- 學位論文
大蒜二丙烯基二硫化物及二丙烯基三硫化物保護血管內皮細胞功能之研究
Protection of diallyl disulfide and diallyl trisulfide on vascular endothelium functions
摘要
氧化低密度脂蛋白(ox-LDL)在粥狀動脈硬化的起始過程中扮演重要的角色，具有誘發細胞激素分泌、激發黏附分子表現、增加淋巴細胞與單核細胞侵入血管壁、刺激血管壁肌肉細胞增生、抑制一氧化氮合成酶活性(eNOS)等作用。已知大蒜擁有多樣的生理活性，然而過去有關大蒜保護心血管功能的研究大都侷限在其降血脂、抗發炎、抗血小板凝集、抗氧化活性上，其他作用機轉則少見。本研究以人類臍靜脈內皮細胞(HUVEC)為模式，探討在ox-LDL刺激下，三種富含於大蒜精油的硫化物diallyl sulfide (DAS)、diallyl disulfide (DADS)及diallyl trisulfide (DATS)抑制血管黏附分子-1 (VCAM-1)和E-選擇素(E-selectin)表現及保護eNOS活性之效應及機轉。 本研究首先將人類臍靜脈內皮細胞分別先以1 mmol/L DAS、200 μmol/L DADS或100 μmol/L DATS預處理16小時，再給予細胞ox-LDL處理24小時，結果顯示：DADS和DATS可有效抑制ox-LDL誘發HUVEC表現VCAM-1與E-selectin，效果以DATS最佳，DADS次之，DAS則最差，因此，HL-60與HUVEC之間的黏附也受到DADS和DATS的抑制；DCF-DA進一步指出DADS及DATS預處理分別抑制ox-LDL誘發胞內活性氧的產生達43%及50%；H89 (PKA抑制劑)和wortmannin (PI3K抑制劑)存在時，DADS和DATS抑制ox-LDL誘發VCAM-1和E-selectin表現的效應則消失；Electrophoretic mobility shift assay (EMSA)結果顯示CREB與DNA的結合和CREB磷酸化的變化一致。這些結果顯示大蒜二丙烯基硫化物可以有效抑制ox-LDL誘發血管內皮細胞黏附分子的表現及其和單核球的黏附，且其作用與PI3K/PKB或PKA/CREB訊號路徑有關。 第二部份，我們進而分析大蒜二丙烯基硫化物是否可以透過PI3K/PKB訊號路徑增加eNOS磷酸化及抑制ox-LDL誘發泛素-蛋白酶體路徑減少eNOS蛋白質的表現。結果顯示：DADS與DATS避免ox-LDL抑制eNOS絲氨酸1177的磷酸化。然而給予PKB抑制劑- wortmannin處理下，則會降低DADS和DATS回復eNOS磷酸化的效應。同樣地，DADS與DATS也可以回復ox-LDL抑制細胞環磷酸鳥苷(cGMP)及一氧化氮的生成(P<0.05)。再者給予wortmannin會抑制DADS與DATS增加一氧化氮的生合成。免疫沉澱法也指出DADS及DATS預處理可以減少ox-LDL對eNOS與窖蛋白-1形成複合物的抑制。另外，DADS與DATS亦可保護ox-LDL抑制胞內eNOS的含量。給予細胞預處理蛋白質合成抑制劑-cycloheximide，DADS與DATS減少eNOS蛋白質降解的效應與蛋白酶體抑制劑-MG132結果一致。大蒜二丙烯基硫化物與MG132顯著抑制ox-LDL誘發chymotrypsin-like proteasome的活性(P<0.05)。這些研究結果顯示DADS與DATS預處理有助於減少ox-LDL誘發VCAM-1和E-selectin表現及對eNOS Ser1177磷酸化的抑制作用，此保護作用與PI3K/PKB或cAMP/PKA及泛素-蛋白酶體路徑有關。
並列摘要
Oxidized low-density lipoprotein (ox-LDL), an independent atherosclerotic risk factor, plays a critical role in the initiation and progression of atherosclerosis. ox-LDL stimulates endothelial cells producing a number of pro-inflammatory cytokines, adhesion molecules, and growth factors, which result in the recruitment of monocytes to the vessel wall, migrating into intima, and subsequently the formation of the foam cells. In addition, ox-LDL inhibits endothelial nitric oxide synthase (eNOS) activity, which affects the vascular tone. Garlic has been reported to be potent on preventing atherosclerosis, however, the protection is focused mainly on its cholesterol lowering, antiinflammation, anti-platelet aggregation, and antioxidative effects. The actual molecular mechanism(s) by which garlic acts has not been fully elucidated. In this study, we intend to examine whether diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), three major organosulfur compounds of garlic oil, reduce vascular cell adhesion molecule 1 (VCAM-1) and E-selectin expression and also enhance eNOS activation in human umbilical vein endothelial cells (HUVECs) by ox-LDL insult, if so, through what mechanism. In the first experiment, human umbilical vein endothelial cells (HUVECs) were preincubated with 1 mmol/L DAS, 200 mol/L DADS, or 100 μmol/L DATS for 16 h and then with 40 μg/ml ox-LDL for an additional 24 h. As results indicated, ox-LDL induction of cellular and cell surface expression of E-selectin and VCAM-1 was suppressed by garlic allyl sulfides in the order DATS>DADS>DAS. The adhesion of HL-60 cells to endothelial cells was also inhibited and the production of cellular peroxides was inhibited 43% and 50% by DADS and DATS, respectively (P<0.05). In the presence of H89 (PKA inhibitor) and wortmannin (PI3K inhibitor), abolished DADS and DATS suppression of ox-LDL-induced VCAM-1 and E-selectin expression. Electrophoretic mobility shift assay (EMSA) indicated that the activation of CREB binding to DNA was consistent with changes in CREB phosphorylation. These results suggest that the suppression ox-LDL-induced E-selectin and VCAM-1 expression by diallyl sulfide and, thus, monocyte adhesion to endothelial cells is likely dependent on the PI3K/PKB or PKA/CREB signaling pathway in an adhesion molecule-specific manner. Next experiment, we further examined whether the protection effect of garlic allyl sulfides on endothelium eNOS activity mediated by PI3K/PKB pathway and eNOS protein context against ox-LDL insult via ubiquitin-proteasome pathway. Results indicated that DADS and DATS reversed the suppression of eNOS Ser1177 phosphorylation by ox-LDL, and wortmannin abolished the reversal by DADS and DATS. Similarly, the inhibition of cellular cGMP and nitric oxide (NO) production by ox-LDL was reversed by DADS and DATS (P<0.05). This increase in NO bioavailability by the allyl sulfides was attenuated by wortmannin. Immunoprecipitation assay revealed that DADS and DATS preserved the interaction of eNOS with caveolin-1 in the membrane. In addition, DADS and DATS suppressed the reduction of the cellular eNOS protein content by ox-LDL. When cycloheximide was added to block protein synthesis, DADS and DATS suppressed eNOS protein degradation similarly to that noted by MG132. ox-LDL increased chymotrypsin-like proteasome activity, and this increase was inhibited by the allyl sulfides and MG132 (P<0.05). These results suggest that DADS and DATS decrease ox-LDL-induced VCAM- and E-selectin expression and protect eNOS activity against ox-LDL insult and this protection can be attributed partly to their mediation of PI3K/PKB signaling and prevention of eNOS degradation.