Statins藥物在心律不整與凝血機制的多效性

Abstract

心房顫動(AF)是臨床上很常遇到心律不整，而且常常會跟栓塞性中風的發生有關。最近的研究指出發炎反應可能會跟AF的發生機轉有關，而C-反應蛋白（CRP）則跟發炎反應有很明顯的相關性。因此我們針對病人體內的CRP濃度，還有CRP的基因多型性設計了這個研究，我們希望藉由這項研究來探討CRP跟AF的發生有無關聯性，還有CRP的基因多型性是否可以用來預測AF的患者併發栓塞性中風的風險。另外針對這項研究的臨床應用方面，由於statin類藥物具有抗發炎反應的效果，在臨床上也被廣為應用，所以我們也進行了細胞學基礎實驗來探討statin類藥物在凝血機制的多效性。 在這個研究的第一部份，我們針對地域上不同的兩個獨立群體進行了CRP的基因多型性與AF之間的遺傳關聯性研究（I：100位AF患者和101位對照組; II：348位AF患者和356位對照組）。我們在CRP基因的1-kb啟動子(promoter)中發現了三種不同的基因多型性（T-861C，A-821G和C-390A / C-390T）。在這些基因多型性裡，我們發現C-390A⁄ C-390T這三種等位基因多型性(triallelic polymorphism)帶有決定全部個體表現型的資訊(haplotype information)，而且會影響CRP基因的啟動子活性與受試者體內的CRP濃度。另外我們也發現C-390A⁄ C-390T這個基因多型性幾乎會跟外顯子2(exon2)的基因多型性G1059C連帶一起遺傳(linkage disequilibrium)。我們也發現帶有-390A這個基因多型性的受試者除了有較高的CRP基因啟動子活性、有較高的CRP濃度，還會跟AF的發生風險有關。我們也發現CRP會增加心房肌細胞(atrial myocytes)的內向L型鈣電流(inward L-type calcium current)，但是不會影響心房纖維母細胞(atrial fibroblasts)的第I型α1（COL1A1）、第III型α1（COL3A1）和第1型α2（COL1A2）等前膠原(procollagens)的表現。這也解釋了CRP誘發AF產生的分子機轉。 在這個研究的第二部份，我們針對AF的患者追蹤超過十年的時間。我們分析了這些人的C-390A⁄ C-390T等位基因多型性，並且依照他們的CRP 濃度分成四組。我們發現具有最高CRP濃度的組別發生腦中風的機率比較高(hazard ratio= 2.27, 95% confidence interval 1.08-4.81; the lowest CRP quartile as the reference group)。除此之外我們也發現，假如AF的患者帶有A-390 或者是 T-390的等位變異基因，他們後續發生腦中風的風險也會提高(3.35±2.71 vs 2.43±2.00 mg/L)。這種由基因多型性所造成的影響在我們把傳統的危險因子納入迴歸分析後也依舊顯著(hazard ratio= 2.07, 95% confidence interval 1.23-3.48)。我們因此證實了CRP 的基因多型性會與AF的患者發生腦中風有關。 在這個研究的第三部份，我們探討了statin類藥物對於抗血栓作用的分子機轉。我們使用了肝癌細胞（HepG2）、protein C基因啟動子，還有螢光素酶檢測法(luciferase reporter)來作statin類藥物調控protein C基因轉錄(transcription)功能的細胞學研究。另外我們也使用了Wistar大鼠，並且餵食牠們simvastatin 藥物（5mg•kg-1•d-1）來進行動物實驗。在細胞學實驗中我們發現simvastatin可以增加肝細胞的protein C表現 (361±64% and 313±59% after 2 hrs and 6 hrs of stimulation, respectively, both p< 0.01)。在動物實驗裡，被餵食simvastatin的大鼠有較高濃度的protein C表現(8.4 ± 3.2 unit/ml in controls vs. 22.7 ± 15.2 unit/ml for 1 week, and 20.3 ± 16.9 unit/ml for 2 weeks, respectively, both p< 0.05)。另外我們也發現，除了protein C啟動子的活性會因為simvastatin的刺激而增加外，肝細胞核因子1α (hepatocyte nuclear factor 1α, HNF1α)也會增加。而在HNF1α不存在或者是Rac1持續活躍時則會抑制simvastatin影響protein C的效果。 我們藉由這個研究找出了誘發AF並且影響AF併發栓塞性腦中風的基因多型性，而這些基因多型性都跟發炎反應有關，除此之外我們也探討了statin類藥物調控凝血機制的分子機轉。我們希望藉由這個研究來提供AF患者後續使用statin類藥物預防AF產生與併發腦中風的理論基礎。

Atrial fibrillation (AF) is the most common arrhythmia in clinical practice, and is associated with thromboembolic stroke. Recent studies have demonstrated the role of inflammation in the mechanism of AF. C-reactive protein (CRP) is closely related with inflammation reaction. Therefore, our project is designed to investigate the effect of serum CRP level and genetic polymorphisms of CRP on the incidence of thromboembolic events in patients with AF. Furthermore, statins are well clinically used for anti-inflammation, we also try to investigate how statins prevent inflammation-related pro-coagulation by basic cellular studies. In the section I of the project, a genetic association study between CRP gene polymorphisms and AF was performed in two independent populations (I: 100 AF patients and 101 controls; II: 348 AF patients and 356 controls). Three polymorphisms (T-861C, A-821G and C-390A⁄ C-390T) were found in the 1-kb promoter of CRP. A triallelic polymorphism (C-390A⁄ C-390T) captured all haplotype information and determined the CRP gene promoter activity and the plasma CRP level, and was in nearly complete linkage disequilibrium with G1059C polymorphism in exon 2. The -390A variant was associated with a higher CRP gene promoter activity, a higher plasma CRP level and a higher risk of AF. Patients with AF also had a higher plasma CRP level than controls. CRP significantly increased the inward L-type calcium current in atrial myocytes with no changes in other ionic currents. CRP did not affect the expressions of type I alpha 1 (COL1A1), type III alpha 1 (COL3A1) and type 1 alpha 2 (COL1A2) procollagens in atrial fibroblasts. In the section II of the project, a total of 725 AF patients were longitudinally followed-up for more than 10 years. CRP gene promoter triallelic polymorphism (C-390A/C-390T) were genotyped and CRP levels were checked. After adjustment for conventional risk factors, patients with higher CRP levels were more likely to develop thromboembolic stroke compared to those with the lowest CRP quartile (hazard ratio= 2.27, 95% confidence interval 1.08-4.81; the lowest CRP quartile as the reference group). Patients carrying the A-390 or T-390 allele had higher CRP levels (3.35±2.71 vs 2.43±2.00 mg/L), and were more likely to develop thromboembolic stroke, even after adjustment for conventional risk factors (hazard ratio= 2.07, 95% confidence interval 1.23-3.48). In the section III of the project, we conducted the study to investigate the potential molecular mechanisms for the anti-thrombotic effect of statins. Cultured human hepatoma cells (HepG2) were used as the in vitro model. The human protein C gene promoter was cloned into the luciferase reporter to study the transcriptional regulation of human protein C gene. Wistar rats fed with simvastatin (5 mg • kg–1 • d–1) were used as the in vivo model. We found simvastatin increased the expression of protein C in hepatocytes (361±64% and 313±59% after 2 hrs and 6 hrs of stimulation, respectively, both p< 0.01). In the animal study, the serum protein C levels were increased in the simvastatin treated group (8.4 ± 3.2 unit/ml in controls vs. 22.7 ± 15.2 unit/ml for 1 week, and 20.3 ± 16.9 unit/ml for 2 weeks, respectively, both p< 0.05). The level of hepatocyte nuclear factor 1α (HNF1α) was also increased in both the in vivo and in vitro models. We found that the protein C promoter activity was increased by simvastatin, and this effect was inhibited by HNF1α knockdown and constitutively active Rac1. Our project identify inflammation-related genetic risk factors for AF occurrence, AF-related thromboembolic events, and establish the beneficial effects of statins in reducing AF complication.