Potential therapeutic role of punicalagin against mechanical-trauma-induced stress urinary incontinence via upregulation of Nrf2 and TGF-β1 signaling : Effect of punicalagin on mechanical trauma induced SUI

Abstract

Introduction and hypothesis: We investigated the effect of punicalagin (PUN; 2,3-hexahydroxydiphenoyl-gallagyl-D-glucose), on mechanical-trauma-induced stress urinary incontinence (SUI) in mouse and the mechanisms underlying any effects.

Methods: Ninety virgin female C57BL/6 mice were randomized into six groups: five groups underwent vaginal distention (VD) for 1 h and leak-point pressure (LPP) was measured on the 1st, 3rd, 7th, 14th, and 28th day following (VD groups 1 d, 3 d, 7 d, 14 d, and 28 d). The sixth group was a noninstrumented control (NC) group. Then, 75 virgin female C57BL/6 mice were randomized into five groups: a VD group (that just underwent VD) and an NC group were orally administered saline every day for 7 days; and three VD + PUN groups that underwent VD and were orally administered PUN respectively at 2.5, 5, and 10 mg/kg every day for 7 days. LPP was tested on the day 7, then all mice were sacrificed and their urethras and anterior vaginal walls harvested for Masson staining, immunohistochemistry study, Western blot analysis, and quantitative polymerase chain reaction (qPCR).

Results: LPPs after VD were significantly lower than the NC group, and the LPPs of mice on days 14 and 28 day after VD were significantly higher than on the days 1, 3, and 7. PUN significantly improved VD-induced drops in LPP and alleviated VD-induced decrease of collagen I, collagen III, α-smooth muscle actin (SMA), transforming growth factor (TGF)-β1, and p-Smad3, nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), and glutathione peroxidase (GPx1) protein levels, and increase of 8-hydroxydeoxyguanosine (OHdG) in urethra and anterior vaginal wall. PUN also up-regulated the expression of manganese superoxide dismutase (MnSOD), whereas protein levels of Smad 2, p-Smad2, and Smad3 were not changed.

Conclusions: PUN exerts certain therapeutic effect on mechanical-trauma-induced SUI in mice, which might be through the activation of TGF-β1/Smad3 and Nrf2/antioxidant response element (ARE) signaling activation.

Keywords: Extracellular matrix; Mechanical trauma; Oxidative damage; Punicalagin; Stress urinary incontinence; Vaginal distension.

Fig. 1

Leak-point pressure (LPP) values: a Mice in vaginal distention (VD) groups 1 d, 3 d, 7 d, 14 d, and 28 d were significantly lower than those in noninstrumented control (NC) group, and LPP recovered from the day 14 after VD. *P < 0.05 vs NC group; ^# P < 0.05 vs VD 1 d group; &P < 0.05 vs VD 3 d group; ^$ P < 0.05 vs VD 7 d group; ^{^} P < 0.05 vs VD 14 d group. b LPP values of mice in VD, VD + punicalagin (PUN) 2.5, VD + PUN 5, and VD + PUN 10 groups were significantly lower than the NC group, whereas, values were significantly increased in VD + PUN 5 and VD + PUN 10 groups compared with the NC group. *P < 0.05 vs NC group; ^# P < 0.05 compared with VD group. Every measurement was repeated five times

Fig. 2

Masson trichrome staining of urethra and anterior vaginal wall: a noninstrumented control (NC) group (a–c), vaginal distention (VD) group (d–f), VD + punicalagin (PUN) 2.5 group (g–i), VD + PUN 5 group (j–l), and VD + PUN 10 group (m–p). Collagen fibers stained blue, muscle white, cellulose and cytoplasm red, nuclei blue-purple. Second column: magnification of the black rectangle in the first column; third column: magnification of the red dotted rectangle in the first column. Original magnification: ×100 (a, d, g, j, m); ×200 (b–c, e–f, h–i, k–l, o–p). b Semiquantitative assay of collagen using quantity one-4.6.2. *P < 0.05. Every experiment was repeated three times

Fig. 3

Protein and messenger RNA (mRNA) expressions of collagen I, collagen III, and α-smooth-muscle actin (SMA) in five groups. a Western blotting was performed to detect protein expression of collagen I, collagen III, and α-SMA in urethra and anterior vaginal wall of mice in five groups, and semiquantitative assay was done using quantity one-4.6.2. b Messenger RNA (mRNA) expression of collagen I, collagen III, and α-SMA in urethra and anterior vaginal wall of mice in five groups were detected by quantitative polymerase chain reaction (q-PCR). *P < 0.05 vs nonintrumented control (NC) group; ^# P < 0.05 compared with vaginal distention (VD) group; every experiment was repeated three times

Fig. 4

Western blotting shows protein expressions of transforming growth factor (TGF)-β1/Smads signaling pathways in urethra and anterior vaginal wall of mice in five groups. Semiquantitative assay was done using quantity one-4.6.2. *P < 0.05 compared with NC group; ^# P < 0.05 compared with VD group. Every experiment was repeated three times

Fig. 5

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling-related proteins and 8-hydroxydeoxyguanosine (8-OHdG) expression in five groups. a Western blotting detected Nrf2, glutathione peroxidase (GPx1), and manganese superoxide dismutase (MnSOD) in urethra and anterior vaginal wall of mice in five groups. Semiquantitative assay was done using quantity one-4.6.2. b Expression of 8-OHdG was detected using immunohistochemistry in urethra and anterior vaginal wall; representative images are shown: negative control (a–b); noninstrumented control (NC) (c–d); vaginal dilation (VD) (e–f2); VD + punicalagin (PUN) 2.5 (g–h); VD + PUN 5 (i–j); VD + PUN 10 (k–l). Images in the second column are magnification of the first column; the images in the forth column are magnification of the third column. Scale = 50 μm. c Semiquantitative assay used Image-Pro Plus 5.1. *P < 0.05 cvs ^# P < 0.05 vs VD. Every experiment was repeated three times