Fig 2: Induction of BDKRB1 and BDKRB2 expression by lipopolysaccharide (LPS) and serum amyloid A1 (SAA1) in human amnion fibroblasts. (A, B) Concentration-dependent induction of BDKRB1 and BDKRB2 mRNA expression by LPS (0, 1, 10, 50 ng/ml, 24 h) (n = 4) and SAA1 (0, 10, 50, 100 ng/ml, 24 h) (n = 5). (C, D) Upregulation of BDKRB1 and BDKRB2 protein levels by LPS (50 ng/ml, 24 h) and SAA1 (50 ng/ml, 24 h) (n = 5). (E, F) The TRL4 inhibitor CLI-095 (5 µM) blocked LPS (n = 3)- and SAA1 (n = 4)-induced BDKRB1 and BDKRB2 mRNA expression. Data are the means ± SEM. Statistical analysis was performed with one-way ANOVA test followed by Tukey test. Top panels are the representative immunoblots. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. 0 ng/ml for (A) and (B); vs. control (CTR) for (C) and (D); vs. group without LPS/SAA1 and CLI-095 treatment for (E) and (F) # p < 0.05, ## p < 0.01 vs. LPS- or SAA1-treated groups.

Fig 3: Pathway of bradykinin synthesis and the expression of the bradykinin system in human amnion. (A) Pathway of bradykinin synthesis. Italic word in brackets is the gene name of the corresponding protein. UD, undetected. (B) FPKM of gene transcripts related to bradykinin synthesis in the amnion at term with labor (TL, n = 3) and without labor (TNL, n = 3) as revealed by transcriptomic sequencing. (C) Immunohistochemical staining of bradykinin B1 receptor (BDKRB1) and bradykinin B2 receptor (BDKRB2) in human amnion, and comparison of BDKRB1 and BDKRB2 mRNA expression in human amnion fibroblasts and epithelial cells (n = 3). ae, amnion epithelial cells; af, amnion fibroblasts; scale bar, 50 µm. Statistical analysis was performed with unpaired Student’s t-test. **p < 0.01 vs. ae.

Fig 4: B1 and B2 receptors (B1R, B2R) and vascular endothelial growth factor A (VEGFA) mRNA expression in human retinae (A–L). Microphotographs showing the mRNA labeling for B1R (green; B,F,J), B2R (red; C,G,K) and VEGFA (purple; D,H,L) in the control (CTL), dry age-related macular degeneration (AMD) and wet AMD retinae. Sections are counterstained for 4',6-diamidino-2-phenylindole (DAPI; blue; A,E,I), which labels cell nuclei. Note that B1R was partially colocalized with B2R in wet AMD in the outer nuclear layer (ONL), and B2R was also partially colocalized with VEGFA in the ONL, however, there was rare colocalization between B1R and VEGFA. Images were obtained at 40×. Scale bar: 20 µm. (M–O) Quantification of the mRNA (mean fluorescence intensity/cell) of B1R, B2R and VEGFA in the different layers of the retina. Data are mean ± SEM of values obtained from five retinae per group and four zones per retina. * p < 0.05 AMD compared with control. CTL: control (O), Dry: dry AMD (?), Wet: wet AMD (?), GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer.

Fig 5: Induction of PTGS2 expression by BK and DABK was mediated by BDKRB2 and BDKRB1, respectively, in human amnion fibroblasts. (A) BDKRB1 inhibitor ELN-441958 failed to block BK (0.1 µM, 4 h)-induced PTGS2 mRNA (n = 4) and protein (n = 3) expression. (B) The BDKRB2 inhibitor icatibant blocked BK (0.1 µM, 4 h)-induced PTGS2 mRNA (n = 4) and protein (n = 3) expression. (C) The BDKRB1 inhibitor ELN-441958 blocked DABK (0.1 µM, 4 h)-induced PTGS2 mRNA (n = 4) and protein (n = 5) expression. (D) The BDKRB2 inhibitor icatibant failed to block DABK (0.1 µM, 4 h)-induced PTGS2 mRNA (n = 5) and protein (n = 6) expression. Statistical analysis was performed with one-way ANOVA test followed by Tukey test. Top panels are the representative immunoblots. *p < 0.05, **p < 0.01 vs. group without BK/DABK and antagonist treatment. # p < 0.05, ## p < 0.01 vs. BK- or DABK-treated groups.