Alpha-lipoic acid inhibits thrombin-induced fetal membrane weakening in vitro

Effect of Increasing Thrombin Dose upon FM Mechanical Properties: Thrombin (0–100 u/ml) induced concentration-dependent decreases in FM rupture strength (upper panel), stiffness (middle panel) and work to rupture (bottom panel). All incubations were for 48 h. The data shown represent triplicate FM cultures repeated three times using three different placentas. (Data are presented mean ± SD, *p < 0.001 vs. no thrombin control).

Thrombin Induces MMP9 and MMP3 in Amnion: Thrombin (0–100 u/ml for 48 h) induced concentration-dependent increases in both the pro-MMP9 (92 kD) and the active MMP9 (78 kD) proteins (Panel A); and in both the pro-MMP3 (57 kD) and the active MMP3 (45 kD) proteins (Panel B); in the amnion component of treated FM explants. The Western blot shown is representative of three replicate experiments with three different placentas. To confirm the significance of qualitative results, triplicate blots were scanned and subjected to densitometric analysis. Each blot was normalized to the zero-thrombin controls as indicated in Methods. (Data are presented mean ± SD, *p < 0.01 vs. no thrombin control).

Thrombin effect upon MMP9 and MMP3 in Choriodecidua: Thrombin (0–100 u/ml for 48 h) failed to show concentration-dependent increases in pro-MMP9 (92 kD) in the choriodecidua component of treated FM explants. The active MMP9 (78 kD) form was not detected (Panel A). Thrombin (0–100 u/ml for 48 h) failed to show concentration-dependent increases in either the pro-MMP3 (57 kD) or the active MMP3 (45 kD) protein in the choriodecidual component of treated FM explants (Panel B). The Western blot shown is representative of three replicate experiments with three different placentas. To confirm the significance of qualitative results, triplicate blots were scanned and subjected to densitometric analysis. Each blot was normalized to the zero-thrombin controls as indicated in Methods. (Data are presented mean ± SD, no significant differences were detected.)

Lipoic Acid (LA) inhibits Thrombin-induced-weakening: FM fragments were pre-incubated with or without LA (0.25 mM) for 6 h, then incubated with or without the addition of Thrombin (10 u/ml) for 48 h. Four groups are displayed: Control, Thrombin alone, LA pretreatment alone, LA pre-treatment + Thrombin. Rupture Strength (upper panel), Stiffness (middle panel) and Work to Rupture (lower panel) were determined as outlined in Methods. Data points represent pooled results of three experiments using three FM fragments per treatment group in each experiment. (Data are presented mean ± SD, *p < 0.001 vs. all other groups).

Lipoic Acid (LA) inhibits Thrombin-induced increases in MMP9 in the amnion component of cultured FM. Intact FM fragments were pre-incubated with or without LA (0.25 mM) for 6 h, then incubated with or without the addition of thrombin (10 u/ml) for 48 h. After the incubations, the amnion was then separated from the choriodecidua and tested. Four groups are displayed: Control, Thrombin alone, LA pre-treatment alone, LA pretreatment + Thrombin. The Western blot shown is representative of three replicate experiments using three different placentas. Both the pro-MMP9 (92 kD) and the active MMP9 (78 kD) proteins in the amnion component of treated FM explants are shown. To confirm the significance of qualitative results, blots were scanned and subjected to densitometric analysis. Each blot was normalized to the zero-thrombin controls as indicated in Methods (Data are presented mean ± SD, *p < 0.01 vs. all other groups).

Lipoic Acid (LA) inhibits Thrombin-induced increases in MMP3 in the amnion component of cultured FM. Intact FM fragments were pre-incubated with or without LA (0.25 mM) for 6 h, then incubated with or without the addition of Thrombin (10 u/ml) for 48 h. After the incubations, the amnion was then peeled from the choriodecidua and tested. Four groups are displayed: Control, Thrombin alone, LA pre-treatment alone, LA pre-treatment + Thrombin. The Western blot shown is representative of three replicate experiments with three different placentas (N = 9).Both the pro-MMP3 (57 kD) and the active MMP3 (45 kD) protein in the amnion component of treated FM explants are shown. To confirm the significance of qualitative results, blots were scanned and subjected to densitometric analysis. Each blot was normalized to the zero-thrombin controls as indicated in Methods (Data are presented mean ± SD, *p < 0.01 from all groups, + p < 0.05 from control).