Effect of mechanical strain-induced oxidative stress on expression of types I and III collagen from fibroblasts in human parametrial ligament

Abstract

Objective To investigate the effect of mechanical strain-induced oxidative stress on expression of types Ⅰ and Ⅲ collagen from fibroblasts in human parametrial ligament. Methods Ten cases of vaginal hysterectomy conducted at our hospital between January and October 2014 were included in this study. At surgery, parametrial ligaments were harvested and subjected to primary culture and identification of fibroblasts. A four-point bending system was used to establish the mechanical strain model in which fibroblasts were stretched as reflected by displacement of culture plate due to strain-induced deformation. A plate displacement of 1 mm corresponded to a mechanical force of 1 333 μstrain. The strains were 0 μstrain (0 mm) , 1 333 μstrain (1 mm) and 5 333 μstrain (4 mm) , loaded with a frequency of 0.5 Hz over a loading time of 4 h. Meanwhile, fibroblasts were incubated in different concentrations of H2O2 to establish the model of oxidative stress. The H2O2 concentrations were 0, 0.2, 0.4, 0.8 and 1.6 mmol/L, respectively, during an incubation time of 4 h. The types Ⅰ and Ⅲ collagen mRNA and protein expression of fibroblasts in both models were determined by real-time fluorescent quantitative PCR and Western blotting. Immunofluorescence was used to detect the levels of 8-hydroxy-deoxyguanosine (8-OHdG) , a product of cellular oxidative damage in the mechanical strain model. Results In the mechanical strain model, real-time PCR and Western blotting showed that compared with the control group (0 μstrain) , 1 333 μstrain mechanical strain resulted in significantly higher expression levels, whereas 5 333 μstrain mechanical strain resulted in significantly lowered expression levels, of both types Ⅰ and Ⅲ collagen mRNA and protein in fibroblasts (all P 0.05) ; Incubation with 0.8 or 1.6 mmol/L H2O2 for 4 h resulted in significantly lowered expression levels of both types Ⅰ and Ⅲ collagen mRNA (all P 0.05) , but significantly increased the type Ⅲ collagen protein expression (P 0.05) ; Incubation in 0.8 or 1.6 mmol/L H2O2 for 4 h resulted in significantly lowered expression levels of both types Ⅰ and Ⅲ collagen proteins (all P<0.05) . Conclusions The mechanical strain and oxidative stress within a certain range can inhibit the expression of collagen types Ⅰ and Ⅲ in fibroblasts of parametrial ligament. Mechanical strain may induce oxidative stress in pelvic supporting tissues and remodeling of extracellular matrix, thereby playing a role in the development of pelvic organ prolapse. Key words: Fibroblasts; Pelvic organ prolapse; Oxidative stress; Collagen type Ⅰ; Collagen type Ⅲ