Yang C, Zhu Y, Cai Z Y, Nian H, Du B, Wei R H
Tianjin Medical University Eye Hospital, Eye Institute and School of Optometry, Tianjin Branch of National Clinical Research Center for Ocular Disease, Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin 300384, China.
Zhonghua Yan Ke Za Zhi. 2025 Aug 11;61(8):609-617. doi: 10.3760/cma.j.cn112142-20240904-00381.
To investigate the effect of dimethyl itaconate (DMI), an NRF2 activator, on hypoxia-induced scleral fibroblasts. 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. After 4 weeks of form deprivation, guinea pigs in the FDM group exhibited a significant myopic shift in refraction [(-3.248±1.501) (4.143±0.350)D; <0.001] and increased axial length [(8.650±0.081) (8.363±0.020) mm; <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 0.796±0.032; <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 0.843±0.053; <0.001) and MMP2 (0.640±0.042 0.355±0.052; <0.05), but decreased COL1A1 (0.451±0.063 0.701±0.043; <0.05). NRF2 protein (0.154±0.023 0.399±0.026, <0.05) and NQO1 mRNA (0.700±0.050 1.000±0.004, <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 0.146±0.023; <0.05) and NQO1 mRNA (1.775±0.028 1.001±0.022; <0.001) expression, enhanced NRF2 nuclear translocation, increased COL1A1 protein (0.842±0.082 0.400±0.054; <0.05), and decreased MMP2 protein (0.547±0.077 0.841±0.039; <0.05). 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.
为研究NRF2激活剂衣康酸二甲酯（DMI）对缺氧诱导的巩膜成纤维细胞的影响。这是一项实验研究。建立了豚鼠形觉剥夺性近视（FDM）模型。分别使用偏心红外验光和A超超声检查测量眼屈光和眼轴长度。采用蛋白质免疫印迹法检测FDM豚鼠巩膜中NRF2蛋白表达。培养原代豚鼠巩膜成纤维细胞（GSFs），并通过波形蛋白和细胞角蛋白的免疫荧光染色进行鉴定。使用第3 - 5代的GSFs进行实验。对于缺氧处理，将GSFs置于含1% O₂、5% CO₂和94% N₂的缺氧培养箱中培养，以在21% O₂条件下培养的细胞作为常氧对照。培养48小时后，进行蛋白质免疫印迹分析以检测GSFs中HIF - 1A、COL1A1、MMP2和NRF2的表达。通过定量实时聚合酶链反应（qRT - PCR）检测NRF2下游靶基因NQO1 mRNA的表达。通过免疫荧光染色评估NRF2的核转位。对于DMI处理，将GSFs用150 μmol/L DMI或等体积的PBS处理3小时，随后进行48小时的缺氧处理。随后按照上述方法进行分析（蛋白质免疫印迹法、qRT - PCR和免疫荧光染色）。形觉剥夺4周后，与正常对照组相比，FDM组豚鼠的屈光出现显著近视偏移[（-3.248±1.501） （4.143±0.350）D；<0.001]，眼轴长度增加[（8.650±0.081） （8.363±0.020）mm；<0.05]。FDM豚鼠巩膜中NRF2蛋白表达显著降低（0.347±0.110 0.796±0.032；<0.05）。培养7 - 9天后，GSFs从巩膜组织块中迁移出来，大约14天后达到汇合状态。低密度时，GSFs呈星状；汇合后，它们排列成束状或漩涡状。免疫荧光结果证实GSFs波形蛋白呈阳性，细胞角蛋白呈阴性。与常氧组相比，缺氧组HIF - 1A（1.361±0.043 0.843±0.053；<0.001）和MMP2（0.640±0.042 0.355±0.052；<0.05）的蛋白表达增加，但COL1A1（0.451±0.063 0.701±0.043；<0.05）的蛋白表达降低。缺氧组NRF2蛋白（0.154±0.023 0.399±0.026，<0.05）和NQO1 mRNA（0.700±0.050 1.000±0.004，<0.05）表达降低，且NRF2核转位减少。与PBS组相比，DMI处理组NRF2蛋白（0.361±0.056 0.146±0.023；<0.05）和NQO1 mRNA（1.775±0.028 1.001±0.022；<0.001）表达增加，NRF2核转位增强，COL1A1蛋白（0.842±0.082 0.400±0.054；<0.05）增加，MMP2蛋白（0.547±0.077 0.841±0.039；<0.05）降低。NRF2的激活通过促进巩膜成纤维细胞中COL1A1的表达和抑制MMP2的表达参与近视中的巩膜重塑，提示NRF2作为近视的潜在治疗靶点。
