Subjects
Blood were obtained from 13 consecutive unselected SLE patients identified according to the Systemic Lupus International Collaborative Clinics (SLICC) classification criteria[9], including 6 patients (46.2%) positive for anti-Sm Ab, 7 patients (53.8%) positive for anti-dsDNA Ab (Table 2), as well as the healthy blood donors. Serum was obtained by allowing whole blood to clot at room temperature for 30 min, then leaving the whole samples stay on ice for another 60 min, followed by centrifugation at 4000 rpm at 4°C for 10 min. The supernatant was collected and stored at − 80°C until use.
Written informed consent was obtained for each participant. This study was approved by the Ethics Committee of Union Hospital at Huazhong University of Science and Technology, and the methods were applied in accordance with the approved guidelines. Disease activity was measured using the Systemic Lupus Erythematosus Disease Activity Index-2000 (SLEDAI-2K).[10]
Routine laboratory investigations
Routine laboratory investigations were performed, including Coagulation Tests(D-Dimer, FDP, ATIII and so on), inflammatory markers(ESR, CRP, PCT), liver and kidney function tests. Immunological examinations such as complement (C3 and C4), cytokines (IL-2,IL-4,IL-6,IL-10) and autoantibody screen were measured by standardized technique.
Measurement of the Complement proteins
The Serum C3a and C1q were detected by western blot. Briefly, serum (0.5ul/sample) was premixed with PBS (7.5ul/sample) and roti-load 1 (2ul/sample), then cooked for 10min at 98°C. The mixed samples were then separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked in 4% skim milk powder and 1% BSA (supplied with 0.05% Tween-20) at 4℃ overnight, folllowed by incubation at room temperature for 1 h with either Rabbit anti-human C3a (1:2000; Cat No. #A218, Complement Technology, Inc.) or Goat anti-human C1q (1:2000; Cat No. #A200, Complement Technology, Inc.). After washing, horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2000; Cat No. SA00001-2, Proteintech, Inc.) and rabbit anti-goat IgG (1:2000; Cat No. SA00001-4, Proteintech, Inc.) were then added as the secondary antibody. Bands were analysed using a ChemiDoc™ XRS+ Imaging System with Image Lab™ Software version 5.2. (Bio-Rad Laboratories, Inc.).
Statistical analyses
Statistical analyses were performed by using GraphPad Prism (version 5.01). Mean serum level of C3a and C1q in two groups was compared by Student’s t test. Correlation of C3a with SLEDAI scores, anti-dsDNA Ab, complement components C3 and C4, IFN-a and IL-6 were analyzed by Pearson correlation test. The results were expressed as median and range. Statistical tests with two-tailed p values less than 0.05 were considered significant.