Animals were distributed into three different groups apoE mice administered with the Amuvatinib c-Met inhibitor PDE5 inhibitor sildenafil, apoE mice administered with vehicle and WT control mice. This dose of sildenafil has been previously used in apoE mice by Dussault et al. and by our labora tory in studies about endothelial dysfunction, based on the fact that this drug has reduced Inhibitors,Modulators,Libraries oral bioavailability by pre systemic hepatic metabolism besides high clear ance in mice. All animals had ad libitum access to water and food during housing and treatment periods. For each protocol were used 6 to 10 animals per group. Samples Animals were euthanized with sodium thiopental overdose. A thoracic incision was performed for blood collection trough intra cardiac puncture. Blood was immediately transferred to a tube containing EDTA.
Per ipheral blood mononuclear cells were isolated by HistopaqueW density gradient centrifugation, according to the manufacturers instructions. The samples were stored at 80 C until further analysis. Liver cells enriched fractions from the mice liver of dif ferent groups were prepared as standardized in our labora tory based on previous studies. The left Inhibitors,Modulators,Libraries lobe of the liver was grossly triturated with surgical scissors and incu bated with an extraction solution containing proteinase K and collagenase type II to dissociate the cells. Then, the cell extract was filtered through a nylon screen to remove cell debris. After, the samples were washed twice in phosphate buffered saline to remove the enzymes. The samples were stored at 80 C until further analysis.
Before performing analyzes of ROS production and genotoxicity was carried out viability test through trypan blue exclusion test and both MNC and liver cells showed 80 90% viability. Measurement of lipid profile The plasma of peripheral blood samples was used to measurement of lipid profile, total plasma cholesterol, Inhibitors,Modulators,Libraries HDL, LDL and triglycerides were determined using com mercial colorimetric assay kits. VLDL and IDL were estimated Inhibitors,Modulators,Libraries by subtracting HDL and LDL from total serum cholesterol. Measurement of cytoplasmic reactive oxygen species by DHE DHE was used for the flow cytometry detection of intra cellular superoxide anion. DHE is freely permeable to cells and is rapidly oxidized, Inhibitors,Modulators,Libraries mostly by superoxide, to ethidium, which binds to DNA and amplifies red fluor escence signal.
To estimate the content of superoxide anion in cell suspension, selelck kinase inhibitor 106 MNC were incubated with 20 uL of DHE for 30 min at 37 C in the dark to load the cells with the dyes. For positive con trol, samples were treated for 5 min with 50 uM H2O2 to create an oxidative stress without being toxic to the cells. Cells were then washed, resuspended in PBS, and kept on ice for an immediate detection by flow cyto metry. Data was acquired and analyzed using the FACSDiva software.