The corneal epithelium is located on the outermost part of the cornea, is directly exposed to the environment, and is most susceptible to damage due to a variety of physical, chemical, and biological factors. Corneal epithelial injury induced by ultraviolet (UV) or ultraviolet radiation is a common ocular surface injury, and if the DNA damage response (DDR) cannot be activated in time to induce apoptosis or repair damaged cells, it can lead to cell mutations, and accumulated mutations can cause delayed healing of corneal damage, structural impairment and cancerous lesions. DDR is a regulatory mechanism triggered by DNA damage that causes a series of related reactions within the cell, repairing damaged cells or inducing apoptosis depending on the degree of DNA damage. While ultraviolet light is considered the most prevalent environmental DNA damaging agent, although the ozone layer absorbs the most dangerous part of the sun’s ultraviolet spectrum (UVC), residual UVA and UVB can induce damage to 100,000 exposed cells per hour (Jackson et al., 2009). Long-term exposure to ultraviolet light can increase cellular oxidative stress levels, resulting in DNA damage leading to corneal lesions and persistent inflammation ( Delic et al., 2017). In addition to causing some degree of vision loss, severe corneal damage may require corneal transplantation or even eye extraction (Volatier et al., 2022). Although the study reported that the overall incidence of ultraviolet keratitis was only 0.06% (McIntosh et al., 2011), and the patients were generally mountaineers, skiers and beach recreationists, the demand for various environmental disinfection increased after the epidemic, ultraviolet rays were used for air and surface disinfection, and corneal damage caused by improper use often occurred (Sengillo et al., 2021). Therefore, clarifying the mechanism of ultraviolet-induced corneal epithelial damage repair can effectively optimize the treatment method and improve the repair efficiency.
HMGB1 (High mobility group box 1) is a conserved non-histone chromatin-associated protein that plays an important role in regulating the tertiary structure of chromatin, affecting a wide range of nuclear functions, including transcription, DNA repair, and genome stability (Travers, 2003, Müller et al., 2001). HMGB1 is involved in many types of DNA repair, such as mismatch repair, nucleotide excision repair, base excision repair, and non-homologous terminal ligation(Lange et al., 2009). HMGB1’s function in DDR is mediated by its ability to bind to damaged DNA and interact with repair enzymes(Liu et al., 2010). Zhang et al. (2019) found that UVB-induced DNA damage can increase HMGB1 time-dependently, and that levels of HMGB1 increase intracellular and extracellular levels over time and further UV exposure. Pasheva et al. (1998) believe that HMGB1 can preferentially bind to UV-induced DNA damage, and they found that HMGB1 prefers to bind to TT and TC oligonucleotides after irradiation, and by preferentially binding to damaged DNA, HMGB1 can form a transient complex that stabilizes the bent DNA at the lesion site. These suggest that HMGB1 plays a role in UV-induced DDR. Johnson et al. (2013) found that UV irradiation stimulates HMGB1 release and pro-inflammatory cytokine secretion in cultured keratinocytes, and acute UV irradiation induces HMGB1 release and dermal inflammation in murine skin. Han et al. (2015) found that UV-induced ROS production and oxidative stress lead to the translocation of HMGB1 protein into the extracellular environment, recruiting leukocytes to UV-exposed sites, thereby causing ocular surface inflammation. We therefore suspect that HMGB1 may be involved in UV-induced corneal epithelial cell damage.
HMGB1 is localized to the nucleus under normal conditions (Erlandsson Harris and Andersson, 2004), moves from the nucleus to the cytoplasm and is released from the cell under conditions of cellular stress or injury, and functions as a damage-associated molecular model (DAMP) (Sims et al., 2010). It can be passively released from necrotic cells or actively secreted in response to angiogenesis and inflammatory signals, and when it is passively released from cells, activates host innate immunity (Hazlett et al., 2021), and can promote dendritic cell maturation, triggering tissue pathogenesis and inflammation (Entezari et al., 2012). Extracellular HMGB1 binds to cell surface receptors, such as advanced glycosylated end-product receptors (RAGE) and toll-like receptors (TLRs), which activate NF-κB (Van Beijnum et al., 2008) thereby inducing the release of pro-inflammatory factors, initiating an inflammatory cascade leading to the pathogenesis of various diseases, including ocular lesions (Cavone et al., 2011). Among them, TLR2, TLR4 and RAGE receptors have been studied more, and some studies have found that HMGB1 induces the polarization of M1 macrophages through TLR2, TLR4 and RAGE/NF-κB signaling pathways, and participates in LPS-induced acute lung injury (Li et al., 2020, Wang et al., 2020). Another study found that mRNA levels of TLR4 are increased in corneal damage, both mechanical and chemical, and they have considered it necessary to study HMGB1 downstream pathways (including RAGE and TLR4) through specific inhibitors and transgenic mouse RAGE (Wang et al., 2022). While the signaling cascade initiated by TLR2 and TLR4 shows a lot of overlap, differentiation of these signaling pathways may occur at the level of conjugate proteins bound to the TIR domain of TLR (Akira et al., 2004). Therefore, we believe that TLR2 may be involved in signaling in UV-induced corneal epithelial cell damage and lead to NF-κB activation and nuclear translocation. In addition, HMGB1 can interact with a variety of pattern-recognition receptors, of which TLR9 has been extensively studied and considered to be an effective HMGB1 receptor (Harris et al., 2012, Guo et al., 2021, Hiraku et al., 2016). Once stimulated by the ligand, TLR9 recruits downstream linker molecules to produce a variety of pro-inflammatory cytokines such as interleukin IL-6 and tumor necrosis factor-α (TNF-α)(Beutler et al., 2004), and studies have shown that when cells are damaged or necrosis, HMGB1 is released from the cell to form a complex (HMGB1-DNA) and interacts with RAGE into neighboring living cells and is recognized by TLR9 in nuclear endosomes (Hiraku et al., 2016). Another study has found that HMGB1 may play an important role in the development and progression of diabetic retinopathy through the TLR9 signaling pathway (Jiang et al., 2017). MyD88, also known as myeloid differentiation factor, is an important connexin in the signaling pathway of TLRs (Bustin et al., 1999). TLR regulates the expression of various inflammatory mediators by NF-κB through the signaling factor MyD88, leading to a cascade of inflammatory responses (Catena et al., 2009). It has been reported that TNF-α secretion in TLR2-mediated macrophages is inhibited by the expression of MyD88 and several downstream proteins, pointing to the TLR2-induced MyD88-dependent pathway, which ultimately leads to NF-κB activation (Underhill et al., 1999); HMGB1 can also activate the NF-κB pathway through the TLR9/MyD88 signaling pathway and participate in the inflammatory response(Guo et al., 2021).
As a potent inhibitor of HMGB1, glycyrrhizin (GLY) is a natural triterpene ethylene glycol conjugate present in the roots and stems of licorice, which can not only inhibit the expression of HMGB1 compared with other anti-HMGB1 monoclonal antibodies, but also has anti-inflammatory, antiviral and other pharmacological activities(Tan et al., 2018). Studies have found that GLY effectively reduces HMGB1 mRNA levels and protein expression in infected or noninfectious corneal injury, thereby improving corneal repair (Ekanayaka et al., 2016, Zhou et al., 2020); Another study found that the targeting of HMGB1-NF-κB by GLY improved corneal injury in a mouse model of alkaline burns(Wang et al., 2022), indicating that the corneal protective effect of GLY deserves further study and has potential clinical translational value. Therefore, we intend to study the alleviation effect of GLY on UV-induced corneal epithelial cell injury and inflammation through the HMGB1-TLR/MyD88-NF-κB signaling pathway through TLR2/TLR9.