Abstract
Ankylosing spondylitis (AS) is a chronic inflammatory disease characterized by abnormal bone metabolism, with few effective treatments available. Danshensu [3-(3,4-dihydroxy-phenyl) lactic acid) is a bioactive compound from traditional Chinese medicine with a variety of pharmacologic effects. In the present study, we investigated the pharmacologic effect and molecular mechanism of Danshensu in AS. Potential targets of Danshensu were identified in four drugs-genes databases; and potential pharmacologic target genes in AS were identified in three diseases-genes databases. Differentially expressed genes related to AS were obtained from the Gene Expression Omnibus database. Overlapping targets of Danshensu and AS were determined and a disease–active ingredient–target interaction network was constructed with Cytoscape software. Enrichment analyses of the common targets were performed using Bioconductor. To test the validity of the constructed network, an in vitro model was established by treating osteoblasts from newborn rats with low concentrations of tumor necrosis factor (TNF)-α. Then, the in vitro model and AS fibroblasts were treated with Danshensu (1–10 μM). Osteogenesis was evaluated by alkaline phosphatase staining and activity assay, alizarin red staining, quantitative PCR, and western blotting. We identified 2944 AS-related genes and 406 Danshensu targets, including 47 that were common to both datasets. The main signaling pathways associated with the targets were the c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways. A low concentration of TNF-α (0.01 ng/ml) promoted the differentiation of osteoblasts; this was inhibited by Danshensu, which had the same effect on AS fibroblasts but had the opposite effect on normal osteoblasts. Danshensu also decreased the phosphorylation of JNK and ERK in AS fibroblasts. There results provide evidence that Danshensu exerts an anti-osteogenic effect via suppression of JNK and ERK signaling, highlighting its therapeutic potential for the treatment of AS.
Highlights
Ankylosing spondylitis (AS) is a chronic progressive inflammatory disease with insidious onset characterized by chronic inflammatory back pain, bone resorption, and new bone formation, with the latter leading to ankylosis and functional disability (Poddubnyy and Sieper, 2017; Lories, 2018)
We identified putative targets of Danshensu (Figure 2A) in BATMAN-TCM along with in TCMSP, 100 in SwissTargetPrediction, and 273 in Pharmmapper (Figure 2B)
To confirm the effect of low tumor necrosis factor (TNF)-α concentrations on osteoblast differentiation, we evaluated the expression of BMP2 and SP7—2 genes related to this process—by qPCR
Summary
Ankylosing spondylitis (AS) is a chronic progressive inflammatory disease with insidious onset characterized by chronic inflammatory back pain, bone resorption, and new bone formation, with the latter leading to ankylosis and functional disability (Poddubnyy and Sieper, 2017; Lories, 2018). Heterotopic ossification involves endochondral and membranous bone formation and cartilage metaplasia, in which fibroblasts and bone cells such as mesenchymal stem cells, osteoblasts, and osteoclasts play an essential role (Van Mechelen et al, 2018; Liu et al, 2021). Inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-17A, and IL-6 are implicated in new bone formation (Li et al, 2016; van Tok et al, 2019; Zou et al, 2020). Low concentration of TNF-α enhanced the osteogenic differentiation of AS fibroblasts, which was accompanied by the expression of osteogenesis markers including Runt-related transcription factor (RUNX), osteopontin (OPN), and osteocalcin (OCN) (Zou et al, 2020)
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