The Flavonoid Naringenin Alleviates Collagen-Induced Arthritis through Curbing the Migration and Polarization of CD4+ T Lymphocyte Driven by Regulating Mitochondrial Fission

Abstract

Rheumatoid arthritis (RA) is a progressive autoimmune disease. Due to local infiltration and damage to the joints, activated CD4+ T cells play a crucial role in the progression of RA. However, the exact regulatory mechanisms are perplexing, which makes the effective management of RA frustrating. This study aimed to investigate the effect of mitochondria fission on the polarization and migration of CD4+ T cells as well as the regulatory mechanism of NAR, so as to provide enlightenment on therapeutic targets and novel strategies for the treatment of RA. In this study, a collagen-induced arthritis (CIA) model was established, and rats were randomly given saline or naringenin (NAR, 10 mg/kg, 20 mg/kg, 50 mg/kg, i.p.) once a day, before being euthanized on the 42nd day of primary immunization. The pain-like behavior, articular index scores, account of synovial-infiltrated CD4+ T cells, and inflammatory factors were investigated in each group. In vitro, spleen CD4+ T lymphocytes were derived from each group. In addition, mitochondrial division inhibitor 1 (Mdivi-1) or NAR was added to the cell medium containing C-X-C motif chemokine ligand 12 (CXCL12) in order to induce CD4+ T lymphocytes, respectively. The polarization capacity of CD4+ T cells was evaluated through the immunofluorescence intensity of the F-actin and myosin light chain phosphorylated at Ser19 (pMLC S19), and the mitochondrial distribution was determined by co-localization analysis of the translocase of outer mitochondrial membrane 20 (TOM20, the mitochondrial marker) and intercellular adhesion molecule 1 (ICAM1, the uropod marker). The mitochondrial fission was investigated by detecting dynamin-related protein 1 (Drp1) and mitochondrial fission protein 1 (Fis1) using Western blot and immunofluorescence. This study revealed that high-dose NAR (50 mg/kg, i.p.) alleviated pain-like behavior and articular index scores, reduced the serum level of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), and accounted for CD4+ T lymphocytes that infiltrated into the synovial membrane of the CIA group. Meanwhile, NAR (50 mg/kg, i.p.) suppressed the polarization of spleen CD4+ T lymphocytes, reduced the redistribution of mitochondria in the uropod, and inhibited the expression of Drp1 and Fis1 in the CIA model. Furthermore, the in vitro experiments confirmed that NAR reduced mitochondrial fission, which in turn inhibited the CXCL12-induced polarization and migration of CD4+ T lymphocytes. Our results demonstrated that the flavonoid NAR was a promising drug for the treatment of RA, which could effectively interfere with mitochondrial fission, thus inhibiting the polarization and migration of CD4+ T cells in the synovial membrane.