This study investigated the potential efficacy of pirarubicin (THP) in modulating rabbit conjunctival fibrosis both in vitro and in vivo and characterized the underlying mechanisms. Primary rabbit conjunctival fibroblasts (RCF) were cultured and treated with THP or mitomycin C (MMC) for 5 min, followed by assaying for cell viability, cell cycle distribution, apoptotic and autophagic pathways. The production of reactive oxygen species (ROS) and chemotaxis of macrophages by RCF were evaluated using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) labeling and transwell migration assay, respectively. Limbal stem cell excision in combination with alkali burn was performed on the rabbits to establish a model of limbal deficiency and conjunctival fibro-vascular invasion. After three months, the modeled fibro-vascular tissue was excised combined with topical subconjunctival 5-min exposure to THP compared with MMC intraoperatively. The recurrence of postoperative fibrosis and the expression of apoptosis, autophagy, and inflammation markers were evaluated by immunohistochemistry. All modeled rabbits developed conjunctival fibro-vascular lesions, which were similar to human recurrent pterygium (HRP). Both THP and MMC inhibited RCF proliferation and arrested cell cycle at the G0/G1 phase. In particular, 7.5 μmol/L THP remarkably promoted RCF autophagy by upregulating the levels of Beclin 1, Atg 5/12 conjugate, and LC3B, whereas, 15 μmol/L THP significantly triggered a cascade of mitochondrial-associated RCF apoptosis. THP induced the production of ROS and enhanced the chemoattraction of macrophages by RCF. Similar to 600 μmol/L MMC, both 7.5 μmol/L and 15 μmol/L THP attenuated postoperative conjunctival fibrosis in the models; 7.5 μmol/L THP preferentially enhanced autophagy while causing fewer side effects. THP exerted its antifibrotic action by modulating autophagy in RCF, inducing cell cycle arrest, and mitochondrial-mediated apoptosis. THP at the dose of 7.5 μmol/L prevented postoperative conjunctival fibrosis in an animal model.

这项研究调查了吡柔比星（THP）在体外和体内调节兔结膜纤维化中的潜在功效并描述了潜在的机制。培养原代兔结膜成纤维细胞（RCF），并用THP或丝裂霉素C（MMC）处理5分钟，然后测定细胞活力，细胞周期分布，凋亡和自噬途径。分别使用2'，7'-二氯荧光素二乙酸酯（DCFH-DA）标记和Transwell迁移分析评估了RCF产生的活性氧（ROS）和巨噬细胞的趋化性。对家兔进行角膜缘干细胞切除联合碱烧伤，建立角膜缘缺损和结膜纤维血管浸润的模型。三个月后，与MMC术中相比，切除了模型化的纤维血管组织，并在结膜下局部暴露了THP 5分钟。通过免疫组织化学评估术后纤维化的复发以及凋亡，自噬和炎症标志物的表达。所有模型兔均发生结膜纤维血管病变，与人复发性翼状（肉（HRP）相似。THP和MMC均抑制RCF增殖并将细胞周期阻滞在G0 / G1期。特别是，7.5μmol/ L的THP通过上调Beclin 1，Atg 5/12共轭物和LC3B的水平显着促进RCF自噬，而15μmol/ L的THP显着触发了线粒体相关的RCF凋亡级联反应。THP诱导了ROS的产生，并增强了RCF对巨噬细胞的化学吸引。与600μmol/ L MMC相似，模型中7.5μmol/ L和15μmol/ L THP均可减轻术后结膜纤维化。7。5μmol/ L THP优先增强自噬，同时减少副作用。THP通过调节RCF中的自噬，诱导细胞周期停滞和线粒体介导的凋亡发挥其抗纤维化作用。在动物模型中，THP的浓度为7.5μmol/ L可以预防术后结膜纤维化。