Interleukin 6 il 6

The paraffin sections were cut from control and treatment groups which underwent immunohistochemistry tests by using antigen retrieval methods. Tissue sections were incubated with primary antibodies for interleukin 6 (IL-6) (Genescript), interleukin 6 (IL-6) (Thermo Fischer), rabbit polyclonal collagens I and III (Thermo Fischer), and tumor necrosis factor TNF-α (Sino Biological) overnight, and then, they were stained with secondary antibodies at 370°C for thirty minutes. The stained sections were developed using diaminobenzidine and hematoxylin was used for counterstaining. All sections were viewed and captured using the ZEISS LSM 780 laser scanning microscope (Zeiss).
Sections were also stained with DAPI after three washings and then mounted on Fluoromount-G (Southern Biotech). Sections from cardiac tissue which were stained with troponin-I initially were analyzed for troponin intensity changes and compared with control, diabetic only, and diabetic rats with treatment for identification of the effects of glycyrrhizin on troponin-I in ventricular cardiac muscle.
Myocardial apoptosis: the effect of glycyrrhizin on apoptosis in cardiomyocytes in the diabetic rats was evaluated using DAPI, caspase, and BrDU staining.

Scutellarin was purchased from Shanghai Rong Wo Pharmaceutical Technology Co. (China). BLM hydrochloride was obtained from Haizheng Pharmaceuticals (Zhejiang, China). Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) ELISA kits were obtained from eBioscience (San Diego, CA, United States). Myeloperoxidase (MPO) and malondialdehyde (MDA) Colorimetric Activity Assay Kits were obtained from Jiancheng Institution of Biotechnology (Nanjing, China). Annexin V-fluorescein isothiocyanate (FITC) apoptosis kit was purchased from KeyGen Biotech (Nanjing, China). TRIzol reagent was obtained from Invitrogen Life Technologies (Shanghai, China). All other chemicals and reagents used in the study were of analytical grade.

After injection with pentobarbital sodium, blood samples were gathered from the carotid artery of AS patients and controls. The serum contents were treated with regional citrate anticoagulation (RCA) and centrifuged at 2500g for 20 min at four ° C. Blood cells were recovered by centrifugation at 700 × g for 20 min at 4° C. The ELISA kit (Cat. No. 210-A-050, R&D Systems, Minneapolis, MN) was conducted to identify the plasma and macrophages expressed according to the kit instructions. The serum levels of inflammatory cytokines interleukin-6 (IL-6) (eBioscience) and tumor necrosis factor-α (TNF-α) (eBioscience) were examined using ELISA kits (RapidBio, CA, USA).

The human cytokines Interleukin 10 (IL-10), Transforming Growth Factor beta-1 (TGFβ1), Transforming Growth Factor beta-2 (TGFβ2), Transforming Growth Factor beta-3 (TGFβ3), and Tumor Necrosis Factor Alpha (TNFα) were purchased from Sigma-Aldrich, Interleukin 4 (IL-4) and Interferon Gamma (IFNy) from R&D Systems/Bio-Techne (R&D Systems/Bio-Techne, Wiesbaden-Nordenstadt, Germany), and Interleukin 6 (IL-6) and Vascular Endothelial Growth Factor165 (VEGF) from PeproTech (PeproTech, Winterhude, Germany). Porcine TGFβ3 was purchased from Biozol (Biozol, Eching, Germany), whereas porcine TGFβ1, TGFβ2, IL-4, IL-6, IL-10, IFNy and TNFα were from R&D System/Bio-Techne. Since there was no porcine VEGF available, the above mentioned human VEGF was also used for stimulation of pRMG.
To diminish the influence of cytokines present in FCS, both confluent pRMG and MIO-M1 cells were rinsed two times with prewarmed serum-free medium, followed by starvation for 1 h at 37°C and 5% CO2 with serum-deprived medium. Afterwards, cells were treated over night with IFNy, IL-4, IL-6, IL-10, TGFβ1, TGFβ2, TGFβ3, TNFα or VEGF165, respectively, in a randomized plate design at a concentration of 5 ng/ml in 2 ml medium without FCS. Untreated cells cultured in serum-free medium served as a control. For this study, cells were treated with each cytokine separately, but not with multiple cytokines in combination.