Abstract：
OBJECTIVE: This study investigated the involvement of discoidin domain receptor (DDR) in dry eye and assessed the potential of specific DDR inhibitors as a therapeutic strategy for dry eye by exploring the underlying mechanism.
METHODS: Dry eye was induced in Wistar rats by applying 0.2% benzalkonium chloride (BAC), after which rats were treated topically for 7 days with DDR1-IN-1, a selective inhibitor of DDR1. Clinical manifestations of dry eye were assessed on Day-7 post-treatment. Histological evaluation of corneal damage was performed using haematoxylin and eosin (H&E) staining. In vitro, immortalized human corneal epithelial cells (HCECs) exposed to hyperosmotic stress (HS) were treated with varying doses of DDR1-IN-1 for 24 h. The levels of lipid peroxidation in dry eye corneas or HS-stimulated HCECs were assessed. Protein levels of DDR1/DDR2 and related pathways were detected by western blotting. The cellular distribution of acyl-CoA synthetase long chain family member 4 (ACSL4) and Yes-associated protein (YAP) was evaluated using immunohistochemistry or immunofluorescent staining.
RESULTS: In dry eye corneas, only DDR1 expression was significantly up-regulated compared with normal controls. DDR1-IN-1 treatment significantly alleviated dry eye symptoms in vivo. The treatment remarkably reduced lipid hydroperoxide (LPO) levels and suppressed the expression of ferroptosis markers, particularly ACSL4. Overexpression or reactivation of YAP diminished the protective effects of DDR1-IN-1, indicating the involvement of the Hippo/YAP pathway in DDR1-targeted therapeutic effects.
CONCLUSIONS: This study confirms the significance of DDR1 in dry eye and highlights the potential of selective DDR1 inhibitor(s) for dry eye treatment.
METHODS: Dry eye was induced in Wistar rats by applying 0.2% benzalkonium chloride (BAC), after which rats were treated topically for 7 days with DDR1-IN-1, a selective inhibitor of DDR1. Clinical manifestations of dry eye were assessed on Day-7 post-treatment. Histological evaluation of corneal damage was performed using haematoxylin and eosin (H&E) staining. In vitro, immortalized human corneal epithelial cells (HCECs) exposed to hyperosmotic stress (HS) were treated with varying doses of DDR1-IN-1 for 24 h. The levels of lipid peroxidation in dry eye corneas or HS-stimulated HCECs were assessed. Protein levels of DDR1/DDR2 and related pathways were detected by western blotting. The cellular distribution of acyl-CoA synthetase long chain family member 4 (ACSL4) and Yes-associated protein (YAP) was evaluated using immunohistochemistry or immunofluorescent staining.
RESULTS: In dry eye corneas, only DDR1 expression was significantly up-regulated compared with normal controls. DDR1-IN-1 treatment significantly alleviated dry eye symptoms in vivo. The treatment remarkably reduced lipid hydroperoxide (LPO) levels and suppressed the expression of ferroptosis markers, particularly ACSL4. Overexpression or reactivation of YAP diminished the protective effects of DDR1-IN-1, indicating the involvement of the Hippo/YAP pathway in DDR1-targeted therapeutic effects.
CONCLUSIONS: This study confirms the significance of DDR1 in dry eye and highlights the potential of selective DDR1 inhibitor(s) for dry eye treatment.
摘要：
目的：本研究调查了盘状结构域受体（DDR）在干眼中的参与，并通过探索潜在的机制评估了特异性DDR抑制剂作为干眼治疗策略的潜力。
方法：应用0.2%苯扎氯铵（BAC）诱导Wistar大鼠干眼，之后用DDR1的选择性抑制剂DDR1-IN-1局部治疗大鼠7天。在治疗后第7天评估干眼的临床表现。使用苏木精和曙红(H&E)染色进行角膜损伤的组织学评估。体外，用不同剂量的DDR1-IN-1处理暴露于高渗应激（HS）的永生化人角膜上皮细胞（HCECs）24小时。评估了干眼角膜或HS刺激的HCECs中脂质过氧化的水平。通过蛋白质印迹法检测DDRl/DDR2的蛋白水平和相关途径。使用免疫组织化学或免疫荧光染色评估酰基辅酶A合成酶长链家族成员4（ACSL4）和Yes相关蛋白（YAP）的细胞分布。
结果：在干眼角膜中，与正常对照组相比，只有DDR1表达显着上调。DDR1-IN-1治疗可显著缓解体内干眼症状。该治疗显着降低了脂质过氧化氢（LPO）水平并抑制了铁凋亡标志物的表达，特别是ACSL4。YAP的过表达或再激活降低了DDRl-IN-1的保护作用，表明Hippo/YAP途径参与了DDRl靶向治疗作用。
结论：该研究证实了DDR1在干眼中的重要性，并强调了选择性DDR1抑制剂用于干眼治疗的潜力。
方法：应用0.2%苯扎氯铵（BAC）诱导Wistar大鼠干眼，之后用DDR1的选择性抑制剂DDR1-IN-1局部治疗大鼠7天。在治疗后第7天评估干眼的临床表现。使用苏木精和曙红(H&E)染色进行角膜损伤的组织学评估。体外，用不同剂量的DDR1-IN-1处理暴露于高渗应激（HS）的永生化人角膜上皮细胞（HCECs）24小时。评估了干眼角膜或HS刺激的HCECs中脂质过氧化的水平。通过蛋白质印迹法检测DDRl/DDR2的蛋白水平和相关途径。使用免疫组织化学或免疫荧光染色评估酰基辅酶A合成酶长链家族成员4（ACSL4）和Yes相关蛋白（YAP）的细胞分布。
结果：在干眼角膜中，与正常对照组相比，只有DDR1表达显着上调。DDR1-IN-1治疗可显著缓解体内干眼症状。该治疗显着降低了脂质过氧化氢（LPO）水平并抑制了铁凋亡标志物的表达，特别是ACSL4。YAP的过表达或再激活降低了DDRl-IN-1的保护作用，表明Hippo/YAP途径参与了DDRl靶向治疗作用。
结论：该研究证实了DDR1在干眼中的重要性，并强调了选择性DDR1抑制剂用于干眼治疗的潜力。
