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標題: | 探討共同處理懸浮微粒和高脂飲食所誘發之心肌受損及其相關機轉 To study the effects of combined exposure to particulate matter 2.5 and high fat diet-induced cardiac damage and the related mechanisms |
作者: | Szu-Ju Fu 傅斯? |
指導教授: | 陳玉怜(Yuh-Lien Chen) 陳玉怜(Yuh-Lien Chen | ylchenv@ntu.edu.tw | ), |
關鍵字: | 棕梠酸,懸浮微粒,細胞凋亡,纖維化,粒線體氧化壓力,粒線體分裂,粒線體自噬,褪黑激素, PA,PM,apoptosis,fibrosis,mitochondrial ROS,mitochondrial fission,mitophagy,melatonin, |
出版年 : | 2022 |
學位: | 碩士 |
摘要: | 高脂飲食是誘發心血管疾病的風險因子之一。根據過去的研究指出,空氣中的細懸浮微粒 (particulate matter,PM) 進入呼吸道後,與肺泡接觸進入血液循環,進而導致心血管疾病的產生。目前對於PM在高脂飲食下對心臟功能及粒線體受損所造成的影響仍不清楚。本研究動物模式利用高脂飲食 (high-fat diet,HFD) 餵食小鼠,並利用氣管內注射PM 10 mg/kg模擬暴露空氣污染之環境。結果發現小鼠在共同處理HFD和PM後,在心臟收縮及舒張功能皆有受損的情形。細胞凋亡相關因子PUMA (p53 upregulated modulator of apoptosis) 及纖維化相關蛋白Fibronectin 表現量皆會因HFD或PM單獨處理而上升;而共同處理HFD及PM時,PUMA及Fibronectin表現量則更顯著增加。此外,我們利用大鼠心肌母細胞 (H9c2) 作為體外細胞模式,並以50 μM 棕梠酸 (palmitic acid,PA) 與10 μg/mL PM模擬高脂飲食與空氣汙染之環境。經由TUNEL及Annexin V/PI方法,發現心肌細胞處理PA及PM會加劇細胞凋亡。根據西方墨點法結果,發現細胞在PA+PM共同處理下,PUMA、CASPASE3顯著上升而BCL2 (B-cell lymphoma 2) 顯著下降。接著,透過MitoSOX Red染色,發現PA及PM處理後會顯著增加粒線體活性氧化物 (mitochondrial reactive oxygen species) 產生。此外,相較於個別處理組別,PA+PM處理後,觀察JC-1染色發現粒線體膜電位下降,且ATP產量減少。同時,在Mitotracker染色發現粒線體在經由PA+PM處理後粒線體長度明顯變短,表示分裂增加。經西方墨點法的實驗結果顯示,粒線體分裂相關蛋白:p-DRP1 (phospho-dynamin-related protein 1) 和FIS1 (mitochondrial fission 1 protein) 的表現與個別處理PA和PM組別相比,顯著上升。而粒線體自噬相關蛋白:p62、LC3B (microtubuleassociated protein 1 light chain 3 Beta)、BNIP3 (BCL2 interacting protein 3) 在PA+PM共同處理下則有表現量顯著增加。在纖維化相關蛋白Fibronectin 和ETS-1 (ETS proto-oncogene 1) 表現量均顯著增加。根據結果得知,高脂與PM的環境下會導致粒線體受損加重,進而使心肌產生凋亡及纖維化。此外,利用粒線體分裂抑制劑Mdivi-1及粒線體氧化壓力抑制劑MitoQ處理,結果發現可顯著減少PA+PM共同處理下導致的細胞凋亡、纖維化。進一步使用氧化壓力抑制劑褪黑激素 (melatonin) 進行處理,發現其可有效地改善PA+PM所造成的粒線體受損,氧化壓力下降和粒線體膜電位增加,而粒線體分裂和自噬相關蛋白p-DRP1、p62、LC3B及BNIP3表現量均會減少,凋亡因子PUMA及纖維化相關蛋白Fibronectin 和ETS-1表現也會下降。由上述結果可以證明,在共同處理高脂及細懸浮微粒下,細胞的氧化壓力、細線體分裂增加並使粒線體功能下降,並且加劇細胞凋亡因子和纖維化蛋白的表現,而褪黑激素則可顯著降低PA+PM所誘發的細胞凋亡及纖維化。未來仍需要更深入探討褪黑激素在心血管疾病中提供保護效用之機制,希望其可成為以後治療高脂及空氣汙染引起心血管疾病用藥的選擇。 High-fat diet (HFD) is a risk factor for cardiovascular disease. According to previous studies, particulate matter (PM) in the air enters the respiratory tract, contacts with the alveoli and enters the blood circulation, leading to the onset of cardiovascular disease. Currently, the affect of PM on cardiac function and mitochondrial damage under a high-fat diet is still unclear. In this study, the experimental animal model was established in mice fed with high-fat chow diet and intratracheal injection of PM 10 mg/kg to simulate the environment exposed to HFD and air pollution. These results showed that both HFD and PM deteriorated the systolic and diastolic cardiac function in mice. Apoptosis-related factor PUMA (p53 upregulated modulator of apoptosis) and fibrosis-related protein Fibronectin were both increased in mice treated either with HFD or PM alone, while in mice co-treated with HFD and PM, the expression of PUMA and Fibronectin were significantly increased. In addition, we used rat cardiomyocytes (H9c2) as an in vitro cell model, using 50 μM palmitic acid (PA) and 10 μg/ml PM to simulate the environment of high-fat diet and air pollution. Apoptosis aggravated in cardiomyocytes treated with PA and PM using TUNEL and Annexin V/PI assays. Western blot results showed that PUMA and CASPASE3 were significantly increased and BCL2 (B-cell lympha 2) was significantly decreased under the combined treatment of PA+PM. Combined exposure to PA and PM increased the production of mitochondrial reactive oxygen species (ROS) by MitoSOX Red staining. Moreover, PA+PM decreased the mitochondrial membrane potential by JC-1 staining and significantly diminished the ATP production compared with the singal treatment group. In addition, PA+PM treatment significantly shortened the mitochondrial length using Mitotracker staining, indicating the mitochondrial fission. The expression of mitochondrial fission-related proteins, p-DRP1 (phospho-dynamin-related protein 1) and FIS1 (mitochondrial fission 1 protein) were significantly increased compared with the PA or PM treated groups alone. Moreover, mitophagy-related proteins, p62, LC3B (microtubule-associated protein 1 light chain 3 beta) and BNIP3 (BCL2 interacting protein 3) were significantly increased under PA+PM co-treatment. Also, the expression of fibrosis-related proteins fibronectin and ETS-1 (ETS proto oncogene 1) were significantly increased in H9c2 cells treated with PA and PM. According to these results, combined exposure to PA and PM deteriorated mitochondrial function and led to cardiac apoptosis and fibrosis. In addition, mitochondrial division inhibitor (Mdivi-1) and mitochondrial ROS inhibitor (MitoQ) significantly reduced PA+PM-induced apoptosis and fibrosis. Finally, melatonin, a ROS scavenger was used in this study. Melatonin effectively improved the mitochondrial dysfunction induced by PA+PM, decreased the mitochondrial ROS and alleviated the collapse of mitochondrial membrane potential. In addition, melatonin decreased the PA+PM-induced upregulation of mitochondrial fission and mitophagy-related proteins p-DRP1, p62, LC3B and BNIP3, and apoptosis-related factors PUMA, CASPASE3 and fibrosis-related proteins Fibronectin and ETS-1. In summary, co-exposure to HFD (PA) and PM increased oxidative stress, mitochondrial fission and mitophagy, decreased mitochondrial function, and further aggravated cardiac apoptosis and fibrosis. Melatonin significantly reduced the cardiac apoptosis and fibrosis induced by HFD+PM or PA+PM. In the future, the mechanism of melatonin underlying the protective effects against HFD and PM-induced cardiovascular diseases warrants further investigation, and melatonin may become a therapeutic intervention against high-fat and air pollution-induced cardiovascular diseases. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/83753 |
DOI: | 10.6342/NTU202202132 |
全文授權: | 未授權 |
顯示於系所單位: | 解剖學暨細胞生物學科所 |
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