BACKGROUND: Parkinson's disease is a neurodegenerative disorder, and recent studies suggested that oxidative stress contributes to the degeneration of dopamine cell in Parkinson's disease. Glutamine also has a positive role in reducing oxidative stress damage. In this study, we hypothesized that glutamine offers protection against oxidative stress injury in 1-methyl-4-phenylpyridinium (MPP)-induced Parkinson's disease cell model. METHODS: MPP was used to induce PD models in PC12 cells and classified into control, M0 (MPP), G0 (glutamine), and M0+G0 groups. CCK-8 and AO/EB staining assays were used to examine cell proliferation and apoptosis, respectively. Western blotting was applied to examine the protein expression of PI3K, P-Akt, Akt, P-mTOR, and mTOR. RESULTS: We showed that glutamine suppressed cytotoxicity induced by MPP in PC12 cells. MPP decreased the superoxide dismutase and glutathione peroxidase activity and increased the malondialdehyde content, which were restored by glutamine. Moreover, MPP increased the expression of PI3K, P-Akt, Akt, P-mTOR, and mTOR, which were inhibited by glutamine. And the antioxidant capacity of glutamine on PC12 cells could be improved by LY294002 and inhibited by IGF-1. CONCLUSION These results suggest that glutamine strengthens the antioxidant capacity in PC12 cells induced by MPP through inhibiting the activation of the PI3K/Akt signaling pathway. The effects of glutamine should be investigated and the protective mechanism of glutamine in PD must be explored in future studies.
1-Methyl-4-phenylpyridinium ; administration & dosage ; Analysis of Variance ; Animals ; Cell Culture Techniques ; Disease Models, Animal ; Glutamine ; pharmacology ; Oxidative Stress ; drug effects ; Parkinson Disease ; Phosphatidylinositol 3-Kinases ; metabolism ; Protective Agents ; pharmacology ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats

OBJECTIVE: To study the effects of myo-inositol and luteolin on human lung cancer A549 cells and explore the possible mechanisms. METHODS: A549 cells were treated with different concentrations of myo-inositol and luteolin, either alone or in combination, and the cell viability was examined using MTT assay. A549 cells and human bronchial epithelial Beas-2B cells were treated for 48 h with 10 mmol/L myo-inositol and 20 μmol/L luteolin, alone or in combination, and the cell proliferation was detected using MTT assay; the colony formation and migration of the cells were examined with colony formation assay and wound healing assay, respectively. The protein expression levels in A549 cells were detected using Western blotting. RESULTS: Both myo-inositol and luteolin could dose-dependently inhibit the viability of A549 cells. Treatments with 10 mmol/L myo-inositol, 20 μmol/L luteolin, and both for 48 h caused significant reduction in the cell viability (92%, 83% and 70% of the control level, respectively) and colony number (79%, 73% and 43%, respectively), and significantly lowered the wound closure rate (24.61%, 13.08% and 8.65%, respectively, as compared with 29.99% in the control group). Similar treatments with myoinositol and luteolin alone or in combination produced no significant inhibitory effect on the growth, colony formation or migration of Beas-2B cells. The expressions of p-PDK1 and p-Akt in myo-inositol-treated A549 cells and the expression of pPDK1 in luteolin-treated cells were significantly decreased ( < 0.05), and the decrements were more obvious in the combined treatment group ( < 0.05). CONCLUSIONS Luteolin combined with myo-inositol can selectively inhibit the proliferation and migration of A549 cells, and these effects are probably mediated, at least in part, by suppressing the activation of PDK1 and Akt.
A549 Cells ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Humans ; Inositol ; administration & dosage ; therapeutic use ; Lung Neoplasms ; drug therapy ; metabolism ; Luteolin ; administration & dosage ; therapeutic use ; Protein-Serine-Threonine Kinases ; drug effects ; metabolism ; Proto-Oncogene Proteins c-akt ; drug effects ; metabolism ; Vitamin B Complex

Angiotensin II (Ang II) is involved in endothelium injury during the development of hypertension. Tribulus terrestris (TT) is used to treat hypertension, arteriosclerosis, and post-stroke syndrome in China. The present study aimed to determine the effects of aqueous TT extracts on endothelial injury in spontaneously hypertensive rats (SHRs) and its protective effects against Ang II-induced injury in human umbilical vein endothelial cells (HUVECs). SHRs were administered intragastrically with TT (17.2 or 8.6 g·kg·d) for 6 weeks, using valsartan (13.5 mg·kg·d) as positive control. Blood pressure, heart rate, endothelial morphology of the thoracic aorta, serum levels of Ang II, endothelin-1 (ET-1), superoxide dismutase (SOD) and malonaldehyde (MDA) were measured. The endothelial injury of HUVECs was induced by 2 × 10 mol·L Ang II. Cell Apoptosisapoptosis, intracellular reactive oxygen species (ROS) was assessed. Endothelial nitric oxide synthase (eNOS), ET-1, SOD, and MDA in the cell culture supernatant and cell migration were assayed. The expression of hypertension-linked genes and proteins were analyzed. TT decreased systolic pressure, diastolic pressure, mean arterial pressure and heart rate, improved endothelial integrity of thoracic aorta, and decreased serum leptin, Ang II, ET-1, NPY, and Hcy, while increased NO in SHRs. TT suppressed Ang II-induced HUVEC proliferation and apoptosis and prolonged the survival, and increased cell migration. TT regulated the ROS, and decreased mRNA expression of Akt1, JAK2, PI3Kα, Erk2, FAK, and NF-κB p65 and protein expression of Erk2, FAK, and NF-κB p65. In conclusion, TT demonstrated anti-hypertensive and endothelial protective effects by regulating Erk2, FAK and NF-κB p65.
Angiotensin II ; metabolism ; Animals ; Antihypertensive Agents ; administration & dosage ; Apoptosis ; drug effects ; Blood Pressure ; drug effects ; Endothelium, Vascular ; drug effects ; metabolism ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Hypertension ; drug therapy ; genetics ; metabolism ; physiopathology ; Male ; NF-kappa B ; genetics ; metabolism ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Oxidative Stress ; drug effects ; Plant Extracts ; administration & dosage ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Reactive Oxygen Species ; metabolism ; Tribulus ; chemistry

Objective: To investigate whether androgens can regulate the expression of eNOS in rat corpus cavernosum through AKT3, PIK3CA, CALM, and CAV1 and influence erectile function. METHODS: Thirty-six 8-week-old male SD rats were randomly divided into groups A (4-week control), B (6-week control), C (4-week castration), D (6-week castration), E (4-week castration + testosterone replacement), and F (6-week castration + testosterone replacement). Both the testis and epididymis were removed from the rats in groups C, D, E and F, and on the second day after surgery, the animals of groups E and F were subcutaneously injected with testosterone propionate at 3 mg per kg of the body weight qd alt while all the others with isodose oil instead. At 4 weeks (for groups A, C and E) and 6 weeks (for groups B, D and F) after treatment, we detected the maximum intracavernous pressure (ICPmax), the mean carotid arterial pressure (MAP) and their ratio (ICPmax/MAP), measured the level of serum testosterone (T), and determined the expressions of eNOS, P-eNOS, AKT3, PIK3CA, CALM and CAV1 in the corpus cavernosum by Western blot and immunohistochemistry. RESULTS: No statistically significant differences were observed in the body weight and MAP among different groups. The serum T level and ICPmax/MAP were remarkably lower in groups C and D than in the other four groups (P<0.01) as well as in groups E and F than in A and B (P<0.05) but exhibited no significant differences either between E and F or between A and B. Immunohistochemistry showed that eNOS and P-eNOS were mainly expressed in the vascular endothelial cell membrane and cavernous vascular lumen, while AKT3, PIK3CA, CALM and CAV1 chiefly in the vascular endothelial cell cytoplasm and membrane, with a few in the smooth muscle cells. Western blot analysis manifested that the expressions of eNOS, P-eNOS, AKT3, PIK3CA, CALM and CAV1 were markedly lower in groups C and D than in A, B, E and F (P<0.01) as well as in D than in C (P<0.05) but those in groups E and F did not showed any significant difference from those in A and B, nor E from F or A from B. CONCLUSIONS Androgens can improve erectile function by upregulating the expressions of AKT3, PIK3CA, CALM and CAV1 protein molecules and activating eNOS after its phosphorylation, though the exact molecular mechanisms are yet to be further studied.
Animals ; Blood Pressure ; Blotting, Western ; Caveolin 1 ; metabolism ; Class I Phosphatidylinositol 3-Kinases ; metabolism ; Erectile Dysfunction ; Hormone Replacement Therapy ; Male ; Monomeric Clathrin Assembly Proteins ; metabolism ; Myocytes, Smooth Muscle ; Nitric Oxide Synthase Type III ; metabolism ; Orchiectomy ; Penile Erection ; physiology ; Penis ; enzymology ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Testosterone Propionate ; administration & dosage

Inflammation plays a critical role in intestinal ischemia reperfusion injury (IRI). Epigallocatechin-3-gallate (EGCG) has been demonstrated to possess anti-inflammatory effect. This study examined the effect of EGCG on intestinal IRI and explored the possible mechanisms. Male Wistar rats were randomly divided into three groups: sham-operated group (Sham), IRI control group (IRI) and IRI-EGCG group (EGCG). Rats in IRI-EGCG group were administered dissolved EGCG in drinking water (0.4 mg/mL) for 14 days prior to IRI induction. A rat model of intestinal IRI was established by ligating the superior mesenteric artery (SMA) for 30 min, followed by reperfusion for 1 h. Intestinal histology, pro-inflammatory cytokines and mediators were examined and the effect of EGCG on PI3K/Akt signalling was assessed. EGCG significantly alleviated the pathological changes of the intestine and suppressed the IRI-induced up-regulation of TNF-α, IL-1 and IL-6 mRNA and protein expression in the serum and intestine. The mechanism might be that EGCG enhanced the activation of PI3K/Akt signalling pathway. In conclusion, the administration of EGCG can significantly mitigate the acute intestinal IRI in rats by enhancing the activation of PI3K/Akt signalling pathway to suppress inflammatory response and might be a promising alternative for the prevention or treatment of intestinal IRI in the clinical practice.
Animals ; Anti-Inflammatory Agents ; administration & dosage ; pharmacology ; Catechin ; administration & dosage ; analogs & derivatives ; pharmacology ; Disease Models, Animal ; Gene Expression Regulation ; drug effects ; Interleukin-1 ; genetics ; metabolism ; Interleukin-6 ; genetics ; metabolism ; Intestines ; drug effects ; pathology ; Male ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Wistar ; Reperfusion Injury ; genetics ; metabolism ; prevention & control ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

OBJECTIVETo investigate the effect of CAL-101, a selective inhibitor of PI3Kδ, in combination with bortezomib on the proliferation and apoptosis in human mantle cell lymphoma cell lines Z138, HBL-2 and Jeko-1 in vitro, to explore its mechanisms and provide the foundation for effective treatment strategies against mantle cell lymphoma.

METHODSMTT assay was applied to detect the inhibitory effects of CAL-101 and bortezomib either alone or combined on Z138, HBL-2 and Jeko-1 cells. Calcusyn software was used to analyze the synergistic cytotoxicity. Western blot was used to detect the expression of PI3K-p110σ and p-Akt, Akt, p-ERK and ERK proteins after the cells were exposed to different concentrations of CAL-101. Flow cytometry was employed to assess the apoptosis rate. NF-κB kit was used to determine the changes of location of NF-κB P65, and Western blot was applied to detect the level of caswpase-3 and the phosphorylation of Akt in different groups.

RESULTSCAL-101 and BTZ inhibited the proliferation of Z138, HBL-2 and Jeko-1 cells in a dose- and time-dependent manner. CAL-101/BTZ combination induced significantly synergistic cytotoxicity in the MCL cells. The results of Western blot assay showed that CAL-101 significantly blocked the phosphorylation of Akt and ERK in the MCL cell lines. In addition, CAL-101 combined with BTZ induced pronounced apoptosis (P < 0.01). For example, after the Z138 cells exposed to the drugs for 48 h, the apoptosis rates of the control, CAL-101, BTZ and CAL-101 + BTZ groups were: (2.6 ± 1.8)%, (40.0 ± 3.0)%, (34.0 ± 1.0)%, and (67.4 ± 1.0)%, respectively; and when drug treatment was given to HBL-2 cells over 96 h, the apoptosis rates of these four cell groups were (7.4 ± 0.6)%, (30.7 ± 5.7)%, (12.0 ± 1.0)%, and (85.0 ± 4.0)%, respectively. The combination therapy contributed to the enhanced inactivity of nuclear factor-κB (NF-κB) and Akt inactivation in the MCL cell lines (P < 0.05), however, the casepase-3 activity was up-regulated.

CONCLUSIONSThe combination of CAL-101 and bortezomib is muchmore effective in inhibiting proliferation and promoting apoptosis of mantle cell lymphoma cell lines (Z138, HBL-2 and Jeko-1), which may be mediated through inhibiting PI3K/Akt signaling pathway and the transcription of NF-κB.

Antineoplastic Agents ; pharmacology ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; Boronic Acids ; Bortezomib ; pharmacology ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Class Ia Phosphatidylinositol 3-Kinase ; antagonists & inhibitors ; Dose-Response Relationship, Drug ; Drug Synergism ; Formazans ; Humans ; Lymphoma, Mantle-Cell ; drug therapy ; pathology ; MAP Kinase Signaling System ; drug effects ; NF-kappa B ; metabolism ; Neoplasm Proteins ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism ; Purines ; administration & dosage ; pharmacology ; Pyrazines ; Quinazolinones ; administration & dosage ; pharmacology ; Signal Transduction ; Software ; Tetrazolium Salts

To investigate the protective effect of preconditioning with hyperoside ( Hyp) against myocardial ischemia-reperfusion injury (MIRI) in rats and the role of PI3K/Akt signaling pathway. MIRI was established by ligation of left anterior descending coronary artery for 30 min followed by reperfusion for 120 min in rats. Male SD rats were randomly divided into five groups: sham group,model group (MIRI),Hyp preconditioning group(Hyp), Hyp preconditioning + LY294002 (a PI3K/Akt signaling pathway inhibitor) group (Hyp + LY), and LY294002 group (LY). At the end of reperfusion, hemodynamic parameters were recorded as left ventricular systolic pressure (LVSP) , left ventricular end-diastolic pressure ( LVEDP) and maximal rate of increase and decrease of left ventricular pressure (± dP/dt(max)). Myocardial infaret size, the oxidative stress markers, myocardial enzymes indicators and inflammatory factors were also analyzed. The expressions of Akt, p-Akt, Bax and Bcl-2 proteins was detected by using Western blot method. The results showed that Hyp preconditioning remarkably improved cardiac constriction and relaxation function, reduced myocardial infarct size and enhanced the activities of oxidative stress markers about correlated to MIRI, such as superoxide dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GSH-Px) and decreased the contents of malondialdehyde (MDA) as compared with MIRI group. Simultaneouly, the levels of myocardial enzymes, i. e. creatine kinase ( CK) and creatine kinase MB isoenzyme (CK-MB), and inflammatory factors, for instance tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were decreased. Hyp pretreatment apparently restrained myocardial apoptosis as evidenced by decreasing the level of Bax expression, increasing the levels of phosphorylation of Akt and Bcl-2 expression. These effects were inhibited by LY294002, a blocker of PI3K/Akt signaling pathway. These findings indicated that the cardioprotection of Hyp preconditioning against MIRI may be related to activating PI3K/Akt signaling pathway, upregulating the expression of BCL-2 protein and down-regulating the expression of Bax protein.
Animals ; Creatine Kinase ; genetics ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Heart ; drug effects ; Humans ; Interleukin-6 ; genetics ; metabolism ; Ischemic Preconditioning, Myocardial ; Male ; Malondialdehyde ; metabolism ; Myocardial Reperfusion Injury ; drug therapy ; enzymology ; genetics ; prevention & control ; Phosphatidylinositol 3-Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Quercetin ; administration & dosage ; analogs & derivatives ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; genetics ; metabolism

PURPOSE: To investigate the effects of resveratrol on the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in human adult retinal pigment epithelial (ARPE-19) cells, and on experimental choroidal neovascularization (CNV) in mice. MATERIALS AND METHODS: ARPE-19 cells were treated with different concentrations of resveratrol and then incubated under hypoxic conditions with subsequent evaluation of cell viability, expression of HIF-1alpha, and expression of VEGF. The effects of resveratrol on the synthesis and degradation of hypoxia-induced HIF-1alpha were evaluated using inhibitors of the PI3K/Akt/mTOR and the ubiquitin proteasome pathways. In animal studies, CNV lesions were induced in C57BL/6 mice by laser photocoagulation. After 7 days of oral administration of resveratrol or vehicle, which began one day after CNV induction, image analysis was used to measure CNV areas on choroidal flat mounts stained with isolectin IB4. RESULTS: In ARPE-19 cells, resveratrol significantly inhibited HIF-1alpha and VEGF in a dose-dependent manner, by blocking the PI3K/Akt/mTOR signaling pathway and by promoting proteasomal HIF-1alpha degradation. In mice experiments, orally administered resveratrol significantly inhibited CNV growth in a dose-dependent manner. CONCLUSION: Resveratrol may have therapeutic value in the management of diseases involving pathological neovascularization.
Adult ; Animals ; Anoxia/metabolism/physiopathology ; Cell Survival/drug effects ; Choroidal Neovascularization/*metabolism/pathology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/*drug effects/metabolism ; Mice ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases/antagonists & inhibitors/*physiology ; Proteasome Endopeptidase Complex ; Proto-Oncogene Proteins c-akt/antagonists & inhibitors/*physiology ; Retinal Pigment Epithelium/*drug effects/metabolism ; Signal Transduction ; Stilbenes/administration & dosage/*pharmacology ; TOR Serine-Threonine Kinases/antagonists & inhibitors/*physiology ; Ubiquitin ; Vascular Endothelial Growth Factor A/*drug effects/metabolism

The effect of Lycii Cortex on the PCOS rat model and the mechanism of action were investigated in the present study. The PCOS rat model was induced with Poretsky methods. Then the rats were randomly divided into four groups: the model group, melbine group (0.45 g x kg(-1)), low (2.5 g x kg(-1) and high (10 g x kg(-1)) dosage group of Lycii Cortex. The animals were orally administrated with the drugs for 14 days. In addition, another control group was added in this study. The rats were weighted before and after drug treatment. After 14 days treatment, oestrous cycle of rats were detected; blood serum was separated to determine T and FINS and rat's uteri were isolated. The mRNA and protein (total and phosphorylated) expressions of PI3K and PKB in uteri were measured with Real-time RT-PCR and Western blot, respectively. Compared with the control rats, the body weight gain and serum level of T and FINS were significantly increased. While, the mRNA and protein (phosphorylated) levels of PI3K and PKB were markedly decreased in PCOS group. Lycii Cortex treatment significantly decreased the body weight gain and serum level of T and FINS in a dose-dependant manner. It also markedly increased the mRNA and protein (phosphorylated) expressions of PI3K and PKB. Meanwhile, the melbine treatment also showed the curative effect. Lycii Cortex can relieve the symptoms of PCOS and the mechanism might be related to PI3K/PKB pathway.
Animals ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Humans ; Lycium ; chemistry ; Phosphatidylinositol 3-Kinases ; genetics ; metabolism ; Phosphorylation ; drug effects ; Polycystic Ovary Syndrome ; drug therapy ; enzymology ; genetics ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects

To establish neonatal rat cardiac fibroblast inflammatory secretion model by using LPS 100 microg x L(-1) combined with ATP 5 mmol x L(-1), in order to study the inhibitory effect of quercetin on the secretion of inflammatory factors TNF-alpha, IL-1beta and IL-6 of cardiac fibroblasts, further investigate the effect of quercetin on the protein expression of p-NF-kappaB p65 (S276) and p-Akt (S473) by western blot, and discuss the inhibitory effect of quercetin on the inflammatory secretion of cardiac fibroblasts. According to the findings, quercetin with the concentrations between 51.74 micromol x L(-1) and 827.81 micromol x L(-1) had no significant effect on the activity of cardiac fibroblasts. Quercetin with the concentrations of 82.78, 41.39, 20.70 micromol x L(-1) could notably inhibit the increase of TNF-alpha and IL-1beta induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 36 h (P < 0.01 or P < 0.05). Quercetin with the concentrations of 82.78, 41.39 micromol x L(-1) could notably inhibit the increase of IL-6 induced LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 36 h (P < 0.05), without any notable effect of quercetin with the concentration of 20.70 micromol x L(-1). Quercetin with the concentrations of 82.78, 41.39, 20. 70 micromol x L(-1) could notably inhibit the NF-kappaB p65 (S276) activation induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 15 min, with the most significant effect in 20.70 micromol x L(-1). Quercetin with the concentrations of 82.78, 41.39, 20.70 micromol x L(-1) could notably inhibit the increase of p-Akt(473) expression induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 240 min (P < 0.05). Therefore, this study believes that quercetin could attenuate the secretion of inflammatory factors TNF-alpha, IL-1beta and IL-6 of cardiac fibroblasts by inhibiting the activation of NF-kappaB p65 (S276) and Akt (473).
Animals ; Cells, Cultured ; Drugs, Chinese Herbal ; administration & dosage ; Endomyocardial Fibrosis ; drug therapy ; genetics ; immunology ; Female ; Fibroblasts ; drug effects ; immunology ; Heart ; drug effects ; Humans ; Interleukin-6 ; genetics ; immunology ; Male ; Proto-Oncogene Proteins c-akt ; genetics ; immunology ; Quercetin ; administration & dosage ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; genetics ; immunology