The present study was approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China).

A total of 25 patients with PM (male:female, 12:13; age range, 40–80 years; average age, 60 years) were recruited from the First Affiliated Hospital of Zhengzhou University between June 2014 and May 2016. All enrolled patients were treated with high-dose dexamethasone, and individual patients were treated with tacrolimus or mercaptopurine. Muscle tissue samples were obtained from each patient: One sample was obtained prior to treatment and the other was obtained after treatment. Four men and six women were included in the healthy control group; these individuals had no clinical signs of muscle disease (mean age, 60 years). The clinical features of the selected patients and healthy control individuals are listed in Table I.

Serum samples were collected from the 25 patients with PM pretreatment and post-treatment. Serum samples were also collected from the healthy controls. Briefly, the blood samples were collected into anticoagulant tubes and centrifuged at 1,000 × g for 10 min at 4°C. The serum samples were then maintained at −80°C for subsequent analyses. Written informed consent was obtained from the participants.

A mouse model of experimental autoimmune myositis (EAM) was generated in the present study. BALB/c mice were obtained from Guangdong Medical Laboratory Animal Center (Foshan, China). The mice were housed under the following conditions: Temperature, 21°C; humidity, 40–60%; 12-h light/dark cycle and free access to food and water. Briefly, 1.5 mg rabbit myosin (M1636) and an equal volume of complete Freund's adjuvant (CFA, 1 mg/ml, F5881) (both from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) were mixed, after which 1 ml CFA mixed with 5 mg Mycobacterium tuberculosis (cat. no. 231141; BD Biosciences, Franklin Lakes, NY, USA) was added to the mixture. The obtained reagent was initially injected into the left hind leg of mice. After 1 week, it was injected into the tail root of mice. Furthermore, all of the mice were intraperitoneally injected with pertussis toxin (PT, cat. no. P2980; Sigma-Aldrich; Merck KGaA; 200 µl normal saline mixed with 500 ng PT) alongside each of the two previous injections. Healthy adult female BALB/c mice (weight, 15–18 g; age, 5–6 weeks) were randomly divided into the following groups (n=5 mice/group): Control group, in which mice were untreated; model group, in which mice underwent EAM induction; anti-IL-17 group, in which EAM mice were injected daily with anti-IL-17 antibody (100 µg/mice, cat. no. 13838; Cell Signaling Technology, Inc., Danvers, MA, USA) 14 days post-EAM induction; and anti-HMGB1 group, in which EAM mice were intraperitoneally injected with anti-HMGB1 antibody daily (100 µg/mice, cat. no. 6893; Cell Signaling Technology, Inc.) 14 days post-EAM induction. All animals were euthanized by rapid cervical dislocation 4 weeks after antibody injection. The animal study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University.

The mouse muscle tissues were fixed with 4% paraformaldehyde for 6 h at room temperature. After dehydration with ethanol (from a low concentration to a high concentration), the tissues were embedded in paraffin. Paraffin-embedded mouse muscle tissues were then sliced into 4–6 µm sections and the sections were mounted onto the slides. The slides were placed into hematoxylin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 10 min at room temperature. After being washed with tap water for 1–2 min, the slides were then placed in 10% glacial acetic acid for 10 sec and placed in 1% ammonia water until the sections turned blue. Subsequently, after being washed with tap water for 1–2 min, the slides were placed in eosin (Beijing Solarbio Science & Technology Co., Ltd.) for 10 sec. After dehydration in a gradual series of ethanol (70, 90, 95 and 100%), the slides were placed in xylene for 2 min; this step was repeated once. Finally, the slides were sealed with neutral gum. The slides were observed under an inverted microscope, in order to count the percentage of inflammatory cells in the whole field. The histopathologic scores were evaluated as follows: 0 score, no inflammatory cells; 1 score, <25% inflammatory cells; 2 score, 25–50% inflammatory cells; 3 score, >75% inflammatory cells.

Muscle tissues obtained from the patients or mice, were fixed in 4% paraformaldehyde and 30% sucrose solution at 4°C for 73 h. The tissue samples were embedded in paraffin and then dissected into sections (4–6 µm). Following treatment with 3% hydrogen peroxide in methanol for 30 min at room temperature, the slides were incubated with primary antibodies against CD163 (1:500 dilution, cat. no. ab182422; Abcam, Cambridge, MA, USA) at 4°C overnight. The tissue samples were then incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibodies (1:1,000 dilution, cat. no. ab6721; Abcam) at room temperature for 2 h. Finally, the tissue samples were treated with DAB (Sigma-Aldrich; Merck KGaA). Cells in the negative group were incubated with nonspecific IgG (1:500 dilution, cat. no. ab108337; Abcam).

As previously described (37), isolation of peritoneal macrophages was conducted. Briefly, the normal mice were euthanatized by rapid cervical dislocation and were disinfected in 70% ethanol for 5 min. Under sterile conditions, a small incision was made along the midline of the mice with sterile scissors. Subsequently, pre-chilled saline (4°C) was injected into the peritoneal cavity; fluid from the peritoneum was aspirated and was transferred into a 50-ml conical polypropylene centrifuge tube on ice. The peritoneal exudate cells were centrifuged at 400 × g for 10 min at 4°C. Following removal of the supernatant, the cells were resuspended in cold Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and were gently pipetted up and down. The cells were then seeded at a density of 1×10⁶/ml in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) and incubated at 37°C in an incubator containing 5% CO₂. Following 2 h incubation, the macrophages were grouped as follows: Control group, which consisted of untreated cells; negative control (NC)-mimics group, in which cells were transfected with NC mimics; mimics group, in which cells were transfected with miR-381 mimics; NC-inhibitors group, in which cells were transfected with NC inhibitors negative control; inhibitors group, in which cells were transfected with miR-381 inhibitors; NC-small interfering (si)RNA, in which cells were transfected with NC siRNA (no specific sequence); and inhibitors + siRNA-HMGB1group, in which cells were co-transfected with miR-381 inhibitors and siRNA-HMGB1.

The cells were seeded into a 6-well plate at a density of 3×10⁴/well. Once cell confluence reached 70%, the cells were transfected with miRNA (50 nM) and/or siRNA (50 pmol) using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C. After 48 h, the cells underwent further experimentation. The miRNAs and siRNA used were as follows: miR-381 mimics (miR10017081-1-5); miRNA negative control (miR01201-1-5); miR-381 inhibitor (miR20017081-1-5); miRNA inhibitor control (miR02201-1-5) (all from Guangzhou RiboBio Co., Ltd., Guangzhou, China); si-HMGB1 (MBS8222378) and siRNA negative control (NC) (MBS8241404) (both from MyBioSource, San Diego, CA, USA).

The secreted levels of IL-17 (mouse, M1700; human, D1700) and ICAM-1 (mouse, MIC100; human, Dy720) were detected using ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA). The mouse serum creatine kinase (s-CK) ELISA kit (CSB-E14407m) was purchased from Cusabio Technology LLC (Houston, TX, USA). The human s-CK ELISA kit (NR-R10105) and the HMGB1 ELISA kits (mouse, NB-S11134; human BG-HUM11133) were purchased from Novatein Biosciences, Inc., (Woburn, MA, USA). All experiments were performed according to the manufacturer's protocols. Finally, the optical density at 450 nm was measured on a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The mouse tissues were ground in liquid nitrogen; total RNA from tissues and cultured cells was extracted by TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, 2 µg RNA was used for cDNA synthesis using first strand cDNA kit (Sigma-Aldrich; Merck KGaA), according to the manufacturer's protocol. RT-qPCR was performed using the SYBR Green Premix Reagent (Takara Bio, Inc., Otsu, Japan) on an ABI 7500 Thermocycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR thermocycling conditions were as follows: 94°C for 5 min; followed by 30 cycles at 94°C for 30 sec and 60°C for 30 sec; and a final extension step at 72°C for 10 min. GAPDH/U6 was used as an internal control. The sequences of the primers (Invitrogen; Thermo Fisher Scientific, Inc.) used for PCR amplification are indicated in Table II.

The collected tissues were ground in liquid nitrogen and proteins were isolated using the total extraction sample kit (Sigma-Aldrich; Merck KGaA). Cultured cells were lysed on ice in radioimmunoprecipitation assay lysis buffer (pH 7.4; 1 mM MgCl₂, 1% Triton X-100, 10 mM Tris-HCl and 0.1% SDS). Protein concentration was determined using the bicinchoninic acid protein assay kit (Bio-Rad Laboratories, Inc.). An equal quantity (50 µg) of protein from each sample was separated by 8% SDS-PAGE and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). Nonspecific antigens were blocked by immersing the membranes in 5% non-fat milk at room temperature for 2 h. Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: Anti-HMGB1 (1:500 dilution, cat. no. ab18256), anti-IL-17 (1:5,000 dilution, cat. no. ab9565), anti-ICAM-1 (1:1,000 dilution, cat. no. ab2213) and anti-GAPDH (1:2,500 dilution, cat. no. ab9485) (all from Abcam). The membranes were then incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:2,000; cat. nos. ab6789 and ab6721; Abcam) at room temperature for 1 h. The signals were detected using an enhanced chemiluminescence reagent (EMD Millipore). The blots were analyzed using Quantity One software version 4.62 (Bio-Rad Laboratories).

Bioinformatics prediction was performed using the online databases TargetScan (http://www.targetscan.org/) and miRDB (http://www.mirdb.org/). The HMGB1-3′ untranslated region (3′UTR) was synthesized by Invitrogen; Thermo Fisher Scientific, Inc. The HMGB-3′UTR was amplified to the downstream site of pGL4 luciferase vectors (Promega Corporation, Madison, WI, USA). The HMGB1-3′UTR mutant (mut) was generated using a rapid site-directed mutagenesis kit (Thermo Fisher Scientific, Inc.). Cells were seeded at 3×10⁴/well in a 24-well plate; after 24 h, the cells were transfected with 1 µg HMGB1-3′UTR (wild-type or mut)-luciferase plasmid, 50 nM miR-381 mimics/miR-NC and 150 ng Renilla luciferase plasmid using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). The cells were then incubated at 37°C for 36 h. Luciferase activity was detected using a dual-luciferase reporter assay kit (Promega Corporation), according to the manufacturer's protocol. All data were normalized to Renilla luciferase activity. The relative luciferase activities were expressed as relative fluorescence units.

Cells (~5×10⁴ cells/ml) were serum-starved for 24 h in serum-free DMEM at 37°C. The cells were then plated into a 24-well Transwell plate (Corning Incorporated, Corning, NY, USA). DMEM supplemented with 15% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) was placed into the lower chamber. After 24 h, cells that penetrated into the lower chamber were fixed with 95% ethyl alcohol and were stained with 0.1% crystal violet at room temperature for 20 min. Finally, the cells were observed under an inverted microscope and the number of migrated cells was counted.

All experimental results are presented as the means ± standard deviation and experiments were repeated at least three times. Kaplan-Meier analysis was used for survival analysis; log rank test was used to compare the differences. The receiver operating characteristic analysis was performed to distinguish the patients with high HMGB1 expression from the patients with low HMGB1 expression. GraphPad Prism 6.0 (GraphPad, Inc., La Jolla, CA, USA) was used to perform data analysis. All assays were analyzed by one-way analysis of variance followed by Turkey's test. P<0.05 was considered to indicate a statistically significant difference.

Compared with in the healthy control individuals, the serum concentrations of s-CK (434±70 vs. 97±11 pg/ml), HMGB1 (348±63 vs. 102±19 pg/ml) and IL-17 (297±38 vs. 89±14 pg/ml) were markedly increased in patients with PM. However, following treatment with a glucocorticoid or other immunosuppressive drugs, s-CK (212±35 vs. 434±70 pg/ml), HMGB1 (220±38 vs. 348±63 pg/ml) and IL-17 (158±21 vs. 297±38 pg/ml) expression levels were decreased, as detected by ELISA (P<0.05; Fig. 1A–C). Furthermore, in patients with PM, the expression levels of HMGB1 were significantly increased, whereas the expression levels of miR-381 were markedly decreased compared with in healthy control individuals. Conversely, the expression levels of HMGB1 and miR-381 were reversed in the post-treatment group (P<0.05; Fig. 1D and E). As shown in Fig. 1F, patients with PM with a high expression of HMGB1 had a worse prognosis (P=0.0204). A receiver operating characteristic curve analysis was conducted to distinguish patients with PM with high HMGB1 expression from patients with PM with low HMGB1 expression. The cutoff value was 14.2 ng/ml. The area under the curve was 0.733 (Fig. 1G).

Muscle samples from control, pretreatment and post-treatment groups were stained with anti-CD163. The expression of CD163 was almost negative in the control group (Fig. 2A). Conversely, strong CD163 staining was identified in the pretreatment group (Fig. 2B), whereas CD163 expression was markedly reduced in the post-treatment group (Fig. 2C).

In the mouse control group, muscle fibers were polygonal in shape and were roughly the same size. A large amount of inflammation and macrophage infiltration was detected, and muscle fibers with varied sizes were observed in the EAM model group. Conversely, inflammation and macrophage infiltration was inhibited in the anti-IL-17 and anti-HMGB1 groups, compared with in the model group (Fig. 3A). Similarly, the histopathologic score in the model group was elevated, whereas treatment with anti-IL-17 and anti-HMGB1 resulted in a reduction in histopathologic score (Fig. 3B; Table III).

CD163 expression was increased in the model group compared with in the control group (Fig. 4A and B). Conversely, following treatment with anti-IL-17 and anti-HMGB1, CD163 expression was markedly reduced (Fig. 4C and D).

The results of an ELISA indicated that, compared with in the control group, the levels of s-CK, HMGB1 and IL-17 were significantly increased in the model group. However, the expression levels of s-CK, HMGB1 and IL-17 were reduced following anti-IL-17 and anti-HMGB1 treatment (Fig. 5A–C). Furthermore, the results of RT-qPCR and western blotting revealed that the expression levels of HMGB1, ICAM-1 and IL-17 exhibited a consistent trend with the results of the ELISA. Furthermore, the expression levels of miR-381 were reduced in the model group, whereas anti-IL-17 and anti-HMGB1 did not restore the expression of miR-381 (Fig. 5D–F).

Bioinformatics analysis identified a possible binding site for miR-381 in the 3′UTR of HMGB1 (Fig. 6A). The results of a luciferase assay in cells indicated that the luciferase activity of HMGB1-3′UTR was decreased in the presence of miR-381 (Fig. 6B, P<0.05).

As shown in Fig. 6C and D, an obvious decrease in the number of migratory cells was observed following transfection with miR-381 mimics (P<0.05). The number of migratory cells in the miR-381 inhibitors group was higher than in the other groups (P<0.05). Conversely, the effects of miR-381 inhibitors were blocked by siRNA-HMGB1.

As shown in Fig. 7A, miR-381 expression in the miR-381 mimics group was significantly increased compared with in the other groups (P<0.05). Furthermore, an obvious decrease in miR-381 expression was observed in the miR-381 inhibitors group (P<0.05). The expression levels of HMGB1, IL-17 and ICAM-1 in macrophages were decreased following transfection with miR-381 mimics, whereas miR-381 inhibitors increased the expression levels of HMGB1, IL-17 and ICAM-1 (Fig. 7B–E). Conversely, the effects of miR-381 inhibitors were inhibited by siRNA-HMGB1.