Modified ZhuJing pill protects retinal pigment epithelium against oxidative stress-induced epithelial-mesenchymal transition through Nrf2-mediated Akt/GSK3β pathway

Abstract

Background: The modified ZhuJing pill (mZJP) has been widely used in China as a classical prescription for treating retinal diseases for years. Our preliminary experiment showed that mZJP exerted an antioxidant effect in treating dry age-related macular degeneration (AMD). Nevertheless, the specific mechanism underpinning the impact of mZJP on dry AMD remains obscure.

Methods: The chemical metabolites of mZJP were qualitatively analyzed using LC-Q-TOF-MS. Dry AMD model mice were used to assess the efficacy of mZJP through optical coherence tomography (OCT), fundus autofluorescence (FAF), and immunofluorescence. Epithelial-mesenchymal transition (EMT) in OxLDL-induced ARPE-19 cells was evaluated by monitoring cellular integrity and quantifying EMT-related markers. Cell migration capacity was determined via wound healing and transwell assays. To investigate molecular mechanisms, cells were transfected with Nrf2 siRNA and analyzed through Western blotting, immunofluorescence, and migration assays under Nrf2 inhibition.

Results: A total of 113 major metabolites were identified in mZJP. Our findings revealed that mZJP alleviated retinal pathological alterations and inhibited EMT progression. Furthermore, mZJP upregulated Nrf2 and HO-1 expression levels while downregulating Akt and GSK-3β phosphorylation levels. Notably, the EMT-suppressing effect of mZJP was significantly attenuated upon Nrf2 silencing, as evidenced by enhanced cell migration, decreased epithelial marker expression (E-cadherin), increased mesenchymal marker expression (vimentin and α-SMA), suppression of the Nrf2 pathway, and activation of the Akt/GSK3β pathway.

Conclusion: Our study suggested that RPE protection by mZJP against oxidative stress induced EMT through Nrf2 activation and inhibition of the Akt/GSK3β pathway. MZJP could be a potential candidate drug for the treatment of dry AMD.

Keywords: Akt/GSK3β pathway; Nrf2 pathway; age-related macular degeneration; epithelial–mesenchymal transition; modified ZhuJing pill.

FIGURE 1

Qualitative analysis of the metabolites in mZJP granules. (A) Identification of the metabolites in mZJP. (B) BPC in positive (top) and negative (below) ion modes of mZJP granules (n = 3 per sample, technical replicates).

FIGURE 2

mZJP alleviated the retinal pathological changes in model mice. (A) Representative images of FAF. The white arrow points to the hyperfluorescence spots. (B) AF intensity to estimate the amounts of lipofuscin. LSD: positive drug control group. Mean ± SD, n = 5 mice per group, ^## p < 0.01; compared with the model group, ^* p < 0.05 and ^** p < 0.01. (C) Representative images of OCT. The black line points to the retinal pigment epithelium layer. (D) Retinal thickness to evaluate the effect of mZJP. Mean ± SD, n = 5 mice per group. ^## P < 0.01; compared with the model group, ^* p < 0.05 and ^** p < 0.01. (E) Immunofluorescence staining of ZO-1 on RPE/choroidal flat mounts. The white arrow points to the typical pathological changes. The images displayed are representative of the results from all mice in the same group (200×).

FIGURE 3

mZJP suppressed EMT and activated the Nrf2 pathway in model mice. (A–E) Protein levels of Nrf2, HO-1, α-SMA, and E-cadherin. GAPDH: internal control. Mean ± SD, n = 3 mice per group. Compared with the control group, ^## p < 0.01; compared with the model group, ^* p < 0.05 and ^** p < 0.01.

FIGURE 4

mZJP inhibited the Akt/GSK-3β pathway in model mice. (A–C) Protein expression of Akt, p-Akt, GSK-3β, and p-GSK-3β. In B and C, mean ± SD, n = 3 mice per group. Compared with the control group, ^## p < 0.01; compared with the model group, ^** p < 0.01.

FIGURE 5

mZJP suppressed OxLDL-induced cell migration in ARPE-19 cells. (A) Images of wound healing assay for ARPE-19 cells migration in six groups were captured at 0 h and after 24 h (40×). (B) Wound closure was measured at 24 h after scratching. LSD: positive control group. Mean ± SD, n = 3. Compared with the control group, ^## p < 0.01; compared with the model group, ^** p < 0.01. (C, D) Transwell assay was used for estimating migration ability in OxLDL-treated ARPE-19 cells with or without mZJP after 48 h treatment (200×), and it was visualized using a graph. MS: medicine serum of mZJP. Mean ± SD, n = 3. Compared with the control group, ^## p < 0.01; compared with the model group, ^* p < 0.05, ^** p < 0.01.

FIGURE 6

mZJP-attenuated OxLDL-induced EMT is associated with Akt/GSK3β-mediated Nrf2 activation (1). ARPE-19 cells were transfected with NC-siRNA or Nrf2-siRNA sequences, incubated with/without 10% mZJP for 24 h, and stimulated with OxLDL for 24 h (A, B). Images of wound healing assay for ARPE-19 cell migration in the six groups were captured at 0 h and after 24 h (40×). Wound closure was measured at 24 h after scratching. Mean ± SD, n = 3. Compared with the control group, ^## p < 0.01; compared with the model group, ^** P < 0.01. (C, D) Transwell assay under Nrf2 silenced condition (200×). It was visualized using graph. Mean ± SD, n = 3. Compared with the control group, ^## p < 0.01; compared with the model group, ^** p < 0.01. (E–G) Immunofluorescence staining of ZO-1 (green) and vimentin (red) was detected by fluorescence microscopy. ImageJ software was used to analyze the fluorescence intensity of ZO-1 and vimentin (200×). MS: mZJP. Mean ± SD, n = 3. Compared with the control group, ^## p < 0.01; compared with the model group, ^** p < 0.01.

FIGURE 7

mZJP-attenuated OxLDL-induced EMT is associated with Akt/GSK3β-mediated Nrf2 activation (2). ARPE-19 cells were transfected with Nrf2 siRNA (+) or siRNA (−) for 24 h, incubated with/without 10% mZJP for 24 h, and stimulated with OxLDL for 24 h (A–E) Nrf2- and EMT-related proteins (Nrf2, HO-1, α-SMA, and E-cadherin) were used for Western blot analysis. (F–H) Expression level of Akt/GSK-3β pathway-related proteins. In (B–E, G–H), mean ± SD, n = 3. Compared with the control group, ^## p < 0.01; compared with the model group, ^* p < 0.05 and ^** p < 0.01.