The Protective Effect of Polygonum cuspidatum (PCE) Aqueous Extract in a Dry Eye Model

Abstract

Dry eyes are caused by highly increased osmolarity of tear film, inflammation, and apoptosis of the ocular surface. In this study, we investigated the effect of Polygonum cuspidatum (PCE) aqueous extract in in vivo and in vitro dry eye models. Dry eye was induced by excision of the lacrimal gland and hyperosmotic media. In vivo, oral administration of PCE in exorbital lacrimal gland-excised rats recovered tear volume and Mucin4 (MUC4) expression by inhibiting corneal irregularity and expression of inflammatory cytokines. In vitro, hyperosmotic media induced human corneal epithelial cell (HCEC) cytotoxicity though increased inflammation, apoptosis, and oxidative stress. PCE treatment significantly inhibited expression of cyclooxygenase-2 and inflammatory cytokines (interleukin-6 and tumor necrosis factor-α), and activation of NF-κB p65 in hyperosmolar stress-induced HCECs. Hyperosmolarity-induced increase in Bcl-2-associated X protein (BAX) expression and activation of cleaved poly (ADP-ribose) polymerase and caspase 3 were attenuated in a concentration-dependent manner by PCE. PCE treatment restored anti-oxidative proteins such as heme oxygenase-1 (HO-1), superoxide dismutase-1 (SOD-1), and glutathione peroxidase (GPx) in hyperosmolar stress-induced HCECs. These data demonstrate that PCE prevents adverse changes in the ocular surface and tear fluid through inhibition of hyperosmolar stress-induced inflammation, apoptosis, and oxidation, suggesting that PCE may have the potential to preserve eye health.

Keywords: Mucin4; Polygonum cuspidatum; apoptosis; dry eye; exorbital lacrimal gland-excised; human corneal epithelial cells; hyperosmolar stress; inflammation; matrix metallopeptidase 9 (MMP9); oxidative stress.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

High-performance liquid chromatography (HPLC) chromatogram of Polygonum cuspidatum aqueous extract. (A) The five standard components and (B) aqueous extract of P. cuspidatum at 330 nm.

Figure 2

Effects of P. cuspidatum (PCE) on tear production in exorbital lacrimal gland-excised rats. Tear volume was determined using the phenol red thread tear test. Tear volume was revealed in mm of thread that became wet by tears and turned red. Normal control (NOR): normal control mice, Dry eye disease (DED): vehicle-treated dry eye rats, PCE-10: 10 mg/kg PCE-treated DED rats, PCE-100: 100 mg/kg PCE-treated DED rats, and PCE-250: 250 mg/kg PCE-treated DED rats. The values in the bar graph represent the mean ± standard error (SE), n = 10. # p < 0.05, significantly different from normal rats. * p < 0.05, significantly different from vehicle-treated dry eye rats.

Figure 3

Effects of P. cuspidatum (PCE) on corneal surface irregularities in exorbital lacrimal gland-excised rats. (A) Reflected images of a white ring from the fiber-optic ring illuminator of a stereomicroscope. Scale bar is 1 mm. (B) Corneal irregularity was graded according to the number of distorted quarters in the reflected white ring as follows: 0, no distortion; 1, distortion in one quarter; 2, distortion in two quarters; 3, distortion in three quarters; 4, distortion in all four quarters; 5, severe distortion, in which no ring could be recognized. The values in the bar graph represent the mean ± SEM, n = 10. # p < 0.05, significantly different from normal rats, * p < 0.05, significantly different from vehicle-treated dry eye rats.

Figure 4

Effects of P. cuspidatum (PCE) on expression of mucin 4 (MUC4) and inflammatory cytokines in exorbital lacrimal gland-excised rats. (A) Immunohistochemical staining for Muc4. (B) Morphometric analysis of Muc4-positive signal density in corneal sections from each group. The values in the bar graph represent the mean ± SEM, n = 10. RNA was extracted from the corneal tissue of exorbital lacrimal gland-excised rats. mRNA levels of (C) MUC4, (D) IL-6, (E) TNF-α, and (F) MMP9 were assessed by real-time PCR assay. GAPDH was considered an internal control. Data are the mean ± SEM of three independent experiments for all groups. # p < 0.05, significantly different from normal mice, * p < 0.05, significantly different from vehicle-treated dry eye mice.

Figure 5

Effects of P. cuspidatum (PCE) on hyperosmolar stress-induced cell death in human corneal epithelial cells (HCECs). (A,B) HCECs were treated with PCE or NaCl at different concentrations for 24 h. (C) HCECs were co-treated with PCE and hyperosmotic media for 24 h. To investigate cell viability, CCK-8 (Cell Counting Kit-8) assay was performed. Data are the mean ± SEM of three independent experiments for all groups. # p < 0.05, significantly different from untreated group, * p < 0.05, significantly different from hyperosmotic-treated group.

Figure 6

Effects of P. cuspidatum (PCE) on inflammation in hyperosmolar stress-stimulated human corneal epithelial cells (HCECs). (A) HCECs were co-treated with the indicated concentrations of PCE and hyperosmotic media (528 mOsM) for 1 h or 8 h. The protein expression of phosphor-p65 and COX-2 (Cyclooxygenase-2) was analyzed by western blot analysis. Cytoplasmic and nuclear levels of NF-κB p65 were detected by western blotting to analyze the translocation of NF-κB. β-actin and Lamin A were used as loading controls. (B–D) The relative intensities are expressed as the ratio of phosphor-p65, nuclear p65, and COX-2 to β-actin or Lamin A. RNA was extracted from hyperosmolar stress-stimulated HCECs, and the mRNA levels of (E) TNF-α, (F) IL-6, and (G) MUC4 were assessed by real-time PCR assay. GAPDH was considered an internal control. Data are the mean ± SEM of three independent experiments for all groups. # p < 0.05, significantly different from untreated group, * p < 0.05, significantly different from hyperosmotic-treated group.

Figure 7

Effects of P. cuspidatum (PCE) on hyperosmolar stress-induced apoptotic cell death in human corneal epithelial cells (HCECs). HCECs were co-treated with the indicated concentrations of PCE and hyperosmotic media for 24 h. (A) The protein expression of BAX, Bcl-2, Caspase 3, and polymerase (PARP) was analyzed by western blot analysis. β-actin was used as a loading control. (B–D) The relative intensities are expressed as the ratio of BAX, Bcl-2, cleaved PARP, and cleaved caspase 3 to β-actin. Data are the mean ± SEM of three independent experiments for all groups. # p < 0.05, significantly different from untreated group, * p < 0.05, significantly different from hyperosmotic-treated group.

Figure 8

Effects of P. cuspidatum (PCE) on oxidative stress in hyperosmolar stress-induced human corneal epithelial cells (HCECs). HCECs were co-treated with the indicated concentrations of PCE and hyperosmotic media for 24 h. (A) The protein expression of HO-1, SOD-1, and GPx was analyzed by western blot analysis. β-actin was used as a loading control. (B–D) The relative intensities are expressed as the ratio of HO-1, SOD-1, and GPx to β-actin. Data are the mean ± SEM of three independent experiments for all groups. Means unlike letters in a column with differ significantly (p < 0.05) # p < 0.05, significantly different from untreated group, * p < 0.05, significantly different from hyperosmotic-treated group.