Mechanical stress influences the morphology and function of human uterosacral ligament fibroblasts and activates the p38 MAPK pathway

Abstract

Introduction and hypothesis: Pelvic organ prolapse (POP) is a common condition in older women that affects quality of life. Mechanical injury of the pelvic floor support system contributes to POP development. In our study, we aimed to examine the mechanical damage to human uterosacral ligament fibroblasts (hUSLFs) to preliminarily explore the mechanism of mechanical transduction in POP.

Methods: hUSLFs were derived from POP and non-POP patients. Mechanical stress was induced by the FX-5000 T-cell stress loading system. Student's t-test was used for comparisons between different groups.

Results: We found that hUSLFs from POP patients were larger and longer than those from non-POP patients and exhibited cytoskeleton F-actin rearrangement. Collagen I and III expression levels were lower and matrix metalloproteinase 1 (MMP1) levels were higher in POP patients than in non-POP patients. Additionally, the apoptosis rate was significantly increased in POP patients compared to non-POP patients. After mechanical stretching, hUSLFs underwent a POP-like transformation. Cells became longer, and the cytoskeleton became thicker and rearranged. The extracellular matrix (ECM) was remodelled because of the upregulation of collagen I and III expression and downregulation of MMP1 expression. Mechanical stress also induced hUSLF apoptosis. Notably, we found that the p38 MAPK pathway was activated by mechanical stretching.

Conclusions: Mechanical stress induced morphological changes in ligament fibroblasts, leading to cytoskeleton and ECM remodelling and cell apoptosis. p38 MAPK might be involved in this process, providing novel insights into the mechanical biology of and possible therapies for this disease.

Keywords: Human uterosacral ligament fibroblasts; Mechanical stress; Pelvic organ prolapse; p38 MAPK pathway.

Fig. 1

Comparison of the morphology of hUSLFs from the non-POP and POP groups. Identification of hUSLFs by immunocytochemical staining for (A) FSP-1, (B) vimentin, (C) α-SMA and (D) CK5/6 (magnification: ×100; scale bar = 10 μm). E–G: Bright-field images of hUSLFs observed at low power (E and G, magnification: ×40; scale bar = 100 μm) and high power (F and H, magnification: ×100; scale bar = 10 μm). The red arrows indicate triangular or polygonal cells. I–L: Images of the hUSLF cytoskeleton by immunofluorescence staining for F-actin (green) and DAPI staining for nuclei (blue). The cells were observed under a confocal laser scanning microscope at low power (I and K, magnification: ×200; scale bar = 100 μm) and high power (J and L, magnification: ×1000; scale bar = 10 μm). The yellow arrows indicate triangular or polygonal cells

Fig. 2

Comparison of ECM-related protein expression in and apoptosis of hUSLFs from the non-POP and POP groups. (A) The levels of the ECM-related proteins collagen I, collagen III, MMP1, MMP2, MMP9, TIMP1 and TIMP2 in hUSLFs from the non-POP and POP groups were examined by qPCR. (B) The levels of the apoptosis-related proteins bad and bax in hUSLFs from the non-POP and POP groups were examined by qPCR. (C) Cells from the non-POP and POP groups were stained with an Annexin V-FITC/PI staining kit and analysed by flow cytometry. (D) Statistical analysis of the apoptosis rate. Unpaired t-tests were performed. The data are presented as the mean ± SD. *P < 0.05

Fig. 3

Mechanical stretching altered the morphology of hUSLFs and induced cytoskeleton remodelling. hUSLFs from the non-POP group were exposed to tension stress (0.1-Hz uniaxial, 10% elongation) for 24 h and then subjected to immunofluorescence staining for F-actin (green) and DAPI staining for nuclei (blue). The cells were observed under a confocal laser scanning microscope at low power (A and C, magnification: ×200; scale bar = 100 μm) and high power (B and D, magnification: ×1000; scale bar = 10 μm)

Fig. 4

Mechanical stretching induced ECM remodelling and apoptosis of hUSLFs. hUSLFs from the non-POP group were exposed to tension stress (0.1-Hz uniaxial, 10% elongation) for 24 h and then subjected to qPCR and flow cytometry. (A) The levels of the ECM-related proteins collagen I, collagen III, MMP1, MMP2, MMP9, TIMP1 and TIMP2 in the static and stretched cells were examined by qPCR. (B) The levels of the apoptosis-related protein bad and bax in static and stretched cells were examined by qPCR. (C) Cells in the static and stretched groups were stained with an annexin V-FITC/PI staining kit and analysed by flow cytometry. (D) Statistical analysis of the apoptosis rate. Unpaired t-tests were performed. The data are presented as the mean ± SD. *P < 0.05

Fig. 5

Mechanical stretching activated the MAPK-p38 pathway in hUSLFs. hUSLFs from the non-POP group were exposed to tension stress (0.1-Hz uniaxial, 10% elongation) for 12 or 24 h, and then p38, ERK and JNK expression and the phosphorylation of these proteins were assessed by Western blotting