OBJECTIVETo investigate the impact of NU7026 and Wortmannin, inhibitors of DNA-dependent protein kinase (DNA-PK), in HL60 cells apoptosis induced by 1, 4-benzoquinone (1, 4-BQ).

METHODSHL60 cells were divided into three groups according to the exposures: the poisoned groups which were treated with 0, 5, 10, 25 and 50 µmol/L 1, 4-BQ for 24 h, respectively, the NU7026 groups which were preincubated with 10 µmol/L NU7026 for 1h prior to the 24h treatment of 0, 5, 10, 25 and 50 µmol/L 1, 4-BQ and the Wortmannin groups which were preincubated with 25 µmol/L Wortmannin for 1h prior to the 24 h treatment of 0, 5, 10, 25 and 50 µmol/L 1, 4-BQ. Then we detected the apoptosis via flowcytometry Annexin V-FITC/PI and the DNA Ladder, respectively. We also tested the expressions of Bax mRNA with Real-Time PCR in HL60 cells which were exposed to 10 µmol/L NU7026 for 24 h, 25 µmol/L Wortmannin 24 h, 10 µmol/L 1, 4-BQ 24 h, 10 µmol/L NU7026 1h+10 µmol/L 1, 4-BQ 24 h and 25 µmol/L Wortmannin 1 h+10 µmol/L 1, 4-BQ 24 h, as well as null (control). We also examed the expressions of p53 in HL60 cells with Western blot.

RESULTSAnnexin V-FITC/PI apoptosis tests suggested that apoptosis rates of NU7026+10 µmol/L 1, 4-BQ group and Wortmannin +10 µmol/L 1, 4-BQ were 17.6±1.19% and 15.2±1.22%, respectively. Both of results were higher than that of 10 µmol/L 1, 4-BQ group (6.3±1.04%); Apoptosis of NU7026+25 µmol/L 1, 4-BQ group was 46.2±3.55%, and Wortmannin +25 µmol/L 1, 4-BQ group 26.9±2.62%. Both of results were also higher than that of 25 µmol/L 1, 4-BQ group (14.1±1.54%); Apoptosis of NU7026+50 µmol/L 1, 4-BQ group (61.8±1.78%) was higher than that of 50 µmol/L 1, 4-BQ group (35.9±4.51%). The above results were all statistically significant (P < 0.05).

RESULTSof DNA-Ladder were basically consistent with those of Annexin V-FITC/PI apoptosis tests. In addition, both NU7026 and Wortmannin pretreatment elicited the higher expression of Bax mRNA in HL60 treated by 1, 4-benzoquinone with statistically significance (P < 0.05). However, p53 protein was not detected in HL60 cells as the western blot indicated.

CONCLUSIONInhibitors of DNA-PK, NU7026 and Wortmannin, promote p53-independent apoptosis induced by 1, 4-benzoquinone in HL60 cells.

Androstadienes ; pharmacology ; Apoptosis ; drug effects ; Benzoquinones ; toxicity ; Chromones ; pharmacology ; DNA-Activated Protein Kinase ; antagonists & inhibitors ; Flow Cytometry ; HL-60 Cells ; Humans ; Morpholines ; pharmacology ; RNA, Messenger ; Tumor Suppressor Protein p53

The present study was to explore the effects of insulin on proliferation of skeletal myoblast cells in rats. Separated and cultured primary skeletal myoblast cells from rats were treated by insulin. By means of the incorporation of (3)H-TdR, BrdU assay and MTT assay, the proliferation of skeletal myoblast cells was detected. Western blot was used to check the phosphorylation of Akt and ERK of myoblast cells. The results showed that insulin significantly promoted the incorporation of (3)H-TdR into cultured skeletal myoblast cells in a dose-dependent manner. MTT assay and BrdU assay also showed insulin promoted the proliferation of skeletal myoblast cells. The promotion of skeletal myoblast cells proliferation by insulin was inhibited by PI3K inhibitor wortmannin or MEK inhibitor U0126, and the same phenomenon was shown in L6 and C2C12 cells. Also, insulin increased the phosphorylation of Akt and ERK in myoblast cells. These results suggest that insulin may promote proliferation of skeletal myoblast cells through PI3K/Akt and MEK/ERK pathways.
Androstadienes ; pharmacology ; Animals ; Butadienes ; pharmacology ; Cell Proliferation ; Cells, Cultured ; Enzyme Inhibitors ; pharmacology ; Insulin ; pharmacology ; MAP Kinase Signaling System ; Myoblasts, Skeletal ; cytology ; drug effects ; Nitriles ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats

BACKGROUND5α-Reductase inhibitors (5α-RI) act by inhibiting the conversion of testosterone to dihydrotestosterone (DHT), thereby preventing DHT induced benign prostatic hyperplasia. The existing 5α-RIs can be classified into two types: competitive and noncompetitive. Currently, limited evidence is available concerning the effect differences between the two types of 5α-RI on androgens. The purpose of this study was to assess the effects of competitive and noncompetitive 5α-RIs on serum and intra-prostatic androgens in beagle dogs.

METHODSTwenty beagles with spontaneous benign prostatic hyperplasia were randomly allocated into two groups: epristeride group (n = 10) in which beagles were treated with epristeride at 1 mg/kg once a day for 3 months, and finasteride group (n = 10) in which beagles were treated with finasteride at 1 mg/kg once a day for 3 months. The levels of intra-prostatic testosterone and DHT were measured before treatment and on day one after three months medication. Serum levels of testosterone and DHT were measured at the same time points. Changes in androgen levels before and after treatment were analyzed, and comparisons were made within each treatment group and between treatment groups.

RESULTSAfter 3-month treatment, serum and intra-prostatic DHT levels all decreased significantly in both the epristeride and finasteride groups. The change of DHT in serum was significantly higher in the finasteride group (-14% and -43% in epristeride and finasteride groups respectively, with P < 0.001); however there was no significant difference in the changes of intra-prostatic DHT between the two groups (-47% and -51% in epristeride and finasteride groups, respectively, P = 0.304). The decreases in DHT levels were accompanied by reciprocal increases in serum and intra-prostatic testosterone levels. Changes of testosterone were significantly higher in finasteride group both in serum (20% and 42% in epristeride and finasteride groups, respectively, P < 0.001) and in prostate tissue (18% and 29% in epristeride and finasteride groups, respectively, P = 0.004).

CONCLUSIONSTwo types of 5α-RI have similar effects in reducing DHT in prostate tissue in beagles. Competitive 5α-RI may reduce serum DHT to a greater scale, and significantly increase testosterone in beagle serum and prostate.

5-alpha Reductase Inhibitors ; pharmacology ; Androgens ; blood ; metabolism ; Androstadienes ; pharmacology ; Animals ; Dihydrotestosterone ; blood ; metabolism ; Dogs ; Finasteride ; pharmacology ; Male ; Prostate ; drug effects ; metabolism

The localization of estrogen (E2) has been clearly shown in hippocampus, called local hippocampal E2. It enhanced neuronal synaptic plasticity and protected neuron form cerebral ischemia, similar to those effects of exogenous E2. However, the interactive function of hippocampal and exogenous E2 on synaptic plasticity activation and neuroprotection is still elusive. By using hippocampal H19-7 cells, we demonstrated the local hippocampal E2 that totally suppressed by aromatase inhibitor anastrozole. Anastrozole also suppressed estrogen receptor (ER)beta, but not ERalpha, expression. Specific agonist of ERalpha (PPT) and ERbeta (DPN) restored ERbeta expression in anastrozole-treated cells. In combinatorial treatment with anastrozole and phosphoinositide kinase-3 (PI-3K) signaling inhibitor wortmannin, PPT could not improve hippocampal ERbeta expression. On the other hand, DPN induced basal ERbeta translocalization into nucleus of anastrozole-treated cells. Exogenous E2 increased synaptic plasticity markers expression in H19-7 cells. However, exogenous E2 could not enhance synaptic plasticity in anastrozole-treated group. Exogenous E2 also increased cell viability and B-cell lymphoma 2 (Bcl2) expression in H2O2-treated cells. In combined treatment of anastrozole and H2O2, exogenous E2 failed to enhance cell viability and Bcl2 expression in hippocampal H19-7 cells. Our results provided the evidence of the priming role of local hippocampal E2 on exogenous E2-enhanced synaptic plasticity and viability of hippocampal neurons.
Androstadienes/pharmacology ; Animals ; Aromatase Inhibitors/pharmacology ; Cell Line ; Cell Survival/drug effects ; Estrogen Receptor alpha/agonists/metabolism ; Estrogen Receptor beta/agonists/metabolism ; Estrogens/*metabolism/pharmacology ; Hippocampus/cytology/*metabolism ; Hydrogen Peroxide/pharmacology ; Nervous System/*drug effects ; Neuronal Plasticity/*drug effects ; *Neuroprotective Agents ; Nitriles/pharmacology ; Phosphatidylinositol 3-Kinase/antagonists & inhibitors ; Proto-Oncogene Proteins c-bcl-2/biosynthesis ; Rats ; Triazoles/pharmacology

Akt/protein kinase B is a well-known cell survival factor and activated by many stimuli including mechanical stretching. Therefore, we evaluated the cardioprotective effect of a brief mechanical stretching of rat hearts and determined whether activation of Akt through phosphatidylinositol 3-kinase (PI3K) is involved in stretch-induced cardioprotection (SIC). Stretch preconditioning reduced infarct size and improved post-ischemic cardiac function compared to the control group. Phosphorylation of Akt and its downstream substrate, GSK-3beta, was increased by mechanical stretching and completely blocked by wortmannin, a PI3K inhibitor. Treatment with lithium or SB216763 (GSK-3beta inhibitors) before ischemia induction mimicked the protective effects of SIC on rat heart. Gadolinium (Gd3+), a blocker of stretch-activated ion channels (SACs), inhibited the stretch-induced phosphorylation of Akt and GSK-3beta. Furthermore, SIC was abrogated by wortmannin and Gd3+. In vivo stretching induced by an aorto-caval shunt increased Akt phosphorylation and reduced myocardial infarction; these effects were diminished by wortmannin and Gd3+ pretreatment. Our results showed that mechanical stretching can provide cardioprotection against ischemia-reperfusion injury. Additionally, the activation of Akt, which might be regulated by SACs and the PI3K pathway, plays an important role in SIC.
Androstadienes/pharmacology ; Animals ; Gadolinium/pharmacology ; Glycogen Synthase Kinase 3/*metabolism ; Indoles/pharmacology ; *Ischemic Preconditioning, Myocardial ; Lithium/pharmacology ; Male ; Maleimides/pharmacology ; Myocardial Reperfusion Injury/enzymology/physiopathology/*prevention & control ; Phosphatidylinositol 3-Kinase/*antagonists & inhibitors/metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt/*metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Specific Pathogen-Free Organisms

Diabetic nephropathy (DN) is a progressive kidney disease that is caused by injury to kidney glomeruli. Podocytes are glomerular epithelial cells and play critical roles in the glomerular filtration barrier. Recent studies have shown the importance of regulating the podocyte actin cytoskeleton in early DN. The phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, simultaneously regulates Rac1 and Cdc42, which destabilize the podocyte actin cytoskeleton during early DN. In this study, in order to evaluate the reno-protective effects of wortmannin in early DN by regulating Rac1 and Cdc42, streptozotocin (STZ)-induced proteinuric renal disease (SPRD) rats were treated with wortmannin. The albuminuria value of the SPRD group was 3.55 +/- 0.56 mg/day, whereas wortmannin group was 1.77 +/- 0.48 mg/day. Also, the albumin to creatinine ratio (ACR) value of the SPRD group was 53.08 +/- 10.82 mg/g, whereas wortmannin group was 20.27 +/- 6.41 mg/g. Changes in the expression level of nephrin, podocin and Rac1/Cdc42, which is related to actin cytoskeleton in podocytes, by wortmannin administration were confirmed by Western blotting. The expression levels of nephrin (79.66 +/- 0.02), podocin (87.81 +/- 0.03) and Rac1/Cdc42 (86.12 +/- 0.02) in the wortmannin group were higher than the expression levels of nephrin (55.32 +/- 0.03), podocin (53.40 +/- 0.06) and Rac1/Cdc42 (54.05 +/- 0.04) in the SPRD group. In addition, expression and localization of nephrin, podocin and desmin were confirmed by immunofluorescence. In summary, we found for the first time that wortmannin has a reno-protective effect on SPRD rats during the early DN. The beneficial effects of wortmannin in SPRD rats indicate that this compound could be used to delay the progression of the disease during the early DN stage.
Albumins/metabolism ; Androstadienes/*administration & dosage/pharmacology ; Animals ; Creatinine/blood ; Desmin/genetics/metabolism ; Diabetes Mellitus, Experimental/*drug therapy/metabolism/pathology ; Diabetic Nephropathies/*drug therapy/metabolism/pathology ; Disease Models, Animal ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/metabolism ; Kidney/*pathology ; Membrane Proteins/genetics/metabolism ; Phosphatidylinositol 3-Kinases/*antagonists & inhibitors ; Podocytes/*drug effects/metabolism/pathology ; Rats ; Rats, Inbred Strains ; cdc42 GTP-Binding Protein/genetics/metabolism ; rac1 GTP-Binding Protein/genetics/metabolism

OBJECTIVE: To investigate the effects of intranasal steroid on nasal mucosa in rat model of allergic rhinitis (AR). METHOD: Thirty-six SD rats were randomly divided into three groups (each included 12 rats): the intranasal steroid group(group A),the allergic rhinitis group(group B) and the normal control group(group C). AR model was reproduced by ovalbumin in group A and B. The control group was allergized by NS instead of ovalbumin. Group A was used steroid spray in nasal and Group B and C was used normal saline. Then ,after the last provocation,the allergic symptoms were evaluated,the number of eosinophil (EOS) of the rats' nasal mucosa was counted under hematoxylineosin staining and the goblet cells was counted under alcian blue-periodic acid-Schiff staining. The morphological changes of the rats' nasal mucosa were observed by scanning electron microscope. RESULT: Compared with group B,the inhibition of nasal symptoms was found after used of steroid spray in group A. The EOS and the goblet cells in group A was significantly different with that in Group B (P < 0.05), but no significant differences compared with group C (P > 0.05). Observed by scanning electron microscope, the cilia of the epithelium in group B were significantly lodging, disoriented, winding and secreting,while the cilia of the epithelium in Group A and C had similar appearance. CONCLUSION Intranasal steroid spray can relieve symptoms and inflammatory conditions of nasal mucosa in allergic rhinitis, and can repair injured nasal mucosa cilia.
Androstadienes ; pharmacology ; Animals ; Anti-Inflammatory Agents ; pharmacology ; Cilia ; ultrastructure ; Disease Models, Animal ; Inflammation ; Nasal Mucosa ; drug effects ; pathology ; Rats ; Rats, Sprague-Dawley ; Rhinitis, Allergic, Perennial ; drug therapy ; pathology

OBJECTIVETo study the effects of PI3K/Akt signaling pathway inhibitor wortmannin on long-term learning and memory abilities in neonatal rats with hypoxic-ischemic brain damage (HIBD).

METHODSForty-eight neonatal rats were randomly assigned to blank control (n=8), sham-operated (n=8), HIBD model (n=10), HIBD+DMSO (dimethyl sulfoxide, n=8) and HIBD+wortmannin groups (n=8). Wortmannin (2 μL) was injected to the left hippocampus 30 minutes before HIBD inducement in the HIBD+wortmannin group. The Morris water maze test was used to examine the long-term learning and memory abilities at the age of 28 days.

RESULTSWith the increased number of swimming, the escape latency was shortened in various groups. From the second day, the escape latency in the HIBD+wortmannin group was significantly longer than that in the sham-operated and the blank control groups (P<0.05), and the differences increased with the time. On the fourth day, there were significant differences in the escape latency between the HIBD+wortmannin group and the HIBD+DMSO group as well as the HIBD model group (P<0.05). On the eighth day (retention trial), there were the most obvious differences in the escape latency between the HIBD+wortmannin group with the other four groups. In the space exploration test, the number of times crossing the former platform location within 120 seconds after removing the platform in the HIBD+DMSO and the HIBD model group was lower than the sham-operated and the blank control groups (P<0.05). The HIBD+wortmannin group showed lower number of times crossing the former platform location compared with the HIBD+DMSO and the HIBD model groups (P<0.05), as well as the sham-operated and the blank control groups (P<0.01).

CONCLUSIONSP13K/Akt signaling pathway inhibitor wortmannin can aggravate the cognitive impairments, thus affecting adversely long-term learning and memory abilities in neonatal rats with HIBD.

Androstadienes ; pharmacology ; Animals ; Animals, Newborn ; Dimethyl Sulfoxide ; pharmacology ; Hypoxia-Ischemia, Brain ; psychology ; Learning ; Maze Learning ; Memory ; Phosphatidylinositol 3-Kinases ; physiology ; Proto-Oncogene Proteins c-akt ; physiology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; physiology

OBJECTIVETo study the regulatory effect of glucagon-like peptide-1 (GLP-1) on cell proliferation of skeletal myoblast strain L6 and its possible signal mechanism.

METHODSL6 cells cultured in DMEM high glucose culture medium containing 10% FBS were divided into control group (C, without addition), GLP-1 group (G, added with 10 nmol/L GLP-1), PI3K inhibitor group (W, added with 50 nmol/L PI3K specific inhibitor wortmannin), and GLP-1 + PI3K inhibitor group (GW, added with 10 nmol/L GLP-1 and 50 nmol/L wortmannin) according to the random number table. Cell proliferation activity was detected with MTT assay at post culture hour (PCH) 24, 48, 72 (denoted as absorbance value). At PCH 24, the change in cell cycle was evaluated with flow cytometer, the expression level of proliferating cell nuclear antigen (PCNA) was determined with immunohistochemical staining, the protein levels of phosphorylated PI3K (p-PI3K) and p-Akt were determined with Western blotting. Data were processed with multi-group analysis of variance.

RESULTS(1) The cell proliferation activity at PCH 48, 72 in G group was respectively 0.660 +/- 0.120, 0.870 +/- 0.240, all significantly higher than those in C group (0.530 +/- 0.060, 0.700 +/- 0.100, with F value respectively 5.46, 5.90, P < 0.05 or P < 0.01). The cell proliferation activity in W group at each time point was lower than that in C group. The cell proliferation activity in GW group at PCH 48, 72 was respectively 0.510 +/- 0.080, 0.740 +/- 0.160, all lower than those in G group (with F value respectively 5.46, 5.90, P < 0.05 or P < 0.01). (2) The percentage of S phase cell in G group at PCH 24 [(15.7 +/- 0.4)%] was significantly higher than that in C group [(13.6 +/- 0.6)%] and GW group [(10.1 +/- 0.6)%], while that in W group [(6.8 +/- 1.2)%] was lower than that in C group (with F values all equal to 15.39, P values all below 0.01). (3) PCNA level in G group at PCH 24 [(51.24 +/- 1.18)%] was markedly higher than that in C group [(36.72 +/- 1.56)%] and GW group [(25.90 +/- 1.22)%], and while in W group [(21.70 +/- 0.09)%] was lower than that in C group (with F values equal to 783.80, P values all below 0.05). (4) The protein level of p-Akt in G group at PCH 24 was significantly higher than that in the other 3 groups, while that in W group was lower than that in C group (with F values equal to 94.43, P values all below 0.01). There was no obvious difference in protein level of p-PI3K at PCH 24 among G, GW, and C groups ( F = 20.94, P > 0.05). The protein level of p-PI3K at PCH 24 in W group was lower than that in C group (F = 20.94, P < 0.05).

CONCLUSIONSGLP-1 can promote cell proliferation of skeletal myoblast by accelerating the progression of cell cycle and increasing the synthesis of DNA, which can be attributed to PI3K/Akt signal pathway.

Androstadienes ; pharmacology ; Animals ; Cell Cycle ; Cell Line ; Cell Proliferation ; drug effects ; DNA ; biosynthesis ; Glucagon-Like Peptide 1 ; pharmacology ; Myoblasts, Skeletal ; drug effects ; metabolism ; pathology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Rats ; Signal Transduction ; drug effects

OBJECTIVE: To investigate the dynamic process of the nasal mucosal remodeling, and the effect of the fluticasone propionate (FP) to remodeling, by establish animal model of allergic rhinitis (AR). METHOD: One hundred and twenty Sprague-Dawley (SD) rats were randomly divided into three groups: the normal Group A used as controls and experimental groups: Group B and C, each group had 40 rats. After the animal model were established successfully by OVA+ Al (OH)3 and disposed, then, the dynamic process of the nasal mucosal remodeling was observed, through HE staining and transmission electron microscopic section in special times. RESULT: The Group B, C nasal epithelium and cilia were not complete, eosinophil-based inflammatory cell infiltration, basement membrane thickening, collagen deposition, and a small amount of fibrosis could be found, but the structure of cells were not damaged. While those changes could not be observed in the Group A. The morphological changes of the nasal mucosa of Group B aggravated gradually under persistent allergen exposure, even stripped to the basement membrane in whole epithelial layers, cell and tissue structure were destroyed seriously. The morphological changes of nasal mucosa of Group C did not further increase, but still showed varying degrees of cilia arranged in uneven fashion, basement membrane thickening, collagen deposition and fibrous hyperplasia after treatment by FP. CONCLUSION Remodeling happens in the nasal mucosa, which would be aggravated, and even becomes irreversible if the allergen exposure continues persistently. The FP can relieve the clinical symptoms, slow down and even reverse the remodeling of AR. And it is ineffective when the changes become irreversible.
Androstadienes ; pharmacology ; Animals ; Disease Models, Animal ; Female ; Fluticasone ; Male ; Nasal Mucosa ; drug effects ; pathology ; Rats ; Rats, Sprague-Dawley ; Rhinitis, Allergic, Perennial ; immunology ; pathology