Fig 2: Rb2 reverses the effect of miR-216a on the Smad3/IκBa signaling pathway. (A) Protein expression levels of Smad3 and IκBα with or without Rb2 treatment in PDL8 HUVECs transfected with miR-216a mimics (n=5). *P<0.05; #P<0.05. (B) mRNA expression levels of Smad3, ICAM1 and VCAM1 after silencing Smad3 in PDL8 HUVECs with or without Rb2 treatment (n=5). (C) Protein expression levels of Smad3, ICAM1 and VCAM1 after silencing Smad3 in PDL8 HUVECs with or without Rb2 treatment (n=5). *P<0.05, **P<0.01. miR-216a, microRNA-216a; NC, negative control; VCAM1, vascular cell adhesion molecule 1; Rb2, ginsenoside Rb2; ICAM1, intercellular adhesion molecule; si, small interfering RNA.

Fig 3: Silencing of TXNIP abolishes, while its overexpression induces, REC adhesion molecule upregulation in an IL-1ß-dependent fashion. (a) Representative WB blots (boxes denote separate lanes from the same gel) and statistical analyses of (b) ICAM-1 and (c) PECAM-1 protein expression showed higher levels in cells incubated with PAL-BSA compared with BSA controls in the scrambled-siRNA group. Silencing TXNIP expression using siRNA abolished such responses. Two-way ANOVA showed significant interaction between Pal-BSA and silencing TXNIP expression across PECAM-1 levels (n = 3–4, each measurement was performed in duplicate using two different cell preparations; * p < 0.05 vs. SCsiRNA + BSA, # vs. SCsiRNA + Pal-BSA). (d) Representative blots and WB analysis of (e) ICAM-1 and (f) PECAM-1 protein expression showed higher levels in TXNIP overexpression (TXNIP ++ + vehicle) group compared with EV-GFP + vehicle control group, respectively. Treatment of TXNIP-overexpressing REC with 100 ng/mL of IL-1RA (TXNIP ++ + IL-1RA) suppressed the effect of TXNIP overexpression on expression of REC adhesion molecules (n = 5–6, each measurement was performed in triplicate using two different cell preparations; * p < 0.05 vs. EV-GFP+ vehicle, # vs. TXNIP + + + vehicle).

Fig 4: Overexpression of Cx43 reversed the protective effect of normal glucose in GDM-HUVECs. (A) Cx43; (B) ICAM-1, VCAM-1 protein expression; (C) cell adhesion. Data are presented as mean ± SEM; n=5, *, P<0.05 vs. GDM, #, P<0.05 vs. GDM-G. Scale bar in C: ×200 and ×400. Cx43, Connexin 43; GDM, gestational diabetes mellitus; ICAM-1, intercellular cell adhesion molecule-1; VCAM-1, vascular cell adhesion protein 1; RA, retinoic acid (Cx43 protein expression fortifier).

Fig 5: The mechanism of hyperglycemia-induced cell adhesion. The effect of hyperglycemia-induced Cx43 on cell adhesion is related to the modulation of PI3K/AKT/NF-?B signaling pathway activity and the regulation of its downstream ICAM-1 and VCAM-1 protein expression. Cx43, Connexin43; ICAM-1, intercellular cell adhesion molecule-1; VCAM-1, vascular cell adhesion protein 1.