Mechanism of Mechanical Trauma-Induced Extracellular Matrix Remodeling of Fibroblasts in Association with Nrf2/ARE Signaling Suppression Mediating TGF- β 1/Smad3 Signaling Inhibition

Abstract

Stress urinary incontinence (SUI) is a common hygienic problem affecting the quality of women's life worldwide. In this research, we revealed the involvement and regulation of extracellular matrix (ECM) remodeling, oxidative damage, and TGF-β1 signaling in the pathological mechanisms of mechanical trauma-induced SUI. We found that excessive mechanical strain significantly increased apoptosis rate, decreased cell viability and ECM production, and broke the balance of MMPs/TIMPs compared with the nonstrain control (NC) group. The expression levels of TGFβ1, p-Smad3, Nrf2, GPx1, and CAT were downregulated, the production of ROS, 8-OHdG, 4-HNE, and MDA was increased, and the nuclear translocation of Smad2/3 was suppressed after 5333 μstrain's treatment. Both mTGF-β1 pretreatment and Nrf2 overexpression could reverse mechanical injury-induced TGFβ1/Smad3 signaling inhibition and ECM remodeling, whereas mTGF-β1 had no effect on Nrf2 expression. Nrf2 overexpression significantly alleviated mechanical injury-induced ROS accumulation and oxidative damage; in contrast, Nrf2 silencing exhibited opposite effects. Besides, vaginal distention- (VD-) induced in vivo SUI model was used to confirm the in vitro results; Nrf2 knockout aggravates mechanical trauma-induced LPP reduction, ECM remodeling, oxidative damage, and TGF-β1/Smad3 suppression in mice. Therefore, we deduce that mechanical injury-induced ECM remodeling might be associated with Nrf2/ARE signaling suppression mediating TGF-β1/Smad3 signaling inhibition. This might reflect a new molecular target for SUI researches.

Figure 1

Experimental diagram and the effect of mechanical strain on L929 fibroblast. (a) Experimental diagram and the simulation diagram of mechanical strain. (b) The effect of mechanical strain on cell viabilities. (c) The effect of mechanical strain on cell apoptosis. ^∗P < 0.05 compared with the control group; every experiment was repeated for 3 times. CON: control group; CMS: cyclic mechanical strain group.

Figure 2

Effect of mechanical strain on ECM and MMPs/TIMPs of L929 fibroblast. (a, b) The effect of mechanical strain on protein and mRNA expression levels of ECM. (c, d) The effect of mechanical strain on protein and mRNA expression levels of MMPs and TIMPs. ^∗P < 0.05 compared with the control group; every experiment was repeated for 3 times. CON: control group; CMS: cyclic mechanical strain.

Figure 3

Effect of mechanical strain on TGF-β1/Smad signaling. (a, b) The effect of mechanical strain on protein and mRNA expression levels of TGF-β1/Smad signaling. (c) The effect of mechanical strain on nuclear translocation of Smad2/3. Original magnification: ×100. ^∗P < 0.05 compared with the control group; every experiment was repeated for 3 times. CON: control group; CMS: cyclic mechanical strain.

Figure 4

The involvement of TGF-β1 signaling in the process of mechanical injury-induced ECM remodeling. ^∗P < 0.05; every experiment was repeated for 3 times. CON: control group; CMS: cyclic mechanical strain; TGF-β1: mTGF-β1 pretreatment.

Figure 5

The effect of mechanical strain on ROS levels and oxidative damage on L929 cells and the involvement of Nrf2. (a) The effect of mechanical strain on ROS levels and the role of Nrf2 in this process. (b, c) The effect of mechanical strain on oxidative damage and the role of Nrf2 in this process. The scale is equal to 20 μm. ^∗P < 0.05; every experiment was repeated for 3 times. CON: control group; CMS: cyclic mechanical strain; shNfe2l2: Lv-shNfe2l2 transfection established Nrf2 silencing L929 cells; LV-Nfe2l2: Lv-Nfe2l2 transfection established Nrf2 overexpressing L929 cells.

Figure 6

The effect of mechanical strain on Nrf2 signaling on L929 cells. (a) The effect of mechanical strain on protein expression levels of Nrf2 signaling. (b) The effect of mechanical strain on mRNA expression levels of Nrf2 signaling. ^∗P < 0.05 compared with control group; every experiment was repeated for 3 times. CON: control group; CMS: cyclic mechanical strain.

Figure 7

The involvement of Nrf2 signaling in the process of mechanical injury-induced ECM remodeling. ^∗P < 0.05; every experiment was repeated for 3 times. CON: control group; CMS: cyclic mechanical strain; LV-Nfe2l2: Lv-Nfe2l2 transfection established Nrf2 overexpressing L929 cells.

Figure 8

The relationship of Nrf2 signaling and TGF-β1 signaling in mechanical injury-induced ECM remodeling. (a) The effect of Nrf2 overexpression and LY2109761 on mechanical strain-induced TGF-β1 signaling inhibition. (b) The effect of Nrf2 knockdown and TGF-β1 on mechanical strain-induced TGF-β1 signaling inhibition. (c) The effect of Nrf2 overexpression on mechanical strain-induced nuclear translocation inhibition of Smad2/3. Original magnification: ×100. ^∗P < 0.05; every experiment was repeated for 3 times. CON: control group; CMS: cyclic mechanical strain; shNfe2l2: Lv-shNfe2l2 transfection established Nrf2 silencing L929 cells; LV-Nfe2l2: Lv-Nfe2l2 transfection established Nrf2 overexpressing L929 cells; TGF-β1: mTGF-β1 pretreatment; LY2109761: TGF-β1 signaling inhibitor LY2109761 pretreatment; CON-CMS: 5333 μstrain mechanical strain-treated normal L929 cells; LV-Nfe2l2-CMS: 5333 μstrain mechanical strain-treated Nrf2 overexpressing L929 cells.

Figure 9

In vivo confirmation on vaginal distension-induced SUI mice model. (a) LPPs of mice in six groups. (b, c) Protein expression levels of TGF-β1/Smad3 signaling and ECM in anterior vaginal walls of mice in six groups. (d) MDA concentrations in anterior vaginal walls of mice in six groups. ^∗P < 0.05 compared with the WT-VD group. ^#P < 0.05 compared with the KO-VD group; every experiment was repeated for 3 times. WT-NC: noninstrumented control group of wild-type mice; WT-Sham: sham-operated group of wild-type mice; WT-VD: VD group of wild-type mice; KO-NC: noninstrumented control group of Nrf2 knockout mice; KO-Sham: sham-operated group of Nrf2 knockout mice; KO-VD: VD group of Nrf2 knockout mice.