Autophagy Activation Protects Ocular Surface from Inflammation in a Dry Eye Model In Vitro

Abstract

Inflammation is the main pathophysiology of dry eye, characterized by tear film instability and hyperosmolarity. The aim of this study was to investigate the association of inflammation and cellular autophagy using an in vitro dry eye model with primary cultured human corneal epithelial cells (HCECs). Primary HCECs cultured with fresh limbal explants from donors were switched to a hyperosmotic medium (450 mOsM) by adding sodium chloride into the culture medium. We observed the stimulated inflammatory mediators, TNF-α, IL-1β, IL-6 and IL-8, as well as the increased expression of autophagy related genes, Ulk1, Beclin1, Atg5 and LC3B, as evaluated by RT-qPCR and ELISA. The immunofluorescent staining of LC3B and Western blotting revealed the activated autophagosome formation and autophagic flux, as evidenced by the increased LC3B autophagic cells with activated Beclin1, Atg5, Atg7 and LC3B proteins, and the decreased levels of P62 protein in HCECs. Interestingly, the autophagy activation was later at 24 h than inflammation induced at 4 h in HCECs exposed to 450 mOsM. Furthermore, application of rapamycin enhanced autophagy activation also reduced the inflammatory mediators and restored cell viability in HCECs exposed to the hyperosmotic medium. Our findings for the first time demonstrate that the autophagy activation is a late phase response to hyperosmotic stress, and is enhanced by rapamycin, which protects HCECs by suppressing inflammation and promoting cells survival, suggesting a new therapeutic potential to treat dry eye diseases.

Random Forest Algorithm-Based Ultrasonic Image in the Diagnosis of Patients with Dry Eye Syndrome and Its Relationship with Tear Osmotic Pressure

“…The study of tear osmotic pressure and dry eye severity showed that with the aggravation of dry eye, tear osmotic pressure increased significantly. This was consistent with the research results of Liu et al (2020) [19]. Additionally, van Setten (2019) [20] proposed that different osmotic pressures may be the key mechanism leading to cell death, inflammation, apoptosis, and goblet cell loss observed in dry eye.…”

Experimental Models, Induction Protocols, and Measured Parameters in Dry Eye Disease: Focusing on Practical Implications for Experimental Research

“…In DED, some established in vitro models have also been established: the Wong Kilbourne derivative of Chang conjunctival epithelial cell line (WKD; clone 1-5c-4) [47][48][49][50][51]; immortalized normal human conjunctival cell line (IOBA-NHC) [47,52,53]; human corneal epithelial cells (HCECs), a human transformed SV40 immortalized corneal epithelial cell line [54,55], and human conjunctival cell line (HCC) [56]. In vitro models with primary cell culture are also available: human corneal epithelial cells (HCECs) [57], rabbit corneal epithelial cells (CECs), or rabbit LG acinar cells (LGACs) [58]. The induction of DED-like symptoms in in vitro culture can be performed by the induction of hyperosmotic (500 mOsM) media in the culture media by the addition of 90 mM sodium chloride in the culture medium, which triggers inflammatory-like symptoms [47,48,55,57,59].…”

“…In vitro models with primary cell culture are also available: human corneal epithelial cells (HCECs) [57], rabbit corneal epithelial cells (CECs), or rabbit LG acinar cells (LGACs) [58]. The induction of DED-like symptoms in in vitro culture can be performed by the induction of hyperosmotic (500 mOsM) media in the culture media by the addition of 90 mM sodium chloride in the culture medium, which triggers inflammatory-like symptoms [47,48,55,57,59]. As in the in vivo experiment, benzalkonium chloride (BAC) (0.0001% (1 µg/mL) was also used to induce inflammatory cascades [47][48][49][50][51][52].…”

Recent Developments About the Pathogenesis of Dry Eye Disease: Based on Immune Inflammatory Mechanisms

“…Liu Zhao et al founded that autophagy activation is a late response of HcECs to hyperosmotic stress after inflammation is triggered, which protects HcECs and promotes survival by reducing inflammatory mediators in an vitro model of dry eye. These protective effects were further enhanced when rapamycin enhanced autophagy activation in hypertonic HCECs (Liu et al, 2020a). In addition, they suggest that trehalose, as an autophagy enhancer, induces autophagy antiinflammation by inhibiting Akt activation of transcription factor EB in primary HCECs exposed to high osmotic stress (Liu et al, 2020b).…”