively reverse these changes.Our results showed that myocardial ischemic post-conditioning attenuated myocardial ischemia/reperfusion injury-induced brain injury by activating glycogen synthase kinase 3 beta.According to these results,glycogen synthase kinase 3 beta appears to be an important factor in brain injury induced by myocardial ischemia/reperfusion injury.

Glycogen Synthase Kinase 3β Inhibitor (2'Z,3'E)-6-Bromo-indirubin-3'-Oxime Enhances Drug Resistance to 5-Fluorouracil Chemotherapy in Colon Cancer Cells

Objective： To explore the effects and mechanism of glycogen synthase kinase 3β （GSK-313） inhibitor （2＇Z,3＇E）-6-bromo-indirubin-3＇-oxime （BIO） on drug resistance in colon cancer cells. Methods： The colon cancer SW480 and SW620 cells were treated with BIO, 5-fluorouracil （5-FU） and BIO/5-FU, separately. Cell cycle distribution, apoptosis level and efflux ability of rhodamine 123 （Rh123） were detected by flow cytometry. The protein expressions of P-glycoprotein （P-gp）, multidrug resistance protein 2 （MRP2）, thymidylate synthase （TS）, β-catenin, E2F-1 and βcl-2 were detected by Western blot. β-catenin and P-gp were stained with double immunofluorescence and observed under a confocal microscope. Results： BIO up-regulated β-catenin, P-gp, MRP2 and TS, enhanced the efflux ability of Rh123, decreased Bcl-2 protein and gave the opposite effect to E2F-1 protein in SW480 and SW620 ceils. Furthermore, BIO significantly inhibited cell apoptosis, increased S and G2/M phase cells, and reduced the cell apoptosis induced by 5-FU in SW480 cells, whereas the effects were slight or not obvious in SW620 cells. Conclusion： GSK-3β was involved in drug resistance regulation, and activation of β-catenin and inhibition of E2F-1 may be the most responsible for the enhancement of 5-FU chemotherapy resistance induced by GSK-β inhibitor β10 in colon cancer.

Protection of INS-1 Cells from Free Fatty Acid-induced Apoptosis by Inhibiting the Glycogen Synthase Kinase-3

To examine the role of glycogen synthase kinase 3 （GSK-3） in the apoptosis of pancreatic β-cells to better understand the pathogenesis and to find new approach to the treatment of type 2 diabetes, apoptosis was induced by oleic acid （OA） in INS-1 cells and the activity of GSK-3 was inhibited by LiCl. The PI staining and flow cytometry were employed for the evaluation of apoptosis. The phosphorylation level of GSK-3 was detected by Western blotting. The results showed that OA at 0.4 mmol/L could cause conspicuous apoptosis of INS- 1 cells and the activity of GSK-3 was significantly increased. After the treatment with 24 mmolFL of LiCl, a inhibitor of GSK-3, the OA-induced apoptosis of INS-1 cells was lessened and the phosphorylation of GSK-3 was increased remarkably. It is concluded that GSK-3 activation plays an important role in OA-induced apoptosis in pancreatic β-cells and inhibition of the GSK-3 activity can effectively protect INS-1 cells from the OA-induced apoptosis. Our study provides a new experimental basis and target for the clinical treatment of type-2 diabetes.

Overexpression of fibroblast growth factor 13 ameliorates amyloid-β-induced neuronal damage

Previous studies have shown that fibroblast growth factor 13 is downregulated in the brain of both Alzheimer’s disease mouse models and patients,and that it plays a vital role in the learning and memory.However,the underlying mechanisms of fibroblast growth factor 13 in Alzheimer’s disease remain unclear.In this study,we established rat models of Alzheimer’s disease by stereotaxic injection of amyloid-β(Aβ_(1-42))-induced into bilateral hippocampus.We also injected lentivirus containing fibroblast growth factor 13 into bilateral hippocampus to overexpress fibroblast growth factor 13.The expression of fibroblast growth factor 13 was downregulated in the brain of the Alzheimer’s disease model rats.After overexpression of fibroblast growth factor 13,learning and memory abilities of the Alzheimer’s disease model rats were remarkably improved.Fibroblast growth factor 13 overexpression increased brain expression levels of oxidative stress-related markers glutathione,superoxide dismutase,phosphatidylinositol-3-kinase,AKT and glycogen synthase kinase 3β,and anti-apoptotic factor BCL.Furthermore,fibroblast growth factor 13 overexpression decreased the number of apoptotic cells,expression of pro-apoptotic factor BAX,cleaved-caspase 3 and amyloid-βexpression,and levels of tau phosphorylation,malondialdehyde,reactive oxygen species and acetylcholinesterase in the brain of Alzheimer’s disease model rats.The changes were reversed by the phosphatidylinositol-3-kinase inhibitor LY294002.These findings suggest that overexpression of fibroblast growth factor 13 improved neuronal damage in a rat model of Alzheimer’s disease through activation of the phosphatidylinositol-3-kinase/AKT/glycogen synthase kinase 3βsignaling pathway.

Anti-diabetic potential of apigenin,luteolin,and baicalein via partially activating PI3K/Akt/GLUT-4 signaling pathways in insulin-resistant HepG2 cells

Dietary flavonoids are abundant in natural plants and possess multiple pharmacological and nutritional activities.In this study,apigenin,luteolin,and baicalein were chosen to evaluate their anti-diabetic effect in high-glucose and dexamethasone induced insulin-resistant(IR)HepG2 cells.All flavonoids improves the glucose consumption and glycogen synthesis abilities in IR-HepG2 cells via activating glucose transporter protein 4(GLUT4)and phosphor-glycogen synthase kinase(GSK-3β).These fl avonoids signifi cantly inhibited the production of reactive oxygen species(ROS)and advanced glycation end-products(AGEs),which were closely related to the suppression of the phosphorylation form of NF-κB and P65.The expression levels of insulin receptor substrate-1(IRS-1),insulin receptor substrate-2(IRS-2)and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)pathway in IR-HepG2 cells were all partially activated by the fl avonoids,with variable effects.Furthermore,the intracellular metabolic conditions of the fl avonoids were also evaluated.

Wnt signaling control of bone cell apoptosis

Wnts are a large family of growth factors that mediate essential biological processes like embryogenesis, morpho- genesis and organogenesis. These proteins also play a role in oncogenesis, and they regulate apoptosis in many tissues. Wnts bind to a membrane receptor complex comprised of a frizzled （FZD） G-protein-coupled receptor and a low-density lipoprotein （LDL） receptor-related protein （LRP）. The formation of this ligand-receptor complex initiates a number of signaling cascades that include the canonical/beta-catenin pathway as well as several noncanonical pathways. In recent years, canonical Wnt signaling has been reported to play a significant role in the control of bone formation. Clinical studies have found that mutations in LRP-5 are associated with reduced bone mineral density （BMD） and fractures. Investigations of knockout and transgenic mouse models of Wnt pathway components have shown that canonical Wnt signaling modulates most aspects ofosteoblast physiology including proliferation, differentiation, function and apoptosis. Transgenic mice expressing a gain of function mutant of LRP-5 in bone, or mice lacking the Wnt antagonist secreted frizzled-related protein-l, exhibit elevated BMD and suppressed osteoblast apoptosis. In addition, preclinical studies with pharmacologic compounds such as those that inhibit glycogen synthase kinase-3β support the importance of the canonical Wnt pathway in modulation of bone formation and osteoblast apoptosis.

Baicalin attenuates high fat diet-induced insulin resistance and ectopic fat storage in skeletal muscle,through modulating the protein kinase B/Glycogen synthase kinase 3 beta pathway

Insulin resistance is the pathophysiological basis of many diseases.Overcoming early insulin resistance highly significant in prevention diabetes,non-alcoholic fatty liver,and atherosclerosis.The present study aimed at evaluating the therapeutic effects of baicalin on insulin resistance and skeletal muscle ectopic fat storage in high fat diet-induced mice,and exploring the potential molecular mechanisms.Insulin resistance in mice was induced with a high fat diet for 16 weeks.Animals were then treated with three different doses of baicalin(100,200,and 400 mg·kg^(-1)·d^(-1)for 14 weeks.Fasting blood glucose,fasting serum insulin,glucose tolerance test(GTT),insulin tolerance test(ITT),and skeletal muscle lipid deposition were measured.Additionally,the AMP-activated protein kinase/acetyl-CoA carboxylase and protein kinase B/Glycogen synthase kinase 3 beta pathways in skeletal muscle were further evaluated.Baicalin significantly reduced the levels of fasting blood glucose and fasting serum insulin and attenuated high fat diet induced glucose tolerance and insulin tolerance.Moreover,insulin resistance was significantly reversed.Pathological analysis revealed baicalin dose-dependently decreased the degree of the ectopic fat storage in skeletal muscle.The properties of baicalin were mediated,at least in part,by inhibition of the AMPK/ACC pathway,a key regulator of de novo lipogenesis and activation of the Akt/GSK-3β pathway,a key regulator of Glycogen synthesis.These data suggest that baicalin,at dose up to 400 mg·kg^(-1)·d^(-1),is safe and able to attenuate insulin resistance and skeletal muscle ectopic fat storage,through modulating the skeletal muscle AMPK/ACC pathway and Akt/GSK-3β pathway.

GSK3β inhibitor promotes myelination and mitigates muscle atrophy after peripheral nerve injury

Delay of axon regeneration after peripheral nerve injury usually leads to progressive muscle atrophy and poor functional recovery. The Wnt/β-catenin signaling pathway is considered to be one of the main molecular mechanisms that lead to skeletal muscle atrophy in the elderly. We hold the hypothesis that the innervation of target muscle can be promoted by accelerating axon regeneration and decelerating muscle cell degeneration so as to improve functional recovery of skeletal muscle following peripheral nerve injury. This process may be associated with the Wnt/β-catenin signaling pathway. Our study designed in vitro cell models to simulate myelin regeneration and muscle atrophy. We investigated the effects of SB216763, a glycogen synthase kinase 3 beta inhibitor, on the two major murine cell lines RSC96 and C2C12 derived from Schwann cells and muscle satellite cells. The results showed that SB216763 stimulated the Schwann cell migra- tion and myotube contraction. Quantitative polymerase chain reaction results demonstrated that myelin related genes, myelin associated glycoprotein and cyclin-D1, muscle related gene myogenin and endplate-associated gene nicotinic acetylcholine receptors levels were stimulated by SB216763. Immunocytochemical staining revealed that the expressions of ^-catenin in the RSC96 and C2C12 cytosolic and nuclear compartments were increased in the SB216763-treated cells. These findings confirm that the glycogen synthase kinase 3 beta in- hibitor, SB216763, promoted the myelination and myotube differentiation through the Wnt/β-catenin signaling pathway and contributed to nerve remyelination and reduced denervated muscle atrophy after peripheral nerve injury.

GSK3β inhibits epithelial-mesenchymal transition via the Wnt/β-catenin and PI3K/Akt pathways

AIM： To investigate the regulatory mechanism of glycogen synthase kinase 3β（GSK3β） in epithelialmesenchymal transition（EMT） process after proliferative vitreoretinopathy（PVR） induction. METHODS： Experimental PVR was induced by intravitreal injection of retinal pigment epithelium（RPE） cells in the eyes of rabbits. A PI3 K/Akt inhibitor（wortmannin） and a GSK3β inhibitor（Li Cl） were also injected at different time during PVR progress. Electroretinogram（ERG）, ocular fundus photographs, and B-scan ultrasonography were used to observe the PVR progress. Western blot test on the extracted retina were performed at 1, 2, 4 wk. The expression of the mesenchymal marker vimentin was determined by immunohistochemistry. Toxicity of wortmannin and Li Cl were evaluated by ERG and Td Tmediated d UTP nick-end labeling（TUNEL） assay. The vitreous was also collected for metabolomic analysis. RESULTS： Experimental PVR could significantly lead to EMT, along with the suppressed expression of GSK3β and the activation of Wnt/β-catenin and PI3 K/Akt pathways. It was verified that upregulating the expression of GSK3β could effectively inhibit EMT process by suppressing Wnt/β-catenin and PI3 K/Akt pathways. CONCLUSION： GSK3β effectively inhibits EMT via the Wnt/β-catenin and PI3 K/Akt pathways. GSK3β may be regarded as a promising target of experimental PVR inhibition.

Amyloid beta-peptide worsens cognitive impairment following cerebral ischemia-reperfusion injury

Amyloid 13-peptide, a major component of senile plaques in Alzheimer＇s disease, has been implicated in neuronal cell death and cognitive impairment. Recently, studies have shown that the pathogenesis of cerebral ischemia is closely linked with Alzheimer＇s disease. In this study, a rat model of global cerebral ischemia-reperfusion injury was established via occlusion of four arteries; meanwhile, fibrillar amyloid [3-peptide was injected into the rat lateral ventricle. The Morris water maze test and histological staining revealed that administration of amyloid 13-peptide could further aggravate impairments to learning and memory and neuronal cell death in the hippocampus of rats subjected to cerebral ischemia-reperfusion injury. Western blot showed that phosphorylation of tau protein and the activity of glycogen synthase kinase 313 were significantly stronger in cerebral ischemia-reperfusion injury rats subjected to amyloid [3-peptide administration than those undergo- ing cerebral ischemia-repetfusion or amyloid 13-peptide administration alone. Conversely, the activ- ity of protein phosphatase 2A was remarkably reduced in rats with cerebral ischemia-reperfusion injury following amyloid 13-peptide administration. These findings suggest that amyloid 13-peptide can potentiate tau phosphorylation induced by cerebral ischemia-reperfusion and thereby aggravate cognitive impairment.

A combination extract of Renshen(Panax Ginseng),Yinyanghuo(Herba Epimedii Brevicornus),Yuanzhi(Radix Palygalae) and Jianghuang(Rhizoma Curcumae Longae) decreases glycogen synthase kinase 3β expression in brain cortex of APPV717I transgenic mice

OBJECTIVE： To investigate the neuroprotective mechanism of combination extract of Renshen （Ponax Ginseng）, Yinyanghuo （Herba Epimedii Brevi-comus）, Yuanzhi （Radix Palygalae） and Jianghuang （Rhizoma Curcumae Longae） （GEPT） in treating AI- zheimer＇s disease on the target of glycogen syn- thase kinase 3β（GSK-3β）. METHODS： Three-month-old APPV7171 transgenic mice were randomly divided into ten groups （n=12 per group） and intragastrically administrated vehi- cle or medicines： APP group was given 0.5% CMC, donepezil group was given donepezil （APP ＋ D group） （0.92 mg/kg-1. day-1）, and GEPT groups were given small dose of GEPT （APP＋Gs group） （0.075 g/ kg-1. day-1）, medium dose （APP＋Gm group） （0.15 g/ kg-1. day-1）, and large dose （APP＋GI group） （0.30 g/ kg-1. day-1） for 4 or 8 months, respectively. Three-month-old C57BL/6J mice as vehicle controls （n=12） were given 0.5% CMC for 4 or 8 months as well. The GSK-3β expression in the cortex of 7- and 11-month-old APPV7171 transgenic mice with and without GEPT or donepezil treatment and normal C57BL/6J mice were measured via Western blotting and Immunohistochemistry. RESULTS： Immunohistochemistry analysis showed significant increase of GSK-3β in the cerebral cortex of 7-month-old APP group （compare to control group P=0.003）, while the GSK-313 expression of donepezil or OEPT group were all significantly de-creased （Donepezil vs APP： P=0.041; GI vs APP： P=0.049, Gm vs APP： P=0.029, Gh vs APP： P=0.036）. Western blot analysis showed similar results. The densitometric measures of GSK-3β in APP mice in- creased significantly as compared with the control group （P=0.008）. And the GSK-3β expression indonepezil and GEPT groups were all decreased. There was significant difference between Gh group or donepezil group and the control group （P=0.05）. Similar findings were shown in the 11-month-old mice in each group, except for greater decrease of GSK-3β in the GEPT group. CONCLUSION： GEPT can effectively decrease the level of GSK-3β expression in the brain cortex of AP- PV7171 transgenic mice, and such effect is more sig- nificant in 11-month-old mice. This partially ex- plains the neuroprotecting mechanism of GEPT in preventing and treating of AD.

TLR7/8 signalling affects X-sperm motility via the GSK3α/β-hexokinase pathway for the efficient production of sexed dairy goat embryos

Background:Goat milk is very similar to human milk in terms of its abundant nutrients and ease of digestion.To derive greater economic benefit,farmers require more female offspring(does);however,the buck-to-doe offspring sex ratio is approximately 50%.At present,artificial insemination after the separation of X/Y sperm using flow cytometry is the primary means of controlling the sex of livestock offspring.However,flow cytometry has not been successfully utilised for the separation of X/Y sperm aimed at sexing control in dairy goats.Results:In this study,a novel,simple goat sperm sexing technology that activates the toll-like receptor 7/8(TLR7/8),thereby inhibiting X-sperm motility,was investigated.Our results showed that the TLR7/8 coding goat Xchromosome was expressed in approximately 50%of round spermatids in the testis and sperm,as measured from cross-sections of the epididymis and ejaculate,respectively.Importantly,TLR7/8 was located at the tail of the Xsperm.Upon TLR7/8 activation,phosphorylated forms of glycogen synthase kinaseα/β(GSK3α/β)and nuclear factor kappa-B(NF-κB)were detected in the X-sperm,causing reduced mitochondrial activity,ATP levels,and sperm motility.High-motility Y-sperm segregated to the upper layer and the low-motility X-sperm,to the lower layer.Following in vitro fertilisation using the TLR7/8-activated sperm from the lower layer,80.52±6.75%of the embryos were XX females.The TLR7/8-activated sperm were subsequently used for in vivo embryo production via the superovulatory response;nine embryos were collected from the uterus of two does that conceived.Eight of these were XX embryos,and one was an XY embryo.Conclusions:Our study reveals a novel TLR7/8 signalling mechanism that affects X-sperm motility via the GSK3α/β-hexokinase pathway;this technique could be used to facilitate the efficient production of sexed dairy goat embryos.

Wnt/Glycogen Synthase Kinase 3β/β-catenin Signaling Activation Mediated Sevoflurane Preconditioning-induced Cardioprotection

Background： Sevoflurane preconditioning （SP） has been shown to invoke potent myocardial protection in animal studies and clinical trials. However, the mechanisms underlying SP are complex and not yet well understood. We investigated the hypothesis that the cardioprotection afforded by SP is mediated via the Writ/glycogen synthase kinase 3β（GSK3β）/β-catenin signaling pathway. Methods： Two models were established： A Langendorffperfused rat heart model and the H9C2 cell hypoxia/reoxygenation model. Both rats and H9C2 cells were randomly divided into 6 groups as follows： S group, ischemia-reperfusion （I/R） group, DMSO group, IWP group, SP group, and SP ＋ IWP group. Hemodynamic parameters, lactate dehydrogenase （LDH） activity in coronary effluent and cell culture supernatant, and the infarct size were measured to evaluate myocardial ischemia-reperfusion injuries. To determine the activity of Wnt/GSK3β/β-catenin signaling pathway, the expressions of Wnt3a, phospho-GSK3β, and β-catenin were measured by Western blotting. Results： SP improved cardiac function recovery, reduced infarct size （18 ±2% in the SP group compared with 35 ± 4% in the 1/R group; P 〈 0.05）, decreased LDH activity in coronary effluent, and culture supematant. IWP-2, an inhibitor of Wnt, abolished the cardioprotection by SR In addition, Western blotting analysis demonstrated that the expressions of Wnt3a, phospho-GSK3β, and β-catenin significantly （P 〈 0.05） increased in the I/R group, compared with the S group; and compared to I/R group, SP significantly （P 〈 0.05） increased Wnt3a, phospho-GSK3 β, and β-catenin expressions. Pretreatment with IWP-2 significantly （P 〈 0.05） abolished SP-induced Wnt/GSK3β/β-catenin signaling activation. Conclusions： The results showed for the first time that cardioprotection afforded by SP may be mediated partly via the Wnt/GSK3β/β-catenin signaling pathway.

Lithium inhibits proliferation of human esophageal cancer cell line Eca-109 by inducing a G_2/M cell cycle arrest

AIM： To investigate the effect of lithium on proliferation of esophageal cancer （EC） cells and its preliminary mechanisms. METHODS： Eca-109 cells were treated with lithium chloride, a highly selective inhibitor of glycogen synthase kinase 31） （GSK-31））, at different concentrations （2-30 mmol/L） and time points （0, 2, 4, 6 and 24 h）. Cell proliferative ability was evaluated by 3-（4,5-Dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay, and Cell cycle distribution was examined by flow cytometry. Expressions of p-GSK-3β, β-catenin, cyclin B1, cdc2 and cyclin D1 protein were detected by Western blotting, and the subcellular localization of β-catenin was determined by immunofluorescence. The mRNA level of of cyclin B1 was detected by reverse transcription polymerase chain reaction （RT-PCR）. RESULTS： Lithium could inhibit the proliferation of Eca-109 cells. Lithium at a concentration of 20 mmol/L lithium for 24 h produced obvious changes in the distribution of cell cycle, and increased the number of cells in G2/M phase （P 〈 0.05 vs control group）. Western blotting showed that lithium inhibited GSK-β） by Ser-9 phosphorylation and stabilized free β-catenin in the cytoplasm. Immunofluorescence further confirmed that free β-catenin actively translocated to the nucleus. Horeover, lithium slightly elevated cyclin D1 protein expression, whereas lowered the cyclin B1 expression after 24 h lithium exposure and no obvious change was observed for cdc2 protein. CONCLUSION： Lithium can inhibit the proliferation of human esophageal cancer cell line Eca-109 by inducing a G2/H cell cycle arrest, which is mainly mediated through the inhibition of lithium-sensitive molecule, GSK-3β, and reduction of cyclin B1 expression.

A retrograde apoptotic signal originating in NGF-deprived distal axons of rat sympathetic neurons in compartmented cultures

Previous investigations of retrograde survival signaling by nerve growth factor （NGF） and other neurotrophins have supported diverse mechanisms, but all proposed mechanisms have in common the generation of survival signals retrogradely transmitted to the neuronal cell bodies. We report the finding of a retrograde apoptotic signal in axons that is suppressed by local NGF signaling. NGF withdrawal from distal axons alone was sufficient to activate the pro-apoptotic transcription factor, c-jun, in the cell bodies. Providing NGF directly to cell bodies, thereby restoring a source of NGF-induced survival signals, could not prevent c-jun activation caused by NGF withdrawal from the distal axons. This is evidence that c-jun is not activated due to loss of survival signals at the cell bodies. Moreover, blocking axonal transport with colchicine inhibited c-jun activation caused by NGF deprivation suggesting that a retrogradely transported pro-apoptotic signal, rather than loss of a retrogradely transported survival signal, caused c-jun activation. Additional experiments showed that activation of c-jun, pro-caspase-3 cleavage, and apoptosis were blocked by the protein kinase C inhibitors, rottlerin and chelerythrine, only when applied to distal axons suggesting that they block the axon-specific pro-apoptotic signal. The rottlerin-sensitive mechanism was found to regulate glyco- gen synthase kinase 3 （GSK3） activity. The effect of siRNA knockdown, and pharmacological inhibition of GSK3 suggests that GSK3 is required for apoptosis caused by NGF deprivation and may function as a retrograde carrier of the axon apoptotic signal. The existence of a retrograde death signaling system in axons that is suppressed by neurotro- phins has broad implications for neurodevelopment and for discovering treatments for neurodegenerative diseases and neurotrauma.

Phosphorylation of tau protein over time in rats subjected to transient brain ischemia

Transient brain ischemia has been shown to induce hyperphosphorylation of the micro- tubule-associated protein tau. To further determine the mechanisms underlying these processes, we investigated the interaction between tau, glycogen synthase kinase （GSK）-313 and protein phos- phatase 2A. The results confirmed that tau protein was dephosphorylated during brain ischemia; in addition, the activity of GSK-3β was increased and the activity of protein phosphatase 2A was de- creased. After reperfusion, tau protein was hyperphosphorylated, the activity of GSK-3β was de- creased and the activity of protein phosphatase 2A remained low. Importantly, the interaction of tau with GSK-3β and protein phosphatase 2A was altered during ischemia and reperfusion. Lithium chloride could affect tau phosphorylation by regulating the interaction of tau with GSK-3β and pro- tein phosphatase 2A, and improve learning and memory ability of rats after transient brain ischemia. The present study demonstrated that it was the interaction of tau with GSK-3β and protein phos- phatase 2A, rather than their individual activities, that dominates the phosphorylation of tau in tran- sient brain ischemia. Hyperphosphorylated tau protein may play an important role in the evolution of brain injury in ischemic stroke. The neuroprotective effects of lithium chloride partly depend on the inhibition of tau phosphorylation during transient brain ischemia.

Adult neural stem cell dysfunction in the subventricular zone of the lateral ventricle leads to diabetic olfactory defects

Sensitive smell discrimination is based on structural plasticity of the olfactory bulb,which depends on migration and integration of newborn neurons from the subventricular zone.In this study,we examined the relationship between neural stem cell status in the subventricular zone and olfactory function in rats with diabetes mellitus.Streptozotocin was injected through the femoral vein to induce type 1 diabetes mellitus in Sprague-Dawley rats.Two months after injection,olfactory sensitivity was decreased in diabetic rats.Meanwhile,the number of Brd U-positive and Brd U＋/DCX＋double-labeled cells was lower in the subventricular zone of diabetic rats compared with agematched normal rats.Western blot results revealed downregulated expression of insulin receptorβ,phosphorylated glycogen synthase kinase 3β,and β-catenin in the subventricular zone of diabetic rats.Altogether,these results indicate that diabetes mellitus causes insulin deficiency,which negatively regulates glycogen synthase kinase 3β and enhances β-catenin degradation,with these changes inhibiting neural stem cell proliferation.Further,these signaling pathways affect proliferation and differentiation of neural stem cells in the subventricular zone.Dysfunction of subventricular zone neural stem cells causes a decline in olfactory bulb structural plasticity and impairs olfactory sensitivity in diabetic rats.

Effect of Lithium on Cell Cycle Progression of Pig Airway Epithelial Cells

To investigate the effect of lithium on cell cycle progression of airway epithelial cells, primary pig tracheobronchial epithelial cells were incubated with lithium chloride (LiCl) at different concentrations (0, 5 mmol/L, and 10 mmol/L) and time (12 h, 16 h and 24 h). After the treatment, cells were counted, cell cycle profile was measured by BrdU labeling and flow cytometry, and expression of cyclin D1 and cyclin B1 were detected by Western blotting. The results showed that after 24h of 10mmol/L but not 5mmol/L LiCl treatment, proliferation of cells was slowed down as manifested by delayed confluence and cell number accumulation (P<0.05). Lithium did not change the percentage of cells in S phase (P>0.05), but 24 h incubation with 10 mmol/L LiCl induced a G 2/M cell cycle arrest. Furthermore, 10mmol/L LiCl elevated cyclin D1 expression after 12h treatment, while expression of cyclin B1 increased more significantly after 24h incubation. These data demonstrate that lithium inhibits proliferation of pig airway epithelial cells by inhibiting cell cycle progression, and suggest that lithium-sensitive molecule(s) such as glycogen synthase kinase 3 may have a role in the regulation of growth of airway epithelial cells.