Jinwujiangu Capsule Treats Fibroblast-Like Synoviocytes of Rheumatoid Arthritis by Inhibiting Pyroptosis via the NLRP3/CAPSES/GSDMD Pathway

Abstract

Jinwujiangu capsule (JWJGC) is a traditional Chinese medicine formula used to treat rheumatoid arthritis (RA). However, whether its mechanism is associated with pyroptosis remains unclear. In this study, the ability of JWJGC to inhibit the growth of fibroblast-like synoviocytes of RA (RA-FLS) through pyroptosis was evaluated. The cells isolated from patients with RA were identified by hematoxylin and eosin (H&E) staining, immunohistochemistry, and flow cytometry. After RA-FLS were treated with different concentrations of JWJGC-containing serum, the cell proliferation inhibition rate, expression of caspase-1/3/4/5, NOD-like receptor protein 3 (NLRP3), gasdermin-D (GSDMD), and apoptosis-associated speck-like protein containing a CARD (ASC), concentrations of interleukin-1β (IL-1β) and interleukin-18 (IL-18), the activity of lactic dehydrogenase (LDH), and pyroptosis were evaluated. The results showed that JWJGC increased the proliferative inhibition rate, decreased the expression of caspase-1/3/4/5, GSDMD, NLRP3, and ASC, suppressed the expression of IL-1β and IL-18, induced the activity of LDH, and downregulated the number of double-positive FITC anti-caspase-1 and PI. Generally, our findings suggest that JWJGC can regulate NLRP3/CAPSES/GSDMD in treating RA-FLS through pyroptosis.

Figure 1

The work flowchart of this study.

Figure 2

(a) (×100) and (b) (×200) showing the H&E staining results. Immunohistochemistry results are shown in (c) (×100) and (d) (×200). (e) The purity of the isolated cells marked with FIDC anti-CD90 (96.6 ± 1.44%; n = 4). (f) The control. (g) The purity of the isolated cells marked with APC anti-VCAM-1 (97.1 ± 0.39%; n = 4). (h) The control.

Figure 3

(a) The cell proliferative inhibition rate after the different treatments for 24 h or 48 h. (b)-(c) The concentrations of IL-1β and IL-18. (d) The concentrations of LDH. Data are expressed as mean ± SEM (n = 5). ^∗P > 0.05, compared with blank control; ^#P < 0.05, compared with the rabbit group. Blank control, blank serum control group; rabbit control, rabbit serum control group; JWJGC-H, JWJGC high-dose group; JWJGC-M, JWJGC medium-dose group; JWJGC-L, JWJGC low-dose group; Leflunomide, leflunomide-positive control group.

Figure 4

Western blot bands of the expression of NLRP3, ASC, GSDMD, and caspase-1/3/4/5 after the different treatments. Data are expressed as mean ± SEM (n = 3). ^∗P > 0.05 compared with blank control; ^#P < 0.05 compared with the rabbit group. Blank control, blank serum control group; rabbit control, rabbit serum control group; JWJGC-H, JWJGC high-dose group; JWJGC-M, JWJGC medium-dose group; JWJGC-L, JWJGC low-dose group; Leflunomide, leflunomide-positive control group.

Figure 5

Double-positive caspase-1/PI is shown in the Q2 of every image. Data are expressed as mean ± SEM (n = 3). ^∗P > 0.05 compared with blank control; ^#P < 0.05 compared with the rabbit group. Blank control, blank serum control group; rabbit control, rabbit serum control group; JWJGC-H, JWJGC high-dose group; JJWJGC-M, JWJGC medium-dose group; JWJGC-L, JWJGC low-dose group; Leflunomide, leflunomide-positive control group.