Increased serum calpain activity is associated with HMGB1 levels in systemic sclerosis

Abstract

Background: Systemic sclerosis (SSc) or scleroderma is an intractable autoimmune disorder that affects multiple organs. The objectives were to investigate clinical correlations of serum calpain activity and high mobility group box 1 (HMGB1) levels with immunological and clinical traits.

Methods: A total of 31 patients with SSc, 20 age- and gender-matched healthy control subjects (HC), and 10 patients with other connective tissue diseases (CTD) were recruited in the study. We measured serum calpain activity and HMGB1 levels and analyzed the datasets (GSE40839, GSE48149, GSE76808, GSE81292, GSE33463, and GSE58095) from Gene Expression Omnibus (GEO) database to explore the potential mechanism by which calpain exerts its function through bioinformatics methods.

Results: Serum calpain activity was significantly increased in patients with SSc compared with those in HC and in patients with CTD and was correlated with serum HMGB1 levels, modified Rodnan skin score, erythrocyte sedimentation rate, mean platelet volume, and plateletcrit. Notably, serum calpain activity and HMGB1 levels in SSc patients with interstitial lung disease (ILD) were significantly higher than those in SSc patients without ILD. Serum calpain activity and HMGB1 levels could be the independent risk factors for SSc-ILD and novel biomarkers in patients with SSc.

Conclusion: This is the first study that reports increased serum calpain activity and the correlation between calpain and HMGB1 in patients with SSc or SSc-ILD. The serum calpain activity and HMGB1 levels may serve as measures of ILD in patients with SSc. Also, calpain and HMGB1 could be potential therapeutic targets for patients with SSc or SSc-ILD in the future.

Keywords: Calpain; HMGB1; Interstitial lung disease; Microarray analysis; Systemic sclerosis.

Fig. 1

Expression of calpain-related genes in patients with SSc or SSc-ILD. a Differentially expressed heatmap of calpain-associated genes in lung samples of patients with SSc-ILD (GSE40839, GSE48149, GSE76808, GSE81292). b Differentially expressed heatmap of calpain-associated genes in PBMC samples of patients with SSc (GSE33463). c Differentially expressed heatmap of calpain-associated genes in skin samples of patients with SSc (GSE58095). d Differentially expressed boxplot of calpain-associated genes in lung samples of patients with SSc-ILD (50 SSc-ILD vs 28 HC). e Differentially expressed boxplot of calpain-associated genes in PBMC samples of patients with SSc (69 SSc vs 41 HC). f Differentially expressed boxplot of calpain-associated genes in skin samples of patients with SSc (59 SSc vs 43 HC). *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: SSc systemic sclerosis, ILD interstitial lung disease, HC healthy control subjects

Fig. 2

Increased serum calpain activity and HMGB1 levels in SSc patients. a Significantly higher serum calpain activity and HMGB1 levels (d) were found in SSc patients than in HC and in patients with other CTD. b Patients with dSSc showed significantly higher serum calpain activity and HMGB1 levels (e) than patients with lSSc. c SSc patients with ILD showed significantly higher serum calpain activity and HMGB1 levels (f) than SSc patients without ILD. g Correlations between serum calpain activity and HMGB1 levels from patients with SSc. The short bar indicates mean ± 2SD in each group. ***< 0.001, **< 0.01, *< 0.05. Abbreviations: SSc systemic sclerosis, ILD interstitial lung disease, CTD connective tissue diseases, HC healthy control subjects, SD standard deviation, dSSc diffuse cutaneous systemic sclerosis, lSSc limited cutaneous systemic sclerosis

Fig. 3

The correlation of serum calpain activity and HMGB1 levels with clinical parameters in patients with SSc. Abbreviations: mRSS modified Rodnan skin score, FVC forced vital capacity, DL_CO diffusing capacity of the lung for carbon monoxide, FEV1 forced the first second of expiratory volume, ESR erythrocyte sedimentation rate, PLT platelet count, PDW platelet distribution width, PCT plateletcrit, MPV mean platelet volume, P-LCR platelet large cell ratio

Fig. 4

Receiver operating characteristic curve (ROC). Serum calpain activity and HMGB1 levels for the diagnosis of patients with SSc (a) or SSc-ILD (b)

Fig. 5

KEGG pathway and GO enrichment analysis. a The top 5 GO enrichment pathways of differentially expressed genes between cluster1 and cluster2. b The size of each circle means the amounts of genes. The different color of each circle means p value. GeneRatio means the number of genes in differentially expressed genes that belong to this pathway divided by the number of genes in the background gene cluster that belong to this pathway. Abbreviations: GO gene ontology, KEGG Kyoto Encyclopedia of Genes and Genomes