Animals at early stage 3 were anaesthetized by incubation in CO2 for 20 min

nterassay and intraassay coefficients of variation for the ELISAs were,10% and,5%, respectively. RT-PCR ELISA Tools, LLC was used to interfere miR-155 and suppressors of cytokine signaling1 interaction, with control-TPmiR-155 as the matched control. CD4+ T cell activation and polarization 4 h after nucleofection, CD4+ T cells were activated by 5 mg/ml plate-bound anti-CD3 and 2 mg/ml soluble anti-CD28. For propagation under Treg condition, 100 u/ml rIL-2, 10 ng/ml rTGF-b1, 10 mg/ml anti-IFN-c, and 10 mg/ml anti-IL-4 were provided. For propagation under Th17 condition, 2.5 ng/ml rTGF-b1, 30 ng/ml rIL-6, 10 mg/ml anti-IFN-c, and 10 mg/ml anti-IL-4 were provided. All antibodies used were purchased from ebioscience. All cytokines used were purchased from Peprotech. Western blotting Protein extracts were prepared in lysis buffer with proteinase inhibitor as well as phosphatase inhibitor. Whole-cell lysates were separated in 9% or 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis gels and then electroblotted onto a nitrocellulose. After incubation for 1 h in 5% skim milk diluted in Tris-buffered saline with Tween 20 at room temperature, the membrane was incubated overnight at 4uC with a dilution between 1/500 and 1/1000 of primary antibodies. Then washed 3615 min with TBST, incubated for 2 h with a 1:2000 dilution of horseradish peroxidaseconjugated goat anti-rabbit or anti-mouse IgG secondary antibody for 2 h at room temperature. The membrane was washed 3615 min with TBST again and the blots were developed by enhanced chemiluminescence. The levels of phosphorylated proteins were normalized to their total protein levels and other proteins were normalized to the signal generated for b-actin. All reagents used were purchased from cell signaling technology. Flow cytometry analysis 4 days after transfection and activation, cells were collected and used for detecting Treg and Th17 cells differentiation. For detecting Th17 cell differentiation, cells were stimulated for 4 h with 25 ng/ml phorbol Piclidenoson myristate acetate and 1 mg/ml ionomycin in the presence of 2 mM monensin in the last 2 h. Cells were collected and stained with FITC-labeled anti-mouse CD4 for 30 min at 4uC. Then, cells were fixed and permeabilized according to the manufacturer’s protocol. Next, cells were incubated with PE-labeled anti-mouse IL-17A in the dark for 20 min. For detecting Treg cells differentiation, cells were stained with FITC-labeled anti-mouse CD4 and APC-labeled anti-mouse CD25 for 30 min at 4uC. After fixation and permeabilization, PElabeled anti-mouse Foxp3 were provided. Finally, stained cells were resuspended in 200 ml washing buffer and analyzed by FACS Calibur Flow Cytometer. All reagents used were purchased from eBioscience. Statistical analysis All values were expressed as mean 6 SD. Statistical significance was determined by performing ANOVAs for multiple comparisons and Student’s t test for two comparisons. P values,0.05 were considered statistically significant and,0.01 were considered highly statistically significant. According to the World Health Organization, over 1.7 billion people worldwide are overweight or obese. Moreover, obesity is one of the major epidemics expanding in Western countries today, and increases the prevalence of most cardiovascular risk factors independently of other associated diseases. One of the most common independent cardiac features in obesity is cardiac hypertrophy . The physiopathology of CH in obesity is complex an