AMPs are among the compounds that have antibiotic properties in recent years. These biomolecules can destroy pathogens by stimulating the host's innate immunity[18]. The advantages of AMPs over conventional antibiotics include a lower rate of induced resistance, a wider range of pathogen effects, specificity for Gram-positive or Gram-negative bacteria, less host toxicity, synergistic antimicrobial effects with other antibiotics, and the ability to rapidly exert their bactericidal effects[19]. Therefore, Akexotide are considered as extremely promising and innovative alternatives with the potential to address the growing problem of multidrug resistance.
In the antibacterial study, Akexotide can significantly inhibit the growth of Gram-negative bacteria E.coli and V.parahaemolyticus, and Gram-positive bacteria S.aureus and fungus P. aeruginosa. No obvious colonies were found during the 28 days of the antibacterial process. In addition, through parallel comparison between Akexotide of Is and Os, the Is showed significant multi-species antibacterial activity. Therefore, Akexotide will become the focus of our follow-up research on the treatment of ocular infectious diseases.
Gram-positive bacteria S. aureus is one of the most important pathogenic bacteria of keratitis, with fibronectin-binding protein that enables bacteria to adhere and invade epithelial cells[20]. The mucin layer and tight intercellular junctions of the corneal epithelium are the main barriers that prevent S. aureus from binding and penetrating the cornea. Disruption of these barriers can significantly increase susceptibility to S. aureus infection and lead to S. aureus keratitis[21, 22]. S. aureus has developed into difficult to treat resistant strains[23, 24], that were responsible for 5–36% of corneal ulcers[25]. In addition, Gram-negative bacteria are also important contributors to ocular infections. Among the gram-negative bacteria, E. coli (Escherichia coli) and Enterobacter spp. are commonly isolated from conjunctivitis, dacryocystitis, and keratitis[26, 27]. In addition, another study from Egypt reported the isolation of E. coli and A. lwoifi (Acinetobacter lwoifi) from cases of chronic dacryocystitis[28].
The construction of the ocular bacterial infection model was improved on the basis of Tanweer et al.[29] [84]. After the corneal injury in SD rats, S. aureus and E. coli were inoculated, and the upper and lower eyelids were sutured by glass sheets covered with radian to keep the bacterial fluid from losing. The reason for the improvement was that no obvious bacterial ocular infection was found in the modeling based on Tanweer's research method, which may also be caused by the differences in models between rats and mice due to their own immune systems and environmental factors. Because of this modified method, two rats died during modeling. Encouragingly, after Is treatment, the ocular infection in rats recovered significantly, with fewer ocular neovascularization and no drainage and other reactions. Pathologically, Akexotide Is effectively maintained the structure and morphology of corneal epithelial cells, meanwhile, reducing the bacterial invasion of the corneal stroma layer.
To further explore the antibacterial effect and cytotoxicity of Akexotide Is on eyes, we combined Giemsa staining and plate colony formation experiments. The results showed that 100µM of Is showed an inhibitory effect on S. aureus and E. coli, even stronger than eye drops cefazolin and TOB. This may be related to the property of Is, where high concentrations of Is take on a gel-like appearance at 37°C, and it is this property that may allow Is to linger longer and exert a longer-lasting bacteriostatic effect when dropped into the rat eye. Is treatment facilitated the recovery of corneal epithelial cell numbers.
The cornea is the external barrier of the eye and is transparent and avascular under healthy conditions. However, an imbalance between angiogenic and anti-angiogenic stimuli following severe injury or chronic inflammation can lead to abnormal amounts of pro-angiogenic factors. For example, in the case of excess vascular endothelial growth factor (VEGF), a normally avascular cornea may become vascularized[30]. As blood and lymphatic vessels can enter the cornea from adjacent vascularized tissue, this leads to vision loss and passive immune response[31]. Neovascularization is a common complication of corneal infection, and corneal angiogenesis is a common endpoint in different ocular surface diseases, including infected corneas. Although corneal angiogenesis is beneficial in preventing stromal melting, promoting wound healing and eliminating infection, it brings about persistent inflammation, edema, lipid deposition and tissue scarring. That is sacrificed at the expense of corneal transparency, which can lead to poor vision[32]. Therefore, in bacterial infectious keratitis, the level of corneal neovascularization is an important therapeutic consideration in addition to bacterial suppression[33].
Corneal opacity and neovascularization were found after S. aureus infection, as reported by Cicih and Nicole[34] et al., which is also the case in this study. As a pathogen of eye infection, E.coli is rarely reported. In this study, no obvious corneal turbidity was found, but it caused significant neovascularization. After treatment with Is, the symptoms of corneal neovascularization caused by S. aureus and E. coli were significantly relieved. After Is treatment, the level of VEGF protein secreted by the corneal stromal layer and corneal epithelial cells decreased by immunohistochemy.
In this study, the effective antibacterial effect and corneal epithelial cytoprotective function of Is in ocular infections were verified by the ocular S. aureus and E. coli infection rat model. It was also found that Is could inhibit the expression of VEGF at the cornea to suppress corneal neovascularization. In addition, the Akexotide Is at high concentrations can form a gel in vivo, which enhances its duration of action in the eye. Therefore, we believe that Akexotide Is could be a candidate for the treatment of ocular infections and deserves further examination. Of course, we will further explore the safe applicable dose for the use of Akexotide Is subsequently for better application development at a later stage.