ObjectiveTo explore the effect of cathepsin L (CTSL) inhibitor on apoptosis of retinal pigment epithelial (RPE) cells and mitochondrial oxidative stress. MethodsRPE cells were cultured in vitro and divided into control group, hydrogen peroxide (H2O2) group, and H2O2+CTSL inhibitor group. The cells of H2O2 group and H2O2+CTSL inhibitor group were incubated in the medium containing 400 μmol/L H2O2 for 24 hours and 10 μmol/L CTSL inhibitor was added in H2O2+CTSL inhibitor group at the same time. The cells of normal group were routinely cultured cells. The follow-up experiment was carried out 24 hours after modeling. The rate of apoptosis was detected by flow cytometry. The expression of CTSL was detected by immunofluorescence staining, Western blot and real time-polymerase chain reaction. The level of mitochondrial super oxide was detected by MitoSOX fluorescent probe, and the mitochondrial structure was observed after MitoTracker staining, the average area, form factors, and branch of mitochondria were quantitatively analyzed. The two groups were compared using two-tailed Student t test, while numerous groups were compared using one-way ANOVA. ResultsCompared with control group, the rate of apoptosis in H2O2 group was significantly higher (t=3.307, P=0.029 7), the expression level of CTSL was significantly increased (t=19.950, 6.916, 14.220; P＜0.05). Compared with H2O2 group, the expression level of CTSL, the rate of apoptosis and the mitochondrial ROS level in H2O2+CTSL inhibitor group were significantly lower (t=11.940, 4.718, 16.680; P＜0.05). The mitochondria of H2O2+CTSL inhibitor group were elongated, oval-shaped or rod-shaped, while the mitochondria of H2O2 group lost their continuous contour shape and complete structure. The differences of the average area, form factors, and brach of mitochondria among 4 groups were statistically significant (F=251.700, 34.010, 60.500; P＜0.000 1). ConclusionsH2O2 can significantly induce apoptosis in RPE cells and increase CTSL expression. CTSL inhibitor can inhibit the H2O2-induced apoptosis of RPE cells, lower the mitochondrial super oxide level, and successfully repair the mitochondrial structure.
The research on vitreous substitutes aims to find materials that can replace the functions of natural vitreous and be used to treat vitreoretinal diseases. Traditional substitutes such as gases and silicone oil have many drawbacks. However, hydrogels are regarded as highly potential substitutes due to their high water content, good biocompatibility, adjustable physical and chemical properties, and potential for controlled drug release. Researchers have developed two types of in-situ cross-linked hydrogels: chemical cross-linking and physical cross-linking. Chemical cross-linked hydrogels achieve in-situ gelation by forming chemical covalent bonds, showing good stability and degradability, but still require precise control of the degradation rate and the safety of degradation products. Physical cross-linked hydrogels utilize physical or supramolecular interactions between polymer chains to achieve in-situ gelation, having low toxicity and self-repairing properties, but they degrade too quickly and require a combination of physical and chemical cross-linking to extend the material's retention time. Additionally, researchers have explored in-situ cross-linked hydrogels loaded with anti-inflammatory, antioxidant, or anti-proliferative drugs for vitreoretinal disease, elevating vitreous substitutes from simple physical filling to an active treatment level. Future research needs to further optimize the comprehensive performance of hydrogels and deeply study their long-term biological activity impact on the intraocular microenvironment to promote their clinical translation.