E el h0134c

Articular cartilage tissue (50–100 mg) was obtained from each KOA patient during artificial joint replacement surgery. Total RNA was extracted using Qiagen 74106 tissue RNA extraction kit (Qiagen, Germany). The mRNA was reverse-transcribed into cDNA using the M-MLV2 reverse transcription kit. RT-PCR was performed in 25 μl reaction volumes containing 2 μl of cDNA, 12.5 μl of 2× PCR buffer, 15 mmol/l MgCl₂, 25 μmol/l each of the four dNTPs, 50 pmol/l primer, and 1 U of Taq enzyme. The primers used for RT-PCR were as follows: IL-6, 5′-GAG CTT CAG GCA GGC AGT ATC-3′ (forward) and 5′-GTA TAG ATT CTT TCC TTT GAG GC-3′ (reverse); IL-8, 5′-AGT GCT AAA GAA CTT AGA TG-3′ (forward) and 5′-TAT GAA TTC TCA GCC CTC TT-3′ (reverse); MMP-13, 5′-GGT CCC AAA CGA ACT TAA CTT ACA-3′ (forward) and 5′-CCT TGA ACG TCA TCA TCA GGA AGC-3′ (reverse); and β-actin, 5′-ACC ACC ATG GAG AAG GCT GG-3′ (forward) and 5′-CTC AGT GTA GCC CAG GAT GC-3′ (reverse). Synovial fluid (1 ml) from each KOA patient was collected and centrifuged at 14000 r/min for 20 min at 4°C, after which the supernatant was isolated for determination of IL-6 (Cat#E-EL-M0044c, Elabscience), IL-8 (Cat# E-EL-H0048c, Elabscience), and MMP-13 (Cat# E-EL-H0134c, Elabscience) levels by ELISA. All procedures were carried out in strict accordance with the manufacturers’ instructions.

The cytokines IL-17A and IL-10 (D1700 and D1000, respectively, purchased from R&D systems, Minneapolis, United States) and MMP13 (E-EL-H0134c, Elabscience Biotechnology, Wuhan, China) were determined using enzyme-linked immunosorbent assay (ELISA). The ELISA was performed according to the manufacturer’s instructions.