Real-time PCR
Total RNA of PBMCs was extracted using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) in accordance with the manufacturer’s instructions. cDNA was synthesized from the total RNA by using PrimeScript Reverse Transcriptase (Takara Bio, Otsu, Japan) according to the manufacturer’s instructions. To confirm the mRNA expression ofCOX2 in PBMCs, real-time PCR was performed with the LightCycler 480 System II (Roche Diagnostic, Mannheim, Germany) using SYBR Premix DimerEraser (Takara Bio) following the manufacturer’s instructions.β-actin (ACTB ) was used as a reference gene. The relative expression levels were calculated by using the ΔΔCt method, and the results were indicated as relative change to no treatment group. Primers were 5’-ACG TTT TCT CGT GAA GCC CT-3’ and 5’-TCT ACC AGA AGG GCG GGA TA-3’ for COX2 , and 5’-TCT TCC AGC CTT CCT TCC TG-3’ and 5’-ACC GTG TTG GCG TAG AGG TC-3’ for ACTB .