小分子化合物ZLN005的心血管应用及机制研究
发布时间：2018-11-28 15:50
【摘要】：人口结构老龄化加剧是全球以及我国都在面对的重大卫生问题。衰老导致生理机能下降,促进一系列衰老相关疾病的发生,其中包括糖尿病和心血管功能紊乱。糖尿病是严重威胁人类健康的主要疾病之一,2011年调查世界人口中约有3.66亿糖尿病患者,并且其发病率近年来仍持续上升。缺血性心脏病目前仍是全世界死亡率来源的首要因素,而在糖尿病患者中心血管并发症也是造成死亡的最主要原因,超过50%糖尿病患者死于心血管并发症。能量限制(CR)是非基因干预途径中最有效的抗衰老方法。研究表明CR能够对多种衰老相关疾病的预防和治疗起到有益作用。SIRT1参与细胞在CR时的代谢反应和调控。SIRT1是一种NAD+依赖的脱乙酰酶,能够对组蛋白和很多转录因子脱乙酰化调节,包括NF-κB、p53、Fox Os、PGC-1α,并从而调节一系列细胞功能如细胞生存、衰老、自噬和代谢。小分子化合物ZLN005[2-(4-叔丁基苯基)苯并咪唑]是一种新发现的PGC-1α转录调节剂,在小鼠糖尿病模型中能够调节代谢水平起到保护作用,但是对于ZLN005是否能在心血管疾病中发挥作用未见研究报道。【研究目的】1.研究ZLN005是否能够保护心肌对抗高糖产生的损害;2.研究ZLN005是否能够调节巨噬细胞极化和胆固醇代谢;3.探讨ZLN005发挥作用的机制,初步做出新药开发的可行性评价。【研究方法】1.培养原代乳鼠心肌细胞,给予高糖刺激并使用ZLN005干预,观察细胞形态,检测细胞活性、细胞氧化应激水平、细胞自噬水平和凋亡比率;2.培养巨噬细胞,给予高胆固醇环境并用ZLN005干预,观察泡沫细胞形成情况、检测细胞内胆固醇含量;3.培养巨噬细胞并给予LPS刺激,检测ZLN005作用下细胞活性变化及M1和M2标志表达的情况;4.研究ZLN005对细胞内SIRT1表达的影响,及在ZLN005作用时特异性抑制SIRT1功能对细胞活力、细胞自噬和凋亡及胆固醇代谢、巨噬细胞极化的影响;5.通过分子对接分析ZLN005激活SIRT1的方式和结合能力并与其它SIRT1激动剂对比。【研究结果】1.在33m M高糖刺激下,培养的原代乳鼠心肌细胞活力下降、细胞氧化应激水平增高、细胞自噬下调,凋亡比率上升,给予ZLN005干预能够上调细胞自噬水平,减少应激,部分对抗高糖引起的细胞损害;2.在ox LDL-c作用下,Thp-1细胞来源的巨噬细胞形成泡沫细胞,同时给予ZLN005可见其减少了泡沫细胞内脂质成分比例,经检测证实ZLN005干预减少细胞内胆固醇含量;3.LPS引起Thp-1细胞来源的M2巨噬细胞向M1分化并降低细胞活力,添加ZLN005减少细胞活力下降的程度并减少M1标志即炎性因子的表达。4.ZLN005能够上调心肌细胞内SIRT1的m RNA和蛋白表达。特异性抑制SIRT1可消除ZLN005对自噬相关蛋白表达的影响,抑制ZLN005对高糖致细胞凋亡的保护作用。5.在Thp-1来源的巨噬细胞中,特异性抑制SIRT1导致ZLN005对LPS损害细胞活力和高脂促进泡沫细胞形成中的保护作用减少,增加了ZLN005作用组细胞内胆固醇水平。抑制SIRT1还对抗了ZLN005对巨噬细胞极化的影响,增加了M1标志的表达。ZLN005在对巨噬细胞中通过SIRT1调节ABCA1和ABCG1的表达;6.通过分子对接得出了ZLN005与SIRT1结合的可能方式和具体结合位置以及与ZLN005分子结构的具体关系,其结合能力强于多种SIRT1激动剂,并有以其结构为基础进行改进的可能,初步判断有进一步进行药物开发的可行性。【结论】1.ZLN005能够通过SIRT1途径对抗高糖造成的心肌细胞损伤,其机制可能与调节自噬抑制凋亡有关;2.ZLN005能够通过SIRT1途径调节Thp-1来源巨噬细胞胆固醇代谢,可能与增加ABCA1和ABCG1表达有关,并进一步减少巨噬细胞源性泡沫细胞内脂滴含量;3.ZLN005能够通过SIRT1途径调控Thp-1来源巨噬细胞极化,抑制其向M1分化;4.ZLN005增加心肌细胞内SIRT1转录和翻译,通过分子对接分析其可直接结合SIRT1起到变构激活效应,推测其结合能力强于多种SIRT1激动剂。
[Abstract]:The increase in the ageing of the population is a major health problem in the world and in our country. Aging results in a decline in physiological function and a series of aging-related diseases, including diabetes and cardiovascular dysfunction. Diabetes is one of the major diseases that pose a serious threat to human health. In 2011, about 36.6 million diabetic patients were surveyed, and the incidence of diabetes has continued to rise in recent years. Ischemic heart disease is still the leading cause of mortality worldwide, and cardiovascular complications in patients with diabetes are also the leading causes of death, and more than 50% of patients with diabetes die from cardiovascular complications. The energy limitation (CR) is the most effective anti-aging method in non-gene intervention. The results show that CR can play a beneficial role in the prevention and treatment of various aging-related diseases. SIRT1 is involved in the metabolic response and regulation of the cells at the time of CR. SIRT1 is a NAD + dependent deacetaminidase capable of deethanizing histone and many transcription factors, including NF-B, p53, Fox Os, PGC-1, and thereby modulating a range of cell functions such as cell survival, aging, autophagy and metabolism. The small-molecule compound ZLN005[2-(4-tert-butylphenyl) benzidine] is a newly discovered PGC-1 transcription regulator, which can regulate the metabolism level to play a protective role in the mouse diabetes model, but has no research report on whether the ZLN005 can play a role in the cardiovascular disease.[Study objective] 1. To study whether ZLN005 can protect the myocardium against high sugar production; Whether ZLN005 is capable of regulating macrophage polarization and cholesterol metabolism; The mechanism of the function of ZLN005 was discussed, and the feasibility of new drug development was preliminarily made.[Study Method] 1. The cell morphology, the cell activity, the level of oxidative stress, the level of autophagy and the rate of apoptosis were observed and the cell morphology, cell activity, level of oxidative stress, autophagy and apoptosis were observed. The macrophage was cultured, the high-cholesterol environment was given, and with the intervention of ZLN005, the formation of the foam cells was observed, and the content of cholesterol in the cells was detected. The changes of cell activity and the expression of M1 and M2 markers under the action of ZLN005 were detected and stimulated by LPS. The effect of ZLN005 on the expression of SIRT1 in the cells and the specific inhibition of the function of the SIRT1 on the cell viability, autophagy and apoptosis and the metabolism of cholesterol and the polarization of macrophages were studied in the case of ZLN005. The method and binding ability of the SIRT1 to activate the SIRT1 were analyzed by molecular docking and compared to other SIRT1 agonists.[Study Results] 1. Under the condition of 33m M high sugar, the cell viability of the cultured primary milk rats decreased, the level of the oxidative stress of the cells increased, the autophagy and the apoptosis rate of the cells increased, and the intervention of the ZLN005 can increase the autophagy level of the cells, reduce the stress and partially resist the cell damage caused by high sugar; Under the action of ox LDL-c, the macrophages of the Thp-1 cell source form a foam cell, and at the same time, ZLN005 is given to reduce the proportion of the lipid components in the foam cell, and the content of the cholesterol in the cell is reduced by the detection of the ZLN005 intervention; 3. The expression of mRNA and protein of SIRT1 in the cardiomyocytes could be up-regulated by the addition of ZLN005 to the differentiation of the M2 macrophages from the Thp-1 cell source to the M1 and to decrease the cell viability, to add to the extent of the reduction of the cell viability by the addition of the ZLN005 and to reduce the expression of the M1-marker, that is, the inflammatory factor. The specific inhibition of SIRT1 can eliminate the effect of ZLN005 on the expression of autophagy-related protein, and inhibit the protective effect of ZLN005 on the apoptosis of high-sugar-induced cell. In the macrophages of Thp-1, the specific inhibition of SIRT1 resulted in a decrease in the protective effect of ZLN005 on LPS-induced cell viability and the formation of high-fat-promoting foam cells, increasing the level of cholesterol in the cell of the ZLN005 group. The inhibition of SIRT1 also antagonized the effect of ZLN005 on the polarization of the macrophage, and the expression of the M1 marker was increased. ZLN005 regulates the expression of ABCA1 and ABCG1 in macrophages by SIRT1; The possible way and the specific binding position of the combination of ZLN005 and SIRT1 and the specific relation with the molecular structure of ZLN005 were obtained by molecular docking. The binding ability of ZLN005 was stronger than that of multiple SIRT1 agonists.[Conclusion] 1. ZLN005 can be used to antagonize the myocardial cell injury caused by high sugar through the SIRT1 pathway, and its mechanism may be related to the regulation of autophagy and apoptosis; 2. ZLN005 can regulate the cholesterol metabolism of the macrophage of the Thp-1 from the SIRT1 pathway, and may be related to the increase of ABCA1 and ABCG1 expression, and the content of the lipid droplets in the macrophage-derived foam cells is further reduced; and the 3. ZLN005 is capable of regulating the polarization of the Thp-1-derived macrophage by the SIRT1 route, inhibiting the polarization of the macrophage to the M1, and increasing the SIRT1 transcription and translation in the cardiac muscle cell by the molecular docking analysis, and can directly bind to the SIRT1 to function as a allosteric activation effect through molecular docking analysis, It is presumed that its binding capacity is stronger than that of multiple SIRT1 agonists.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R54
，
本文编号：2363358
[Abstract]:The increase in the ageing of the population is a major health problem in the world and in our country. Aging results in a decline in physiological function and a series of aging-related diseases, including diabetes and cardiovascular dysfunction. Diabetes is one of the major diseases that pose a serious threat to human health. In 2011, about 36.6 million diabetic patients were surveyed, and the incidence of diabetes has continued to rise in recent years. Ischemic heart disease is still the leading cause of mortality worldwide, and cardiovascular complications in patients with diabetes are also the leading causes of death, and more than 50% of patients with diabetes die from cardiovascular complications. The energy limitation (CR) is the most effective anti-aging method in non-gene intervention. The results show that CR can play a beneficial role in the prevention and treatment of various aging-related diseases. SIRT1 is involved in the metabolic response and regulation of the cells at the time of CR. SIRT1 is a NAD + dependent deacetaminidase capable of deethanizing histone and many transcription factors, including NF-B, p53, Fox Os, PGC-1, and thereby modulating a range of cell functions such as cell survival, aging, autophagy and metabolism. The small-molecule compound ZLN005[2-(4-tert-butylphenyl) benzidine] is a newly discovered PGC-1 transcription regulator, which can regulate the metabolism level to play a protective role in the mouse diabetes model, but has no research report on whether the ZLN005 can play a role in the cardiovascular disease.[Study objective] 1. To study whether ZLN005 can protect the myocardium against high sugar production; Whether ZLN005 is capable of regulating macrophage polarization and cholesterol metabolism; The mechanism of the function of ZLN005 was discussed, and the feasibility of new drug development was preliminarily made.[Study Method] 1. The cell morphology, the cell activity, the level of oxidative stress, the level of autophagy and the rate of apoptosis were observed and the cell morphology, cell activity, level of oxidative stress, autophagy and apoptosis were observed. The macrophage was cultured, the high-cholesterol environment was given, and with the intervention of ZLN005, the formation of the foam cells was observed, and the content of cholesterol in the cells was detected. The changes of cell activity and the expression of M1 and M2 markers under the action of ZLN005 were detected and stimulated by LPS. The effect of ZLN005 on the expression of SIRT1 in the cells and the specific inhibition of the function of the SIRT1 on the cell viability, autophagy and apoptosis and the metabolism of cholesterol and the polarization of macrophages were studied in the case of ZLN005. The method and binding ability of the SIRT1 to activate the SIRT1 were analyzed by molecular docking and compared to other SIRT1 agonists.[Study Results] 1. Under the condition of 33m M high sugar, the cell viability of the cultured primary milk rats decreased, the level of the oxidative stress of the cells increased, the autophagy and the apoptosis rate of the cells increased, and the intervention of the ZLN005 can increase the autophagy level of the cells, reduce the stress and partially resist the cell damage caused by high sugar; Under the action of ox LDL-c, the macrophages of the Thp-1 cell source form a foam cell, and at the same time, ZLN005 is given to reduce the proportion of the lipid components in the foam cell, and the content of the cholesterol in the cell is reduced by the detection of the ZLN005 intervention; 3. The expression of mRNA and protein of SIRT1 in the cardiomyocytes could be up-regulated by the addition of ZLN005 to the differentiation of the M2 macrophages from the Thp-1 cell source to the M1 and to decrease the cell viability, to add to the extent of the reduction of the cell viability by the addition of the ZLN005 and to reduce the expression of the M1-marker, that is, the inflammatory factor. The specific inhibition of SIRT1 can eliminate the effect of ZLN005 on the expression of autophagy-related protein, and inhibit the protective effect of ZLN005 on the apoptosis of high-sugar-induced cell. In the macrophages of Thp-1, the specific inhibition of SIRT1 resulted in a decrease in the protective effect of ZLN005 on LPS-induced cell viability and the formation of high-fat-promoting foam cells, increasing the level of cholesterol in the cell of the ZLN005 group. The inhibition of SIRT1 also antagonized the effect of ZLN005 on the polarization of the macrophage, and the expression of the M1 marker was increased. ZLN005 regulates the expression of ABCA1 and ABCG1 in macrophages by SIRT1; The possible way and the specific binding position of the combination of ZLN005 and SIRT1 and the specific relation with the molecular structure of ZLN005 were obtained by molecular docking. The binding ability of ZLN005 was stronger than that of multiple SIRT1 agonists.[Conclusion] 1. ZLN005 can be used to antagonize the myocardial cell injury caused by high sugar through the SIRT1 pathway, and its mechanism may be related to the regulation of autophagy and apoptosis; 2. ZLN005 can regulate the cholesterol metabolism of the macrophage of the Thp-1 from the SIRT1 pathway, and may be related to the increase of ABCA1 and ABCG1 expression, and the content of the lipid droplets in the macrophage-derived foam cells is further reduced; and the 3. ZLN005 is capable of regulating the polarization of the Thp-1-derived macrophage by the SIRT1 route, inhibiting the polarization of the macrophage to the M1, and increasing the SIRT1 transcription and translation in the cardiac muscle cell by the molecular docking analysis, and can directly bind to the SIRT1 to function as a allosteric activation effect through molecular docking analysis, It is presumed that its binding capacity is stronger than that of multiple SIRT1 agonists.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R54
，
本文编号：2363358
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