A Novel Phytochemical, DIM, Inhibits Proliferation, Migration, Invasion and TNF-α Induced Inflammatory Cytokine Production of Synovial Fibroblasts From Rheumatoid Arthritis Patients by Targeting MAPK and AKT/mTOR Signal Pathway

Abstract

In rheumatoid arthritis(RA) pathogenesis, activated RA fibroblast-like synoviocytes (RA-FLSs) exhibit similar proliferative features as tumor cells and subsequent erosion to cartilage will eventually lead to joint destruction. Therefore, it is imperative to search for compounds, which can effectively inhibit the abnormal activation of RA-FLSs, and retard RA progression.3′3-Diindolylmethane (DIM), the major product of the acid-catalyzed oligomerization of indole-3-carbinol from cruciferous vegetables, has been reported to be functionally relevant to inhibition of migration, invasion and carcinogenesis in some solid tumors. In this study, we explored the anti-proliferation, anti-metastasis and anti-inflammation effects of DIM on RA-FLSs as well as the underlying molecular mechanisms. To do this, primary RA-FLSs were isolated from RA patients and an animal model. Cell proliferation, migration and invasion were measured using CCK-8, scratch, and Transwell assays, respectively. The effects of DIM on Matrix metalloproteinases (MMPs) and some inflammatory factors mRNA and key molecules such as some inflammatory factors and those involved in aberrantly-activated signaling pathway in response to tumor necrosis factor α(TNF-α), a typical characteristic mediator in RA-FLS, were quantitatively measured by real-time PCR and western blotting. Moreover, the effect of DIM on adjuvant induced arthritis(AIA) models was evaluated with C57BL/6 mice in vivo . The results showed that DIM inhibited proliferation, migration and invasion of RA-FLS in vitro . Meanwhile, DIM dramatically suppressed TNF-α–induced increases in the mRNA levels of MMP-2, MMP-3, MMP-8 , and MMP-9 ; as well as the proinflammatory factors IL-6, IL-8 , and IL-1 β. Mechanistic studies revealed that DIM is able to suppress phosphorylated activation not only of p38, JNK in MAPK pathway but of AKT, mTOR and downstream molecules in the AKT/mTOR pathway. Moreover, DIM treatment decreased expression levels of proinflammatory cytokines in the serum and alleviated arthritis severity in the knee joints of AIA mice. Taken together, our findings demonstrate that DIM could inhibit proliferation, migration and invasion of RA-FLSs and reduce proinflammatory factors induced by TNF-α in vitro by blocking MAPK and AKT/mTOR pathway and prevent inflammation and knee joint destruction in vivo , which suggests that DIM might have therapeutic potential for RA.

Knockdown of YAP/TAZ Inhibits the Migration and Invasion of Fibroblast Synovial Cells in Rheumatoid Arthritis by Regulating Autophagy

“…In the current study, we clearly demonstrated that YAP and TAZ in RA-FLS exhibited higher expression than those in normal FLS, which was consistent with the results in multiple human cancer cells, implying that YAP and TAZ might serve function as the regulators in RA-FLS. Since RA-FLS can migrate and invade the articular cartilage and bone, thereby leading to destruction of cartilage and bone [ 6 , 7 ], we firstly established RA-FLS stable knockdown of YAP or TAZ using the lentiviral-mediated YAP or TAZ knockdown approaches to evaluate the effect of YAP or TAZ knockdown on migration and invasion of RA-FLS by wound healing and Transwell migration and invasion assays. Our data demonstrated that either knockdown of YAP or TAZ markedly decreased cell motility, indicating the role of loss of YAP or TAZ resulting in the inhibition of the aggressive invasive ability of RA-FLS.…”

“…Evidence is accumulating that the fibroblast-like synoviocytes from RA patients (RA-FLS) play a pivotal role in the pathogenesis of RA [ 1 ]. RA-FLS exhibit some tumor cell-like characteristics, such as excessive proliferation, migration, and invasion; anchorage-independent proliferation; and lack of contact inhibition in vitro [ 2 – 4 ], and can migrate and invade the articular cartilage and bone [ 5 ], contributing to the destruction of cartilage and bone and joint damage through secretion of proinflammation cytokines and production of the proteolytic enzymes such as matrix metalloproteinases (MMPs) [ 6 , 7 ]. Therefore, understanding and investigating the factors that regulate the migration and invasion of RA-FLS might provide novel targets for RA therapy.…”

Identification of drug targets and potential molecular mechanisms of Wantong Jingu Tablet in treatment of rats with collagen-induced arthritis based on 16S rDNA high-throughput sequencing and metabolomic analysis

“…The results of clinical manifestations and pathological changes demonstrated that WJT played an important role in suppressing the pathological progression of arthritis in CIA rats, which laid a foundation for the further study. In addition, The aberrant release and recruitment of pro-in ammatory cytokines (TNF-α, IL-1β, and IL-6) are essential to the pathogenesis of RA [18,20,21]. It's well known that both TNFα and IL-1β play roles in the activation and in ltration of immune cells, and contribute to production of in ammatory cytokines and chemokines, thereby resulting in a complex network that promotes in ammatory reactions, cartilage damage and autoimmune pathology [18,22].…”

Identification of drug targets and potential molecular mechanisms for Wantong Jingu Tablet extract in treatment of rheumatoid arthritis: bioinformatics analysis of fibroblast-like synoviocytes

“…Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation, synovial hyperplasia, and bone hyperplasia and destruction [1,2]. Synoviocytes are the major cells in the synovial membrane, and these are the terminal targets in the pathogenesis of RA [3,4]. Synovial hyperplasia, which contributes to the progression and development of RA, mainly results from the active proliferation and aggressive tumor-like characteristics of rheumatoid arthritis-broblast-like synoviocytes (RA-FLSs) [5,6].…”