Tanshinone IIA ameliorates chronic arthritis in mice by modulating neutrophil activities

Abstract

Rheumatoid arthritis (RA) is a chronic immune inflammatory disease mediated by the influx of immune cells into the synovial joint space. As Tanshinone IIA (TIIA) has potent anti-oxidant and anti-inflammatory activities, we used the adjuvant-induced arthritis (AA) murine model of RA to investigate the impact of TIIA on RA and immune cell activation. The anti-arthritic activity of TIIA was investigated in an adjuvant-induced arthritis model of RA in mice. Myeloperoxidase and neutrophil elastase expression levels were assessed in ankle joints by immunohistochemistry analysis. Immune cell infiltration was evaluated in air pouch experiments. Proinflammatory cytokines expression levels were determined by quantitative real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays. Neutrophil extracellular traps (NETs) were assessed by immunostaining and confocal microscopy. Treatment with TIIA alleviated cartilage erosion and neutrophil infiltration in the ankle joints of AA mice and reduced proinflammatory cytokine expression levels in sera. TIIA suppressed interleukin-6 and tumour necrosis factor-α expression and release in neutrophils and promoted neutrophil apoptosis. TIIA also inhibited the NET formation of neutrophils. Our findings demonstrated that TIIA can ameliorate RA effectively by targeting neutrophils, indicating that TIIA may act as a potential therapeutic for RA.

Keywords: arthritis; inflammation; neutrophil; tanshinone IIA.

Figure 1

Alleviation of adjuvant‐induced arthritis following the treatment of Tanshinone IIA (TIIA) in mice. (a) Macroscopic observation of severity of inflammation of paw joints from each group on day 30 after Freund's complete adjuvant (FCA) immunization. (b) Joint swelling was assessed by measuring ankle joint diameter. Values are the mean ± standard error of the mean (s.e.m.). **P < 0·01 when comparing antigen‐induced arthritis treated with the TIIA (RA + TIIA) group with antigen‐induced arthritis (RA) group (n = 10). (c) The severity of arthritis was graded on a scale of 0–4. Values are the mean ± s.e.m. **P < 0·01 when comparing the RA + TIIA group with the RA group (n = 10). (d) Histological assessment was performed using haematoxylin and eosin‐stained ankle joint sections from each treatment groups. Representative sections are shown. [Colour figure can be viewed at wileyonlinelibrary.com].

Figure 2

Immunohistochemistry analyses of myeloperoxidase (MPO) and neutrophil elastase (NE) in ankle joints of antigen‐induced arthritis mice. Immunohistochemistry analysis was used to assess the expression of MPO and NE in the ankle joints of mice treated with phosphate‐buffered saline (PBS) (control, a,d), antigen‐induced arthritis group (RA, b,e) and antigen‐induced arthritis treated with Tanshinone IIA (TIIA) group (RA + TIIA, c,f). The arrow indicated positive signal. The intensity of the staining signal was measured (g,f). [Colour figure can be viewed at wileyonlinelibrary.com].

Figure 3

Tanshinone IIA (TIIA) inhibited lipopolysaccharide (LPS)‐induced neutrophil influx in vivo. Air pouches were created and TIIA or phosphate‐buffered saline (PBS) was administered intraperitoneally 30 min prior to the injection of LPS into pouches, exudates were harvested 6 h later and the number and identification of leucocytes were determined by cytology as described in Materials and methods. (a) The number of total leucocytes in the air pouch are expressed as means ± standard error of the mean (s.e.m.) (n = 5). **P < 0·01 versus corresponding group. (b) The number of neutrophil in the air pouch are expressed as means ± s.e.m. (n = 5). **P < 0·01 versus corresponding group.

Figure 4

Tanshinone IIA (TIIA) induces the apoptosis of lipopolysaccharide (LPS)‐treated neutrophils. (a) Western blot analyses of B‐cell lymphoma 2 (Bcl‐2), Bax and cleaved caspase 3. (b) Density analyses of Bcl‐2, Bax and cleaved caspase 3 expressions in (a). (c) The effects of TIIA on neutrophils apoptosis by flow cytometry. (d) The percentage of cells labelled as annexin V (+), propidium iodide (PI) (−) and annexin V (+) PI (+) was investigated. The data were obtained from three independent experiments. Data are expressed as means ± standard error of the mean (s.e.m.). *P < 0·05, LPS‐treated cells versus TIIA‐treated cells. [Colour figure can be viewed at wileyonlinelibrary.com].

Figure 5

Tanshinone IIA (TIIA) decreases cytokine productions in lipopolysaccharide (LPS)‐treated neutrophils in vitro. (a,b) Quantitative real time–polymerase chain reaction (qRT–PCR) analyses of tumour necrosis factor (TNF)‐α (a) and interleukin (IL)‐6 (b) mRNA level in neutrophils. (c,d) Enzyme‐linked immunosorbent assay (ELISA) analyses of TNF‐α (c) and IL‐6 (d) proteins in the culture media of neutrophil. (e,f) ELISA analyses of TNF‐α (e) and IL‐6 (f) proteins in the plasma of antigen‐induced arthritis (RA) mice and adjuvant‐induced arthritis treated with TIIA (RA + TIIA) mice (n = 10). Values are the means ± standard error of the mean (s.e.m.).*P < 0·05; **P < 0·001.

Figure 6

Tanshinone IIA (TIIA) inhibits neutrophil extracellular traps (NETs) formation and myeloperoxidase (MPO) and neutrophil elastase (NE) release in vitro. Phorbol 12‐myristate 13‐acetate (PMA)‐induced (NETs) formation was visualized by staining with anti‐MPO (red, a), anti‐NE (green, b) and 4',6‐diamidino‐2‐phenylindole (DAPI) (blue) and observed by immunofluorescence confocal microscopy. The data were obtained from three independent experiments. [Colour figure can be viewed at wileyonlinelibrary.com].