Study population
From June 2019 to September, twenty-eight newly diagnosed and untreated IIM patients (20 with DM and 8 with ASS) (Table 1) who visited the Department of Rheumatology and Immunology of the First Hospital of Shanxi Medical University were enrolled in the study. All patients fulfilled the diagnostic criteria established by the 239th ENMC International Workshop in 201912. We also enrolled 12 age and sex-matched patients who underwent muscle biopsy to exclude neuromuscular disease based on clinical suspicion as healthy controls (HCs). Patients and HCs were excluded if they had an acute infection, malignant tumors, heart diseases, severe liver and kidney diseases, infectious diseases, other connective tissue diseases, and endocrine metabolic diseases. Disease activity was evaluated using visual analog scales for myositis disease activity (MYOACT) on admission (0–10); higher scores were considered indicative of greater disease activity. ILD was detected based on fibrosis on high-resolution computed tomography scans. The study was approved by the ethics committee of the First Hospital of Shanxi Medical University. Written informed consent was obtained from all patients and HCs.
Table 1
Clinical characteristics of the enrolled patients and controls.
Clinical Characteristics | IIM (n = 28) | HCs (n = 12) | P |
Age, years | 53.21 ± 15.33 | 51.92 ± 13.45 | 0.801 |
Female, number (%) | 20 (71) | 9 (75) | 0.817 |
BMI | 21.23 ± 3.03 | 20.82 ± 6.46 | 0.785 |
DM, ASS, number Disease duration, months Interstitial lung disease, number (%) | 20, 8 7.88 ± 12.56 9 (32) | (-) (-) (-) | |
Dermatomyositis |
Anti-TIF1-γ, number (%) | 5 (18) | | |
Anti-MDA-5, number (%) | 3 (11) | | |
Anti-Mi-2, number (%) | 4 (14) | | |
Anti-NXP2, number (%) | 4 (14) | | |
Antibody-negative dermatomyositis | 4 (14) | | |
Anti-synthetase syndrome |
Anti-Jo-1, number (%) | 6 (21) | | |
Anti-Pl-7, number (%) | 1 (4) | | |
Anti-Pl-12, number (%) | 1 (4) | | |
IIM, idiopathic inflammatory myopathy; HCs, healthy controls; BMI, body mass index; DM, dermatomyositis; ASS, Anti-synthetase syndrome; TIF1-γ, transcription intermediary factor 1 gamma; MDA-5, melanoma differentiation-associated protein 5; NXP2, nuclear matrix protein 2; Jo-1, histidyl tRNA synthetase; PL-7, threonyl tRNA synthetase; PL-12, alanyl tRNA synthetase. P < 0.05 was considered indicative of statistical significance. |
Tissue Lysis And Western Blotting Analysis
Isolated muscle tissue was stored in liquid nitrogen. Muscle tissue samples were collected and lysed using radioimmunoprecipitation assay lysis buffer (10 µL/mg) with 1% phosphatase and 1% protease inhibitor (Boster Biological Technology, Wuhan, China). Total protein concentration was detected using the BCA method (Boster Biological Technology). Aliquots of 25 µg of protein from each sample were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 30 min at 90 V followed by 60 min at 120 V. Proteins were then electrotransferred onto polyvinylidene difluoride membranes (Trans-Blot Turbo Transfer; Bio-Rad, Hercules, CA, USA). The membranes were blocked by incubation for 2 h at room temperature with 5% skimmed milk powder in phosphate-buffered saline (PBS) and then incubated overnight at 4°C in the same buffer with the primary antibodies (anti-p-STAT3, 1:1000; Abcam, Cambridge, UK; anti-IL-17, 1:1000; ABclonal Technology, Wuhan, China; anti-β-actin, 1:10,000; Bioworld Technology, Beijing, China), washed three times with PBS and incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobin G secondary antibody (1:10,000; Boster Biological Technology) at 37°C for 2 h. The membranes were next exposed to X-rays and imaged after soaking in enhanced chemiluminescence reagent (Boster Biological Technology) for 1 min. Experiments were performed with at least two replicates. Then images were processed with ImageJ (NIH, Bethesda, MD, USA), data analysis was performed using SPSS 26.0 Software (SPSSInc., Chicago, Ill., USA) and graphs were performed using Prism (GraphPad Software, San Diego, CA, USA).
Immunohistochemistry
Immunohistochemical analyses for p-STAT3 and IL-17 were performed using formalin-fixed, paraffin-embedded 5-µm-thick sections cut from muscle tissue and breast cancer tissue (used as positive controls) samples as described previously (19). Isotype-matched irrelevant antibodies were used as negative controls. The slides were boiled in 600 mL of 10 mM sodium citrate buffer (pH 6.0) for 15 min for antigen retrieval. The primary antibodies (rabbit anti-human IL-17, 1:100 dilution; ABclonal Technology; rabbit anti-human p-STAT3, 1:100 dilution; Abcam) were added, and incubation was continued for 12 h at 4°C. In the control experiments, each isotype-matched antibody was applied at the same concentration as the primary antibodies. The scores representing p-STAT3- and IL-17-expressing cells were determined by combining the average percentage score of positive cells with the average intensity score in three representative high-power microscopic fields (original magnification 400×) using CaseViewer (3DHISTECH, Budapest, Hungary). The average percentage of positive cells was determined from the number of brown-stained cells compared to the number of total cells in each image, in accordance with Fromowitz’s scoring criteria (0, < 10%; 1, ≥ 10 to < 50%; 2, ≥ 25 to < 50%; 3, ≥ 50 to < 75%; and 4, ≥ 75%). The staining intensity was recorded as mean RGB score (0, RGB > 245.75; 1, RGB ≤ 245.75; 2, RGB ≤ 227.25; 3, RGB ≤ 208.75). The RGB score was multiplied by the number of positive cells (< 3, negative; ≥ 3, positive).
Enzyme-linked Immunosorbent Assays (Elisas)
Serum was separated from venous blood and placed at room temperature for 10–20 min. The supernatant was centrifuged at 12000 r/min for 15 min, then stored at − 20℃. IL-6 levels were determined using Human IL-6 ELISA kits (Abcam, Cambridge, UK), in accordance with the manufacturer’s instructions, then quantified using a microplate reader (450 nm).
Statistical analysis
Continuous variables were expressed as means ± standard deviations. Categorical variables were expressed as median and compared by the Mann–Whitney rank sum test. Spearman's correlation analysis and Bonferroni correction were performed to analyze the correlation. A P value of < 0.05 was considered statistically significant. All statistical analyses were performed using IBM SPSS Statistics 26.0 (SPSS, Chicago, IL, USA).