Although many experts and scholars have researched the pathogenesis of RA from genetic, pathophysiological, immunological and other aspects for many years, the specific pathogenesis of RA has not yet been fully elucidated(16–18). Studies have shown that fibroblast-like synoviocytes (FLS) is the main cell in synovial hyperplasia. The normal synovial tissue is usually composed of a 1–3 layers of cells; however, through a variety of stimulating activation, proliferation and expansion, this tissue reaches 10–15 layers of cells with the infiltration of a variety of inflammatory cells(19). The proliferation of activated synovial cells continues to produce a variety of inflammatory factors and proteases, such as matrix metalloproteinases (MMP), accelerating the migration and invasion of FLS and the destruction of articular cartilage. At the cellular level, the process of synovial cell proliferation is divided into three steps: first, the extracellular stimulus signal; then intracellular signal transduction; and finally, nuclear translocation and transcriptional activation in the nucleus. IL-1, TNF-α, EGF, TGF-β and other cytokines can stimulate the proliferation of synovial cells, and the gene expression induced by different stimuli is not the same, due to the signal transduction pathway that is initiated. Studies have shown that the abnormal activation of signal transduction pathways is an important factor in the abnormal proliferation of synovial cells and the excessive secretion of inflammatory factors, which plays an important role in the pathogenesis of RA(19).
Mitogen-activated protein kinases (MAPK) are a group of proteins that is commonly found in most cells and can be stimulated by a variety of different extracellular stimuli, such as cytokines, hormones, neurotransmitters, cell adhesion and cell activated serine-threonine protein kinase. These proteins regulate cell proliferation and differentiation, apoptosis, growth, cytokine secretion, cellular recombination and metabolism and other important biological activities. At present, a large number of studies have confirmed that the synovial cells of RA patients exhibit abnormal activation of the MAPK signaling pathway. The main members of the MAPK pathway, p38, ERK1/2 and JNK, can be found in RA patients with joint synovial lesions, and these proteins are phosphorylated. Studies have shown that the MAPK signal transduction pathway regulated the induction of inflammatory cytokine secretion, aggregation of chemotactic inflammatory cells, and promotion of T cell activation, which in turn have a regulatory role in the pathogenesis of RA and affects the proliferation, differentiation and apoptosis a variety of cells (such as synovial cells, osteoblasts, osteoclasts)(10).
Effects of Daphnetin on MAPK Signaling Pathway in Synoviocytes of CIA Rats Induced by TNF – α On the expression of MAPK related cytokines
IL-6 is a multicellular cytokine that is a key cytokine in RA. The pathogenic effects of IL-6 on RA mainly promote the differentiation of T cells and B cells associated with inflammation. The acute phase of this response promotes protein synthesis induces osteoclast differentiation and the loss of joint tissue, and IL-1 is synergistically induced by the production of MMP, resulting in increased bone and cartilage damage(20). Studies have shown that RA patients with significantly increased serum levels of TGF-β. TGF-β is a growth factor with multiple effects that can induce the secretion of the extracellular matrix and is involved in the expression of inflammatory factors and the differentiation of a variety of immune cells. The activation and proliferation of macrophages and fibroblasts have an important impact promoting angiogenesis, pannus formation, and synovial fibrosis(21). The expression of MMP-3 and MMP-13 in the synovial fluid and synovium of patients with active RA was significantly increased compared to controls. MMP-3, also known as matrix lysin 1, is secreted by synovial cells, chondrocytes, etc., can make collagen types II, III, IV, IX, and XI and multiple proteoglycan matrices, degrades laminin, and is positively correlated with RA. MMP-13, also known as collagenase-3, is mainly secreted by chondrocytes and has the highest degradation efficiency for type II collagen, which is the most abundant collagen in the cartilage matrix. Therefore, MMP-13 is also the limiting enzyme in the speed of matrix collagen degradation. IL-6, TGF-β, MMP-3 and MMP-13 are all cytokines regulated by the MAPK signaling pathway.
Our study found that daphnetin was able to significantly downregulate the expression of IL-6, TGF-β, MMP-3 and MMP-13 mRNA in the synovial cells of CIA rats induced by TNF-α. Additionally, we detected the secretion of IL-6, TGF-β, MMP-3 and MMP-13 in the supernatant of the synovial cells of CIA rats with ELISA and found that the results were consistent with the gene expression analysis. This reduction of the inflammatory response and tissue cell damage by daphnetin is of great significance.
Effects of daphnetin on MAPK pathway protein expression, phosphorylation and nuclear translocation in CIA rat synoviocytes induced by TNF-α
The MAPK signaling pathway undergoes stepwise activation after various stimuli. The phosphorylation of the pathway protein is transferred to the nucleus to be activated, and the activated protein regulates the transcription, translation and synthesis of the downstream proteins and participates in the important life processes in the cell. We investigated the effects of daphnetin on the protein expression and nuclear translocation of the MAPK signaling pathway molecules p-p38, p-ERK1/2, p-JNK in the synovial cells of CIA rats induced by TNF-α with an immunofluorescence assay.
The results showed that the p38, ERK1/2, JNK proteins were significantly activated in the model group and the C group. Additionally, phosphorylated p38, ERK1/2 and JNK were translocated into the nucleus, and their expression levels in the nucleus were stronger than those in the cytoplasm. The expression of p-p38, p-ERK1/2 and p-JNK was significantly decreased in the nucleus compared with that in the cytoplasm and compared with the expression of p38, ERK1/2 and JNK, respectively. The p-ERK1/2-FITC fluorescence intensity was significantly lower in the nucleus than in the cytoplasm. The expression and phosphorylation of MAPK pathway proteins were detected by Western blot analysis. We found that the p-p38, p-ERK1/2, and p-JNK protein expression levels in the model group were significantly increased, while daphnetin significantly reduced the expression of p38, ERK1/2, and JNK in the synovial cells of CIA rats induced by TNF- α. The phosphorylation levels of ERK1/2 and JNK were lower in the DAP group than in the C and TP groups, and there was no significant difference in p38 phosphorylation level between the C and TP groups.
p-p38, p-ERK1/2, p-JNK immunofluorescence and Western blot results confirmed that daphnetin could inhibit the phosphorylation and activation of the MAPK signal transduction pathway, which affected the regulation of various biological activities of cells by MAPK and was closely related to the pharmacological effects of daphnetin.