Beijing Institute of Ophthalmology, Beijing TongRen Eye Center, Beijing Key Laboratory of Ophthalmology and Visual Sciences, Beijing Tongren Hospital, Capital Medical University, Beijing, 100005, China.
Department of Ophthalmology, The Third Medical Center of Chinese People's Liberation Army General Hospital, Beijing, 100039, China.
Free Radic Biol Med. 2024 Sep;222:106-121. doi: 10.1016/j.freeradbiomed.2024.05.032. Epub 2024 May 24.
Severe dry eye disease causes ocular surface damage, which is highly associated with mitochondrial dysfunction. Mitochondrial transcription factor A (TFAM) is essential for packaging mitochondrial DNA (mtDNA) and is crucial for maintaining mitochondrial function. Herein, we aimed to explore the effect of a decreased TFAM expression on ocular surface damage.
Female C57BL/6 mice were induced ocular surface injury by topical administrating benzalkonium chloride (BAC). Immortalized human corneal epithelial cells (HCECs) were stimulated by tert-butyl hydroperoxide (t-BHP) to create oxidative stress damage. HCECs with TFAM knockdown were established. RNA sequencing was employed to analyze the whole-genome expression. Mitochondrial changes were measured by transmission electron microscopy, Seahorse metabolic flux analysis, mitochondrial membrane potential, and mtDNA copy number. TFAM expression and inflammatory cytokines were determined using RT-qPCR, immunohistochemistry, immunofluorescence, and immunoblotting.
In both the corneas of BAC-treated mice and t-BHP-induced HCECs, we observed impaired TFAM expression, accompanied by mitochondrial structure and function defects. TFAM downregulation in HCECs suppressed mitochondrial respiratory capacity, reduced mtDNA content, induced mtDNA leakage into the cytoplasm, and led to inflammation. RNA sequencing revealed the absent in melanoma 2 (AIM2) inflammasome was activated in the corneas of BAC-treated mice. The AIM2 inflammasome activation was confirmed in TFAM knockdown HCECs. TFAM knockdown in t-BHP-stimulated HCECs aggravated mitochondrial dysfunction and the AIM2 inflammasome activation, thereby further triggering the secretion of inflammatory factors such as interleukin (IL) -1β and IL-18.
TFAM reduction impaired mitochondrial function, activated AIM2 inflammasome and promoted ocular surface inflammation, revealing an underlying molecular mechanism for ocular surface disorders.
严重的干眼疾病会导致眼表面损伤，这与线粒体功能障碍高度相关。线粒体转录因子 A（TFAM）对于包装线粒体 DNA（mtDNA）至关重要，是维持线粒体功能所必需的。在此，我们旨在探索 TFAM 表达降低对眼表面损伤的影响。
通过局部给予苯扎氯铵（BAC）诱导雌性 C57BL/6 小鼠眼表面损伤。用叔丁基过氧化氢（t-BHP）刺激永生化人角膜上皮细胞（HCEC）以产生氧化应激损伤。建立 TFAM 敲低的 HCEC。采用 RNA 测序分析全基因组表达。通过透射电子显微镜、 Seahorse 代谢通量分析、线粒体膜电位和 mtDNA 拷贝数测量线粒体变化。采用 RT-qPCR、免疫组织化学、免疫荧光和免疫印迹法测定 TFAM 表达和炎症细胞因子。
在 BAC 处理的小鼠角膜和 t-BHP 诱导的 HCEC 中，我们观察到 TFAM 表达受损，伴有线粒体结构和功能缺陷。HCEC 中的 TFAM 下调抑制了线粒体呼吸能力，降低了 mtDNA 含量，诱导 mtDNA 漏入细胞质，并导致炎症。RNA 测序显示，在 BAC 处理的小鼠角膜中缺失黑色素瘤 2（AIM2）炎性小体被激活。在 TFAM 敲低的 HCEC 中证实了 AIM2 炎性小体的激活。在 t-BHP 刺激的 HCEC 中，TFAM 敲低加剧了线粒体功能障碍和 AIM2 炎性小体的激活，从而进一步触发白细胞介素（IL）-1β和 IL-18 等炎症因子的分泌。
TFAM 减少损害了线粒体功能，激活了 AIM2 炎性小体并促进了眼表面炎症，揭示了眼表面疾病的潜在分子机制。
