Il 6 and tnf α

Cell lysates were rinsed with ice-cold PBS and prepared using lysis buffer with 1% Triton, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), and 1% deoxycholate and protease inhibitors cocktail (Roche). Protein concentration was determined by Bradford protein assay kit (Bio-Rad) and equal amount of protein was separated by 12% SDS-PAGE using Bio-Rad apparatus. The membranes were blocked for 2 hours in 5% skim milk at room temperate after protein was transferred to PVDF membrane (Millipore, Billerica, MA, USA). The proteins were incubated with the following primary antibodies: IL-6 and TNF-α (Merck Millipore, Bedford, MA, USA), ICAM-1, Hes5 and MCP-1 (Abcam, Cambridge, Mass, USA), Hes1 (OriGene Technologies, Rockville, MD, USA), Hey1 and Hey2 (Proteintech Group, Chicago, IL, USA), and phospho-p65 (CST, Chicago, IL, USA). Anti-GAPDH antibody (Bioworld, Nanjing, China) was used as the loading control in all western blots. The secondary antibodies, anti-mouse and anti-rabbit IgGs conjugated to horseradish peroxidase, were obtained from Kangchen (Shanghai, China). Finally, protein bands were visualized using Supersignal West Femto Substrate (Pierce, Rockford, IL, USA).

Fasting blood samples (5 mL anticoagulant and 5 mL non-anticoagulant) were collected from 8:00 to 9: 00 on the day at the end of each intervention. IL-6, TNF-α, CRP, FIB, CD62P, CD40L, ICAM-1, NO, ET-1, MMP-2 and MMP-9 levels were tested by a professional testing institution. CHO, TG, HDL-C and LDL-C levels were tested by the laboratory of the hospital. IL-6, TNF-α, CD62P, CD40L and ICAM-1 was detected by a bead-based multiple flow cytometry on Luminex 200 system (Luminex Corporation, Austin, TX, USA) with chips (IL-6 and TNF-α: Merck Millipore, Germany; CD62P, CD40L and ICAM-1: R&D, USA). CRP, NO, MMP-2, MMP-9, FIB and ET-1 was separately detected by ELISA kit (CRP, NO, MMP-2, MMP-9: R&D, USA; FIB: NOVUS, USA; ET-1: Elabscience Biotechnology Co. Ltd, Wuhan, China).
In addition, a compound index-atherogenic index of plasma (AIP) was calculated by the results of TG and HDL-C levels and the formula: $\text{AIP}=\text{log}\left(\frac{\text{TG}}{\text{HDL}-\text{C}}\right)$ .