New England College of Optometry, Boston, MA, United States of America.
PLoS One. 2024 Oct 31;19(10):e0313027. doi: 10.1371/journal.pone.0313027. eCollection 2024.
Osteopontin (OPN) is a glycosylated, secreted phosphoprotein known to be elevated in both human and mouse retinas during various stages of diabetic retinopathy. However, its specific roles in modulating ocular surface dynamics and immune responses in diabetes remain unexplored. This study aims to investigate the role of OPN in the development of ocular surface disease (OSD) in type 2 diabetic (T2D) mice.
Three- to four-week-old C57BL/6 wild-type (WT) and OPN-knockout (OPN-/-) mice were fed a high-fat diet (HFD) and were rendered diabetic by streptozotocin (STZ; 40 mg/kg body weight) in citrate buffer (vehicle); non-diabetic controls were injected with vehicle alone. Diabetes was confirmed if blood glucose levels were >200 mg/dL, measured 1-2 weeks post-STZ injection. Control, age- and sex-matched db/db diabetic mice fed a standard chow diet were also included in this study. Ocular surface inflammation was assessed using ELISA to quantify inflammatory cytokine proteins and wheat germ agglutinin (WGA) staining was utilized to highlight corneal surface irregularities. Clinical signs were evaluated by corneal fluorescein staining, tear production measurements, and tear sodium (Na+) concentration assessments. These evaluations were conducted 4, 6, 8 and 16-weeks post-diabetes onset in WT and OPN-/- mice and were compared to those obtained in non-diabetic controls. Statistical analysis was performed using a two-way ANOVA, with significance set at P < 0.05.
Both WT and OPN-/- mice developed T2D within 4 and 8 weeks, respectively, following HFD + STZ treatment. Corneal OPN levels in WT diabetic mice increased ~2-fold at 2 weeks and ~4-fold at 16 weeks compared to non-diabetic controls, with similar elevations observed in their tear fluid. Diabetic db/db mice also exhibited elevated OPN levels in the blood and ocular surface, which persisted as diabetes progressed. Enhanced fluorescein staining, indicating corneal irregularities, appeared in WT mice at 8 weeks and in OPN-/- mice at 10 weeks post-T2D induction. Additionally, WGA staining showed a significant reduction in fluorescence intensity in WT mice treated with HFD and STZ, confirming corneal surface irregularities that were delayed in OPN-/- mice. Elevated tear sodium concentration was observed in both WT and OPN-/- diabetic mice without affecting tear production rates. Notably, OPN levels increased early, at week 2, following HFD and STZ treatment, preceding changes in interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and matrix metalloproteinase-9 (MMP-9). Upregulation of IL-6 became apparent at 6 weeks in WT mice and was delayed until 10 weeks in OPN-/- mice post-T2D induction.
Elevated OPN levels were detected early post-T2D induction in diabetic WT and db/db mice corneas without initial subclinical changes. This early increase in OPN precedes other proinflammatory cytokines associated with eventual ocular surface inflammation as diabetes progresses. Persistence of OPN also correlated with clinical signs such as increased corneal surface irregularities and elevated tear Na+ concentration. Future research will explore OPN's role as a biomarker in ocular surface disease (OSD), including dry eye disease (DED), and investigate its impact on inflammatory processes and other mechanistic pathways in diabetic ocular complications.
骨桥蛋白（OPN）是一种糖基化的分泌型磷酸蛋白，已知在各种糖尿病视网膜病变阶段，在人和小鼠的视网膜中均升高。然而，其在调节糖尿病患者眼表动力学和免疫反应中的具体作用仍未得到探索。本研究旨在研究 OPN 在 2 型糖尿病（T2D）小鼠眼表疾病（OSD）发展中的作用。
3-4 周龄 C57BL/6 野生型（WT）和 OPN 敲除（OPN-/-）小鼠喂食高脂肪饮食（HFD），并用链脲佐菌素（STZ；40mg/kg 体重）在柠檬酸盐缓冲液（载体）中致糖尿病；非糖尿病对照仅注射载体。如果血糖水平>200mg/dL，即在 STZ 注射后 1-2 周测量，则确认糖尿病。本研究还包括喂食标准玉米淀粉饮食的 db/db 糖尿病模型小鼠。使用酶联免疫吸附试验（ELISA）定量炎性细胞因子蛋白，并用小麦胚凝集素（WGA）染色突出角膜表面不规则性，评估眼表炎症。通过角膜荧光素染色、泪液产生测量和泪液钠（Na+）浓度评估来评估临床症状。在 WT 和 OPN-/-小鼠糖尿病发病后 4、6、8 和 16 周进行这些评估，并与非糖尿病对照组进行比较。使用双因素方差分析进行统计分析，显著性水平设为 P<0.05。
WT 和 OPN-/- 小鼠分别在 HFD+STZ 治疗后 4 周和 8 周内发展为 T2D。WT 糖尿病小鼠的角膜 OPN 水平在 2 周时增加了约 2 倍，在 16 周时增加了约 4 倍，与非糖尿病对照组相比，其泪液中的 OPN 水平也有类似的升高。糖尿病 db/db 小鼠的血液和眼表也表现出 OPN 水平升高，随着糖尿病的进展而持续升高。增强的荧光素染色表明角膜不规则，在 WT 小鼠中在 8 周时出现，在 OPN-/- 小鼠中在 10 周时出现，在 T2D 诱导后出现。此外，WGA 染色显示 HFD 和 STZ 处理的 WT 小鼠的荧光强度显著降低，证实了角膜表面不规则性，而 OPN-/- 小鼠的角膜表面不规则性则延迟。WT 和 OPN-/- 糖尿病小鼠的泪液钠浓度升高，但不影响泪液产生率。值得注意的是，OPN 水平在 HFD 和 STZ 治疗后第 2 周就升高，早于白细胞介素 6（IL-6）、肿瘤坏死因子-α（TNF-α）和基质金属蛋白酶 9（MMP-9）的变化。WT 小鼠在 6 周时出现 IL-6 的上调，而 OPN-/- 小鼠直到 T2D 诱导后 10 周才出现这种上调。
在 WT 和 db/db 糖尿病小鼠的角膜中，在 T2D 诱导后的早期就检测到 OPN 水平升高，而没有初始的亚临床变化。这种 OPN 的早期增加先于与最终眼表炎症相关的其他促炎细胞因子，随着糖尿病的进展而出现。OPN 的持续存在也与临床症状相关，如角膜表面不规则性增加和泪液 Na+浓度升高。未来的研究将探索 OPN 作为眼表疾病（OSD）的生物标志物的作用，包括干眼症（DED），并研究其对糖尿病眼部并发症中炎症过程和其他机制途径的影响。
