Fig 2: Suppression of ATP decreases the pro-inflammatory effect of miRNA-20b in cerebral ischemia. (A) ATP levels were determined using ELISA; densitometric analysis of (B) NLRP3, (C) caspase-1, (D) IL-18 and (E) IL-1ß protein and (F) western blot analysis of NLRP3, caspase-1, IL-18 and IL-1 protein expression following miRNA-20b overexpression and ATP inhibition. Serum levels of (G) IL-1ß and (H) IL-18 following miRNA-20b overexpression and ATP inhibition. **P<0.01 vs. negative control group. ##P<0.01 vs. miRNA-20b overexpression group. miRNA, microRNA; ATP, adenosine 5'-triphosphate; NLRP3, NOD-like receptor pyrin domain containing 3; IL, interleukin; control, negative control group; miRNA-20b, miRNA-20b overexpression group; ATP inhibitor, 10 µM INF39 and miRNA-20b overexpression group.

Fig 3: PLP, Caffeic acid, and P-coumaric acid slowed down the loss of tight junction proteins in colonic epithelial tissue and the infiltration of immune cells, and they inhibited the secretion of pro-inflammatory factors and the activation of NF-?B cell signaling in the colon tissue of DSS-induced colitis mice (A–N). Representative histopathological images at 100× magnification (A–E). Colonic MPO activity (F). ZO-1, occludin, and claudin 1 mRNA in colon tissue were determined by real-time PCR (G–I). p-p65, p65, and IL-1b were detected by Western blot (J–L). IL-6 and IL-1ß mRNA in colon tissue were determined by real-time PCR (M,N). Nor: normal control mice; DSS: colitis model control mice fed dextran sulphate sodium salt; PLP-200: pineapple leaf phenols-treated (200 mg/kg) DSS mice; Caffeic acid: Caffeic acid-treated (50 mg/kg) DSS mice; P-coumaric acid: P-coumaric acid-treated (50 mg/kg) DSS mice. Data are expressed as the mean ± SD (n = 7); * p < 0.05, ** p < 0.01 compared to the mice treated with DSS alone.

Fig 4: A20 is a functional miR-21 target for regulation of NF-?B and NLRP3 inflammasomes.a–e A20 (a), Nlrp3 (b), Asc (c), Casp1 (d), and IL-1b (e) mRNA levels in LPS-treated WT and miR-21 KO BMDMs. f A20, IL-1b and NLRP3 inflammasome protein levels in LPS-treated WT and miR-21 KO BMDMs after 4 h. g Sequence alignment of the A20 3'-UTR with miR-21 across multiple species. h Activity of luciferase reporters containing WT or mutant A20 3'-UTRs that were used with miR-21 mimics or respective controls to cotransfect HEK-293T cells. i BMDMs were treated with 500 ng/ml LPS for different lengths of time. Cell extracts were immunoblotted for IKBa and p-IKBa. j, k WT and miR-21 KO BMDMs were left unstimulated (control) or stimulated with LPS (500 ng/ml), ATP (5 mM), or nigericin (20 mM) alone or stimulated with LPS (500 ng/ml) for 4 h and then treated with 5 mM ATP or 20 mM nigericin for 30 min. Culture supernatants were analyzed for IL-6 secretion (j) and TNF secretion (k). The data represent the mean ± SD of one among three biological replicates, with three technical replicates each (*P < 0.05; **P < 0.01; Student’s t test)

Fig 5: Suppression of NLRP3 decreases the pro-inflammatory effect of miRNA-20b in cerebral ischemia. Densitometric analysis of (A) NLRP3, (B) caspase-1, (C) IL-18 and (D) IL-1ß protein and (E) western blot analysis of NLRP3, caspase-1, IL-18 and IL-1 protein expression. Serum levels of (F) IL-1ß and (G) IL-18 levels. **P<0.01 vs. negative control group. ##P<0.01 vs. miRNA-20b overexpression group. miRNA, microRNA; NLRP3, NOD-like receptor pyrin domain containing 3; IL, interleukin; control, control negative group; miRNA-20b, miRNA-20b overexpression group; NLRP3 inhibitor, 5 nM MCC950 and miRNA-20b overexpression group.