Suppression of Inflammation by Si Miao San in Experimental Rheumatoid Arthritis Through Modulation of the AKT/ROS/Autophagy Axis

Abstract

Objective: Si Miao San is effective in ameliorating rheumatoid arthritis (RA) both clinically and experimentally. NETs play a fundamental role in the onset and progression of RA. The goal of this study was to explore the therapeutic effects of Si Miao San (SMS) on adjuvant-induced arthritis in mice and the regulatory mechanisms of NETs both in vivo and in vitro.

Methods: SMS decoctions were identified using LC-MS/MS analysis and TCMSP. In the adjuvant induced RA murine model, SMS decoction and methotrexate were administered orally. Disease progression was analysed by assessing arthritic scores and joint diameter, H&E staining, safranin O-fast green staining, toluidine blue staining and micro-CT analysis. The expression of NE, MPO, PAD4, LC3B, CitH3, p-AKT and p-PI3K and the production of ROS were detected using IHC, WB and IF analyses. Cytokines in the sera of the mice were detected using cytometric bead arrays. After the in vitro culture of neutrophils, NE, MPO, PAD4, LC3B, CitH3, ROS, p-PI3K and p-AKT were measured using IF and WB analyses. Autophagy was further observed with TEM.

Results: SMS decoction compounds were first identified. Compared with the model group, SMS significantly inhibited joint swelling, inflammation progression and bone destruction. The levels of NE, MPO, PAD4, CitH3, LC3B, ROS production and relative expression of p-AKT and p-PI3K in joint tissues were significantly reduced in the SMS group compared to the model group (P < 0.05). In vitro culture, SMS-containing serum significantly reduced the LC3B-II/LC3B-I ratio and the relative expression levels of p-AKT and p-PI3K, as well as the levels of ROS, NE, MPO, PAD4, and CitH3 compared with those in the PMA-treated group (P < 0.05), which was abolished by the treatment with the AKT activator SC79.

Conclusion: The SMS-induced suppression of inflammation in experimental RA occurred through the modulation of the AKT/ROS/autophagy axis.

Keywords: autophagy; neutrophil extracellular traps; protein kinase B; reactive oxygen species; rheumatoid arthritis.

Graphical abstract

Figure 1

SMS attenuated the severity of arthritis in AA mice. (A) Schematic representation of the animal experiment. (B) Joint swelling was assessed by measuring the ankle joint diameter (n = 8). (C) Arthritis severity was graded on a scale of 0–4. (D) Representative images of arthritic paws from each group at the end of the experiments. (E) Changes in the body weights of the mice (n = 8). (F) Mouse spleen index (n = 8). (G) H&E images. Scale bars, 100 and 200 μm. (H) Histological scores (n = 5). The values are presented as the means ± SDs. *P<0.05, **P<0.01, ^nsP > 0.05.

Figure 2

Bone destruction in mice with RA was ameliorated by SMS. Representative joints with (A) safranin O-fast green staining and (B) toluidine blue staining. Scale bars, 100 and 200 μm. (C) Histological scores (n = 5). (D) Representative micro-CT images of the mice. The red arrows indicate bone damage. (E) BV/TV of each group (n = 5). (F) IL-6 concentration in the serum of each group (n = 5). (G) TNF-α concentration in the serum of each group (n = 5). The values are presented as means ± SDs. *P<0.05, **P<0.01, ^nsP > 0.05.

Figure 3

SMS inhibited local NET formation in joints. (A) IHC was used to assess MPO protein expression in mouse ankle joints. Scale bars, 100 and 200 μm. (B) Quantitative analysis of (A) (n = 3). (C) IHC was used to assess PAD4 protein expression in mouse ankle joints. Scale bars, 100 and 200 μm. (D) Quantitative analysis of (C) (n = 3). (E) Immunofluorescence analysis was used to detect the expression levels of CitH3 and NE proteins in mouse joints. (F) Quantitative analysis of NE (n = 3). (G) Quantitative analysis of CitH3 (n = 3). The values are presented as the means ± SDs. **P<0.01.

Figure 4

SMS inhibited local NET formation in joints through PI3K/AKT/ROS/autophagy axis. (A) FACS analysis of ROS production. (B) Quantitative analysis of (A). (C) WB analysis of the expression of p-AKT and AKT in periarticular muscle tissues. (D) WB analysis of the expression of p-PI3K and PI3K in periarticular muscle tissues. (E) Quantitative analysis of p-AKT in (C) (n = 3). (F) Quantitative analysis of p-PI3K in (D) (n = 3). (G) IHC analysis of LC3B protein expression. Scale bars, 100 and 200 μm. (H) Quantitative analysis of LC3B in G (n = 3). The values are presented as means ± SD. *P<0.05, **P<0.01.

Figure 5

SMS inhibited the PI3K/AKT signalling pathway and NET production in vitro. Neutrophils extracted from the peritoneal cavity of mice were subjected to different interventions in the following groups: NC, SMS, NC+PMA, SMS+PMA, and SMS+PMA+SC79. (A) WB analysis of the expression of p-AKT and AKT in the neutrophils of each group. (B) Quantitative analysis of the relative expression of p-AKT in (A). (C) WB analysis of the expression of p-PI3K and PI3K in the neutrophils of each group. (D) Quantitative analysis of the relative expression of p-PI3K in (C). (E) IF analysis of MPO expression. (F) IF analysis of NE expression. (G) IF analysis of PAD4 expression. (H) IF analysis of CitH3 expression. (I) Quantitative analysis of MPO in (E) (n = 3). (J) Quantitative analysis of NE in (F) (n = 3). (K) Quantitative analysis of PAD4 in (G) (n = 3). (L) Quantitative analysis of CitH3 in (H) (n = 3). (M)Neutrophil nuclear area of (E) (n = 20). (N) Neutrophil nuclear area of (F) (n = 20). (O) Neutrophil nuclear area of (G) (n = 20). (P) Neutrophil nuclear area of (H) (n = 20). The values are presented as the means ± SDs. *P<0.05, **P<0.01, ^nsP > 0.05.

Figure 6

SMS inhibited ROS production and autophagy through AKT signalling in vitro. Neutrophils extracted from the peritoneal cavity of mice were subjected to different interventions in the following groups: NC, SMS, NC+PMA, SMS+PMA, and SMS+PMA+SC79. (A) IF images of neutrophils from each group after being stained with DCFH-DA for 4 h. (B) Quantitative analysis of ROS in (A) (n = 3). (C) WB analysis of the expression of ATG5, Beclin-1, and LC3B in the neutrophils of each group. (D) Quantitative analysis of ATG5 in (C) (n = 3). (E) Quantitative analysis of Beclin-1 in (C) (n = 3). (F) Quantitative analysis of LC3B in (C) (n = 3). (G) IF analysis of the expression of LC3B in the neutrophils of each group. (H) Quantitative analysis of LC3B in (G) (n = 3). (I) Transmission electron microscopy results for each group. The Orange arrows indicate autophagosomes. The values are presented as the means ± SDs. *P<0.05, **P<0.01.