嚼食檳榔與心血管疾病之探討—從臨床至基礎醫學

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Title:	嚼食檳榔與心血管疾病之探討—從臨床至基礎醫學 The Survey Between Betel Nut Chewing and Cardiovascular Disease- From Clinical to Basic Medical Science
Authors:	林文元;Wen-Yuan Lin
Contributors:	醫學院臨床醫學研究所博士班
Keywords:	檳榔;心血管疾病;肥胖;死亡率;心肌肥大;凋亡;纖維化;betel nut;cardiovascular disease;obesiy;mortality;cardiac hypertrophy;apoptosis;fibrosis
Date:	2010
Issue Date:	2010-09-29 12:16:44 (UTC+8)
Abstract:	Part I臨床醫學嚼檳榔已成為全球第四大成癮習慣，預估目前全球有超過10%以上（約6億）的人口有此習慣。近年來的研究正逐漸發現嚼食檳榔與許多慢性病有關（如心血管疾病、糖尿病、慢性腎病、代謝症候群等），其中心臟疾病、腦血管疾病、糖尿病、慢性腎病皆名列我國前十大死因（2006年分居第2、3、4、8位）其所產生的嚴重性，早已超過癌症。根據國內的臨床流行病學資料已發現嚼食檳榔會增加得到肥胖、第二型糖尿病、心臟病及代謝症候群的機率。在臨床醫學研究方面，首先我們使用1998至1999年在全國四家美兆健檢中心共56,116位20歲以上男性受試者的資料研究，經八年的追蹤後，發現心血管疾病死亡率及任何原因死亡率，經調整年齡、身體質量指數、糖尿病、高血壓、血脂、抽菸、喝酒、運動習慣、收入及教育程度後，比起“從未嚼食檳榔者”，心血管疾病死亡率及任何原因死亡率在“已戒除嚼食檳榔者”分別為1.56（1.02 ~2.38）及1.40（1.17 ~1.68），而在“目前正嚼食檳榔者”則各為2.02（1.31 ~3.13）及1.40（1.16 ~1.70），同時隨著嚼食頻率的增加，死亡率也隨之增加，顯見有“劑量效應”，故嚼食檳榔會增加心血管疾病死亡率及任何原因死亡率的增加。那究竟是何種原因導致心血管疾病死亡率的增加？我們另外使用“台中社區健康調查”，此調查乃針對台中市40歲以上的居民，作二階段隨機分層抽樣，共有2,359位居民參與此研究，同理，我們只分析1,049位男性，去探討嚼食檳榔的累積量與全身性肥胖及中央性肥胖的相關，結果，我們使用多重邏輯式回歸分析及多重線性回歸分析皆發現，嚼食檳榔與全身性/中央性肥胖呈現正相關，經調整年齡、糖尿病、高血壓、血脂、抽菸、喝酒、運動習慣、收入及教育程度後，與“從未嚼食檳榔者”相比，低度累積檳榔者的勝算比各為1.78（1.07 ~2.96）及1.19（0.70 ~2.02），而在高度累積檳榔者的勝算比為2.01（1.18 ~3.41）及1.89（1.10~ 3.23），同時隨著劑量的增加而可看到肥胖的比率也隨之增加，顯示有“劑量效應”，而在多重線性回歸分析中也發現經調整干擾因子後，嚼食檳榔累積量與身體質量指數及腰圍皆呈現正相關，由此可知肥胖與嚼食量有莫大的正相關。由上述兩種研究我們發現，嚼食檳榔可能是先透過身體體重及脂肪的增加，接著再使得心臟的負荷增加或心血管粥狀硬化，導致心血管疾病的死亡率也隨之大增，再加上檳榔引起之癌症增加，並進一步增加任何死因死亡率。 Part II基礎醫學由文獻中我們發現嚼食檳榔可能會透過影響心血管疾病而增加心血管疾病死亡率，但其確切機轉十分不明朗，而過去關於檳榔的研究多著重於癌症的病理機制，對於心臟方面的影響並不多，故本研究以動物實驗先了解嚼食檳榔所引起的型態上的改變，包括心臟重量的變化、血管病變的變化、心肌細胞型態改變、甚至細胞凋亡及纖維化產生而誘發心臟疾病之罹患，接著再進一步探討檳榔主要成分(arecoline)誘導心肌細胞走向程式性細胞凋亡（apoptosis）時，所透過之訊息傳遞途徑變化及分子機制，藉以了解檳榔主要成份影響心肌功能之機轉。首先，我們將SD大白鼠分成三組，每組各八隻，第一組是控制組（control group），第二組及第三組為實驗組，以連續每天腹腔注射檳榔鹼（arecoline）誘發大白鼠傷害之動物模式，來觀察arecoline對心肌組織的影響，實驗組分為每天注射5mg/kg為低劑量組（low group）及每天注射50mg/kg為高劑量組（high group），每天記錄其體重變化，試驗期間為二週，二週後進行心臟超音波檢查後，採斷頭犧牲並收集心臟組織以進行分析實驗結果。我們將大白鼠心臟組織切片後，分別以H&E染色觀察心臟腔室與壁厚的變化及細胞型態上的改變，以TUNEL/DAPI染色觀察心肌細胞凋亡程度，並以Trichrome染色觀察心肌纖維化的變化，再以western blotting來探討arecoline所影響心肌組織肥大、凋亡及纖維化之相關訊息傳遞路徑。從心臟組織切片H&E染色觀察可發現arecoline誘導之大白鼠心臟整體有變大的趨勢，呈現拉長型肥大的現象，其左心室的心肌組織有受拉扯的情形而使得左心室腔壁厚度變薄、腔室也變得較寬較大，由western blotting分析發現大白鼠心臟組織之MEK5-ERk5及pJAK-stat3蛋白表現量皆有上升趨勢，因此我們初步判斷arecoline會活化心臟組織之IL-6-MEK5-ERk5及IL-6-pJAK-stat3肥大路徑來造成心肌細胞拉長型肥大，同時arecoline也會影響calcineurin-NFAT3- GATA4的蛋白表現量上升而導致心肌肥大。由心臟組織切片經TUNEL染色我們觀察到arecoline會導致心臟組織的細胞凋亡現象，接著再以western blotting檢測其所透過之訊息傳遞途徑，發現arecoline會經由death receptor-dependent外在訊息途徑活化FasL、Fas、FADD的蛋白表現，並活化下游的caspase 8及caspase 3而誘導心肌細胞走細胞凋亡，同時也會經由活化death receptor-independent內在途徑，增加促凋亡蛋白Bak的表現及抑制抗凋亡蛋白Bcl2、Bclxl的表現，而造成cytochrome c自粒線體中釋放大量增加，而活化caspase 9、caspase 3等切割蛋白，造成細胞凋亡的發生。由Trichrome染色中也可發現arecoline誘導傷害之大白鼠心肌組織有較多不規則藍色膠原蛋白纖維產生，顯示arecoline會促進心臟纖維化的情形發生，再經由western blotting分析可得知arecoline會活化纖維化訊息傳遞路徑之TGFβ-pSmad-CTGF蛋白的表現量上升，進而造成心臟纖維化的傷害，同時我們也發現tPA、uPA及MMP9蛋白表現量皆有上升的趨勢，顯示arecoline的確會造成心肌組織的纖維化。由上述結果我們可知，經由腹腔注射檳榔鹼（arecoline）之SD大白鼠在二週的實驗期間內，低劑量（5mg/kg/day）檳榔鹼即會誘發大白鼠心臟組織拉長型肥大的現象、同時造成心肌凋亡及心臟纖維化的傷害，而高劑量（50mg/kg/day）檳榔鹼造成的傷害也越加嚴重。 Part I: Clinical Medical Science Betel nut (Areca catechu) is the fourth most widely used addictive substance in the world. There are ~600 million people (10 % of all the people in the world) who chew betel nut. Recent studies had reported that betel nut chewing is associated with the increased risk of many chronic diseases, such as cardiovascular disease (CVD), diabetes, chronic kidney disease, and metabolic syndrome). Coronary heart disease, cerebral vascular disease, diabetes, and chronic kidney disease were among top 10 causes of death (2nd, 3rd, 4th, and 8th cause of death in 2008). The burden of these diseases was superior to cancer-related death. Previous studies in Taiwan had found that betel nut chewing increased the risk of obesity, type 2 diabetes, coronary heart disease, and metabolic syndrome. In the first study, we aimed to to investigate the association between betel nut chewing and CVD and all-cause mortality. A baseline cohort of 56,116 male participants, aged 20 and above, was recruited from four nationwide health screening centers in Taiwan from 1998 to 1999. Cox proportional hazards regression analyses were used to estimate the relative risks (RRs) of CVD and all-cause mortality for betel nut chewers during an 8-year follow-up period. There were 1,549 deaths during the follow-up period; of these, 309 deaths were due to CVD. Adjusted for age, BMI, diabetes, hypertension, lipids, smoking, alcohol drinking, physical activity, income, and education level, the RRs (95% CI) of CVD and all-cause mortality among the former betel nut chewers were 1.56(1.02,2.38) and 1.40(1.17,1.68), respectively, and 2.02(1.31,3.13) and 1.40(1.16,1.70), respectively among current chewers compared with individuals who had never chewed betel quid. Current and former betel nut chewers had higher risk of CVD mortality (RR=2.10, P<0.05) than current and former smokers. Furthermore, increased betel nut chewing frequency was associated with increased CVD and all-cause mortality. In the second study, we investigated the association between betel nut chewing and general obesity (body mass index≥25 kg/m2) and central obesity (waist circumference ≥ 90cm). A total of 1049 male subjects, aged 40 years and above, were recruited from Taichung city in Taiwan in 2004. The relationships between betel nut chewing and general and central obesity were studied by multiple linear and logistic regression analyses. The prevalence of current and former betel nut chewing was 7.0% and 10.5% in our male Taiwanese cohort. Current/former betel nut chewers had a higher prevalence of general and central obesity than never chewers. Adjusted for age, diabetes, hypertension, lipids, smoking, alcohol drinking, physical activity, income, and education level, the odds ratios(95% CIs) of general and central obesity among the lower consumption of betel nut chewers were 1.78(1.07,2.96) and 1.19(0.70,2.02), respectively, compared to 2.01(1.18,3.41) and 1.89(1.10,3.23), respectively among higher consumption chewers compared to individuals who had never chewed betel. The increasing odds ratios of general and central obesity with higher betel nut consumption revealed dose-response effects. Using multiple linear regression analyses, after adjusting for potential confounders, betel nut consumption was statistically significantly associated with body mass index and waist circumference. From these two studies, we proposed that betel nut chewing increased body weight and body fat, then it may increase heart burden or induce atherosclerosis. Finally, betel nut chewing increased the risk of CVD mortality. Furthermore, betel nut chewing also increased the risk of cancer death. Therefore, betel nut chewing increased all-cause mortality. Part II: Basic Medical Science From the literatures review, we found that betel nut chewing increased the risk of cardiovascular disease (CVD) mortality, but the exact mechanism is still unclear. Previous studies regarding to betel nut focused on the pathophysiology of betel nut related cancers, not on the heart. Therefore, this animal study focused on the morphologic change on heart and cardiac-related function, such as the weight change of heart, morphologic change of cardiomyocyte, cell apoptosis, and fibrosis. Then we further investigated the signal pathway of cell apoptosis and fibrosis on cardiomyocyte using the major component (arecoline) of betel nut. In order to identify the effects of betel nut on the mechanisms behind the development of pathologic hypertrophy, apoptosis and cardiac fibrosis of cardiomyocytes, we evaluate the acute effect of betel nut on rats with arecoline for 14 days to investigate the heart function by analyses including hypertrophy detection(ratio of heart weight/body weight), apoptosis detection(Western blot, TUNEL, DAPI assays), cardiac fibrosis detection (Immunochemistry assay), and the related signal pathways involving in hypertrophy, apoptosis and fibrosis. SD rats were divided into 3 groups. Each group has 8 rats. Arecoline was injected into SD rat via intra-peritoneal (IP) method to see the effects of arecoline on cardiomyocyte. First group is control group (IP normal saline). Second group (low dose group) was injected via IP with 5 mg/kg arecoline. Third group (high dose group) was injected via IP with 50mg/kg arecoline. Weight was recorded everyday for 2 weeks. After 2 weeks, we did cardiac echo for every SD rat, then these rats were scarified and collected their heart tissue to do further evaluation. There are three major findings in this study. First, from the result of H& E stain on heart tissue biopsy, we found that arecoline induced the increase of heart volume which showed eccentric hypertrophy. The wall of left ventricle got thin and the chamber got wide and enlarged. The protein of MEK5-ERk5 and pJAK-stat3 of heart tissue increased using western blotting analysis. Therefore, we proposed that arecoline activated the hypertrophic pathway of IL-6MEK5-ERK5 and IL-6-pJAK-stat3, and then induced the hypertrophy of cardiomyocyte. Meanwhile, arecoline induced cardiac hypertrophy via calcineurin-NFAT3-GATA4 pathway. Second, TUNEL stain also found that arecoline induced cell apoptosis on cardiac tissue. We also used western blotting to test the signal pathway which showed that arecoline activated the protein expression of FasL, Fas, and FADD (Death receptor-dependent pathway), then activated caspase 8 and caspase 3 and lead to cell apoptosis of cardiomyocyte. Arecoline also activated death-receptor independent pathway (increased the expression of Bak and inhibited the expression of Bcl2 and Bclxl, then enhanced the cytochrome c release from mitochondria. Finally, arecoline activated caspase 9 and caspase 3 to induce cell apoptosis. Third, arecoline induced cardiomyocyte fibrosis from the Trichrome stain (increased blue irregular collagen fiber). From western blotting analyses, we found that arecoline induced the increase of expression of TGFβ-pSmad-CTGF which caused cardiac fibrosis. We also found that the protein expression of tPA, uPA, and MMP9 increased. From above findings, we proposed that arecoline did increase the fibrosis of cardiac tissue. In summary, we used IP injected SD rats for 2 weeks. We found that low-dose arecoline (5mg/kg) induced eccentric hypertrophy of cardiac tissue, caused cell apoptosis of cardiomyocyte and finally resulted in the fibrosis of cardiomyocyte. High-dose arecoline also caused more damage than low-dose arecoline in SD rats.
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