Figure 1.
Spectral-domain optical coherence tomography images of CNV.
(A) Control group. (B) Low-dose M. officinalis extract (50 mg/kg) group. (C) High-dose M. officinalis extract (100 mg/kg) group. White arrows indicate a spindle-shaped choroidal neovascularization (CNV) lesion. (D) Measurement of CNV thickness, in vivo, showed significantly smaller CNV in M. officinalis extract-treated rats than in control rats. The response was dose dependent. Data are presented as mean ± standard deviation. *indicates statistical significance (P<0.05).
Figure 2.
Fluorescent images of CNV in retinal pigment epithelium-choroid-sclera flat mounts.
(A, D) Control group. (B, E) Low-dose M. officinalis extract (50 mg/kg) group. (C, F) High-dose M. officinalis extract (100 mg/kg) group. (A, B, C) Representative images of fluorescein isothiocyanate (FITC)-dextran staining (green). (D, E, F) Representative images of tetramethylrhodamine isothiocyanate (TRITC)-conjugated isolectin B4 staining (red). White arrows indicate laser-induced choroidal neovascularization (CNV) size. The scale bar represents 100 µm. (D) Mean CNV area was significantly lower in M. officinalis extract-treated rats than in control rats. The response was dose dependent. Data are presented as mean ± standard deviation. *indicates statistical significance (P<0.05).
Figure 3.
Hematoxylin and eosin staining of paraffin-embedded rat eye sections through areas of CNV.
(A) Control group. (B) Low-dose M. officinalis extract (50 mg/kg) group. (C) High-dose M. officinalis extract (100 mg/kg) group. Black arrows indicate choroidal neovascularization (CNV) lesion margins. Scale bar indicates 50 µm. (D) Rats treated with M. officinalis extract had significantly smaller CNV lesion than controls. The response was dose dependent. (E) Mean CNV/choroid thickness ratios were significantly lower in M. officinalis extract-treated rats than in control rats. Data are presented as mean ± standard deviation. *indicates statistical significance (P<0.05).
Figure 4.
Fluorescein angiographic images.
Early (A, C, E) and late (B, D, F) phase fluorescein angiographic images taken 14 days after laser injury to induce choroidal neovascularization (CNV). (A, B) Control group. (C, D) Low-dose M. officinalis extract (50 mg/kg) group. (E, F) High-dose M. officinalis extract (100 mg/kg) group. White arrows indicate laser treatment location. (G) Histogram of angiographic leakage grades. Significantly fewer grade 2B lesions were observed in the high-dose group than in the control group.
Figure 5.
Quantification of VEGF, MMP-2, and MMP-9 levels in choroidal-scleral complex and retina.
Levels of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, and MMP-9 were evaluated in the choroidal-scleral complex (A, C, E) and retina (B, D, F). (A, B) Mean normalized VEGF levels in choroidal-scleral and retinal tissues were significantly lower in high-dose M. officinalis extract (100 mg/kg) group than in controls. The low-dose M. officinalis extract (50 mg/kg) group only had significantly lower VEGF levels than controls in choroidal-scleral tissues. (C, D) Mean normalized MMP-2 levels in choroidal-scleral and retinal tissues were significantly lower in high-dose extract rats than in controls. (E, F) Mean normalized MMP-9 levels in choroidal-scleral and retinal tissues were significantly lower than controls in both high- and low-dose groups. Data are presented as mean ± standard deviation. #, *indicate significant difference (P<0.05) in comparison to negative control or the vehicle-treated group, respectively.
Figure 6.
Quantification of VEGF secretion after treatment of t-BH on ARPE-19 cells.
Mean normalized tertiary-butylhydroperoxide (t-BH)-induced vascular endothelial growth factor (VEGF) expression was significantly lower with cotreatment of 25 µg/mL of M. officinalis extract. Data are presented as mean ± standard deviation. #, *indicate significant difference (P<0.05) in comparison to negative control or the t-BH alone group, respectively.
Figure 7.
VEGF, MMP-2, and MMP-9 expression after treatment of t-BH on ARPE-19 cells and HUVECs.
Expression of mRNAs for vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, and MMP-9 determined by real-time quantitative polymerase chain reaction (PCR) were evaluated in human retinal pigment epithelial (ARPE-19) cells (A, C, E) and in human umbilical vein endothelial cells (HUVECs) (B, D, F). (A, B) The relative VEGF mRNA levels, which are induced by tertiary-butylhydroperoxide (t-BH), were reduced significantly with cotreatment of 25 µg/mL of M. officinalis extract. (C, D) The expression of MMP-2 mRNA did not change in the t-BH treated cells, nor did that in the t-BH and M. officinalis extract treated cells. (E, F) The relative MMP-9 mRNA levels decreased significantly with cotreatment of 25 µg/mL of M. officinalis extract. Data are presented as mean ± standard deviation. #, *indicate significant difference (P<0.05) in comparison to negative control or the t-BH alone group, respectively.