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Aim To study the protective effect of quercetin ( Que) on vascular endothelial progenitor cells ( EPCs) under oxidative stress and the underlying mechanisms. Methods The human umbilical cord blood EPCs were separated .differentiated and randomized into two groups, namely, hydrogen peroxide (H202) group, in which the oxidative stress damage EPCs model was established by adding H202 of 500 jjunol • L"1 ,and Que intervention group,in which Que pretreated with a concentration of 60 jimol • L~' and 90 p.mol • L'1 was added for 30 min respectively, and then 500 jimol • L"1 H202 was added for co-culture. WST-1 method was used to assess the proliferation of EPCs after 12,24 and 48 h culture. Western blot was used to detect the expression levels of Akt and phos- phorylated Akt. Real-time fluorescence RT-PCR was employed to determine the expression levels of PI3K and AKT3 mRNA. Results Compared with blank control group,the proliferation capacity of EPCs treated with hydrogen peroxide was significantly reduced. The expression of Akt protein in EPCs decreased significantly, and the relative expression of PI3K and AKT3 mRNA decreased significantly ( P < 0. 01, P < 0. 05 ). After 60,90 jtmol • L"1 Que was added for 12,24 and 48 h,respectively,the proliferation capacity of damaged EPCs cells increased significantly, and the difference was statistically significant compared with the model group (fcO. 05). At the same time, the protein expression of p-Akt in EPCs significantly increased ( P < 0.05),and mRNA expression of PI3K and AKT3 increased ( P < 0. 01 ). Conclusions Que can fix the EPCs oxidative stress induced by H202 ,and its mecha-nism may be through the activation of PI3K/AKT signaling pathway, reducing the influence of oxidative stress on cerebrovascular lesions,and promoting the proliferation and differentiation of cerebrovascular EPCs.
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Objective To observe of the effects of tacrolimus on blood glucose,insulin secretion and the expression of phosphorylated AKT in rats in order to study the mechanism of diabetogenic effects of tacrolimus.Methods 40 male SD rats were randomly divided into two groups.The rats in tacrolimus group were delivered tacrolimus at a dose of 4mg/kg· d.The rats in the control group were given the same amount of saline solution in the same way.The body weights,fasting blood glucose levels and blood concentrations of tacrolimus were measured monthly.After 5 months,all rats were killed.Pancreas and liver tissue were stored in 4％ paraformaldehyde solution.Serum insulin levels were detected by radioimmunoassay method.The expression of phosphorylated AKT in liver were measured by immunohisto-chemical method.Results ①The body weights in tacrolimus group in the 3rd,4th,and 5th month were significantly lower than those in the control group (P＜0.01).②The blood glucose levels in tacrolimus group in the 3rd,4th,and 5th month were significantly higher than those in the control group (P＜0.05).③The insulin secretion and insulin sensitivity index in tacrolimus group were significantly lower than those in the control group (P＜0.01).④The rats in tacrolimus group showed varying degrees of damage in pancreatic duct and pancreatic islet cells.⑤The expression of phosphorylated AKT in liver cells in tacrolimus group were significantly lower than those in the control group (P＜0.05).Conclusions Tacrolimus can induce pancreatic islet cells necrosis,decrease the number of islet cells,reduce insulin secretion and insulin sensitivity,which lead to blood hyperglycemia in rats.In addition,we also find that tacrolimus can reduce expression of phosphorylated AKT in hepatic tissue,which indicates that tacrolimus results insulin resistance through interfering PI3K/ AKT signal transduction pathways.
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Objective To observe the relationship between Toll-like receptor 4 (TLR4) and the sensitivity of PANC1 cells to gemcitabine (GEM),and to analyze the potential mechanism.Methods PANC1 cells were divided into GEM group,lipopolysaccharide (LPS) + GEM group and TLR4-siRNA + GEM group.GEM group was treated by GEM alone.LPS + GEM group was pretreated with 1 mg/L LPS for 4 h and then treated by GEM.TLR4-siRNA + GEM group was transfected with 100 pmol/mL TLR4-siRNA for 4 h and then treated by GEM.The untreated cells were used as the control group.MTT method was used to detect the cell proliferation.Morphological changes and apoptosis rate of the cells were examined by Hoechst33258 staining and flow cytometry,respectively.The protein expression of TLR4,phosphorylated AKT (p-AKT) and activated Caspase-3 were detected by Western blot.Results The median inhibition concentration (ICs0) of GEM in the GEM group,LPS + GEM group and TLR4-siRNA + GEM group was (8.9 ± 0.32),(14.21 ±0.95),(3.96 ± 0.27) mg/L,respectively.The IC50 in LPS + GEM group was significantly higher than that in GEM group (P ＜ 0.01),and the IC50 of GEM in TLR4-siRNA + GEM group was significantly lower than that in GEM group (P ＜0.01).Compared with that in GEM group,the cells with typical apoptotic morphological changes were decreased in LPS + GEM group,which was increased in TLR4-siRNA + GEM group.The apoptotic rate in control group,GEM group,LPS + GEM group,TLR4-siRNA + GEM group was (2.1 ± 0.3) ％,(15.1 ± 2.3) ％,(9.8 ± 1.5) ％,(22.9 ± 3.1) ％,respectively.Compared with that in GEM group,the cells apoptotic rate was significantly reduced in LPS + GEM group (P ＜0.01),which was significantly increased in TLR4-siRNA + GEM group (P ＜0.01).TLR4 protein level in the 4 groups was 0.83 ±0.08,0.81 ±0.07,0.85 ±0.07 and 0.16 ±0.03;p-AKT protein level 0.61 ±0.05,0.36 ±0.03,0.73 ± 0.07 and 0.21 ± 0.02;activated Caspase-3 protein level was 0.66 ± 0.05,0.73 ± 0.07,0.45 ± 0.04 and 0.91 ± 0.07,respectively.The expression of TLR4 and p-AKT in TLR4-siRNA + GEM group was significantly lower than that in GEM group (P ＜0.01),while the expression of activated Caspase-3 protein was increased significantly (P ＜ 0.05).Compared with the GEM group,the expression of p-AKT protein in LPS + GEM group was significantly increased (P＜0.01),and the expression of activated Caspase-3 protein was significantly decreased (P＜0.01).Conclusions TLR4 can inhibit the sensitivity of pancreatic cancer PNAC1 cells to GEM,and the mechanism is related to the activation of PI3K/AKT pathway and downregulation of activated Caspase-3.
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INTRODUCTION: Tyrosine kinase inhibitors have revolutionized the treatment of metastatic lung cancer in patients with epidermal growth factor receptor (EGFR) mutations. Amplified refractory mutation system (ARMS)‑reverse transcription‑polymerase chain reaction (RT‑PCR), the current standard for detecting EGFR mutation status is time‑consuming and highly expensive. Consequently any surrogate test which are cheaper, faster and as accurate as the PCR method will help in early diagnosis and management of patients with lung cancer, especially in resource‑limited settings. MATERIALS AND METHODS: Eighty‑five patients, all of South Indian origin, with adenocarcinoma of lung, registered between October 2009 and January 2013, were evaluated for EGFR mutation status by using scorpion probe based ARMS RT‑PCR method. Immunohistochemical (IHC) was performed using the phosphorylated AKT (P‑AKT) and thyroid transcription factor‑1 (TTF‑1) on above patient’s sample, and the results were compared with EGFR mutation tests. RESULTS: EGFR mutation was positive in 34 of 85 patients (40%). P‑AKT and TTF‑1 were positive in 50 (58.8%) and 68 (80%) patients respectively. Both P‑AKT and TTF‑1 had statistically significant correlation with EGFR mutation status. Positive and negative predictive value of P‑AKT in diagnosing EGFR mutation was 58% and 85.5% and that for TTF‑1 was 48.5% and 94.1%, respectively. The problem of low positive predictive value can partly be overcome by testing P‑AKT and TTF‑1 simultaneously. CONCLUSION: P‑AKT and TTF‑1 using IHC had statistically significant correlation with EGFR mutation with high negative predictive value. In the case of urgency of starting treatment, EGFR mutation testing may be avoided in those patients who are negative for these IHC markers and can be started on chemotherapy.
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Objective To observe the expressions of Akt and ERK phosphorylation in abdominal aortic aneurysm of rat model, and explore the pathogenesis of abdominal aortic aneurysm. Methods The rat model of abdominal aortic aneu-rysm was established. The diameter of abdominal aorta was measured and the extended rate of the aorta was calculated. HE staining was used to observe the change of pathology. Immunohistochemistry and Western blot methods were used to detect the expressions of Akt and ERK phosphorylation in the level of protein. Results The dilation of aorta was significantly high-er in abdominal aortic aneurysm group than that of saline group and normal group (P＜0.05). HE staining showed structural disorder and inflammatory cell infiltration in abdominal aortic aneurysm group. The results of immunohistochemistry and Western blot results showed that phosphorylation of Akt expression was significantly higher in abdominal aortic aneurysm group than that of saline group and normal group (P＜0.05). There was no significant difference in phosphorylation of ERK expression between three groups (P>0.05). Conclusion PI3K/Akt signaling pathway may be involved in the development of abdominal aortic aneurysm.
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PURPOSE: Mounting evidence suggests that alterations of Akt/protein kinase B (PKB) play an important role in tumorigenesis. Phosphorylated Akt regulates many of the key effector molecules involved in apoptosis, angiogenesis, and cell-cycle progression during tumorigenesis. The expression of phosphorylated Akt has been described in some human malignancies, but not in primary human gastric cancer. The purpose of this study was to explore the expression status of phosphorylated Akt protein in gastric carcinomas. MATERIALS AND METHODS: In the current study, we analyzed the expression of phosphorylated Akt protein in 60 advanced gastric adenocarcinomas by using immunohistochemistry and a tissue microarray approach. RESULTS: Immunopositivity (defined as > or =30%) was observed for the phosphorylated Akt in 42 (70%) of the 60 cancers. Normal gastric mucosal cells showed no or weak expression of phosphorylated Akt protein. CONCLUSION: Taken together, these results indicate that Akt is frequently activated in gastric adenocarcinoma cells and suggest that phosphorylayed Akt may play a role in the development of human gastric adenocarcinomas.