Assay of isometric force in Rat learn more aorta rings The isolated

aortic rings were cleaned to remove the adherent tissues and hung in 10-ml organ bath with Krebs’ solution at 37°C, pH 7.4, and containing 95% O2 and 5% CO2. The modified Krebs’ solution was composed of the following components: 110 mM NaCl, 4.6 mM KCl, 2.5 mM CaCl2, 24.8 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 5.6 g glucose. The tissue’s isometric tension was measured with force transducers that connected with a BL-420E+ biological function experimental system (Chengdu Technology and Market, Chengdu, China). The vessel rings were equilibrated for 1 hour with the tension of 2.0 g and pre-contracted with KCl (60 mM) to produce the maximal KCL-induced contractile plateau. Subsequently the cumulative dose–response curve for noradrenaline (NA) (10-10-10-5M) was obtained. The values of the NA-induced contraction were expressed as a percentage of maximal contraction induced by KCl. Measurement of SOD, MDA and nitrite/nitrate (NOx) levels in plasma The oxidative

stress indices were measured to explore whether LBP could reduce exhaustive exercise-induced oxidative stress. The levels of SOD, MDA and NOx (NO2- and NO3-) were determined by using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) EPZ015938 according to the manufacturer’s instructions. HSP70 determination The plasma level of HSP70 was detected by a commercially available ELISA kit (Cusabio Biotechnology, Wuhan, China). The amount Mirabegron of HSP70 in plasma was estimated from the calibration curve ranging from 62.5 to 4000 pg/ml. RT-PCR analysis Total RNA was prepared from the thoracic aorta using RNA AxyPrep Pure RNA isolation kit (www.selleckchem.com/products/XL880(GSK1363089,EXEL-2880).html AXYGEN, USA) according to the manufacturer’s instructions. The purity and concentration of RNA was determined by spectrophotometry at 260 nm and 280 nm. Complementary DNA (cDNA) was synthesized using a reverse transcription kit (TransGen

Biotechnology, Beijing). Quantitative PCR was performed using a quantitect SYBR green PCR kit (TransGen Biotechnology, Beijing) as follows: 35 cycles of denaturation at 94°C for 30 sec, annealing at 62°C for 30 sec and extension at 72°C for 30 sec. Primers used for the PCR were shown in Table 1. Relative gene expression levels were determined using the 2—△△Ct method. Table 1 GenBank accession code, primer sequences, and predicted size of the amplified product Gene Primer sequences GenBank bp eNOS Forward primer: 5′-CACACTGCTAGAGGTGCTGGAA-3′ NM_021838 109 Reverse primer: 5′-TGCTGAGCTGACAGAGTAGTAC-3′   β-actin Forward primer: 5′-TCATGAAGTGTGACGTTGACATCCGT-3′   285 Reverse primer: 5′-CCTAGAAGCATTTGCGGTGCAGGATG-3′   Statistical analysis Results were presented as the mean ± SD. Two-way ANOVA was used to evaluate any differences between the two sets of dose–response curves. The remaining data were evaluated by one-way ANOVA and Student’s t-test. The statistical analyses were performed by SPSS for Windows 11.5.0 software. P<0.