Kim Seok-Jo, Cheresh Paul, Jablonski Renea P, Morales-Nebreda Luisa, Cheng Yuan, Hogan Erin, Yeldandi Anjana, Chi Monica, Piseaux Raul, Ridge Karen, Michael Hart C, Chandel Navdeep, Scott Budinger G R, Kamp David W
Department of Medicine, Division of Pulmonary & Critical Care Medicine, Jesse Brown VA Medical Center, Chicago, IL, United States; Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, United States.
Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, United States.
Free Radic Biol Med. 2016 Dec;101:482-490. doi: 10.1016/j.freeradbiomed.2016.11.007. Epub 2016 Nov 11.
Alveolar epithelial cell (AEC) injury and mitochondrial dysfunction are important in the development of lung fibrosis. Our group has shown that in the asbestos exposed lung, the generation of mitochondrial reactive oxygen species (ROS) in AEC mediate mitochondrial DNA (mtDNA) damage and apoptosis which are necessary for lung fibrosis. These data suggest that mitochondrial-targeted antioxidants should ameliorate asbestos-induced lung.
To determine whether transgenic mice that express mitochondrial-targeted catalase (MCAT) have reduced lung fibrosis following exposure to asbestos or bleomycin and, if so, whether this occurs in association with reduced AEC mtDNA damage and apoptosis.
Crocidolite asbestos (100µg/50µL), TiO (negative control), bleomycin (0.025 units/50µL), or PBS was instilled intratracheally in 8-10 week-old wild-type (WT - C57Bl/6J) or MCAT mice. The lungs were harvested at 21d. Lung fibrosis was quantified by collagen levels (Sircol) and lung fibrosis scores. AEC apoptosis was assessed by cleaved caspase-3 (CC-3)/Surfactant protein C (SFTPC) immunohistochemistry (IHC) and semi-quantitative analysis. AEC (primary AT2 cells from WT and MCAT mice and MLE-12 cells) mtDNA damage was assessed by a quantitative PCR-based assay, apoptosis was assessed by DNA fragmentation, and ROS production was assessed by a Mito-Sox assay.
Compared to WT, crocidolite-exposed MCAT mice exhibit reduced pulmonary fibrosis as measured by lung collagen levels and lung fibrosis score. The protective effects in MCAT mice were accompanied by reduced AEC mtDNA damage and apoptosis. Similar findings were noted following bleomycin exposure. Euk-134, a mitochondrial SOD/catalase mimetic, attenuated MLE-12 cell DNA damage and apoptosis. Finally, compared to WT, asbestos-induced MCAT AT2 cell ROS production was reduced.
Our finding that MCAT mice have reduced pulmonary fibrosis, AEC mtDNA damage and apoptosis following exposure to asbestos or bleomycin suggests an important role for AEC mitochondrial HO-induced mtDNA damage in promoting lung fibrosis. We reason that strategies aimed at limiting AEC mtDNA damage arising from excess mitochondrial HO production may be a novel therapeutic target for mitigating pulmonary fibrosis.
肺泡上皮细胞（AEC）损伤和线粒体功能障碍在肺纤维化的发展过程中起重要作用。我们的研究小组已经表明，在接触石棉的肺组织中，AEC中线粒体活性氧（ROS）的产生介导了线粒体DNA（mtDNA）损伤和细胞凋亡，而这两者是肺纤维化所必需的。这些数据表明，靶向线粒体的抗氧化剂应该能够改善石棉诱导的肺损伤。
确定表达靶向线粒体过氧化氢酶（MCAT）的转基因小鼠在接触石棉或博来霉素后肺纤维化是否减轻，如果是，这是否与AEC的mtDNA损伤和细胞凋亡减少有关。
将青石棉（100µg/50µL）、二氧化钛（阴性对照）、博来霉素（0.025单位/50µL）或磷酸盐缓冲液经气管内注入8-10周龄的野生型（WT - C57Bl/6J）或MCAT小鼠体内。在第21天收获肺组织。通过胶原蛋白水平（Sircol）和肺纤维化评分对肺纤维化进行定量。通过裂解的半胱天冬酶-3（CC-3）/表面活性蛋白C（SFTPC）免疫组织化学（IHC）和半定量分析评估AEC凋亡。通过基于定量PCR的检测方法评估AEC（来自WT和MCAT小鼠的原代AT2细胞以及MLE-12细胞）的mtDNA损伤，通过DNA片段化评估细胞凋亡，并通过Mito-Sox检测评估ROS的产生。
与WT相比，通过肺胶原蛋白水平和肺纤维化评分测量，接触青石棉的MCAT小鼠肺纤维化减轻。MCAT小鼠中的保护作用伴随着AEC的mtDNA损伤和细胞凋亡减少。博来霉素暴露后也观察到类似的结果。线粒体超氧化物歧化酶/过氧化氢酶模拟物Euk-134减轻了MLE-12细胞的DNA损伤和细胞凋亡。最后，与WT相比，石棉诱导的MCAT AT2细胞ROS产生减少。
我们的研究结果表明，MCAT小鼠在接触石棉或博来霉素后肺纤维化、AEC的mtDNA损伤和细胞凋亡减少，这表明AEC线粒体HO诱导的mtDNA损伤在促进肺纤维化中起重要作用。我们推断，旨在限制过量线粒体HO产生引起的AEC mtDNA损伤的策略可能是减轻肺纤维化的一种新的治疗靶点。
