Of 0 to 3, with 0 = negative; 1 = weak; 2 = moderate, and 3 = strong.Determination of E-sel
Of 0 to 3, with 0 = negative; 1 = weak; 2 = moderate, and 3 = strong.Determination of E-sel, ICAM-1, VCAM-1 and MCP-1 gene expression94 , 35 cycles of denaturation at 94 for 30 sec, annealing at the temperature indicated in Table 1 for 1 min, and extension at 72 for 30 sec, followed by a final extension step for 10 min at 72 . Electrophoresis was carried out at 5 V/cm for 30 min on a 2 agarose gel and PCR products were visualized with silver staining. Absorbances of each band were determined by densitometric analysis using the one-Dscan gel analysis software (Scanalytics, Billerica, USA). mRNA levels were expressed as the ratios between target genes and -actin.Statistical analysisResults were expressed as mean D. Statistic analysis was carried out using the SPSS statistical package version 10.0 (SPSS Inc., USA). Student’s t-test was performed to compare means between two groups. The p value<0.05 or 0.01 was considered to be statistically significant.Total RNA was isolated from the second portion of aorta using TRIZOL (DingGuo Biotechnology Co. Ltd, Beijing, China). To avoid the interference of DNA, 4 L of the obtained RNA was dealt with DNase (Promega, Madison, USA) in 10 L reaction mixture before reverse transcription. Subsequently, 4 L of the DNA-free RNA were reverse transcribed into cDNA in 20 L reaction mixture using a reverse transcriptase (Toyobo, Osaka, Japan). Reverse transcription was carried out for 10 min at 30 , 60 min at 42 , followed by an inactivation step at 99 for 5 min. Target gene expressions were determined by semi-quantitative PCR with -actin as an PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26778282 internal standard. PCR amplification was performed on an Gene Cycler (Bio-Rad, USA) in a total volume of 25 L, which was composed of 1.0 L of cDNA template, 10 ?PCR buffer (50 mM KCl, 10 mM Tris Cl, 2.5 mMMgCl2, pH 8.3), 0.2 mM dNTPs (Genview scientific Inc., USA), 20 pmol/L forward primers, 20 pmol/L reverse primers,1.0 U of Taq DNA polymerase (TaKaRa Co. Ltd., Tokyo, Japan). The amplification procedure consisted of an initial denaturation step for 2 min atTable 1 Primers sequence of target genesTarget genes -Actin E-sel Gene bank accession no. NM_001101683.1 PrimersResultsBody weight and serum lipids analysisBodyweight and serum lipids were measured after six and ten weeks respectively, and the obtained data were shown in Table 2. According to Table 2, there was no significant difference in bodyweight among any of the groups at t = 6 week and at t = 10 week (p > 0.05). After order ML390 feeding on high cholesterol diet for 6 and 10 weeks, serum lipid levels including total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) of model rabbits all increased greatly (P<0.01) compared with control rabbits. Treatment of fenofibrate (12 mg/kg) or kaempferol (30 mg/kg and 150 mg/kg) for six and ten weeks significantly lowered TC, TG, HDL-C and LDL-C levels of rabbits in comparison to the model group (p < 0.01 or P<0.05).Serum inflammatory factors analysisAs has mentioned in Background, TNF- and IL-1 are two important inflammatory factors in the progressionAnnealing temperature ( )Size (bp)5 -TTCCAGCCCTCCTTCCT- 3 5 -GCCCGACTCGTCATACT-NM_001082312.5 - AATGGCAGATACAGAGAACT- 3 5 -TGGCTTGGAAGAGAATAACT-ICAM-AB128157.5 -GACATTCTTGAACAGTGACAG- 3 5 -CGGACACAGCTCTCAGTA-VCAM-NM_001082152.5 -GGAGACACTGTCATTATCTCCTG- 3 5 -TCCTTTCATGTTGGCTTTTCTTGC-MCP-M28883.5 -GGTGTAAAGGCAGGTGTG- 3 5 -AGGATAGGAAAGGATGGG-Kong et al. Lipi.