鼠動脈平滑肌細胞中待匹力達抑制骨質素分泌及基因表現的機制

Dipyridamole Suppresses High Glucose-Induced Osteopontin Secretion and mRNA Expression in Rat Aortic Smooth Muscle Cells

謝銘松(Ming-Song Hsieh)

指導教授 ： 李宏謨

臺北醫學大學/藥學院/藥學系(博士班)/博士(2010年)

https://doi.org/10.6831/TMU.2010.00106

摘要

中文摘要
第一部份
實驗目的是利用體外的鼠動脈平滑肌細胞培養當實驗模式，探討在高糖狀態下，待匹力達(Dp)透過抑制PDE而使骨質素分泌降低的訊息傳導路徑。利用體外的鼠動脈平滑肌細胞培養當實驗材料，探討(一)在高糖狀態下，骨質素是否會增加分泌? (二) 待匹力達(Dp)在細胞處於高糖狀態下如何抑制骨質素? (三)探討待匹力達(Dp)抑制骨質素分泌降低的機轉為何? (四)利用多種抑制劑來驗證待匹力達(Dp)抑制骨質素的機轉。 在高糖血管細胞分子訊息研究，結果已強力證實鼠動脈平滑肌細胞(RASMCs)在高糖狀態下，確實是經由ROS而使骨質素分泌增加；待匹力達(Dp)的藥理作用是透過抑制cAMP與cGMP的分解,進而提高cAMP與cGMP 的濃度，接著透過PKA及PKG提升thioredoxin的蛋白濃度，而達到抑制ROS，而使骨質素分泌降低，而且我們使用的抗氧化劑l-NAC，抑制劑H89、KT8523、DNCB等多方面的實驗都產生抑制性的結果。結論是待匹力達(Dp)可以表現抗血小板、抗發炎及抗纖維化反應， 因此，待匹力達(Dp)也許可以對糖尿病病人的血管粥樣病變的防範或治療提供有效的參考。
第二部份
實驗目的是Arachidonate 5-lipoxygenase-activating protein (ALOX5AP)的基因變異性跟阻塞性腦血管中風之危險性有很高的關係；因此，我們希望評估PDE4D及ALOX5AP基因的變異多樣性分析，探討血管粥樣化的原因；同時也研究洗腎病人糖尿病組、非糖尿病組與骨質素的關係。 實驗材料是收集洗腎病人對照組49人、糖尿病洗腎組54人、非糖尿病洗腎組45人，性別、年齡配對；利用酵素免疫分析法分析血液中的骨質素濃度，及利用自動生化分析儀檢驗血中無機磷酸鹽及鈣的濃度、以放射免疫法分析血中副甲狀腺素濃度。選擇270位健康的志願者當對照組，100位罹阻塞性腦中風病人當實驗組，性別、年齡配對；抽取志願者及病人全血之白血球抽取血球的genomic DNA，利用高速能的TaqMan PCR方法及DNA序列分析，讀取PDE4D基因特定片段中的SNP87 (rs2910829) 及SNP41 (rs152312)兩個特定之單核苷多型性之位點(SNPs)；及ALOX5AP基因HapA (SG13S89and SG13S32)與HapB (SG13S41 and SG13S35) 4個單核苷多型性之位點(SNPs)；單核苷多型性之位點出現的頻率作多樣變異作統計分析。結果顯示洗腎患者血液中骨質素的濃度比對照組高，而且洗腎後血中骨質素的濃度比洗腎前略高。洗腎患者血中的無機磷酸鹽與副甲狀腺素濃度都與骨質素的濃度呈現正相關性。在腦血管中風基因的研究，我們選取的SNP87（rs2910829）及SNP41（rs152312）；HapA (SG13S89 and SG13S32)與HapB (SG13S41 and SG13S35)等這六個單核苷位置，在台灣族群中，並沒有很大的關聯性；但與英、日、韓的結果相同。結論為臺灣人腦血管中風病人的6個SNPs的變異與出現頻率分析顯示與正常對照組並無明顯差異；但是從洗腎病人血中發現骨質素的濃度偏高；洗腎患者血中的無機磷酸鹽與副甲狀腺素濃度都與骨質素的濃度呈現正相關性。

關鍵字

關鍵詞: 腦中風 ； 單一核苷多樣化(SNP) ； 糖尿病 ； 高糖 ； ALOX5AP ； PDE4D ； 待匹力達 ； 骨質素蛋白 ； 血液透析 ； 訊號傳遞

並列摘要

Abstract
First part
Diabetic patients are frequently afflicted with medial artery calcification, a predictor of cardiovascular mortality. Diabetes induced expression of osteopontin in arterial vasculature is an indicator of disease progression in artery calcification and vascular stiffness. Here, we explored signal transduction and strategies that suppress high glucose-induced osteopontin expression in arterial vascular smooth muscle cells. The incubation of rat aortic smooth muscle cells under high glucose concentration increased osteopontin protein secretion and mRNA expression. Treatment with dipyridamole decreased high glucose-induced osteopontin expression and secretion. Dipyridamole decreased glucose-induced osteopontin through inhibition of phosphodiesterase, thereby increasing intracellular levels of cyclic AMP (cAMP) and cyclic GMP (cGMP), and increased thioredoxin expression to inhibit the reactive oxygen species (ROS) system. Induction of osteopontin were reversed when cells were pretreated with N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulfonamide (H89, PKA inhibitor), KT5823 (cGMP dependent protein kinase inhibitor), or dinitrochlorobenzene (thioredoxin reductase inhibitor). Antioxidant N-acetyl-L-cysteine suppressed glucose-induced osteopontin expression by decreasing ROS generation. H89 and KT5823 downregulated thioredoxin expression. These results suggest a novel effect for dipyridamole to suppress high glucose-induced osteopontin protein secretion and mRNA expression. Dipyridamole has antioxidant properties and a phosphodiesterase inhibitor activity, which might be useful to ameliorate diabetic vasculopathy and its cardiovascular complications.

Second part
We evaluated whether PDE4D or ALOX5AP gene polymorphisms confers a risk of ischemic stoke in Taiwanese patients. We also studied the relationship between plasma OPN and calcium-phosphate imbalance in hemodialysis (HD) patients. HD patients (54 diabetes and 45 non-diabetes ) and in 49 healthy volunteers. The concentrations of inorganic phosphate、calcium and parathyroid hormone (PTH) in blood were examined. The results demonstrated that the concentration of OPN in HD patients was higher than healthy volunteers and was positively correlated with inorganic phosphate and PTH levels. Two single-nucleotide polymorphisms (SNPs) covering the PDE4D gene and four single-nucleotide polymorphisms (SNPs) covering the ALOX5AP gene were genotyped using genomic DNA sequencing and a high throughput TaqMan® PCR assay in 100 patients who had suffered an ischemic stroke and 270 healthy individuals. No significant associations with ischemic stroke were observed with the SNP87 (rs2910829) and SNP41 (rs152312) SNPs from PDE4D, HapA (SG13S32 and SG13S89) and HapB (SG13S41 and SG13S35) from ALOX5AP. Although, no significant associations with ischemic stroke were observed with, but the concentration of OPN in HD patients was higher than healthy volunteers and was positively correlated with inorganic phosphate and PTH levels.

並列關鍵字

Keywords: Stroke ； Single Nucleotide ； Polymorphism (SNP) ； ALOX5AP ； PDE4D ； Diabetes Mellitus ； High Glucose ； Signal Transduction ； Dipyridamole ； Osteopontin ； Hemodialysis

參考文獻

Agnihotri R, Crawford, HC, Haro H. Osteopontin, a novel substrate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin). J Biol Chem 2001; 276: 28261-2826.

Alberts MJ: Diagnosis and treatment of ischemic stroke. Am J Med 1999; 106:211-221.

Andoh T, Chiueh CC, Chock PB. Cyclic GMP-dependent protein kinase regulates the expression of thioredoxin and thioredoxin peroxidase-1 during hormesis in response to oxidative stress-induced apoptosis. J Biol Chem 2003; 278: 885-90.

Ariga M, Neitzert B, Nakae S, Mottin G, Bertrand C, Pruniaux MP, Jin SL,Conti M. Nonredundant function of phosphodiesterases 4D and 4B in neutrophil recruitment to the site of inflammation. J Immunol 2004; 173:7531-8.

Arnér E, Holmgren A. Physiological functions of thioredoxin and thioredoxinreductase. Eur J Biochem 2000; 267: 6102–9.