[The experimental study on the effect of the NRF2 activator dimethyl itaconate on hypoxia-induced scleral...

Objective: To investigate the effect of dimethyl itaconate (DMI), an NRF2 activator, on hypoxia-induced scleral fibroblasts. Methods: It was a experimental study. A guinea pig model of form-deprivation myopia (FDM) was established. Ocular refraction and axial length were measured using eccentric infrared photorefraction and A-scan ultrasonography, respectively. Western blot was used to detect NRF2 protein expression in the sclera of FDM guinea pigs. Primary guinea pig scleral fibroblasts (GSFs) were cultured and identified via immunofluorescence staining for vimentin and cytokeratin. GSFs at passages 3-5 were used for experiments. For the hypoxic treatment, GSFs were cultured in a hypoxia incubator chamber with 1% O₂, 5% CO₂, and 94% N₂, with cells cultured under 21% O₂ serving as the normoxia control. After 48 h of culture, Western blot analysis was performed to detect the expression of HIF-1A, COL1A1, MMP2, and NRF2 in GSFs. The mRNA expression of the NRF2 downstream target gene NQO1 mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Nuclear translocation of NRF2 was assessed by immunofluorescence staining. For the DMI treatment, GSFs were treated with 150 μmol/L DMI or an equal volume of PBS for 3 h, followed by the hypoxic treatment for 48 h. Subsequent analyses (Western blot, qRT-PCR, and immunofluorescence staining) were performed as described above. Results: After 4 weeks of form deprivation, guinea pigs in the FDM group exhibited a significant myopic shift in refraction [(-3.248±1.501) vs. (4.143±0.350)D; P<0.001] and increased axial length [(8.650±0.081) vs. (8.363±0.020) mm; P<0.05] compared to the normal control group. NRF2 protein expression in the sclera of FDM guinea pigs was significantly decreased (0.347±0.110 vs. 0.796±0.032; P<0.05). GSFs migrated from scleral tissue pieces 7-9 days after culture and reached confluence after approximately 14 days. At low density, GSFs were stellate-shaped; after confluence, they organized into bundle-or swirl-shaped patterns. Immunofluorescence results confirmed GSFs were positive for vimentin and negative for cytokeratin. Compared with the normoxia group, the hypoxia group showed increased protein expression of HIF-1A (1.361±0.043 vs. 0.843±0.053; P<0.001) and MMP2 (0.640±0.042 vs. 0.355±0.052; P<0.05), but decreased COL1A1 (0.451±0.063 vs. 0.701±0.043; P<0.05). NRF2 protein (0.154±0.023 vs. 0.399±0.026, P<0.05) and NQO1 mRNA (0.700±0.050 vs. 1.000±0.004, P<0.05) expression was reduced, and the NRF2 nuclear translocation was diminished in the hypoxia group. Compared with the PBS group, the DMI-treated group showed increased NRF2 protein (0.361±0.056 vs. 0.146±0.023; P<0.05) and NQO1 mRNA (1.775±0.028 vs. 1.001±0.022; P<0.001) expression, enhanced NRF2 nuclear translocation, increased COL1A1 protein (0.842±0.082 vs. 0.400±0.054; P<0.05), and decreased MMP2 protein (0.547±0.077 vs. 0.841±0.039; P<0.05). Conclusion: Activation of NRF2 participates in scleral remodeling in myopia by promoting COL1A1 expression and inhibiting MMP2 expression in scleral fibroblasts, suggesting NRF2 as a potential therapeutic target for myopia.