苯芘對於缺氧狀態下所誘導的血管新生的影響：多環芳香烴受體角色之探討

Abstract

苯芘屬於多環芳香烴的一員，而多環芳香烴是一種人體經常接觸到的環境污染物，主要是由有機物質的不完全燃燒所產生，其暴露來源主要有交通運輸廢氣、工廠廢氣、香菸及碳烤食物等。過去文獻指出多環芳香烴與缺血性心血管疾病發生有相關性。而血管新生作用在缺血性心血管疾病中扮演著相當重要的角色，因為血管新生作用可避免組織因過度缺血氧而壞死。而且多環芳香烴受體也已被證實會參與在血管新生的調控。此外，過去研究文獻指出苯芘的代謝活化是透過多環芳香烴受體而去啟動下游代謝酵素CYP1A1、CYP1B1的基因轉錄活化，將其代謝成為benzo[a]pyrene-7,8- dihydrodiol-9,10-epoxide (BPDE)。因此，本篇的研究目的欲探討在苯芘前處理的人類臍靜脈內皮細胞是否透過AhR去影響缺氧狀態下的血管新生作用。 本實驗將人類臍靜脈內皮細胞以不同濃度的苯芘處理觀察細胞經由苯芘處理後，對於缺氧狀態下(0.5% O2)所誘導的管狀構造形成作用是否有影響，並且以缺氧狀態培養的方式，來觀察前處理苯芘的人類臍靜脈內皮細胞對於缺氧狀態所造成的缺氧誘導因子-1 alpha累積現象的影響並藉由HRE promoter reporter assay觀察苯芘所抑制的缺氧誘導因子-1 alpha累積是否會影響其轉錄活化的能力。接著，以缺氧狀態培養的方式，來觀察苯芘前處理人類臍靜脈內皮細胞對於缺氧狀態所促進HIF-1 alpha下游基因VEGF的表現量的影響。此外，以不同濃度及不同時間點的苯芘處理，藉由西方墨點法觀察苯芘的處理是否會造成人類臍靜脈內皮細胞的AhR活化及誘導其下游CYP1A1、CYP1B1蛋白質表現，同時也進一步去觀察AhR的蛋白質表現。接著，再以AhR拮抗劑alpha-naphthoflavone (alpha-NF)前處理人類臍靜脈內皮細胞的方式來進行實驗，觀察抑制AhR活化對於苯芘所減少缺氧狀態下的管狀構造形成及HRE 活性是否有所影響。此外，也利用AhR shRNA將人類臍靜脈內皮細胞進行基因靜默的方式來進行實驗，觀察AhR靜默後對於苯芘所減少缺氧狀態下的管狀構造形成是否有影響。最後，本實驗利用XRE及HRE promoter reporter assay觀察人類臍靜脈內皮細胞前處理苯芘後，再置於缺氧狀態下，XRE及HRE兩者活性的消長關係並且利用免疫沉澱法觀察苯芘處理是否會影響HIF-1 alpha與ARNT (HIF-1 alpha)之間的鍵結程度。 本研究結果顯示，苯芘能抑制缺氧狀態所誘導的人類臍靜脈內皮細胞管狀構造形成作用，並且抑制缺氧狀態所造成的缺氧誘導因子-1 alpha累積之現象，同時也抑制缺氧狀態所活化的HRE活性，進而抑制缺氧誘導因子-1 alpha下游基因VEGF mRNA表現。而且，苯芘能誘導人類臍靜脈內皮細胞的AhR活化及誘導其下游CYP1A1、CYP1B1蛋白質表現，同時也造成AhR的蛋白質表現減少。此外，利用AhR的拮抗劑alpha-NF及AhR shRNA可以回復苯芘所減少缺氧狀態下所誘導的管狀構造形成。因此，證明了AhR的確參與在苯芘所減少缺氧狀態下所誘導的管狀構造形成當中。然而，在HRE/XRE冷光酶活性分析法及免疫沉澱法等實驗結果得知苯芘所抑制的缺氧狀態活化之HRE活性不是經由AhR與HIF-1 alpha競爭ARNT (HIF-1 beta)。

Benzo[a]pyrene (B[a]P) belongs to one of polycyclic aromatic hydrocarbons (PAHs), which is produced during incomplete combustion of organic materials and found in diesel exhaust, charcoal-broiled food, industrial waste, and cigarette smoke. Several toxicological and epidemiological studies indicate that exposure to PAHs is a risk factor for ischemic heart disease. In physiology, angiogenesis is thought as a protective defense mechanism against tissue ischemia. Some researches show that aryl hydrocarbon receptor (AhR) is involved in regulation of angiogenesis. B[a]P binds to AhR and subsequent translocates to the nucleus to activate the downstream gene expression, such as CYP1A1 and CYP1B1, to metabolite B[a]P to benzo[a]pyrene-7,8- dihydrodiol-9,10-epoxide (BPDE). We have investigated whether AhR involved in the effects of B[a]P on hypoxia-induced angiogenesis. First, we pretreated HUVECs with different concentration of B[a]P (0.3, 1, 3, 10 uM) and then under hypoxia condition (0.5% O2) to observe the tube formation assay, HIF-1 alpha protein accumulation, HRE promoter reporter assay and the downstream VEGF mRNA expression. Second, we treated human umbilical vein endothelial cells (HUVECs) with different concentration of B[a]P (0.3, 1, 3, 10 uM) in different time points (3, 6, 12, 24, 48 hours) to observe the AhR, CYP1A1 and CYP1B1protein expression. In order to investigate whether AhR involved in the effects of B[a]P on hypoxia-induced angiogenesis, We used the AhR antagonist, alpha-naphthoflavone (alpha-NF), to observe the effects of B[a]P on the hypoxia-induced tube formation and HRE activity. Besides, we also used AhR shRNA to observe the effects of B[a]P on the hypoxia-induced tube formation. In addition, we used XRE/ HRE promoter reporter assay and immunoprecipitation assay to observe AhR, HIF-1 alpha and ARNT (HIF-1 alpha) interation. In these results, we found that B[a]P 10 uM inhibited the hypoxia-induced tube formation in HUVECs. In the western blotting, B[a]P 10 uM decreased the HIF-1 alpha protein accumulation in HUVECs under hypoxia. B[a]P 10 uM decreased HRE activity and down-regulated VEGF mRNA expression in HUVECs. These results show that B[a]P impaired HIF-1 alpha protein accumulation to decrease HRE activity and VEGF mRNA expression, and then these effects cause the hypoxia-induced tube formation inhibited by B[a]P. As we known B[a]P induces CYP1A1, CYP1B1 through binding to and activating AhR. We also observe that B[a]P through activating AhR to induce CYP1A1 and CYP1B1 protein expression and down-regulate AhR protein expression in the time- and dose-dependent response in HUVECs. In the results, alpha-NF can reverse the inhibition of B[a]P on the hypoxia-induced tube formation and HRE activity. Besides, AhR shRNA also can reverse the inhibition of B[a]P on the hypoxia-induced tube formation. Therefore, these results suggest that AhR is involved in B[a]P-inhibited hypoxia-induced tube formation. In addition, the XRE/ HRE promoter reporter assay and immunoprecipitation assay showed that the inhibition of B[a]P on the hypoxia-induced HRE activity is not due to ARNT (HIF-1 beta) competion.