Quercetin Alleviates Scleral Remodeling Through Inhibiting the PERK-EIF2α Axis in Experiment Myopia

Abstract

Purpose: This study aims to investigate the effect of quercetin (QUE) on scleral remodeling by inhibiting the PERK-EIF2α signaling pathway and to evaluate its potential role in slowing myopia.

Methods: Lens-induced myopia (LIM) guinea pigs were obtained and treated with QUE. After 4 and 6 weeks of treatments, ocular biological measurements were conducted. Hematoxylin and eosin (H&E) staining was used to observe the changes in scleral morphology and thickness, and Masson staining was used to examine scleral collagen fiber arrangement. Quantitative PCR (qPCR) and Western bolt were utilized to detect the mRNA and protein expression of PERK, EIF2α, MMP-2, TIMP-2, and collagen I in the scleral tissues. Calcium ion flow in each group was measured using noninvasive micro-test technology, and reactive oxygen species levels were detected by flow cytometry.

Results: Compared with the LIM group, the ocular measurements showed that the refractive errors and axial length of the eyes were significantly reduced in the LIM + QUE group (P < 0.01). H&E and Masson staining showed that sclera in the LIM + QUE group was thickened, collagen was dense, and the fiber gap was reduced. In the LIM + QUE group, the expression levels of PERK, EIF2α, and MMP-2 were decreased, whereas the expression levels of TIMP-2 and collagen I were increased. Calcium release and reactive oxygen species (ROS) in the LIM + QUE group were decreased.

Conclusions: Quercetin ameliorates scleral remodeling in myopic guinea pigs by inhibiting the PERK-EIF2α signaling pathway, thereby alleviating the progression of myopia. These findings provide new experimental evidence for the potential application of quercetin in myopia prevention and treatment.

Figure 1.

Chemical structural formula of quercetin.

Figure 2.

Comparison of right eye axial length (A) and refraction (B) in guinea pigs at 0, 4, and 6 weeks across the NC, LIM, LIM + SHAM, and LIM + QUE groups. The LIM group was compared to the NC group, as well as to the LIM + SHAM group (****P < 0.0001 and ***P < 0.001).

Figure 3.

(A) Interactions are known: the compounds form hydrogen bonding interactions with ASP164, ASN167, LYS169, and ILE170 of the protein and Pi-Alkyl interactions with LYS162. (B) Interactions are known: compounds form hydrogen bonding interactions with GLY215, GLU194, and GLY193 of proteins, Pi-Anion interactions with GLU194, Pi-Sigma interactions with GLU194, and Pi-Alkyl interactions with ALA189. (C) CO-IP detects the direct interaction between PERK and EIF2α. (D) STRING predicts PERK interacting with EIF2α (EIF2S1).

Figure 4.

The mRNA levels of PERK (A, B) and EIF2α (C, D) genes in guinea pig sclera were quantified using qPCR at 4 and 6 weeks in the NC, LIM, LIM + SHAM, and LIM + QUE groups. Western blot analysis was performed for PERK and EIF2α (E, F), with β-actin as an internal reference. Relative levels of scleral target proteins PERK and EIF2α (G–J) in guinea pigs were assessed in the NC, LIM, LIM + SHAM, and LIM + QUE groups at 4 and 6 weeks. The LIM group was compared to the NC group, as well as to the LIM + SHAM group (***P < 0.001, **P < 0.01, and *P < 0.05; n = 6).

Figure 5.

Immunofluorescence showed the expression level of PERK (A), EIF2α (B) in scleral tissue. Magnification = 40×. Measurement of PERK (C, D) and Eif2α (E, F) expression at protein level by immunofluorescence in the sclera of the guinea pig. For the comparison between LIM and NC groups, **P < 0.01. For the comparison between LIM and LIM + QUE groups (***P < 0.001 and *P < 0.05; n = 3).

Figure 6.

ROS and calcium ion levels after 4 and 6 weeks of myopia induction and quercetin treatment. We found that the proportion of ROS in the guinea pig sclera in the LIM group, the original balance of ROS and antioxidants was disrupted and oxidative stress occurred. This phenomenon also appeared in the LIM + SHAM group. Whereas quercetin was effective in inhibiting the development of myopia, ROS levels in the LIM + QUE group decreased compared with the LIM and LIM + SHAM groups (A). The results of the study found that after 4 and 6 weeks of induction, calcium release was increased in the LIM and LIM + SHAM groups compared to the NC group, and decreased after quercetin treatment (B, C). (n = 3).

Figure 7.

The mRNA levels of collagen I (A), TIMP-2 (B), and MMP-2 (C) genes in guinea pig sclera were assessed via qPCR at 4 and 6 weeks in the NC, LIM, LIM + SHAM, and LIM + QUE groups. Western blot analysis was performed for collagen I, TIMP-2, and MMP-2 (D), with β-actin as an internal reference. Relative levels of scleral target proteins collagen I, TIMP-2, and MMP-2 (E–G) in guinea pigs were assessed in the NC, LIM, LIM + SHAM, and LIM + QUE groups at 4 and 6 weeks. The LIM group was compared with both the NC group and the LIM + SHAM group (***P < 0.001, **P < 0.01, and *P < 0.05; n = 6).

Figure 8.

Immunofluorescence showed the expression level of collagen I (A, D, E), TIMP-2 (B, F, G) and MMP-2 (C, H, I) in scleral tissue. At a magnification of 40×. The LIM group was compared with both the NC group and the LIM + SHAM group (***P < 0.001, **P < 0.01, and *P < 0.05; n = 3).

Figure 9.

Masson (A) and H&E staining (B) of ocular tissue sections showing histopathological features of the sclera of guinea pigs at 4 and 6 weeks (n = 3). The results showed that the scleral collagen fibers in the NC group were uniformly arranged and tightly packed, whereas the scleral collagen fibers in the LIM group were loosely arranged with enlarged gaps, and the scleral collagen fibers in the LIM + QUE group were tightly packed with smaller gaps compared with those in the LIM group.

Figure 10.

Illustration of the role of quercetin and ROS-based ER stress-mediated scleral remodeling through the PERK-EIF2α signaling pathway.