Advanced glycation end products decrease collagen I levels in fibroblasts from the vaginal wall of patients with POP via the RAGE, MAPK and NF-κB pathways

Abstract

The present study was carried out to observe the impact of advanced glycation end products (AGEs) on collagen I derived from vaginal fibroblasts in the context of pelvic organ prolapse (POP), and explore the downstream effects on MAPK and nuclear factor-κB (NF-κB) signaling. After treating primary cultured human vaginal fibroblasts (HVFs) derived from POP and non-POP cases with AGEs, cell counting was carried out by sulforhodamine B. The expression levels of collagen I, receptor of advanced glycation end products (RAGE), matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were detected by western blot analysis and PCR. RAGE, MAPK and NF-κB were molecularly and pharmacologically-inhibited by siRNA, SB203580 and PDTC, respectively, and downstream changes were detected by western blot analysis and PCR. Inhibition of HVF proliferation by AGEs occurred more readily in POP patients than that noted in the controls. After treatment with AGEs, collagen I levels decreased and MMP-1 levels increased to a greater extent in the HVFs of POP than that noted in the controls. During this same period, RAGE and TIMP-1 levels remained stable. Following treatment with AGEs and RAGE pathway inhibitors by siRNA, SB203580 and PDTC, the impact induced by AGEs was diminished. The inhibition of p-p38 MAPK alone was not able to block the promoting effect of AGEs on the levels of NF-κB, which suggests that AGEs may function through other pathways, as well as p-p38 MAPK. On the whole, this study demonstrated that AGEs inhibited HVF proliferation in POP cases and decreased the expression of collagen I through RAGE and/or p-p38 MAPK and NF-κB-p-p65 pathways. Our results provide important insights into the collagen I metabolism in HVFs in POP.

Figure 1

Human vaginal fibroblasts (HVFs) cultured and identified by immunohistochemistory. The primary cultured fibroblasts in vaginal wall of pelvic organ prolapse (POP) patients are shown. HVFs were identified by an antibody against anti-vimentin: (A) magnification ×100 and (B) ×400.

Figure 2

Comparison of the effects of advanced glycation end products (AGEs) on the proliferation of human vaginal fibroblasts (HVFs). The effects were detected by sulforhodamine B. (A and B) Proliferation curves of HVFs of pelvic organ prolapse (POP) and the control group cultured under different concentrations of AGEs (at doses of 0, 25, 50, 75, 100 and 150 mg/l, 48 h). (C and D) Proliferation curves of HVFs of POP and the control group cultured for various time periods (treatment time, 0, 12, 24, 48, 72 and 96 h, at 50 mg/l). 'POP-1, 2, 3' belong to the POP groups' 'non-POP-1, 2, 3' belong to the control groups. Proliferation of the HVFs of the POP groups was more easily inhibited than that of the control groups.

Figure 3

Treatment with advanced glycation end products (AGEs) was shown to affect the expression levels of collagen I, matrix metalloproteinases-1 (MMP-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and receptor of advanced glycation end products (RAGE) in human vaginal fibroblasts (HVFs) by western blot analysis. (A) Representative western blots; (B–E) Protein levels of collagen I, MMP-1, TIMP-1 and RAGE, respectively. HVFs of pelvic organ prolapse (POP) and control groups were cultured under different concentrations of AGEs (at the dose of 0, 25, 50, 75, 100 and 150 mg/l, respectively, 48 h). Data are presented as the means ± SEM. ^*p<0.05 vs. control (0 mg/l). ^#P<0.05 as compared with POP group.

Figure 4

Treatment with advanced glycation end products (AGEs) for different culture times (treatment time, 0, 24, 48, 72 and 96 h) was shown to impact the protein expression levels of collagen I, matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and receptor of advanced glycation end products (RAGE) in human vaginal fibroblasts (HVFs) by western blotting. HVFs of pelvic organ prolapse (POP) and control were cultured under different culture times. The expression levels of collagen I, MMP-1, TIMP-1 and RAGE are shown.

Figure 5

Treatment with advanced glycation end products (AGEs) was shown to impact the mRNA expression of (A) collagen I, (B) matrix metalloproteinase-1 (MMP-1), (C) tissue inhibitor of metalloproteinase-1 (TIMP-1) and (D) receptor of advanced glycation end products (RAGE) by real-time PCR in human vaginal fibroblasts (HVFs). mRNA expression of collagen I, MMP-1, TIMP-1 and RAGE cultured under AGEs (50 mg/l) for different culture times in HVFs of pelvic organ prolapse (POP) and non-POP groups. The culture times were 0, 1, 2, 4, 8, 16, 24 and 48 h, respectively. Peak mRNA levels occurred at 4 h after AGE treatment in the POP groups, and 16 h in the control groups respectively. Data are presented as the means ± SEM (n=3), ^*p<0.06 compared to the control (blank, 0 mg/l) in their own group, respectively.

Figure 6

Effects on the mRNA expression of (A) collagen I, (B) matrix metalloproteinase-1 (MMP-1), (C) tissue inhibitor of metalloproteinase-1 (TIMP-1) and (D) receptor of advanced glycation end products (RAGE) at 4 h after treatment with advanced glycation end products (AGEs). mRNA expression of collagen I, MMP-1, TIMP-1 and RAGE was detected by real-time PCR after human vaginal fibroblasts (HVFs) of pelvic organ prolapse (POP) and control group were cultured with AGEs at different concentrations (0, 25, 50, 75, 100 and 150 mg/l, respectively). (A-D) Collagen I, MMP-1, TIMP and RAGE groups, respectively. Data are presented as the mean ± SEM (n=3), ^*p<0.05 compared to the control (0 mg/l) in their own group, respectively.

Figure 7

Effect on the mRNA expression of (A) collagen I, (B) matrix metalloproteinase-1 (MMP-1), (C) tissue inhibitor of metalloproteinase-1 (TIMP-1) and (D) receptor of advanced glycation end products (RAGE) at 16 h after treatment with advanced glycation end products (AGEs) is shown. mRNA expression of collagen I, MMP-1, TIMP-1 and RAGE was detected by real-time PCR after human vaginal fibroblasts (HVFs) of pelvic organ prolapse (POP) and control group were cultured under AGEs at different concentrations (0, 25, 50, 75, 100 and 150 mg/l, respectively). (A-D) Collagen I, MMP-1, TIMP and RAGE, respectively. Data are presented as the mean ± SEM (n=3), ^*p<0.06 compared to the control (0 mg/l, respectively).

Figure 8

Change in (A) MAPK and (B) nuclear factor-κB (NF-κB) expression in human vaginal fibroblasts (HVFs) following treatment with advanced glycation end products (AGEs) at different times (0, 8, 16, 30 and 60 min respectively for MAPK; 0, 0.5, 1, 2 and 4 h, respectively for NF-κB). Data are presented as the mean ± SEM (n=3) (n=3). ^*p<0.05 compared to the control (0 min or hour, respectively).

Figure 9

Ttransfection of siRNA with fluorescence and receptor of advanced glycation end products (RAGE) expression in human vaginal fibroblasts (HVFs) transfected with different siRNAs. (A) Images in white and green light after transfection. (B and C) Western blot and histogram of RAGE expression after transfection. Data are presented as the mean ± SEM (n=3), ^*p<0.05 compared to the control (blank). All the signals were obtained from the same membrane.

Figure 10

Quantity of siRNA2 added to dishes with different amount of cells in DMEM. Dish with (A) 12,500, (B) 9,000 and (C) 7,000 cells. Data are presented as the mean ± SEM (n=3). ^*p<0.05 compared to the control (blank). All the signals were obtained from the same membrane.

Figure 11

Detection of p-p38 MAPK after receptor of advanced glycation end products (RAGE) was blocked by siRNA for 16 min. The concentration of advanced glycation end products (AGEs) was 50 mg/l, and the quantity of siRNA was 7.5 μl/dish with 2 ml DMEM. Data are presented as the mean ± SEM (n=3). ^*p<0.05 compared to the control (treatment 1, respectively). All the signals were obtained from the same membrane. The numbering of the bars (–4) is the same as that in the top

Figure 12

Detection of Nf-κB-p-p65 after receptor of advanced glycation end products (RAGE) was blocked by siRNA and p38 was inhibited by SB203580 for 1 h. The concentration of advanced glycation end products (AGEs) was 50 mg/l, and SB203580 was 10 μM. Data are represented as the mean ± SEM (n=3). ^*p<0.05 compared to the control (treatment 1, respectively). All the signals were obtained from the same membrane. The numbering of the bars (–6) is the same as that in the top

Figure 13

Detection of collagen I and matrix metalloproteinase-1 (MMP-1) after receptor of advanced glycation end products (RAGE), p38 and p65 were blocked by siRNA (7.5 μl/dish), SB203580 (20 μM) and PDTC (10 μM), respectively, or in combination. The concentration of advanced glycation end products (AGEs) was 50 mg/l, and siRNA was added after AGE treatment for 48 h. The blocking time was 48 h. Data are presented as the mean ± SEM (n=3). The numbering of the bars (–9) is the same as that in the top. ^*p<0.05 compared to the control (blank,respectively).

Figure 14

mRNA expression of collagen I and matrix metalloproteinase-1 (MMP-1) was detected by real-time PCR. Human vaginal fibroblasts (HVFs) were cultured with advanced glycation end products (AGEs) (50 mg/l) and cell signaling was blocked by siRNA, SB203580 and PDTC, respectively, or a combination. The quantity of siRNA2 [blocker of receptor of advanced glycation end products (RAGE)] was 7.5 μl/dish with 2 ml DMEM, the concentration of SB203580 (inhibitor of MAPK) was 10 μM, PDTC [inhibitor of nuclear factor-κB (NF-κB)] was 20 μM. siRNA was added after AGEs treating for 48 h, the block time was 24 h.