Abstract
Rheumatoid arthritis (RA) is a common autoimmune disease worldwide. Neutrophils play critical roles in the onset and development of RA and are the promising target for RA treatment. Tetrandrine is a bis-benzyl isoquinoline alkaloid derived from the traditional Chinese herbal Stephania tetrandra S. Moore. Tetrandrine is effective in alleviating RA by inhibiting macrophage inflammatory response, fibroblast overproliferation, and pannus formation. However, whether tetrandrine regulates the activities of neutrophils in RA is largely unknown. In this study, we adopted adjuvant-induced arthritis (AA) murine model to explore the effect of tetrandrine on RA and neutrophils. Twenty-eight mice were divided into four groups. The control group was injected with PBS in the limbs and treated with PBS by intraperitoneal injection (i.p.) from Day 10 to Day 37. The arthritis murine model was induced by injecting FCA into the ankle joints of hind limbs. The AA group, the AA + TET group, and the AA + DEX group mice were treated with PBS, tetrandrine (6 mg/kg), or dexamethasone (1 mg/kg) i.p. daily, respectively. Arthritic scores were evaluated, and the joint diameter was measured every three days. A cytometric bead assay was performed to measure the concentrations of IFN-γ, TNF-α, and IL-6 in the serum. H&E staining and Safranin O-fast staining were adopted to monitor the tissue changes in the joint. Immunohistochemistry assays were applied to detect the MPO, NE, CitH3, and PAD4 expression levels. To assess the effect of tetrandrine on neutrophil activities in vitro, CCK8 tests were applied to determine cell viability. The qPCR and ELISA were performed to determine IL-1β and IL-6 expression levels. Immunofluorescence assays were performed to measure the formation of NETs. The results indicated that tetrandrine significantly alleviated the symptoms of RA in terms of the ankle diameter (from 4.629 ± 2.729 to 3.957 ± 0.257; P < 0.01) and ankle score (from 4.000 ± 0.000 to 3.286 ± 0.756; P < 0.05). Tetrandrine treatment significantly increased the cartilage areas and decreased serum IL-6 significantly (from 5.954 ± 2.127 to 2.882 ± 2.013; P < 0.01). The immunohistochemistry assays also showed decreased expression levels of NE, MPO, PAD4, and CitH3 induced by tetrandrine in comparison with the AA group (P < 0.01). The qPCR assays and ELISAs showed that tetrandrine had an anti-inflammatory effect in vitro by significantly inhibiting IL-6 (P < 0.01). The immunofluorescence assays showed that NET formation induced by PMA could be reduced by tetrandrine (P < 0.01). In conclusion, tetrandrine has good efficacy in treating RA by regulating neutrophil-involved inflammation and NET formation.
Highlights
Rheumatoid arthritis (RA) is a common inflammatory disease that results in continuous inflammation, progressive articular damage, and eventually disability. e clinical symptoms include joint stiffness, pain, and swelling.e onset and progression of RA involve activation of the immune system
Anti-neutrophil elastase (NE), anti-MPO, anti-citrullinated histone H3 (CitH3), and anti-β-actin antibodies were obtained from Abcam (Cambridge, MA, USA); anti-peptidyl arginine deiminase 4 (PAD4) antibody was obtained from ProteinTech Antibody Group (Chicago, IL, USA)
Our study clarified the effect of tetrandrine on adjuvant-induced arthritis (AA) mice and neutrophils
Summary
E onset and progression of RA involve activation of the immune system. Recent studies have found that innate immunity plays a critical role in the progression of RA with macrophage and neutrophil involvement [1]. Evidence-Based Complementary and Alternative Medicine of RA, neutrophils are activated and recruited to the joint cavity. Is process involves the activation of myeloperoxidase (MPO) [4]. MPO mediates the oxidative activation of neutrophil elastase (NE), which in turn translocates to the nucleus and promotes its proteolysis. Activated peptidyl arginine deiminase 4 (PAD4) participates in the emergence of citrullinated histone H3 (CitH3) by mediating the conversion from arginine to citrulline. Nucleic acid depolymerization is triggered [5, 6]. Intracellular proteins and nucleic acids are released from the cell. Citrullinated proteins can induce the production of anticitrullinated protein antibodies (ACPAs) and other autoantibodies [7]. erefore, neutrophils can be a primary target for RA treatment strategies [8, 9]
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