Salubrinal Ameliorates Inflammation and Neovascularization via the Caspase 3/Enos Signaling in an Alkaline-Induced Rat Corneal Neovascularization Model

Abstract

Background and Objectives: Ocular alkaline burn is a clinical emergency that can cause permanent vision loss due to limbal stem cell deficiency and corneal neovascularization (CNV). Although the basic pathogenetic mechanisms are considered to be acute oxidative stress and corneal neovascularization triggered by inflammation, the underlying intracellular mechanisms have not been clearly elucidated. The aim of this study was to investigate the role of endoplasmic reticulum (ER) stress on inflammation and neovascularization, and the effect of the ER stress inhibitor salubrinal (SLB), as a novel treatment in a corneal alkaline burn model in rats. Methods: Chemical burns were created by cautery for 4 s using a rod coated with 75% silver nitrate and 25% potassium nitrate in the corneal center for the corneal neovascularization (CNV) model. Twenty-eight Wistar albino rats were divided into four groups: SHAM, CNV, CNV + SLB, and CNV + bevacizumab (BVC). After the CNV model was applied to the right eye, a single subconjunctival dose (0.05 mL) of 1 mg/kg salubrinal was injected into both eyes in the CNV + SLB group. A total of 1.25 mg/mL of subconjunctival BVC was administered to the CNV + BVC group. Fourteen days after experimental modeling and drug administration, half of the globes were placed in liquid nitrogen and stored at −20 °C until biochemical analysis. The remaining tissues were collected and fixed in 10% buffered formalin for histopathological and immunohistochemical analysis. Three qualitative agents from three different pathways were chosen: TNFR for inflammation, endothelial nitric oxide synthase (e-NOS) for vascular endothelial growth factor (VEGF)-mediated vascular permeability, and caspase-3 for cellular apoptosis. Results: Significantly lower caspase-3 and eNOS levels were detected in the CNV + SLB and CNV + BVC groups than in the CNV group. Additionally, histopathological evaluation revealed a significant decrease in neovascularization, inflammatory cell infiltration, and fibroblast activity in the CNV + SLB and CNV + BVC groups. The endoplasmic reticulum stress inhibitor, salubrinal, administered to the treatment group, attenuated apoptosis (caspase-3) and inflammation (e-NOS). In the control group (left eyes of the SLB group), salubrinal did not have a toxic effect on the healthy corneas. Conclusion: The ER stress pathway plays an important role in angiogenesis after alkaline corneal burns, and treatment with SLB modulates this pathway, reducing caspase-3 and eNOS levels. Further studies are needed to understand the molecular mechanisms altered by SLB-mediated therapy. The fact that more than one mechanism plays a role in the pathogenesis of CNV may require the use of more than one molecule in treatment. SLB has the potential to affect multiple steps in CNV pathogenesis, both in terms of reducing ER stress and regulating cellular homeostasis by inhibiting the core event of integrated stress response (ISR). Therefore, it can be used as a new treatment option and as a strengthening agent for existing treatments. Although blockade of intracellular organelle stress pathways has shown promising results in experimental studies, more in-depth research is needed before it can be used in routine practice. To the best of our knowledge, this study is the first to report the role of ER stress in corneal injury.

The Role of SLIT3–ROBO4 Signaling in Endoplasmic Reticulum Stress–Induced Delayed Corneal Epithelial and Nerve Regeneration

“…8 Inhibiting ER stress mitigates DES [9][10][11] and protects against alkaline burn induced corneal injuries. 12,13 One of our previous studies confirmed excessive ER stress (marked by a higher level of XBP1 and CHOP expression) in the process of corneal wound healing in diabetes; specifically, inhibiting ER stress with 4-phenylbutyric acid (4-PBA) significantly accelerates corneal epithelial and nerve regeneration. 14 Although the primary downstream mechanisms are considered to be excessive inflammation and programmed cell death triggered by ER stress, the underlying molecular mechanism of ER stress affecting corneal epithelial and nerve repair have not been elucidated.…”

Methylation in cornea and corneal diseases: a systematic review

“…Clinically, corneal alkali burns (CAB) can lead to a range of complications such as delayed epithelial healing, conjunctival scar formation, dry eye disease, vascularization and corneal clouding [ 177 ]. The major pathological features following CAB are corneal vascularization, inflammation, and fibrosis [ 178 , 179 ]. Several studies have shown that rapamycin, a mTOR receptor inhibitor, can reduce corneal turbidity and neovascularization in CAB by various signaling pathways like TGF-1/ERK [ 180 , 181 ].…”

Endoplasmic reticulum stress: a novel targeted approach to repair bone defects by regulating osteogenesis and angiogenesis

“…ER stress can induce nucleotide-binding domain and leucine-rich repeat containing (NLRP3) inflammasome through PERK and IRElα pathways, regulate the release of inflammatory cytokines, and trigger inflammatory response [ 196 ]. Wang et al [ 197 ] demonstrated that there is a positive feedback loop between interleukin-17A (IL-17A) and ER stress, and that inhibition of ER stress or IL-17A can reduce the neovascularization area of DR. At present, inhibition of ER stress can alleviate inflammation and inhibit angiogenesis, which has been proved in both cell and animal experiments [ 186 , 198 ]. Although ER stress pathway shows great potential in anti-inflammatory and anti-vascular therapy, more in-depth mechanism studies are needed before clinical trials.…”