Effects of diosgenin on myometrial matrix metalloproteinase-2 and -9 activity and expression in ovariectomized rats

Abstract

Diosgenin, a traditional Yam extraction, has been used in hormone replacement for menopausal women. We aimed to investigate the influences of diosgenin administration upon the MMP-2 and -9 activity and expression and reproductive hormones of ovariectomized (OVX) rats, a model of menopausal status. Seven-week old female Wistar rats with bilateral OVX or sham operation (controls) were divided and administered different dosages of diosgenin (0, 10, 50, or 100 mg/kg/day) for 8 weeks. Serum was then sampled for progesterone (P4) and estradiol (E2) assay and uterine horns harvested. Myometrial MMP-2 and -9 activity and expression were surveyed and myometrial collagen expression was also assayed. The results show higher body weight in OVX rats across the 8 weeks post surgery and no significant differences were noted among OVX or Sham rats with diosgenin supplements. There were lower P4 and E2 concentrations in OVX rats compared to Sham rats, and higher P4 concentration of Sham rats post diosgenin supplement. MMP-2 and -9 mRNA expression and activity was lower in OVX rats, although higher MMP-2 and lower MMP-9 activity/mRNA expression was observed in OVX rats post diosgenin supplementation. Collagen mRNA expression was higher in OVX rats compared to Sham controls, and diosgenin administration decreased collagen mRNA expression in OVX rats. In conclusion, diosgenin is associated with gelatinase expression and collagen metabolism in OVX rats. Diosgenin administration can partially reverse the effects of OVX upon MMP functions and hormone status. Adequate diosgenin supplement might modulate myometrial gelatinase expression and collagen metabolism in menopausal subjects.

Keywords: diosgenin; gelatinase; matrix metalloproteinase; myometrium; ovariectomization.

Figure 1

Comparisons of progesterone and estradiol concentrations between controls (Sham) and ovariectomized (OVX) rats after diosgenin supplementation (0, 10, 50, and 100 mg/kg/day). The serum estradiol (E2) and progesterone (P4) concentrations were measured in duplicate aliquots of each sample. Bar graphs of all values were expressed as the mean ± SEM from six subjects. * p < 0.05 and ** p < 0.01 vs. the Sham groups; † p < 0.05 vs. Diosgenin-0 in the same group.

Figure 2

Comparisons of MMP-2 activity between controls (Sham) and ovariectomized (OVX) rats after diosgenin supplementation (0, 10, 50, or 100 mg/kg/day). (A), MMP-2 activity was determined by gelatin zymography assay. The molecular weight of MMP-2 detected is approximately 72 kDa shown on the gel. The bands of enzymatic activity shown are representatives of six identical determinations. (B), the relative MMP-2 activity affected by ovariectomization and diosgenin treatment was assayed. Bar graphs of all values were expressed as the mean ± SEM from six subjects. ** p < 0.01 vs. the Sham group; ‡ p < 0.01 vs. Diosgenin-0 in the same group.

Figure 3

Comparisons of MMP-2 gene expression between controls (Sham) and ovariectomized (OVX) rats after diosgenin supplementation (0, 10, 50, or 100 mg/kg/day). (A), MMP-2 gene expression was determined by semi-quantitative RT-PCR. The length of the PCR products for MMP-2 and GAPDH were 456 bp and 413 bp, respectively. Amplification of GAPDH was determined as an internal control. The bands of PCR DNA shown are representatives of six identical determinations. (B), the relative MMP-2 gene expression affected by ovariectomization and diosgenin treatment was assayed. Bar graphs of all values were expressed as the mean ± SEM from six subjects. ** p < 0.01 vs. Sham group; † p < 0.05 vs. Diosgenin-0 in the same group.

Figure 4

Comparisons of MMP-9 activity between controls (Sham) and ovariectomized (OVX) rats after diosgenin supplementation (0, 10, 50, or 100 mg/kg/day). (A), MMP-9 activity was determined by gelatin zymography assay. The molecular weight of MMP-9 detected is approximately 95 kDa shown on the gel. The bands of enzymatic activity shown are representatives of six identical determinations. (B), the relative MMP-9 activity affected by ovariectomization and diosgenin treatment was assayed. Bar graphs of all values were expressed as the mean ± SEM from six subjects. ** p < 0.01 vs. Sham group ; † p < 0.05 and ‡ p < 0.01 vs. Diosgenin-0 in the same group.

Figure 5

Comparisons of MMP-9 gene expression between controls (Sham) and ovariectomized (OVX) rats after diosgenin supplementation (0, 10, 50, or 100 mg/kg/day). (A), MMP-9 gene expression was determined by semi-quantitative RT-PCR. The length of the PCR products for MMP-9 and GAPDH were 458 bp and 413 bp, respectively. Amplification of GAPDH was determined as an internal control. The bands of PCR DNA shown are representatives of six identical determinations. (B), the relative MMP-9 gene expression affected by ovariectomization and diosgenin treatment was assayed. Bar graphs of all values were expressed as the mean ± SEM from six subjects. ** p < 0.01 vs. Sham groups; † p < 0.05 vs. Diosgenin-0 in the same group.

Figure 6

Comparisons of type I collagen expression between controls (Sham) and ovariectomized (OVX) rats after diosgenin treatment (0, 10, 50, or 100 mg/kg/day). (A), collagen expression was determined by Western blotting assay. The molecular weight of collagen detected is approximately 72 kDa. The Western blots shown are representatives of six identical determinations. (B), the relative collagen expression affected by ovariectomization and diosgenin treatment was assayed. Bar graphs of all values were expressed as the mean ± SEM from six subjects. * p < 0.05 and ** p < 0.01 vs. Sham groups; † p < 0.05 vs. Diosgenin-0 in the same group.