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.

Protective effects of panax notoginseng saponin on dextran sulfate sodium-induced colitis in rats through phosphoinositide-3-kinase protein kinase B signaling pathway inhibition

BACKGROUND Intestinal inflammation is a common digestive tract disease, which is usually treated with hormone medicines. Hormone medicines are effective to some extent, but long-term use of them may bring about many complications.AIM To explore the protective effects of panax notoginseng saponin(PNS) against dextran sulfate sodium(DSS)-induced intestinal inflammatory injury through phosphoinositide-3-kinase protein kinase B(PI3K/AKT) signaling pathway inhibition in rats.METHODS Colitis rat models were generated via DSS induction, and rats were divided into control(no modeling), DSS, DSS + PNS 50 mg/k, and DSS + PNS 100 mg/kg groups. Then, the intestinal injury, oxidative stress parameters, inflammatory indices, tight junction proteins, apoptosis, macrophage polarization, and TLR4/AKT signaling pathway in colon tissues from rats in each of the groups were detected. The PI3 K/AKT signaling pathway in the colon tissue of rats was blocked using the PI3K/AKT signaling pathway inhibitor, LY294002.RESULTS Compared with rats in the control group, rats in the DSS group showed significantly shortened colon lengths, and significantly increased disease activity indices, oxidative stress reactions and inflammatory indices, as well as significantly decreased expression of tight junction-associated proteins. In addition, the DSS group showed significantly increased apoptotic cell numbers,and showed significantly increased M1 macrophages in spleen and colon tissues.They also showed significantly decreased M2 macrophages in colon tissues, as well as activation of the PI3K/AKT signaling pathway(all P < 0.05). Compared with rats in the DSS group, rats in the DSS + PNS group showed significantly lengthened colon lengths, decreased disease activity indices, and significantly alleviated oxidative stress reactions and inflammatory responses. In addition, this group showed significantly increased expression of tight junction-associated proteins, significantly decreased apoptotic cell numbers, and significantly decreased M1 macrophages in spleen and colon tissues. This group further showed significantly increased M2 macrophages in colon tissues, and significantly suppressed activation of the PI3K/AKT signaling pathway, as well as a dose dependency(all P < 0.05). When the PI3K/AKT signaling pathway was inhibited, the apoptosis rate of colon tissue cells in the DSS + LY294002 group was significantly lower than that of the DSS group(P < 0.05).CONCLUSION PNS can protect rats against DSS-induced intestinal inflammatory injury by inhibiting the PI3K/AKT signaling pathway, and therefore may be potentially used in the future as a drug for colitis.

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.

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.

Impaired PI3K/Akt signal pathway and hepatocellular injury in high-fat fed rats

AIM:To determine whether mitochondrial dysfunction resulting from high-fat diet is related to impairment of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt,also known as PKB) pathway. METHODS:Rat models of nonalcoholic fatty liver were established by high-fat diet feeding. The expression of total and phosphorylated P13K and Akt proteins in hepatocytes was determined by Western blotting. Degree of fat accumulation in liver was measured by hepatic triglyceride. Mitochondrial number and size were determined using quantitative morphometric analysis under transmission electron microscope. The permeability of the outer mitochondrial membrane was assessed by determining the potential gradient across this membrane.RESULTS:After Wistar rats were fed with high-fat diet for 16 wk,their hepatocytes displayed an accumulation of fat (103.1 ± 12.6 vs 421.5 ± 19.7,P < 0.01),deformed mitochondria (9.0% ± 4.3% vs 83.0% ± 10.9%,P < 0.05),and a reduction in the mitochondrial membrane potential (389.385% ± 18.612% vs 249.121% ± 13.526%,P < 0.05). In addition,the expression of the phosphorylated P13K and Akt proteins in hepatocytes was reduced,as was the expression of the anti-apoptotic protein Bcl-2,while expression of the pro-apoptotic protein caspase-3 was increased. When animals were treated with pharmacological inhibitors of P13K or Akt,instead of high-fat diet,a similar pattern of hepatocellular fat accumulation,mitochondrial impairment,and change in the levels of PI3K,Akt,Bcl-2 was observed. CONCLUSION:High-fat diet appears to inhibit the PI3K/Akt signaling pathway,which may lead to hepa-tocellular injury through activation of the mitochondrial membrane pathway of apoptosis.

Influence of Phosphatidylinositol-3-Kinase/Protein Kinase B-Mammalian Target of Rapamycin Signaling Pathway on the Neuropathic Pain Complicated by Nucleoside Reverse Transcriptase Inhibitors for the Treatment of HIV Infection

Background： Nucleoside reverse transcriptase inhibitors （NRTIs） are the earliest and most commonly used anti-human immunodeficiency virus drugs and play an important role in high active antiretroviral therapy. However, NRTI drug therapy can cause peripheral neuropathic pain. In this study, we aimed to investigate the mechanisms ofrapamycin on the pain sensitization of model mice by in vivo experiments to explore the effect of mammalian target of rapamycin （mTOR） in the pathogenesis ofneuropathic pain caused by NRTIs. Methods： Male Kun Ming （KM） mice weighing 20-2 g were divided into control, 2 mg/kg rapamycin, 12 mg/kg stavudine, and CMC-Na groups. Drugs were orally administered to mice for 42 consecutive days. The von Frey filament detection and thermal pain tests were conducted on day 7, 14, 21, 28, 35, and 42 after drug administration. After the last behavioral tests, immunohistochemistry and western blotting assay were used for the measurement of mTOR and other biomarkers. Multivariate analysis of variance was used. Results： The beneficial effects ofrapamycin on neuropathic pain were attributed to a reduction in mammalian target of rapamycin sensitive complex 1 （mTORC1）-positive cells （70.80± 2.41 vs. 112.30 ± 5.66, F = 34.36, P 〈 0.01 ） and mTORC1 activity in the mouse spinal cord. Mechanistic studies revealed that Protein Kinase B （Akt）/mTOR signaling pathway blockade with rapamycin prevented the phosphorylation of mTORC1 in stavudine-intoxicated mice （0.72 ± 0.04 vs. 0.86 ± 0.03, F=4.24, P = 0.045）, as well as decreased the expression of phospho-pTOS6K （0.47 ± 0.01 vs. 0.68 ± 0.03, F=6.01, P = 0.022） and phospho-4EBP1 （0.90 ± 0.04 vs. 0.94 ± 0.06, F= 0.28, P = 0.646）. Conclusions： Taken together, these results suggest that stavudine elevates the expression and activity of mTORC1 in the spinal cord through activating the Akt/mTOR signaling pathway. The data also provide evidence that rapamycin might be useful for the treatment of peripheral neuropathic pain.

Bone marrow-derived mesenchymal stem cells modulate autophagy in RAW264.7 macrophages via the phosphoinositide 3-kinase/protein kinase B/heme oxygenase-1 signaling pathway under oxygen-glucose deprivation/restoration conditions

Background: Autophagy of alveolar macrophages is a crucial process in ischemia/reperfusion injury-induced acute lung injury (ALI). Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent cells with the potential for repairing injured sites and regulating autophagy. This study was to investigate the influence of BM-MSCs on autophagy of macrophages in the oxygen-glucose deprivation/restoration (OGD/R) microenvironment and to explore the potential mechanism.Methods: We established a co-culture system of macrophages (RAW264.7) with BM-MSCs under OGD/R conditionsin vitro. RAW264.7 cells were transfected with recombinant adenovirus (Ad-mCherry-GFP-LC3B) and autophagic status of RAW264.7 cells was observed under a fluorescence microscope. Autophagy-related proteins light chain 3 (LC3)-I, LC3-II, and p62 in RAW264.7 cells were detected by Western blotting. We used microarray expression analysis to identify the differently expressed genes between OGD/R treated macrophages and macrophages co-culture with BM-MSCs. We investigated the gene heme oxygenase-1 (HO-1), which is downstream of the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway.Results: The ratio of LC3-II/LC3-I of OGD/R treated RAW264.7 cells was increased (1.27 ± 0.20vs. 0.44 ± 0.08,t = 6.67,P < 0.05), while the expression of p62 was decreased (0.77 ± 0.04vs. 0.95 ± 0.10,t = 2.90,P < 0.05), and PI3K (0.40 ± 0.06vs. 0.63 ± 0.10,t = 3.42,P < 0.05) and p-Akt/Akt ratio was also decreased (0.39 ± 0.02vs. 0.58 ± 0.03,t = 9.13,P < 0.05). BM-MSCs reduced the LC3-II/LC3-I ratio of OGD/R treated RAW264.7 cells (0.68 ± 0.14vs. 1.27 ± 0.20,t = 4.12,P < 0.05), up-regulated p62 expression (1.10 ± 0.20vs. 0.77 ± 0.04,t = 2.80,P < 0.05), and up-regulated PI3K (0.54 ± 0.05vs. 0.40 ± 0.06,t = 3.11,P < 0.05) and p-Akt/Akt ratios (0.52 ± 0.05vs. 0.39 ± 0.02,t = 9.13,P < 0.05). A whole-genome microarray assay screened the differentially expressed geneHO-1, which is downstream of the PI3K/Akt signaling pathway, and the alteration ofHO-1 mRNA and protein expression was consistent with the data on PI3K/Akt pathway.Conclusions: Our results suggest the existence of the PI3K/Akt/HO-1 signaling pathway in RAW264.7 cells under OGD/R circumstancesin vitro, revealing the mechanism underlying BM-MSC-mediated regulation of autophagy and enriching the understanding of potential therapeutic targets for the treatment of ALI.

Puerarin partly counteracts the inflammatory response after cerebral ischemia/reperfusion via activating the cholinergic anti-inflammatory pathway

Puerarin, a major isoflavonoid derived from the Chinese medical herb radix puerariae （Gegen）, has been reported to inhibit neuronal apoptosis and play an anti-inflammatory role in focal cerebral ischemia model rats. Recent findings regarding stroke pathophysiology have recognized that anti-inflammation is an important target for the treatment of ischemic stroke. The cholinergic anti-inflammatory pathway is a highly robust neural-immune mechanism for inflammation control. This study was to investigate whether activating the cholinergic anti-inflammatory pathway can be involved in the mechanism of inhibiting the inflammatory response during puerarin-induced cerebral ischemia/reperfusion in rats. Results showed that puerarin pretreatment （intravenous injection） re- duced the ischemic infarct volume, improved neurological deficit after cerebral ischemia/reperfusion and decreased the levels of interleukin-1β, interleukin-6 and tumor necrosis factor-a in brain tissue. Pretreatment with puerarin （intravenous injection） attenuated the inflammatory response in rats, which was accompanied by janus-activated kinase 2 （JAK2） and signal transducers and activators of transcription 3 （STAT3） activation and nuclear factor kappa B （NF-KB） inhibition. These observa- tions were inhibited by the alpha7 nicotinic acetylcholine receptor （a7nAchR） antagonist a-bungarotoxin （a-BGT）. In addition, puerarin pretreatment increased the expression of a7nAchR mRNA in ischemic cerebral tissue. These data demonstrate that puerarin pretreatment strongly protects the brain against cerebral ischemia/reperfusion injury and inhibits the inflammatory re- sponse. Our results also indicated that the anti-inflammatory effect of puerarin may partly be medi- ated through the activation of the cholinergic anti-inflammatory pathway.

Treating non-small cell lung cancer by targeting the PI3K signaling pathway

The phosphosphatidylinositol-3-kinase(PI3K)signaling pathway is one of the most important intracellular signal transduction pathways affecting cell functions,such as apoptosis,translation,metabolism,and angiogenesis.Lung cancer is a malignant tumor with the highest morbidity and mortality rates in the world.It can be divided into two groups,non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).NSCLC accounts for>85%of all lung cancers.There are currently many clinical treatment options for NSCLC;however,traditional methods such as surgery,chemotherapy,and radiotherapy have not been able to provide patients with good survival benefits.The emergence of molecular target therapy has improved the survival and prognosis of patients with NSCLC.In recent years,there have been an increasing number of studies on NSCLC and PI3K signaling pathways.Inhibitors of various parts of the PI3K pathway have appeared in various phases of clinical trials with NSCLC as an indication.This article focuses on the role of the PI3K signaling pathway in the occurrence and development of NSCLC and summarizes the current clinical research progress and possible development strategies.

Integration of network and experimental pharmacology to decipher the antidiabetic action of Duranta repens L.

Objective:Duranta repens is reported to contain a wide array of secondary metabolites,including aamylase and a-glucosidase inhibitors,and-has potent antioxidant activity.The present study evaluated the network pharmacology of D.repens(whole plant)with targets related to diabetes mellitus and assessed its outcome by evaluating the effects of the hydroalcoholic extract of D.repens in streptozotocin-nicotinamide-induced diabetes mellitus in rats.Methods:Phytoconstituents of D.repens were retrieved from an open-source database and published literature,and their targets were predicted for diabetes mellitus using Binding DB and the therapeutic target database.Protein-protein interaction was predicted using STRING,and pathways involved in diabetes mellitus were identified using the Kyoto Encyclopedia of Genes and Genomes pathway browser.Druglikeness,ADMET profile(absorption,distribution,metabolism,excretion and toxicity)and cytotoxicity of compounds modulating proteins involved in diabetes were predicted using Mol Soft,admet SAR2.0 and CLC-Pred,respectively.The interaction network among phytoconstituents,proteins and pathways was constructed using Cytoscape,and the docking study was performed using Auto Dock4.0.The hydroalcoholic extract of D.repens was evaluated using streptozotocin-nicotinamide-induced diabetes mellitus animal model for 28 d,followed by an oral glucose tolerance test.At the end of the study,biochemical parameters like glycogen content,hepatic enzymes,antioxidant biomarkers and lipid profiles were quantified.Further,the liver and pancreas were collected for a histopathology study.Results:Thirty-six different secondary metabolites from D.repens were identified to regulate thirty-one targets involved in diabetes mellitus,in which protein-tyrosine phosphatase 1 B(PTP1 B)was primarily targeted.Enrichment analysis of modulated proteins identified 12 different pathways in diabetic pathogenesis in which the phosphatidylinositol 3-kinase-protein kinase B(PI3 K-Akt)signaling pathway was chiefly regulated.The docking study found that durantanin I possessed the highest binding affinity(à8.9 kcal/mol)with PTP1 B.Similarly,ADMET profiling showed that the majority of bioactive constituents from D.repens had higher human intestinal absorptivity and minimal cytotoxicity to normal cell lines,than tumor cell lines.Further,an in vivo animal study reflected the efficacy of the hydroalcoholic extract of D.repens to lower the elevated blood glucose level by stimulating insulin secretion,maintaining pancreatic b cell mass,regulating glycolysis/gluconeogenesis and enhancing the glucose uptake in skeletal muscles.Conclusion:The present study reflected the probable network interaction of bioactive constituents from D.repens,their targets and modulated pathways,which identified the prime regulation of the PI3 K-Akt signaling pathway and PTP1 B protein.Modulation of PTP1 B protein and PI3 K-Akt signaling pathway could contribute to enhancing glucose uptake,insulin production and glycolysis and decreasing gluconeogenesis in diabetes,which was evaluated via the experimental study.