柴油引擎懸浮微粒對心肌細胞致毒機制之探討

Abstract

近年來，流行病學研究顯示空氣懸浮微粒的增加及其所含之化學成分與心血管疾病致死率有關。粒徑小於2.5微米的懸浮微粒(particulate matter; PM2.5)可深入肺部並累積於體內，與血栓的形成和發炎反應具高度相關性。柴油引擎懸浮微粒(diesel exhaust particles, DEPs)的平均直徑約為0.2微米，且為PM2.5之主要成分。柴油引擎車的使用量逐年漸增，因柴油引擎的效率較汽油引擎高，因而降低石油消耗速度及二氧化碳的排放量，較不易加劇溫室效應，但氮氧化物與懸浮微粒的排放量較汽油引擎相對得多。過去研究顯示柴油引擎懸浮微粒萃取物(DEP extracts, DEPEs)可降低心肌細胞存活率，且可能導因於活性氧分子的生成，但其造成的心血管系統毒性的確切機制尚未清楚。因此，本研究主要探討DEPEs對心肌細胞的毒性及參與的機制。 首先，利用初生大鼠心肌細胞探討DEPEs對心肌細胞的毒性。將初生大鼠心肌細胞處理DEPEs (1-15 μg/mL) 24小時，發現對細胞存活率沒有顯著的影響，但細胞大小與細胞中蛋白質含量則有增加，且細胞肥大指標因子[腦鈉素(BNP)及肌凝蛋白質重鏈(β-MHC)]的基因表現量上升，顯示DEPEs具引起心肌細胞肥大的作用。另外，DEPEs亦可活化mitogen-activated protein kinases (MAPKs)，即extracellular signal-regulated kinase、c-Jun-N-terminal kinase (JNK)和p38，及其上游調控分子protein kinase C (PKC)的磷酸化程度也增加。結果顯示DEPEs可能經由PKC調控之MAPKs路徑導致初生大鼠心肌細胞肥大。 接著，利用H9c2心肌細胞株探討DEPEs (3-25 μg/mL)造成的心肌細胞毒性。將細胞處理25 μg/mL DEPEs 24小時後，細胞存活率降低，並且利用流式細胞儀技術發現，細胞會走向凋亡與壞死。同時，細胞內ATP降低，乳酸脫氫酶釋放情形也增加。利用西方墨點法顯示處理DEPEs於短時間內可使JNK磷酸化增加。內質網壓力恆定相關蛋白glucose-regulated protein 78 (GRP78)和C/EBP-homologous protein (CHOP)的表現量亦會上升，並且可被JNK抑制劑回復。結果指出DEPEs可使心肌細胞凋亡與壞死，並且可經JNK調控引起內質網壓力，但此內質網壓力在DEPEs造成的心肌細胞死亡中所扮演的角色仍須進一步探討。 綜合上述結果顯示，DEPEs可造成初生大鼠心肌細胞肥大，亦可使H9c2心肌細胞走向凋亡與壞死。此研究有助於進一步地了解DEPs造成的心血管毒性作用及參與之機制。

Evidences from epidemiological studies indicate that exposure to particulate matter (PM) air pollution contributes to cardiovascular morbidity and mortality. Fine particles with a diameter <2.5 μm (PM2.5) have an important role in triggering biological responses such as thrombosis and inflammation. Diesel exhaust particles (DEPs) with a mean size of about 0.2 &micro;m are a major component of ambient PM2.5. Moreover, the use of diesel engine powered cars has recently been increasing in the world, because diesel engines offer better fuel efficiency and lower emissions of carbon dioxide than gasoline engines, which means that vehicles running on diesel are less responsible for global warming. However, diesel engines emit more nitrogen oxides and particles than do gasoline engines. It was reported that DEP extracts (DEPEs) decreased cardiomyocytes viability and it might be due mainly to reactive oxygen species formation. However, the mechanisms by which DEPs produce adverse cardiovascular effects at the cellular level are not fully understood. In this study, we used rat neonatal cardiomyocytes and H9c2 cardiac myoblasts to investigate the effects of DEPEs. First, we used neonatal rat cardiomyocytes to determine the effects of DEPEs on cardiomyocyte hypertrophic response. Exposure to DEPEs (1-15 μg/mL) had no effects on cardiomyocyte viability. DEPEs treatment was found to elevate not only the cell size but also the ratio of total protein/cell number; additionally, the gene expression of hypertrophy markers including brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) but not atrial natriuretic peptide (ANP) in neonatal rat cardiomyocytes was also increased, which indicated that DEPEs might induce cardiomyocyte hypertrophy. Besides, DEPEs also caused an increase in phosphorylation of extracellular signal-regulated kinases, c-Jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs), upstream regulators of ANP, BNP and β-MHC. And protein kinase C (PKC), one of the upstream regulators of these MAPKs, was also activated. These results suggested that DEPEs was capable of inducing cardiomyocyte hypertrophy and implied that a PKC-regulated MAPK pathway may be involved. On the other hand, we also investigate the effects of DEPEs on H9c2 myoblast death. After DEPEs (3-25 μg/mL) treatment for 24 hours, cell viability significantly decreased. DEPEs also increased annexin-V and propidium iodide binding, indicating that DEPEs could induce apoptosis and necrosis in H9c2 cells. Exposure to DEPEs also caused an increase in phosphorylation of JNK. Expression of endoplasmic reticulum (ER) chaperone protein glucose-regulated protein 78 and pro-apoptotic factor C/EBP-homologous protein was augmented after exposure to DEPEs, which could be reversed by JNK inhibitor. Meanwhile, DEPEs triggered the depletion of intracellular ATP and increased lactate dehydrogenase release. Collectively, these data showed that DEPEs could lead to not only apoptosis but also necrosis and induce ER stress through JNK pathway. The present study demonstrated that DEPEs prompted neonatal rat cardiomyocyte hypertrophy and also induced apoptosis as well as necrosis in H9c2 myoblasts. These data provide the better understanding of potential mechanisms that link DEPs to toxicological regulation of cardiomyocytes.