Drp300

Fibroblast growth factor-21 (FGF-21), leptin, and adiponectin were analyzed using commercially available ELISA kits (Art. No DF2100, DLP00 and DRP300 respectively, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. All other blood samples were analyzed at the central laboratory of Uppsala University Hospital according to clinical routine.

The levels of inflammatory cytokines and chemokines were established in plasma samples and the levels of cortisol and adiponectin were established in serum samples. Undiluted samples were used for inflammatory cytokines and chemokines, and samples for CRP and adiponectin were diluted 200-fold. All biomarkers were analyzed in duplicate using the following enzyme-linked immunosorbent assay (ELISA) kits: IL-1β (KET6013; Abbkine, Wuhan, China), IL-6 (KET6017; Abbkine, Wuhan, China), TNF-α (ADI-900-099; Enzo, Madison Avenue, New York, NY, USA), IL-17 (KET6022; Abbkine, Wuhan, China), CRP (DCRP00; R&D Systems, Minneapolis, MN, USA), adiponectin (DRP300; R&D Systems, Minneapolis, MN, USA), cortisol (ADI-900-071; Enzo, New York, NY, USA), CCL1 (MBS824930; MyBioSource, San Diego, CA, USA), and CCL2 (LS-F146; LSBio, Seattle, WA, USA), according to the manufacturer’s instructions. The standard solutions were respectively prepared using the reagents provided in each kit. Plates were read at 450 nm using a VICTOR X4 Multimode Plate Reader (PerkinElmer, Waltham, MA, USA). Protein quantification was performed using the mean value of the duplicate samples.

The concentrations of IL-6 and adipokines (adiponectin, leptin) in the culture medium was evaluated by enzyme-linked immunosorbent assay test systems (R&D Systems, Canada: D6050, DRP300, DLP00), according to the manufacturers’ instructions.

Various laboratory indices were also measured before and after treatment for all the patients. Briefly, fasting venous blood samples were collected on the second morning to determine their levels of glucose (fasting plasma glucose (FPG) and 2hPG), HbA1c, triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), total cholesterol (TC), C-reactive protein (CRP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and insulin (Fins). All measurements were performed in a blinded manner by independent medical technicians who used a conventional automated analyzer in the biochemistry laboratory of our hospital. Additionally, adiponectin (APN) levels were determined using an ELISA kit (Cat.: DRP300; R&D, Wiesbaden-Nordenstadt, Germany) according to the manufacturer’s protocol. The homeostasis model assessment-insulin resistance index (HOMA-IR) was calculated as FPG × Fins/22.5. Differences in the index before and after treatment were calculated as Δindex = after_index − before_index.