| Abstract: | Acarbose為一種α-glucosidase inhibitor，已有研究指出，其可降低無症狀心肌梗塞的發生，以及減緩葡萄糖耐受性不良患者動脈壁內膜增厚之症狀。本次實驗中我們研究acarbose對於動脈粥狀硬化的影響，分別以體內和體外模式探討其對動脈粥狀硬化病程起始和發展的潛在機制。
第一部分，在動物實驗中以高膽固醇飲食(HCD)餵食紐西蘭大白兔，並給予acarbose (2.5-5.0 mg kg-1)。利用免疫組織化學染色法(IHC)評估動脈粥狀硬化病變iNOS、Ras、 PCNA、IL-6、β-galactosidase及p-AMPK之表現。實驗結果發現，在餵食HCD的紐西蘭大白兔並給予acarbose，會顯著性降低了主動脈粥狀硬化之嚴重程度與減少α-actin、PCNA、IL-6、TNF-α、Ras和β-galactosidase表現，並增加iNOS和p-AMPK的表達，但血糖、血中總膽固醇與LDL不受影響。在細胞實驗中，透過西方墨點法的結果分析顯示，以TNF-α誘導的血管平滑肌細胞(VSMC)，並加入acarbose處理，其會降低β-galactosidase和Ras並增加p-AMPK之表達，且具有劑量依賴性。此外，acarbose可恢復以AMPK抑製劑及iNOS抑製劑處理的A7r5細胞中p-AMPK和iNOS蛋白之表現，藉此探究其上下游機轉為何。綜合上述，acarbose可透過活化AMPK信號以減緩發炎反應及抑制血管平滑肌細胞之增生與爬行作用，進而減緩動脈粥狀硬化之進展。
第二部分，血管平滑肌細胞增生與爬行作用在動脈粥狀硬化中扮演關鍵的角色；血管平滑肌細胞從血管中層遷移到內膜及其隨後的增生作用是動脈粥狀硬化中新生內膜形成和冠狀動脈治療後再狹窄的重要過程。延續第一部分實驗之目的是確認acarbose於A7r5細胞中是否具有抑製Ras表達的作用，故透過轉染k-RasG12V A7r5細胞，評估acarbose對抑制Ras表現之影響。實驗結果發現，acarbose能以劑量依賴的方式抑制FAK和Akt的磷酸化，並能降低基質金屬蛋白酶MMP-2和MMP-9的活性及small G proteins (Ras，Cdc42，RhoA和Rac1)的蛋白表現。因此，本次實驗證明acarbose可以藉由阻斷small G proteins及PI3K / Akt信號傳導之路徑，有效抑制轉染k-RasG12V A7r5細胞之增生與爬行作用。綜合上述實驗結果，acarbose可透過靶向Ras信號傳導路徑來減緩動脈粥狀硬化。
第三部分，動脈粥狀硬化是心血管疾病(CVD)的主要原因，VSMC之增生與爬行作用為動脈粥狀硬化和斑塊破裂病程機制中的重要過程。上述兩部分實驗結果指出，acarbose可以減少動脈粥狀硬化的進展，其可透過靶向k-RasG12V A7r5細胞中的Ras信號傳導來減緩動脈粥狀硬化。近期許多研究主要以microRNA探討動脈粥狀硬化的進展，而acarbose作用於miRNA對動脈粥狀硬化進展的機制尚未釐清，故本研究目的為評估acarbose對糖尿病環境下誘導的A7r5細胞增生與爬行作用相關microRNA調控之影響。此次實驗以wound healing及transwell試驗測定A7r5細胞爬行作用，並透過BrdU試驗評估A7r5細胞的增生能力。結果顯示，acarbose可透過抑制FAK和PI3K/Akt，並降低Ras和small G proteins的磷酸化，以減緩細胞爬行和增生之作用；其亦上調控miR-143的表現以減少糖尿病環境下誘導的A7r5細胞爬行作用，並減少以antagomiR-143和糖尿環境下誘導的A7r5細胞中Ras、PCNA和p-FAK之表現。綜合上述實驗結果，acarbose透過上調節miR-143的表現進而抑制Ras信號傳導之路徑以減少VSMC爬行和增生之作用，進而減緩動脈粥狀硬化之病程。
First of all, acarbose (an α-glucosidase inhibitor), is reported to reduce the incidence of silent myocardial infarction and slow the progression of intima-media thickening in patients with glucose intolerance. Here we investigate other impacts of acarbose on atherosclerosis development and the underlying mechanisms of atherosclerosis initiation and progression in vivo and in vitro. Rabbits fed a high cholesterol diet (HCD) were treated with acarbose (2.5-5.0 mg kg-1). Immunohistochemistry was used to assess the expression of inducible nitric oxide synthase (iNOS), Ras, proliferating cell nuclear antigen (PCNA), IL-6, β-galactosidase, and p-AMPK in atherosclerotic lesions. Treatment with acarbose in HCD-fed rabbits was found to significantly reduce the severity of aortic atheroma and neointimal expression of α-actin, PCNA, IL-6, TNF-α, Ras, and β-galactosidase; to significantly increase expression of iNOS and p-AMPK, but not to affect serum levels of glucose, total cholesterol, and LDL. Western blot analysis showed acarbose dose-dependently decreased β-galactosidase and Ras expression and increased p-AMPK expression in TNF-α-treated A7r5 cells. In addition, acarbose restored p-AMPK and iNOS levels in AMPK inhibitor- and iNOS inhibitor-treated A7r5 cells, respectively. In conclusion, acarbose can pleiotropically inhibit rabbit atherosclerosis by reducing inflammation, senescence, and VSMCs proliferation/migration via upregulating AMPK signals.
Secondly, atherosclerosis includes proliferation and migration of vascular smooth muscle cell (VSMC). Migration of VSMC from the media into the intima and their subsequent proliferation are important processes in neointima formation in atherosclerosis and restenosis after percutaneous coronary interventions. Acarbose, an alpha-glucosidase inhibitor, was demonstrated not to affect serum levels of glucose, and decreased the progression of intima-media thickness in HCD-fed rabbits. It is worth noting finding in our previous experiment was increased Ras protein levels to enhanced migration in TNF-α treated A7r5 cells. The aim of this study was to determine the inhibitory effects of acarbose on Ras expression inA7r5 cells. By utilizing the RasG12V A7r5 cell, we tested the effect of acarbose on inhibition Ras expression. Acarbose also inhibited the phosphorylation of FAK and Akt, activities of matrix metalloproteinases (MMPs) MMP-2 and MMP-9, and protein expressions of small G proteins (Ras, Cdc42, RhoA, and Rac1) in a dose-dependent manner. Finally, we evidence that acarbose could effectively inhibit the proliferation and migration of RasG12V A7r5 cell by blocking small G proteins and PI3K/Akt signaling. These studies demonstrate that acarbose could theoretically produce a decrease in atherosclerosis by targeting Ras signals.
Finally, atherosclerosis is the primary cause of cardiovascular disease (CVD). VSMC migration and proliferation is an important process in the pathogenesis of atherosclerosis and plaque rupture. Acarbose has been shown to reduce the progression of atherosclerosis without affecting serum levels of glucose in rabbits fed a high cholesterol diet. In our previous study, acarbose suppressed atherosclerosis by targeting Ras signaling in k-RasG12V A7r5 cells. The main focus of recent atherosclerosis studies has been microRNA targets. However, the mechanism by which acarbose targets miRNA-mediated atherosclerosis remains unclear.
This study aimed to evaluate the effect of acarbose on microRNA-related regulation of A7r5 cell migration and proliferation induced by diabetic conditions. Rat aortic vascular smooth cell line was cultured with acarbose. Then, cell migration was measured by wound healing and transwell assay. The proliferative abilities of A7r5 cells were evaluated by BrdU cell proliferation assay. The expression of protein and levels of mRNA were measured by western blot analysis and real-time PCR.
Acarbose inhibited the phosphorylation of focal adhesion kinase (FAK) and phosphoinositide 3-kinases (PI3K)/ protein kinase B (Akt), Ras signals, and small GTPase proteins expression to attenuate cell migration and proliferation. Acarbose also upregulated the expression of miR-143 to decrease the migration of diabetic condition-induced A7r5 cells and reduced Ras, PCNA, and p-FAK expression in A7r5 cells co-treated with antagomiR-143 and oleic acid/high glucose.
In conclusion, acarbose reduces VSMC migration and proliferation via upregulating miR-143 to inhibit Ras signaling, and can thereby potentially decrease atherosclerosis. |
