Knockdown of nrf2 Exacerbates TNF-α-Induced Proliferation and Invasion of Rheumatoid Arthritis Fibroblast-Like Synoviocytes through Activating JNK Pathway.

Na Tian,Sheng-Ming Dai,Meng Lu,Xian-Long Zhang,Yu Du,Qian Wang

doi:10.1155/2020/6670464

Abstract

Fibroblast-like synoviocytes (FLS) in the synovial tissue of rheumatoid arthritis (RA) exhibit over-proliferative and aggressive phenotypes, which participate in the pathophysiology of RA. In RA, little is known about the nonantioxidant effect of nuclear factor erythroid 2-related factor 2 (nrf2), the master regulator of redox homeostasis. In this study, we aimed to explore the expression and upstream regulatory factors of nrf2 and revealed its functions in modulating the proliferation and invasion in RA-FLS. FLS were isolated from RA and osteoarthritis patients. Expression of nrf2 in the synovial tissues and FLS was analyzed by immunohistochemistry, real-time PCR, Western blotting, and immunofluorescence staining. Cell proliferation was examined by Cell Counting Kit-8. Cell invasion was tested by transwell assay. Phosphorylation of JNK was determined by Western blotting. The results showed that nrf2 expression in the RA synovial tissues was upregulated. TNF-α promoted expression and nuclear translocation of nrf2 in RA-FLS and increased the intracellular reactive oxygen species (ROS) level. Nrf2 nuclear translocation was blocked by ROS inhibitor N-acetylcysteine. Both knockdown of nrf2 by siRNA and inhibition of nrf2 by ML385 significantly promoted the TNF-α-induced proliferation and invasion of RA-FLS. Activation of nrf2 by sulforaphane (SFN) profoundly inhibited the TNF-α-induced proliferation and invasion of RA-FLS. Knockdown of nrf2 also enhanced the TNF-α-induced matrix metalloproteinases (MMPs) expression and phosphorylation of JNK in RA-FLS. Proliferation and invasion of RA-FLS incubated with TNF-α and nrf2 siRNA were inhibited by pretreatment with JNK inhibitor SP600125. In conclusion, nrf2 is overexpressed in synovial tissues of RA patients, which may be promoted by TNF-α and ROS levels. Activation of nrf2 may suppress TNF-α-induced proliferation, invasion, and MMPs expression in RA-FLS by inhibiting JNK activation, indicating that nrf2 plays a protective role in relieving the severity of synovitis in RA. Our results might provide novel insights into the treatment of RA.

Highlights

Rheumatoid arthritis (RA) is an autoimmune disease that primarily affects the joints, characterized by synovial inflammation and hyperplasia, autoantibody production, and cartilage and bone destruction, which would lead to irreversible functional loss [1]
Nrf2 expression was significantly upregulated in the RA synovium compared with OA; synovial hyperplasia was observed in RA with hematoxylin and eosin (HE) staining (Figure 1)
The nuclear protein level of nrf2 was increased in RA-fibroblastlike synoviocytes (FLS) after stimulation with TNF-α (25 ng/mL) at different time points (Figures 2(b) and 2(c)), indicating that TNFα could promote nrf2 nuclear translocation

Summary

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease that primarily affects the joints, characterized by synovial inflammation and hyperplasia, autoantibody production, and cartilage and bone destruction, which would lead to irreversible functional loss [1]. Major progress has been made in RA treatment, there are still concerns about incomplete responses and toxicity. Further elucidation of RA pathogenesis and the development of effective therapies remain an urgent need. Synovium comprises an intimal lining layer and a sublining layer, where two major cell types are located: fibroblastlike synoviocytes (FLS) and macrophage-like synoviocytes. Normal synovium has a thin intimal lining layer consisting of two to three layers of cells; the intimal lining layer in RA becomes inflamed, hyperplastic with expansion in both cell types, and invasive of cartilage and bone [2, 3]

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