However, the mechanism by which PAE regulates the expression of inflammatory mediators in FK still remains unknown. Ishida et al.
31 found that β-glucan mediated HO-1 expression in the oral epithelium infected by
C. albicans through Nrf2. β-Glucan is an intrinsic component of fungal cell wall
43 and mediates the innate immune response of the body against fungal infections,
44 which suggests that the Nrf2/HO-1 signaling pathway may play a role in the immune response to protect body against fungal infection. Ryuhei Hayashi has demonstrated that Nrf2 signaling was activated throughout the corneal epithelial wound-healing process and its activation plays a protective role in corneal wound healing.
25 It has been confirmed that HO-1 induction attenuated inflammation endangered by corneal epithelial injury, such as keratitis, and accelerated wound healing and represents a fundamental protective system in the cornea,
30 which collectively suggested that Nrf2/HO-1 may play a role in corneal defense to fungal infection. Moreover, It has been proved that PAE can activate Nrf2/HO-1 signaling pathway and exert antioxidant activities in human keratinocytes.
20 To determine the role of the Nrf2/HO-1 pathway, we first examined the expression of Nrf2 and HO-1 in corneas of mice 5 days post-infection. The expression of Nrf2 and HO-1 in the infected corneas increased after stimulation with
A. fumigatus, which suggests that the Nrf2/HO-1 signaling pathway may participate in the immune response in FK. We then treated infected corneas with PAE and found that PAE treatment upregulated Nrf2 and HO-1 expression induced by
A. fumigatus in infected C57BL/6 mice cornea, which confirmed that PAE could upregulate Nrf2 and HO-1 expression and Nrf2/HO-1 signaling pathway may participate in anti-inflammatory effect of PAE in FK. These results were also confirmed in HCECs. To further determine the mechanism by which PAE regulates the Nrf2/HO-1 signaling pathway, we detected the localization of Nrf2 in HCECs. Previous studies have pointed out that PAE activates the activation of Nrf2-KEAP1 system and leads to antioxidant response,
45 illustrating an activation effect of PAE on Nrf2 stimulation. Surprisingly, our immunofluorescence analysis showed that Nrf2 aggregated from the cytoplasm to the nucleus upon PAE treatment compared with fungal stimulation alone, suggesting that PAE could activate the anti-inflammatory effect of Nrf2 by promoting its nuclear translocation of Nrf2 in infected HCECs. The nuclear translocation of Nrf2 plays a key role in increasing its target gene HO-1 expression and inhibiting the subsequent inflammatory response.
46 Under circumstances of no stress, Nrf2 is located in the cytoplasm. When Nrf2 is activated, it translocates into the nucleus and binds with ARE to mediate the expression of HO-1. The HO-1 catalyzes heme to CO with anti-inflammatory effect, and also directly inhibits the expression of pro-inflammatory mediators.
47 Inhibition of Nrf2 by the use of a specific Nrf2 inhibitor, BT, has been reported to attenuate Nrf2-mediated defense mechanisms and impair their antioxidant capacity,
48 therefore, we next pretreat HCECs with BT (Nrf2 inhibitor) and ZnPP (HO-1 inhibitor) and found a sharp decrease in Nrf2 and HO-1 expression. In addition, PAE-induced Nrf2 and HO-1 expression were downregulated, and the production of inflammatory mediators, such as IL-6, IL-8, and TNF-α, was increased, indicating that the inhibition of Nrf2 or HO-1 with specific inhibitors BT or ZnPP, respectively, could partially weaken the suppressive effect of PAE on the expression of
A. fumigatus-induced pro-inflammatory cytokines, such as IL-6, IL-8, and TNF-α. It was further suggested that the Nrf2/HO-1 signaling pathway participated in the anti-inflammatory effect of PAE during corneal fungal infection. The above evidence indicates that PAE suppresses the excessive inflammatory response partly by activating the Nrf2/HO-1 signaling pathway in
A. fumigatus keratitis. However, after applying Nrf2 or HO-1 inhibitors to infected HCECs, the level of pro-inflammatory factors of PAE pretreatment group did not completely restore the inflammation to the levels seen in the infected HCECs with no treatment, which suggested that not all the anti-inflammatory effect of PAE was attributed to Nrf2/HO-1 signaling pathway during corneal fungal infection; there may also be other possible mechanisms participating in the anti-inflammatory effect of PAE in
A. fumigatus keratitis.