Osteopontin promotes gastric cancer progression via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effects of OPN on the proliferation,invasion and migration of GC cells and its possible mechanism.METHODS The mRNA and protein expression of OPN in the GC cells were analyzed by realtime quantitative-reverse transcription polymerase chain reaction and western blotting,and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC.Next,the effects of OPN knockdown on GC cells migration and invasion were examined.The short hairpin RNA(shRNA)and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer’s instructions.Non transfected cells were classified as control in the identical transfecting process.24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay,and cell invasiveness and migration were detected by Trans well assay.Meanwhile,the expression of protein kinase B(AKT),matrix metalloproteinase 2(MMP-2)and vascular endothelial growth factor(VEGF)in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting.RESULTS The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells.OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation,invasion and migration of SGC-7901 cells.Moreover,in the experiments of investigating the underlying mechanism,results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF,it also decreased the phosphorylation of AKT.Meanwhile,the protein expression levels of MMP-2,VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002).CONCLUSION These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to upregulate MMP-2 and VEGF expression,which contribute SGC-7901 cells to proliferation,invasion and migration.Thus,our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.

Fenofibrate Pre-treatment Suppressed Inflammation by Activating Phosphoinositide 3 Kinase/Protein Kinase B(PI3K/Akt) Signaling in Renal Ischemia-Reperfusion Injury

The aim of this study was to investigate the possible beneficial effects of Fenofibrate on renal ischemia-reperfusion injury（IRI） in mice and its potential mechanism. IRI was induced by bilateral renal ischemia for 60 min followed by reperfusion for 24 h. Eighteen male C57BL/6 mice were randomly divided into three groups： sham-operated group（sham）, IRI＋saline group（IRI group）, IRI＋Fenofibrate（FEN） group. Normal saline or Fenofibrate（3 mg/kg） was intravenously injected 60 min before renal ischemia in IRI group and FEN group, respectively. Blood samples and renal tissues were collected at the end of reperfusion. The renal function, histopathologic changes, and the expression levels of pro-inflammatory cytokines [interleukin-8（IL-8）, tumor necrosis factor alpha（TNF-α） and IL-6] in serum and renal tissue homogenate were assessed. Moreover, the effects of Fenofibrate on activating phosphoinositide 3 kinase/protein kinase B（PI3K/Akt） signaling and peroxisome proliferator-activated receptor-α（PPAR-α） were also measured in renal IRI. The results showed that plasma levels of blood urea nitrogen and creatinine, histopathologic scores and the expression levels of TNF-α, IL-8 and IL-6 were significantly lower in FEN group than in IRI group. Moreover, Fenofibrate pretreatment could further induce PI3K/Akt signal pathway and PPAR-α activation following renal IRI. These findings indicated PPAR-α activation by Fenofibrate exerts protective effects on renal IRI in mice by suppressing inflammation via PI3K/Akt activation. Thus, Fenofibrate could be a novel therapeutic alternative in renal IRI.

Poly(A)-specific ribonuclease protein promotes the proliferation,invasion and migration of esophageal cancer cells

BACKGROUND Bioinformatics analysis showed that the expression of the poly(A)-specific ribonuclease(PARN)gene in gastric cancer,head and neck squamous cell carcinoma,melanoma,cervical cancer and lung squamous cell carcinoma tissues was significantly higher than that in normal tissues and was associated with high stage and poor prognosis.The expression of the PARN gene in esophageal cancer(EC)tissue is also significantly higher than that in normal tissues,but the effect of PARN on the proliferation,migration and invasion of EC cells remains unclear.AIM To investigate the relationship between PARN and the proliferation,migration and invasion of EC cells.METHODS The EC tissues of 91 patients after EC surgery and 63 paired precancerous healthy tissues were collected.PARN mRNA levels were measured using a tissue microarray,and the PARN expression level was evaluated using immunohistochemistry to analyze the relationship between PARN expression and clinicopathologic features as well as the survival and prognosis of patients.In addition,the effects of PARN gene knockout on tumor cell proliferation,invasion and migration were studied by using shRNA during the in vitro culture of EC cell lines Eca-109 and TE-1,and the effects of the PARN gene on tumor growth in vivo were verified by a xenotransplantation nude mice model.RESULTS The expression of PARN in EC tissues was higher than that in adjacent normal tissues,and the level of PARN expression was significantly positively correlated with lymphatic metastasis.Patients with high PARN levels had poor overall survival.BIM,IGFBP-5 and p21 levels were significantly increased in the PARN knockout group,while the expression levels of the antiapoptotic proteins Survivin and sTNF-R1 were significantly decreased in the apoptotic antibody array data.In addition,the expression levels of Akt,p-Akt,PIK3CA and CCND1 in the downstream signaling pathway regulating EC progression were significantly decreased.The culture of EC cell lines confirmed that the apoptosis rate of EC cells was significantly increased,the growth and proliferation of tumor cells were significantly inhibited,and the invasion and migration ability of tumor cells were significantly decreased after PARN gene knockout.In vivo experiments of BALB/c nude mice transfected with Eca-109 cells expressing control shRNA(sh-NC)and PARN shRNA(sh-PARN)showed that the tumor volume and weight of nude mice treated with sh-PARN were significantly decreased compared with those of nude mice treated with sh-NC,indicating that PARN knockdown significantly inhibited tumor growth in vivo.CONCLUSION PARN has antiapoptotic effects on EC cells and promotes their proliferation,invasion and migration,which is associated with the development of EC and poor patient prognosis.PARN may become a potential target for the diagnosis,prognosis prediction and treatment of EC.

Cytotoxicity of nonylphenol on spermatogonial stem cells via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway

BACKGROUND With continuous advancement of industrial society,environmental pollution has become more and more serious.There has been an increase in infertility caused by environmental factors.Nonylphenol(NP)is a stable degradation product widely used in daily life and production and has been proven to affect male fertility.However,the underlying mechanisms therein are unclear.Thus,it is necessary to study the effect and mechanism of NP on spermatogonial stem cells(SSCs).AIM To investigate the cytotoxic effect of NP on SSCs via the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway.METHODS SSCs were treated with NP at 0,10,20 or 30μmol.MTT assay was performed to evaluate the effect of NP on the proliferation of SSCs.Flow cytometry was conducted to measure SSC apoptosis.The expression of Bad,Bcl-2,cytochrome-c,pro-Caspase 9,SOX-2,OCT-4,Nanog,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,PLZF and PI3K/AKT/mTOR-related proteins was observed by western blot,and the mRNA expression of SOX-2,OCT-4 and Nanog was detected by quantitative reverse transcription polymerase chain reaction.RESULTS Compared with untreated cells(0μmol NP),SSCs treated with NP at all concentrations showed a decrease in cell proliferation and expression of Bcl-2,Nanog,OCT-4,SOX-2,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,and PLZF(P<0.05),whereas the expression of Bad,cytochrome-c,and pro-Caspase 9 increased significantly(P<0.05).We further examined the PI3K/AKT/mTOR pathway and found that the phosphorylation of PI3K,AKT,mTORC1,and S6K was significantly decreased by NP at all concentrations compared to that in untreated SSCs(P<0.05).NP exerted the greatest effect at 30μmol among all NP concentrations.CONCLUSION NP attenuated the proliferation,differentiation and stemness maintenance of SSCs while promoting apoptosis and oxidative stress.The associated mechanism may be related to the PI3K/AKT/mTOR pathway.

Hippocampal activation of c-Jun N-terminal kinase,protein kinase B,and p38 mitogen-activated protein kinase in a chronic stress rat model of depression

Recent studies have shown that varied stress stimuli activate c-Jun N-terminal kinase （JNK）, protein kinase B （Akt）, and p38 mitogen-activated protein kinase （p38） signal transduction pathway, and also regulate various apoptotic cascades. JNK and p38 promote apoptosis, but Akt protects against apoptosis, in hippocampal neurons. However, changes in the transduction pathway in different regions of brain tissues in a chronic stress rat model of depression remain poorly understood. Results from this study showed that JNK phosphorylation levels were significantly greater in the stress group hippocampus compared with the control group （P 〈 0.05）. No significant difference in JNK phosphorylation levels was detected in the rat cerebral cortex between stress and control groups, and no significant difference in Akt and p38 phosphorylation levels was detected in the rat hippocampus and cerebral cortex between stress and control groups （P 〉 0.05）. These results suggested that the JNK signal pathway is activated by JNK phosphorylation and participates in pathophysiological changes in rat models of depression.

Xuebijing improves intestinal microcirculation dysfunction in septic rats by regulating the VEGF-A/PI3K/Akt signaling pathway

BACKGROUND:This study aims to explore whether Xuebijing(XBJ) can improve intestinal microcirculation dysfunction in sepsis and its mechanism.METHODS:A rat model of sepsis was established by cecal ligation and puncture(CLP).A total of 30 male SD rats were divided into four groups:sham group,CLP group,XBJ + axitinib group,and XBJ group.XBJ was intraperitoneally injected 2 h before CLP.Hemodynamic data(blood pressure and heart rate) were recorded.The intestinal microcirculation data of the rats were analyzed via microcirculation imaging.Enzyme-linked immunosorbent assay(ELISA) kits were used to detect the serum levels of interleukin-6(IL-6),C-reactive protein(CRP),and tumor necrosis factor-α(TNF-α) in the rats.Histological analysis and transmission electron microscopy were used to analyze the injury of small intestinal microvascular endothelial cells and small intestinal mucosa in rats.The expression of vascular endothelial growth factor A(VEGF-A),phosphoinositide 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(Akt),and phosphorylated Akt(p-Akt) in the small intestine was analyzed via Western blotting.RESULTS:XBJ improved intestinal microcirculation dysfunction in septic rats,alleviated the injury of small intestinal microvascular endothelial cells and small intestinal mucosa,and reduced the systemic inflammatory response.Moreover,XBJ upregulated the expression of VEGF-A,p-PI3K/total PI3K,and p-Akt/total Akt in the rat small intestine.CONCLUSION:XBJ may improve intestinal microcirculation dysfunction in septic rats possibly through the VEGF-A/PI3K/Akt signaling pathway.

Inhibition of EGFR attenuates EGF-induced activation of retinal pigment epithelium cell via EGFR/AKT signaling pathway

AIM:To explore the effect of epidermal growth factor receptor(EGFR)inhibition by erlotinib and EGFR siRNA on epidermal growth factor(EGF)-induced activation of retinal pigment epithelium(RPE)cells.METHODS:Human RPE cell line(ARPE-19 cells)was activated by 100 ng/mL EGF.Erlotinib and EGFR siRNA were used to intervene EGF treatment.Cellular viability,proliferation,and migration were detected by methyl thiazolyl tetrazolium(MTT)assay,bromodeoxyuridine(BrdU)staining assay and wound healing assay,respectively.EGFR/protein kinase B(AKT)pathway proteins and N-cadherin,α-smooth muscle actin(α-SMA),and vimentin were tested by Western blot assay.EGFR was also determined by immunofluorescence staining.RESULTS:EGF treatment for 24h induced a significant increase of ARPE-19 cells’viability,proliferation and migration,phosphorylation of EGFR/AKT proteins,and decreased total EGFR expression.Erlotinib suppressed ARPE-19 cells’viability,proliferation and migration through down regulating total EGFR and AKT protein expressions.Erlotinib also inhibited EGF-induced an increase of proliferative and migrative ability in ARPE-19 cells and clearly suppressed EGF-induced EGFR/AKT proteins phosphorylation and decreased expression of N-cadherin,α-SMA,and vimentin proteins.Similarly,EGFR inhibition by EGFR siRNA significantly affected EGF-induced an increase of cell proliferation,viability,and migration,phosphorylation of EGFR/AKT proteins,and up-regulation of N-cadherin,α-SMA,and vimentin proteins.CONCLUSION:Erlotinib and EGFR-knockdown suppress EGF-induced cell viability,proliferation,and migration via EGFR/AKT pathway in RPE cells.EGFR inhibition may be a possible therapeutic approach for proliferative vitreoretinopathy(PVR).

Mechanism of stilbene glycosides on apoptosis of SH-SY5Y cells via regulating PI3K/AKT signaling pathway

Objective:To investigate the effects of stilbene glycoside(TSG)on okadaic acid-induced apoptosis in human neuroblastoma cells(SH-SY5Y)via the PI3K/AKT pathway.Methods:The optimal concentration of OA was screened by CCK-8 assay,and SH-SY5Y cells were divided into control group,model group,TSG group,LY294002 group and LY294002+TSG group.The proliferation and apoptosis in each group were detected by CCK-8 and TUNEL assays;Western blotting method and real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of PI3K,P-PI3K(Y607),AKT,P-AKT(Ser473),Bcl-2 and Bax proteins.The relative protein expression was represented by P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax gray ratio.Results:CCK-8 screened the optimal concentration of OA as 40 nmol/L.Compared with the control group,the model group increased relative cell viability,decreased apoptosis rate,the pathway and apoptotic proteins expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were decreased,and the mRNA expression levels of PI3K,AKT and Bcl-2 were decreased.Bax mRNA expression level increased(P<0.05);Compared with model group,TSG group increased relative cell viability,decreased apoptosis rate,increased protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT,Bcl-2/Bax,and increased mRNA expression levels of PI3K,AKT,and Bcl-2.Bax mRNA expression decreased(P<0.05),LY294002 group decreased relative cell viability,increased apoptosis rate,P-PI3K(Y607)/PI3K protein expression levels were significantly decreased(P<0.05),P-AKT(Ser473)/AKT and Bcl-2/Bax protein expression levels were significantly decreased,but there was no statistical significance,PI3K,AKT and Bcl-2 mRNA expression levels were decreased,and Bax mRNA expression levels were increased(all P<0.05);Compared with LY294002 group,LY294002+TSG group increased relative cell viability,decreased apoptosis rate,and the protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were increased.The mRNA expression levels of PI3K,AKT,Bcl-2 were increased,Bax was decreased(all P<0.05).Conclusion:Stilbene glycoside may alleviate okadaic acid-induced apoptosis in SH-SY5Y cells by interfering with the PI3K/AKT signaling pathway,which in turn regulates the expression of apoptotic factors such as Bcl-2 and Bax.

Telencephalin protects PAJU cells from amyloid beta protein-induced apoptosis by activating the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway

Telencephalin is a neural glycoprotein that reduces apoptosis induced by amyloid beta protein in the human neural tumor cell line PAJU. In this study, we examined the role of the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway in this process. Western blot analysis demonstrated that telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B were not expressed in PAJU cells transfected with empty plasmid, while they were expressed in PAJU cells transfected with a telencephalin expression plasmid. After treatment with 1.0 nM amyloid beta protein 42, expression of telencephalin and phosphorylated phosphatidylinositol-3-kinase/protein kinase B in the transfected cells gradually diminished, while levels of phosphorylated ezrin/radixin/moesin increased. In addition, the high levels of telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B expression in PAJU cells transfected with a telencephalin expression plasmid could be suppressed by the phosphatidylinositol-3-kinase inhibitor LY294002. These findings indicate that telencephalin activates the ezrin/radixin/moesin family/phosphatidylinositol-3-kinase/protein kinase B pathway and protects PAJU cells from amyloid beta protein-induced apoptosis.

Salvianolate increases heat shock protein expression in a cerebral ischemia-reperfusion injury model

Stroke remains a worldwide health problem. Salvianolate exerts a protective effect in various mi- crocirculatory disturbance-related diseases, but studies of the mechanisms underlying its protective action have mainly focused on the myocardium, whereas little research has been carried out in brain tissue following ischemia-reperfusion. We assessed the neuroprotective effects of salvianolate in a rat model of cerebral ischemia-reperfusion injury induced using the suture method. At onset and 24 and 48 hours after reperfusion, rats were intraperitoneally injected with salvianolate （18 mg/kg） or saline. Neurological deficit scores at 72 hours showed that the neurological functions of rats that had received salvianolate were significantly better than those of the rats that had received saline. 2,3,5-Triphenyltetrazolium chloride was used to stain cerebral tissue to determine the extent of the infarct area. A significantly smaller infarct area and a significantly lower number of apoptotic cells were observed after treatment with salvianolate compared with the saline treatment. Expression of heat shock protein 22 and phosphorylated protein kinase B in ischemic brain tissue was significantly greater in rats treated with salvianolate compared with rats treated with saline. Our findings suggest that salvianolate provides neuroprotective effects against cerebral ischemia-reperfusion injury by upregulating heat shock protein 22 and phosphorylated protein kinase B expression.

Y-box binding protein 1 augments sorafenib resistance via the PI3K/Akt signaling pathway in hepatocellular carcinoma

BACKGROUND Sorafenib is the first-line treatment for patients with advanced hepatocellular carcinoma(HCC).Y-box binding protein 1(YB-1)is closely correlated with tumors and drug resistance.However,the relationship between YB-1 and sorafenib resistance and the underlying mechanism in HCC remain unknown.AIM To explore the role and related mechanisms of YB-1 in mediating sorafenib resistance in HCC.METHODS The protein expression levels of YB-1 were assessed in human HCC tissues and adjacent nontumor tissues.Next,we constructed YB-1 overexpression and knockdown hepatocarcinoma cell lines with lentiviruses and stimulated these cell lines with different concentrations of sorafenib.Then,we detected the proliferation and apoptosis in these cells by terminal deoxynucleotidyl transferase dUTP nick end labeling,flow cytometry and Western blotting assays.We also constructed a xenograft tumor model to explore the effect of YB-1 on the efficacy of sorafenib in vivo.Moreover,we studied and verified the specific molecular mechanism of YB-1 mediating sorafenib resistance in hepatoma cells by digital gene expression sequencing(DGE-seq).RESULTS YB-1 protein levels were found to be higher in HCC tissues than in corresponding nontumor tissues.YB-1 suppressed the effect of sorafenib on cell proliferation and apoptosis.Consistently,the efficacy of sorafenib in vivo was enhanced after YB-1 was knocked down.Furthermore,KEGG pathway enrichment analysis of DGEseq demonstrated that the phosphoinositide-3-kinase(PI3K)/protein kinase B(Akt)signaling pathway was essential for the sorafenib resistance induced by YB-1.Subsequently,YB-1 interacted with two key proteins of the PI3K/Akt signaling pathway(Akt1 and PIK3R1)as shown by searching the BioGRID and HitPredict websites.Finally,YB-1 suppressed the inactivation of the PI3K/Akt signaling pathway induced by sorafenib,and the blockade of the PI3K/Akt signaling pathway by LY294002 mitigated YB-1-induced sorafenib resistance.CONCLUSION Overall,we concluded that YB-1 augments sorafenib resistance through the PI3K/Akt signaling pathway in HCC and suggest that YB-1 is a key drug resistance-related gene,which is of great significance for the application of sorafenib in advanced-stage HCC.

Expression of phosphatidylinositol-3 kinase and effects of inhibitor Wortmannin on expression of tumor necrosis factor-α in severe acute pancreatitis associated with acute lung injury

BACKGROUND: Acute lung injury(ALI) is a common and serious complication of severe acute pancreatitis(SAP). The study aimed to investigate the protective effect and mechanism of phosphatidylinositol-3 kinase(PI3K) inhibitor Wortmannin in SAP associated with ALI.METHODS: Ninety rats were randomly divided into three groups: sham operation(SO) group(n=30), SAP group(n=30), and SAP+Wortmannin(SAP+W) group(n=30). SAP model was induced by retrograde injection of 4% sodium taurocholate into the biliopancreatic duct of rats. The rate of lung water content, myeloperoxidase(MPO), matrix metalloproteinase 9(MMP-9), protein kinase B(PKB), abdphosphorylation of protein kinase B(P-PKB) activity in the lung tissue were evaluated.RESULTS: In the SAP group, the p-PKB expression in the lung tissue began to rise at 3 hours after modeling, and peaked at 12 hours(P<0.05); the rate of lung water content, MPO and TNF-α activity were also gradually increased, and the degree of lung lesion gradually increased(P<0.05). In the SAP+Wortmannin group, the p-PKB expression in the lung tissue began to rise at 3 hours after modeling, and peaked at 12 hours; it was higher than that in the SO group(P<0.05), but significantly lower than that in the SAP group(P<0.05). The rest indicators in the SAP+Wortmannin group were also significantly decreased as compared with the SAP group(P<0.05).CONCLUSIONS: The expression of phosphatidylinositol-3 kinase/protein kinase B was elevated in severe pancreatitis rats with lung injury. This suggested that PI3 K signal transduction pathway is involved in the control and release of proinfl ammatory cytokines TNF-α, which may play an important role in the pathogenesis of severe acute pancreatitis associated with lung injury. This finding indicated that Wortmannin can block the PI3 K signal transduction pathway, and inhibit the release of infl ammatory factor TNF-α.

AB020.Inhibition of cyclic-AMP-response element binding protein and its impact on corneal wound healing in vitro and in vivo

Background:The cornea composes the outer surface of the eye and its transparency is required to allow light transmission to the retina.However,because of its position,the cornea is subjected to chemical and mechanical injuries that may lead to blindness.Our studies conducted using the human tissue-engineered cornea(hTEC)as a model provided evidence that the cyclic-AMP-response element binding protein(CREB)pathway is repressed during closure of corneal wounds.Based on these results,we hypothesized that closure of corneal wounds can be enhanced by preventing activation of CREB with the pharmacological inhibitor C646.Our goals were to proceed to the pharmacological inhibition of CREB(I)in vitro using the hTECs as a model,and then(II)in vivo using the rabbit as a model.Methods:The self-assembly approach was used to create hTECs,that were then wounded with an 8-mm diameter biopsy punch to create an epithelial defect.The tissues were then incubated with 10μM of C646(n=8).DMSO was used alone as a negative control(n=4).Closure of the wounds was monitored over a period of 5 days.Besides,the cornea of New Zealand white rabbits was debrided with an ethanol 70%solution to create an epithelial defect of 8-mm diameter.Several concentrations of C646(1,10,100μM et 1 mM)were applied as eye drops 3 times a day for up to 7 days.The wounded corneas(n=4 per concentration)were stained with fluorescein and photographed every day.Results:In vitro pharmacological inhibition of CREB with C646 considerably accelerated wound closure of all treated hTECs(4 days)compared to the control group(7 days).Moreover,the in vivo C646 treatment also accelerated wound healing of the corneas compared to the control group.The most effective concentration of C646 tested was the lowest(1μM),as it considerably enhanced the wound healing process.Conclusions:This study demonstrates that wound healing both in vitro and in vivo can be enhanced by preventing activation of CREB using a pharmacological inhibition approach.Most of all,this experiment suggests mediators from the CREB pathway as potential therapeutic targets on which we may influence to alter the wound healing dynamic of the cornea.We believe this study will lead to significant advancements in the clinical field of corneal defects.

Specific effects of c-Jun NH2-terminal kinaseinteracting protein 1 in neuronal axons

c-Jun NH2-terminal kinase（JNK）-interacting protein 3 plays an important role in brain-derived neurotrophic factor/tropomyosin-related kinase B（Trk B） anterograde axonal transport. It remains unclear whether JNK-interacting protein 1 mediates similar effects, or whether JNK-interacting protein 1 affects the regulation of Trk B anterograde axonal transport. In this study, we isolated rat embryonic hippocampus and cultured hippocampal neurons in vitro. Coimmunoprecipitation results demonstrated that JNK-interacting protein 1 formed Trk B complexes in vitro and in vivo. Immunocytochemistry results showed that when JNK-interacting protein 1 was highly expressed, the distribution of Trk B gradually increased in axon terminals. However, the distribution of Trk B reduced in axon terminals after knocking out JNK-interacting protein 1. In addition, there were differences in distribution of Trk B after JNK-interacting protein 1 was knocked out compared with not. However, knockout of JNK-interacting protein 1 did not affect the distribution of Trk B in dendrites. These findings confirm that JNK-interacting protein 1 can interact with Trk B in neuronal cells, and can regulate the transport of Trk B in axons, but not in dendrites.

Bornyl acetate extracted from Sharen(Fructus Amomi)inhibits proliferation,invasion and induces apoptosis by suppressing phosphatidylinositol-3-kinase/protein kinase B signaling in colorectal cancer

OBJECTIVE:To investigate the antitumor effects of bornyl acetate(BA)isolated from Sharen(Fructus Amomi)in colorectal cancer(CRC)and the underlying mechanisms.METHODS:SW480 and HT29 cells were treated with increasing doses of BA in order to determine its antitumor effects in vitro.Cell viability,colony formation,cell cycle,and apoptosis as well as migration and invasion were assessed using various assays.In addition,the in vivo antitumor effects of BA were assessed using a xenograft mouse model.We then assessed the mechanism of action of BA by conducting pathway activator-mediated rescue experiments and assessed the protein levels by Western blot analysis.RESULTS:BA showed anti-CRC tumor activities in vitro by suppressing cell proliferation and colony formation,inducing apoptosis,blocking cell cycle,and inhibiting migration and invasion.These effects were mediated via suppression of the phosphatidylinositol-3-kinase/protein kinase B(PI3K/AKT)pathway.In the tumor xenograft experiment,BA was found to repress tumor growth in vivo with low toxicity.CONCLUSIONS:The results demonstrated that BA exerts antitumor effects by suppressing the PI3K/AKT pathway,with low toxicity.Thus,BA might be a potential novel therapeutic agent for CRC.

Ginkgo biloba leaf extract improves the cognitive abilities of rats with D-galactose induced dementia

Standardized Ginkgo biloba leaf extract has been used in clinical trials for its beneficial effects on brain func- tions, particularly in dementia. Substantial experimental evidences indicated that Ginkgo biloba leaf extract （EGB） protected neuronal cells from a variety of insults. We investigated the effect of EGB on cognitive ability and protein kinase B （PKB） activity in hippocampal neuronal cells of dementia model rats. Rats received an intra- peritoneal injection of D-galactose to induce dementia. Forty-eight Spraque-Dawley rats were randomly divided into six groups, including the control group, D-galactose group （Gal）, low-dose EGB group （EGB-L）, mid-dose EGB group （EGB-M）, high-dose EGB group （EGB-H） and treatment group. The EGB-L, EGB-M and EGB-H groups were administered with EGB and D-galactose simultaneously. Y-maze, cresyl violet staining, TUNEL assays and immunohistochemistry staining were performed to detect learning and memory abilities, morpho- logical changes in the hippocampus, neuronal apoptosis and the expressing level of phospho-PKB, respectively. Rats in the Gal group showed decreased abilities of learning and memory, and hippocampal pyramidal cell layer was damaged, while EGB administration improved learning and memory abilities. The Gal group exhibited many stained, condensed nuclei and micronuclei, either isolated or within the cytoplasm of cells （39.5 ± 1.4）. Apoptotic cells decreased in the groups of EGB-L （35.9±0.9）, EGB-M （16.8± 1.0） and EGB-H （10.1±0.8）, and there were statistical significances compared with the Gal group. Immunoreactivity of phospho-PKB was localized diffusely throughout the cytosol of cells in all groups, while the immunoreactivity of the Gal group was weak. EGB signifi- cantly attenuated learning and memory impairment in a dose-dependent manner, while it could decrease the nmber of TUNEL-positive cells, and increase the activity of PKB. Our results demonstrated that EGB attenuated memory impairment and cell apoptosis in galactose-induced dementia model rats by activating PKB.

Insensitivity of PI3K/Akt/GSK3 signaling in peripheral blood mononuclear cells of age-related macular degeneration patients

Our recent studies with cultured retinal pigment epithelium cells suggested that overexpression of interleukin 17 receptor C（IL-17RC）,a phenomenon observed in peripheral blood and chorioretinal tissues with age-related macular degeneration（AMD）,was associated with altered activation of phosphatidylinositide 3-kinase（PI3K）,Akt,and glycogen synthase kinase 3（GSK3）.We wondered whether or not altered PI3 K,Akt,and GSK3 activities could be detected in peripheral blood mononuclear cells（PBMC） obtained from AMD patients.In the patients＇ PBMC,absent or reduced serine-phosphorylation of GSK3α or GSK3β was observed,which was accompanied with increased phosphorylation of GSK3 substrates（e.g.CCAAT enhancer binding protein a,insulin receptor substrate 1,and TAU）,indicative of enhanced GSK3 activation.In addition,decreased protein mass of PI3K85α and tyrosinephosphorylation of PI3K50α was present in PBMC of the AMD patients,suggesting impaired PI3 K activation.Moreover,abnormally lowered molecular weight forms of Akt and GSK3 were detected in PBMC of the AMD patients.These data demonstrate that despite the presence of high levels of IL-17 RC,Wnt-3a and vascular endothelial growth factor,the PI3K/Akt/GSK3 signaling pathway is insensitive to these stimuli in PBMC of the AMD patients.Thus,measurement of PI3K/Akt/GSK3 expression and activity in PBMC may serve as a surrogate biomarker for AMD.

LY294002 Enhances Inhibitory Effect of Gemcitabine on Proliferation of Human Pancreatic Carcinoma PANC-1 Cells

Phosphatidylinositide 3-kinase （PI3K）/protein kinase B （PKB, Akt） pathway plays a major role in proliferation and survival of many types of cells. The inhibitory effect of LY294002, widely ap- plied as an inhibitor of PI3K, in combination with gemcitabine on proliferation of PANC-1 ceils was investigated. The expression of PI3K, phosphorylated AM （p-Akt） and multidrng-resistance like protein （MRP） in normal pancreas tissues, chronic pancreatitis tissues and pancreatic carcinoma tissues was de- tected. The effects of LY294002 combined with gemcitabine on proliferation of PANC-1 cells and pro- tein levels of p-Akt and MRP were detected. The results showed that the positive expression rate of PI3K, p-Akt and MRP in pancreatic carcinoma tissues was significantly higher than that in normal pan- creas tissues and chronic pancreatitis tissues （P〈0.01 and P〈0.05 respectively）. LY294002 could effec- tively enhance the inhibitory effect of gemcitabine on proliferation of PANC-1 cells. Furthermore, Western blotting revealed that LY294002 combined with gemcitabine reduced the protein levels of p-Akt and MRP, which contributed to the inhibition of proliferation. It is concluded that LY294002 in combination with gemcitabine may represent an alternative therapy for pancreatic carcinoma.

Regulatory Effects of Zuogui Pill on Apoptosis of Follicles in Rats Injured by 60Co-γRays Based on PI3K/Akt/m TOR Signaling Pathway

[Objectives]To explore the protective effects of Zuogui Pill on ^(60)Co-γ-ray-induced premature aging of rats based on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway.[Methods]Sixty sexually mature female SD rats were irradiated with ^(60)Co-γ-ray(6.0 Gy,LD 40)for 24 h at one time.These rats were randomly divided into model group,Progynova group[0.18(g·kg)/d],Progynova[0.09(g·kg)/d]+Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill medium dose[9.45(g·kg)/d)]group and Zuogui Pill low dose[4.725(g·kg)/d]group.The administration(once a day)lasted 21 d.The rat serum[follicle-stimulating hormone(FSH),luteinizing hormone(LH)and estradiol(E_(2))]were detected by Enzyme-linked immunosorbent assay(ELISA).The morphological changes of ovary were observed by hematoxylin-eosin(HE)staining.The apoptosis rate of granulosa cells was detected by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL).The protein expression of phosphorylated(p)-PI3K,p-Akt,p-mTOR,B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax)in ovarian tissues were detected by Western blot.[Results]Compared with the normal group,the model group showed significant increase in the serum FSH(P<0.01),significant decrease in serum E_(2)(P<0.05),and decrease in the number of early follicles and luteum in the ovary(P<0.01).Besides,the apoptosis rate of granulosa cells increased significantly(P<0.01);the expression of p-PI3K,p-Akt,p-mTOR and Bcl-2 in ovarian tissue decreased significantly,while the expression of Bax increased significantly(P<0.01).Compared with the model group,the number of early follicles in the ovary increased and the apoptosis rate of granulosa cells decreased after intervention in each administration group.In addition,the protein expressions of p-PI3K,p-Akt,p-mTOR and Bcl-2 increased,while the expression of Bax decreased,especially in Progynova+Zuogui Pill high dose group,the differences were statistically significant(P<0.05,P<0.01).[Conclusions]Zuogui Pill may protect the radiation-injured ovary through activating the expression of PI3K/Akt/mTOR protein in ovarian tissue,increasing the amount of Bcl-2 protein and inhibiting the expression of Bax protein.

Regulation of enolase activation to promote neural protection and regeneration in spinal cord injury

Spinal cord injury(SCI)is a debilitating condition characterized by damage to the spinal cord resulting in loss of function,mobility,and sensation with no U.S.Food and Drug Administration-approved cure.Enolase,a multifunctional glycolytic enzyme upregulated after SCI,promotes pro-and anti-inflammatory events and regulates functional recovery in SCI.Enolase is normally expressed in the cytosol,but the expression is upregulated at the cell surface following cellular injury,promoting glial cell activation and signal transduction pathway activation.SCI-induced microglia activation triggers pro-inflammatory mediators at the injury site,activating other immune cells and metabolic events,i.e.,Rho-associated kinase,contributing to the neuroinflammation found in SCI.Enolase surface expression also activates cathepsin X,resulting in cleavage of the C-terminal end of neuron-specific enolase(NSE)and non-neuronal enolase(NNE).Fully functional enolase is necessary as NSE/NNE C-terminal proteins activate many neurotrophic processes,i.e.,the plasminogen activation system,phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B,and mitogen-activated protein kinase/extracellular signal-regulated kinase.Studies here suggest an enolase inhibitor,ENOblock,attenuates the activation of Rho-associated kinase,which may decrease glial cell activation and promote functional recovery following SCI.Also,ENOblock inhibits cathepsin X,which may help prevent the cleavage of the neurotrophic C-terminal protein allowing full plasminogen activation and phosphatidylinositol-4,5-bisphosphate 3-kinase/mitogen-activated protein kinase activity.The combined NSE/cathepsin X inhibition may serve as a potential therapeutic strategy for preventing neuroinflammation/degeneration and promoting neural cell regeneration and recovery following SCI.The role of cell membrane-expressed enolase and associated metabolic events should be investigated to determine if the same strategies can be applied to other neurodegenerative diseases.Hence,this review discusses the importance of enolase activation and inhibition as a potential therapeutic target following SCI to promote neuronal survival and regeneration.