【作者】 熊宇芳；

【导师】 屈伸；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 内皮功能紊乱是动脉粥样硬化(AS)发生过程中最早和最有决定性的事件之一。氧化低密度脂蛋白(ox-LDL)通过降低eNOS的活性,NO产生减少从而导致内皮功能紊乱是不争的事实。凝集素样氧化低密度脂蛋白受体(LOX-1)是内皮细胞膜上特异性识别摄取ox-LDL的最主要受体,LOX-1在介导ox-LDL导致内皮功能紊乱的过程中起关键作用。蛋白激酶Akt磷酸化eNOS的丝氨酸1179／1177(牛／人)位点是调控eNOS活性的关键因素。最新研究显示：ox-LDL可促发内皮细胞的内质网应激反应(ER stress),从而引起内皮功能紊乱。然而,ox-LDL导致eNOS活性下降的具体分子机制仍不十分清楚,是否与ox-LDL促发内质网应激反应有关,仍有待进一步研究。因此,在本研究中我们采用ox-LDL(200μg/ml)孵育人脐静脉内皮细胞(HUVEC), Western blotting检测内质网应激标志物PERK(PRK-like endoplasmic reticulum kinase)的磷酸化程度逐渐上升,特别是ox-LDL孵育半小时后。p-PERK的磷酸化程度和葡萄糖调节蛋白78(GRP78)的蛋白表达水平,以此来评估内质网应激反应的发生。研究发现,ox-LDL孵育HUVEC0.5小时即可诱发内质网应激反应(P<0.01,n=3)。进一步评估eNOS的磷酸化程度发现,ox-LDL可致eNOS的Ser1177位点的磷酸化程度显著降低,并呈时间依赖性,而eNOS的Thr 497位点的磷酸化程度以及eNOS的蛋白表达水平均无改变。这一结果提示：ox-LDL导致的eNOS的Ser1177位点的去磷酸化具有显著位点特异性。同时,我们也检测了eNOS的酶活性,结果显示,和对照组相比,ox-LDL可显著降低eNOS的酶活性(P<0.05,n=3),且呈时间依赖性,该结果与ox-LDL导致的eNOS的Ser1177位点的去磷酸化程度一致。同时我们发现：ox-LDL处理组的Akt的Thr308和Ser473位点的磷酸化程度也显著下降,该结果提示：Akt的活性显著降低。而细胞总蛋白抽提物中蛋白磷酸酶PP2A和PP1的表达均无改变。我们进一步采用Pull-down技术证明,在ox-LDL短期孵育的HUVEC中,Akt失活导致eNOS Ser1177位点去磷酸化与PP2A和PP1与eNOS的相互作用程度无关。我们进一步采用化学伴侣分子PBA(可降低ER stress)预处理HUVEC14小时,再用ox-LDL孵育0-4小时。结果显示,PBA处理组可显著降低ox-LDL诱导的p-PERK和GRP78的表达(P<0.01),即PBA降低了HUVEC的内质网应激反应。同时,PBA可显著逆转ox-LDL导致的eNOS Ser1177位点和Akt Thr308、Akt Ser473位点的去磷酸化程度。为进一步证明内质网应激在ox-LDL导致的eNOS活性下降中起关键作用,我们采用内质网应激诱导剂BFA(5μg/ml)处理HUVEC,结果发现：Akt和eNOS的磷酸化程度显著下降,且呈时间依赖性。这一结果证明：内质网应激可导致Akt和eNOS Ser1177位点的去磷酸化。我们进一步探讨内质网应激反应是否通过PI3K/Akt信号通路下调eNOS的磷酸化程度,我们采用PI3K抑制剂LY294002(0.5nM)和PI3K激活剂IGF-1(100ng／ml)分别处理HUVEC,结果显示：内质网应激诱导剂BFA可直接影响PI3K/Akt信号通路,进而导致eNOS Ser1177位点的去磷酸化。我们也检测了是否是LOX-1介导ox-LDL促发内质网应激反应。我们采用LOX-1封闭抗体JTX20处理细胞,结果发现：JTX20预处理HUVEC可显著逆转ox-LDL导致的eNOS和Akt的去磷酸化。我们进一步评估HUVEC内质网应激反应的程度,发现JTX20也逆转了细胞内p-PERK和GRP78的表达(P<0.01),即JTX20可降低HUVEC的内质网应激反应。上述结果提示：ox-LDL经LOX-1介导可促发内质网应激反应,并通过Akt信号途径下调eNOS的磷酸化程度,从而导致eNOS的功能下降。更多还原

【Abstract】 Endothelial dysfunction is one of the early and decisive event in the development of the atherosclerosis. There is no doubt that the ox-LDL can cause the endothelial dysfunction via the reduced activity of eNOS and the reduced production of NO. LOX-1 was identified from endothelial cells as the main receptor specially recognizing and uptaking the ox-LDL, LOX-1 plays a key role in the endothelial dysfunction triggered by ox-LDL. Phosphorylation of endothelial nitric oxide synthase (eNOS) serine 1179/1177(bovine/human) by protein kinase Akt is a central mechanism of eNOS regulation. Recently, the report showed:ox-LDL can cause ER stress of the endotheliocyte, so cause the endothelial dysfunction. However, the molecular mechanism of the reduced activity of eNOS caused by ox-LDL is not clear, we donot know whether there is a connection with the ER stress caused by ox-LDL, so we need to do research to clarify these questions.To address these issues, we treated the human umbilical venous endothelial cells (HUVEC) with ox-LDL(200μg/ml). The ER stress reaction was estimated by the phosporylation of the PERK(one of the ER stress marker) and the expression level of the glucose-regulated protein 78 (GRP78, ER stress marker) via Western blotting method. The result showed, the ER stress reaction can be caused by ox-LDL just after 0.5hrs treatment(P<0.01, n=3). Further detection of the phosphorylation of eNOS revealed that ox-LDL induced a rapid reduced phosphorylation of eNOS Ser1177 site, and it is time-dependent. This time-dependent action of ER stress reaction was highly specific because neither eNOS Thr 497 phosphorylation nor the total eNOS level in HUVECs was changed. At the same time, we also detected the enzymatic activity of the eNOS, the result showed:Compared with the control group, the enzymatic activity of the eNOS could be markedly reduced by the treatment of ox-LDL(P<0.05, n=3), and it was time-dependent, this result is consistent with the dephosphorylation of the eNOS Ser1177 site caused by ox-LDL. We also found:the phosphorylation of the Thr308 and Ser473 sites of Akt was markedly reduced by the treatment of ox-LDL, this result indicated the activity of Akt was markedly reduced. But the expression of the protein phosphatase 2A(PP2A)and protein phosphatase 1(PP1) in the total proteins of the HUVEC wasn’t changed. We further utilized Pull-down technology to prove that in the ox-LDL-treated HUVEC, the de-phosphorylation of eNOS Ser1177 site was caused by the inactivation of Akt, not due to the elevated association of PP2A and PP1 with eNOS.We further utilized the chemical chaperone PBA (which can decrease the ER stress) to pretreat the HUVEC for 14 hours, then incubated cells with ox-LDL for 0-4 hours. The result indicated:PBA treatment group could markedly decrease the expression of the p-PERK and GRP78 caused by the ox-LDL (P<0.01), that is to say the PBA reduced the ER stress reaction of HUVEC. At the same time, PBA could markedly reverse the dephosphorylation of the eNOS Ser1177 site and the Akt Thr308、Akt Ser473 sites caused by ox-LDL. In order to further prove the key role of the ER stress in the process of the reduced activity of eNOS caused by ox-LDL, we used the inducer of ER stress BFA(5μg/ml) to treat the HUVEC, we found that the phosphorylation of Akt and eNOS were markedly reduced, and it was time-dependent. This result proves:The ER stress can cause the dephosphorylation of Akt and eNOS Ser1177 site.We further investigated whether the ER stress reduced the phosphorylation of eNOS through the PI3K/Akt signal transduction pathway, so we used PI3K inhibitor LY294002(0.5nM) or PI3K inducer IGF-1(100ng/ml) to treat the HUVEC, the result indicated:the ER stress inducer BFA can directly influnce the PI3K/Akt signal transduction pathway, so caused the dephosphorylation of eNOS Ser1177 site.We also detected whether the LOX-1 mediated the ER stress reaction caused by ox-LDL. We used LOX-1 blocking antibody JTX20 to treat the cells, the result proved: the JTX20 pretreatment could markedly reverse the dephosphorylation of the eNOS and Akt caused by ox-LDL. We further estimated the ER stress reaction in HUVEC, we found JTX20 could also reverse the expression of the p-PERK and GRP78(P<0.01), that is to say JTX20 can decrease the ER stress reaction.So, our result indicated:ox-LDL can cause the ER stress reaction via LOX-1, and can reduce the phosphorylation of eNOS through the Akt signal transduction pathway, so reduce the function of eNOS.更多还原