探討褪黑激素對懸浮微粒誘發心臟損傷及粒線體失能的影響

Abstract

空氣污染是目前人類健康的主要威脅，而其中的有害成分PM2.5與空氣污染物導致的心血管疾病的風險增加。在過去研究中，已證實PM2.5會藉由影響細胞內的活性氧化物堆積，進而造成心肌纖維化。在本研究中，我將探討抗氧化藥物，褪黑激素對PM2.5所造成細胞凋亡與纖維化的相關機轉。此外，由於先前本實驗室曾發表心肌纖維化受miR-221/222調控，故在本研究中也將探討miR-221/222所扮演的角色及其是否與melatonin引發的機轉有關。首先，在動物實驗中，我利用C57BL/6 (WT) 及 miR-221/222 KO 小鼠，以進行氣管內注射10 mg/kg PM2.5及管餵20 mg/kg褪黑激素方式建立動物模型。PM2.5處理後的小鼠心臟收縮及舒張功能都有所損傷，而褪黑激素不論是WT或KO小鼠皆能一定程度上恢復其心臟功能。在小鼠心肌細胞凋亡與纖維化方面，通過TUNEL和Fibronectin、ETS-1免疫組織染色實驗，證明PM2.5分別促進WT及 miR-221/222 KO 小鼠的心臟功能損傷。進一步使用會使膠原蛋白呈現紅色的Sirius Red染色，同樣也證實PM2.5處理後會導致小鼠心臟纖維化增加，並且miR-221/222 KO 小鼠的心臟功能損傷和纖維化更為嚴重，而褪黑激素能夠恢復PM2.5造成的傷害。 在細胞實驗中，我利用H9c2大鼠心肌母細胞作為體外細胞模式，使用20 g/mL PM2.5處理H9c2細胞24小時以模擬空氣污染之環境。利用qPCR發現PM2.5會降低miR-221/222表達，並且褪黑激素可恢復miR-221/222的表達。細胞存活率、TUNEL測定和Fibronectin免疫螢光染色，結果顯示褪黑激素可顯著恢復PM2.5造成的細胞活性降低與細胞凋亡和纖維化的增加。Western blot實驗結果證實褪黑激素可顯著恢復PM2.5造成的細胞凋亡相關蛋白PUMA和纖維化相關蛋白Fibronectin、ETS-1的表達之增加。此外，透過 MitoSOX Red 與流式細胞儀共同顯示褪黑激素處理恢復PM2.5誘發的粒線體活性氧化物 (Reactive oxygen species, ROS)表現增加。JC-1染色結果表明褪黑激素處理會恢復PM2.5 造成的心肌細胞中的粒線體膜電位（Δψm）降低。在自噬方面，吖啶橙(Acridine Orange)螢光染色與Western blots實驗共同證實褪黑激素處理減輕PM2.5誘發的細胞自噬，並恢復了PM2.5處理後自噬相關蛋白p62和LC3B、BNIP3表現的增加。綜上所述，PM2.5增加粒線體ROS產生並誘發粒線體功能障礙，增加線粒體裂解及線粒體自噬，進而導致心臟細胞凋亡和纖維化最終導致心臟功能障礙，而褪黑激素能夠恢復PM2.5所造成的傷害。期待在未來褪黑激素可成為預防空氣污染所造成心臟損傷的治療方針。

Air pollution is currently a major threat to human health, with the harmful component PM2.5 contributing to increased cardiovascular diseases risks associated with air pollution. Previous research has confirmed that PM2.5 leads to myocardial fibrosis by influencing the accumulation of intracellular reactive oxygen species (ROS). In this study, I aim to explore the underlying mechanisms of cellular apoptosis and fibrosis caused by PM2.5 and their modulation by antioxidant agents, specifically melatonin. Considering what our lab has already found about how miR-221/222 controls cardiac fibrosis, this study will also look at the role of miR-221/222 and how it might interact with the mechanisms that melatonin sets off. First, C57BL/6 (WT) and miR-221/222 knockout (KO) mice were used to make an animal model by injecting 10 mg/kg PM2.5 into the trachea and giving 20 mg/kg melatonin by mouth. The cardiac contraction and relaxation functions of the mice were impaired following PM2.5 exposure, while melatonin administration partially restored cardiac function in both WT and KO mice. TUNEL assay and immunohistochemical staining for Fibronectin and ETS-1 confirmed the detrimental effects on cardiac function induced by PM2.5, observed in both WT and miR-221/222 KO mice. Furthermore, Sirius Red staining, which stains collagen red, confirmed increased cardiac fibrosis following PM2.5 exposure, with miR-221/222 KO mice exhibiting more severe cardiac dysfunction and fibrosis. The administration of melatonin attenuated the adverse effects caused by PM2.5. In cellular experiments, H9c2 rat cardiomyocytes were employed as an in vitro cellular model and subjected to 20 g/mL PM2.5 treatment for 24 hours to simulate air pollution conditions. qPCR analysis revealed a downregulation of miR-221/222 expression induced by PM2.5, which was restored by melatonin administration. Cell viability assay, TUNEL staining, and Fibronectin immunofluorescence staining demonstrated that melatonin significantly mitigated the decrease in cell viability, the increase in apoptosis, and the fibrotic response induced by PM2.5. Western blot analysis confirmed that melatonin administration effectively restored the elevated expression of the apoptosis-related protein PUMA and the fibrosis-related proteins Fibronectin and ETS-1 triggered by PM2.5. Additionally, co-staining with MitoSOX Red and flow cytometry revealed that melatonin treatment restored the heightened production of mitochondrial reactive oxygen species (ROS) induced by PM2.5. JC-1 staining indicated that melatonin treatment restored the decreased mitochondrial membrane potential (Δψm) observed in PM2.5-treated cardiomyocytes. Regarding autophagy, Acridine Orange staining and Western blot analysis collectively demonstrated that melatonin treatment alleviated PM2.5-induced cellular autophagy and restored the increased expression of autophagy-related proteins p62, LC3B, and BNIP3 induced by PM2.5. In conclusion, PM2.5 increases the production of ROS, which leads to mitochondrial dysfunction, mitochondrial fission, and mitochondrial autophagy. This, in turn, causes cardiac cell apoptosis and fibrosis, which lowers the function of the heart. However, the administration of melatonin effectively mitigates the deleterious effects caused by PM2.5 exposure. It is envisaged that melatonin may hold promise as a therapeutic strategy for preventing cardiac damage resulting from air pollution in the future.