Leonurine Regulates Treg/Th17 Balance to Attenuate Rheumatoid Arthritis Through Inhibition of TAZ Expression

Abstract

Leonurine, an active alkaloid extracted from Herba leonuri, is reported to have potent anti-inflammatory activity against rheumatoid arthritis (RA). However, the molecular mechanism of action of leonurine in RA remains poorly understood. In this study, we detected 3,425 mRNAs differentially expressed between CD4⁺ T cells of RA patients and those of healthy individuals using microarray raw data mining. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that transcriptional coactivator with PDZ-binding motif (TAZ) regulates a variety of biological processes including T-helper (Th)-17 cell development, and was thus selected for functional verification. In a naïve CD4⁺ T cell differentiation assay, we found that TAZ overexpression was associated with impaired balance between T regulatory (Treg) and Th17 cells in vitro. TAZ overexpression increased the levels of the pro-inflammatory cytokines interleukin (IL)-17, IL-1β, and tumor necrosis factor (TNF)-α and decreased that of the anti-inflammatory cytokine IL-10. Leonurine treatment had a direct recovery effect on the impaired balance and reduced the expression of TAZ and led to normalization of IL-17, IL-1β, and TNF-α and IL-10. Furthermore, IL-6 was found to promote the expression of TAZ and receptor activator of nuclear factor kappa-B ligand (RANKL), and RANK. Leonurine significantly inhibited TAZ-mediated expression of RANKL, and RANK and IL-6 in synovial fibroblasts. We conclude that the therapeutic effect of leonurine was through suppression of TAZ led to restoration of Treg/Th17 balance and suppression of synovial fibroblast action.

Keywords: TAZ; Treg Th17; balance; leonurine; rheumatoid arthritis.

Figure 1

Highly expressed TAZ screened in CD4⁺ T cells of rheumatoid arthritis patients using microarray raw data analysis. (A) Heatmap of the hierarchical cluster analysis of differentially expressed miRNAs between 13 CD4⁺ T cells of rheumatoid arthritis patients and 9 CD4⁺ T cells of healthy individuals using P < 0.001 and |FC| > 1.5 as the cutoff (bright blue, under-expression; white, no change; bright red, over-expression). (B,C) Chromosome summary plot and volcano plot of the microarray data. (D) The expression of TAZ was higher in CD4⁺ T cells of rheumatoid arthritis patients. (E) Top 20 statistics of KEGG pathway enrichment analysis. ***P < 0.001.

Figure 2

Leonurine treatment reduced the expression of TAZ in T cells. (A) 293 T-cells stably expressing TAZ previously established by transfection were used as a positive control (ov-TAZ) and a negative control (ov-NC) (magnification, × 100). (B) MTT assays showed that leonurine had the least cytotoxicity at 20 μM. There was no difference in suppression of proliferation between the leonurine 20 and 10 μM groups (P > 0.05), while leonurine 30 μM group was significantly different from the leonurine 20 μM group (P < 0.05), and the leonurine 0 μM groupwas used as a negative control. (C,D) qPCR and Western blot results showed that the expression of TAZ in the ov-TAZ group was higher than that in the ov-NC group. Leonurine treatment reduced the expression of TAZ. Furthermore, leonurine was able to suppress TAZ expression in TAZ-overexpressed T cells. Results are expressed as average of 3 experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3

Flow cytometry results showed that the frequency of Treg cells (expressing Foxp3) in the ov-TAZ group was lower than that in the ov-NC group, whereas the frequency of Th17 cells (RORγt expressing) in the ov-TAZ group was higher than that in the ov-NC group. When leonurine was added, it increased the Treg cell frequency and decreased that of Th17 cells. Furthermore, leonurine increased Treg cell frequency in the ov-TAZ group and decreased that of Th17 cells. Results are expressed as average of 3 experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4

ELISA assay showing TAZ-mediated inflammatory cytokine production by CD4⁺ T cells and suppression by leonurine. Compared with those in the ov-NC group or NC group, the protein levels of the pro-inflammatory cytokines IL-17, IL-1β, and TNF-α in the ov-TAZ group increased, whereas those of the anti-inflammatory cytokine IL-10 decreased. After leonurine treatment, the protein levels of IL-17, IL-1β, and TNF-α in the control group and ov-TAZ group decreased, whereas that of IL-10 increased. Results are expressed as average of 3 experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5

Suppressive effect of leonurine on TAZ-mediated migration and invasion of synovial fibroblast-like cells. (A,B) qPCR and Western blot analysis showed that compared with that in the control group, TAZ, RANKL, and RANK by synovial fibroblast-like cells in the IL-6 group were all up-regulated. However, leonurine treatment (10 and 20 μM) reduced the effects of IL-6 on TAZ/RANKL/RANK in a dose dependent manner. (C) Transwell (magnification, × 200) analysis showed that IL-6 promoted the migration and invasion ability of fibroblast-like cells, whereas leonurine treatment reduced the effect of IL-6. Results are expressed as the average of 3 experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.