Zheng Tao, Zhao Chuchu, Zhao Baowen, Liu Hanruo, Wang Shijian, Wang Liyuan, Liu Ping
Department of Ophthalmology, The First Affiliated Hospital of Harbin Medical University, Harbin, 150001 China.
The Beijing Institute of Ophthalmology, Beijing Tongren Hospital, Capital Medical University, Beijing, 100730 China.
Cell Death Discov. 2020 Sep 12;6(1):85. doi: 10.1038/s41420-020-00320-z. eCollection 2020.
Macular corneal dystrophy (MCD) is ascribed to mutations in the carbohydrate sulfotransferase () gene affecting keratan sulfate (KS) hydrophilicity and causing non-sulfated KS to precipitate in keratocytes and the corneal stroma. We investigated roles for inflammatory responses in MCD pathogenesis by examining the lysosomal-autophagy pathway and activation of pyroptosis in MCD keratocytes. Normal and lesioned keratocytes were obtained from MCD patients undergoing corneal transplantation. The keratocytes were subjected to gene sequencing, RT-PCR, western blotting, transmission electron microscopy, histological staining, induction and inhibition assays of autophagy and pyroptosis, CCK-8 and LysoTracker Green DND-26 labeling, and flow cytometry. A novel homozygous MCD mutation was identified in a family from Northeast China; the mutation was distinguished by cytoplasmic vacuolation, cell membrane disruption, electron dense deposits, and deposition of a band of Periodic acid-Schiff and Alcian blue-positive material in the keratocytes and stroma layer. KS protein levels were decreased, expression of p62 and LC3-II proteins was enhanced, cathepsin D expression was declined and the LysoTracker Green DND-26 signal was dramatically reduced in MCD keratocytes. Bafilomycin-A1 treatment significantly increased caspase-1 and Pro-IL-1β expression in normal and MCD keratocytes. Nod-like receptors pyrins-3 (NLRP3), caspase-1, Pro-IL-1β, and IL-1β levels were pronouncedly elevated in cells exposed to HO. Ac-YVAD-CMK treatment reversed this expression in normal and MCD keratocytes. Suppression of the autophagic degradation of non-sulfated KS by impaired autophagic flux in MCD keratocytes triggers pyroptosis. Amelioration of impaired autophagy and restraint of pyroptosis may, therefore, have therapeutic efficacy in the treatment of MCD.
黄斑角膜营养不良（MCD）归因于碳水化合物硫酸转移酶（）基因突变，该突变影响硫酸角质素（KS）的亲水性，导致非硫酸化的KS在角膜细胞和角膜基质中沉淀。我们通过检查MCD角膜细胞中的溶酶体自噬途径和焦亡激活情况，研究炎症反应在MCD发病机制中的作用。从接受角膜移植的MCD患者中获取正常和病变的角膜细胞。对角膜细胞进行基因测序、逆转录-聚合酶链反应（RT-PCR）、蛋白质免疫印迹、透射电子显微镜检查、组织学染色、自噬和焦亡的诱导及抑制试验、细胞计数试剂盒-8（CCK-8）和溶酶体示踪剂绿色DND-26标记以及流式细胞术检测。在中国东北地区的一个家族中鉴定出一种新的纯合MCD突变；该突变的特征是细胞质空泡化、细胞膜破坏、电子致密沉积物以及角膜细胞和基质层中一条高碘酸-希夫和阿尔辛蓝阳性物质带的沉积。MCD角膜细胞中KS蛋白水平降低，p62和LC3-II蛋白表达增强，组织蛋白酶D表达下降，溶酶体示踪剂绿色DND-26信号显著降低。巴弗洛霉素A1处理显著增加正常和MCD角膜细胞中半胱天冬酶-1和前白细胞介素-1β的表达。在暴露于过氧化氢（HO）的细胞中，NOD样受体吡啉-3（NLRP3）、半胱天冬酶-1、前白细胞介素-1β和白细胞介素-1β水平明显升高。Ac-YVAD-CMK处理可逆转正常和MCD角膜细胞中的这种表达。MCD角膜细胞中自噬通量受损导致非硫酸化KS的自噬降解受到抑制，从而引发焦亡。因此，改善自噬受损和抑制焦亡可能对MCD的治疗具有疗效。
