小分子化合物ZLN005的心血管應(yīng)用及機制研究
發(fā)布時間：2018-11-28 15:50
【摘要】：人口結(jié)構(gòu)老齡化加劇是全球以及我國都在面對的重大衛(wèi)生問題。衰老導(dǎo)致生理機能下降,促進一系列衰老相關(guān)疾病的發(fā)生,其中包括糖尿病和心血管功能紊亂。糖尿病是嚴(yán)重威脅人類健康的主要疾病之一,2011年調(diào)查世界人口中約有3.66億糖尿病患者,并且其發(fā)病率近年來仍持續(xù)上升。缺血性心臟病目前仍是全世界死亡率來源的首要因素,而在糖尿病患者中心血管并發(fā)癥也是造成死亡的最主要原因,超過50%糖尿病患者死于心血管并發(fā)癥。能量限制(CR)是非基因干預(yù)途徑中最有效的抗衰老方法。研究表明CR能夠?qū)Χ喾N衰老相關(guān)疾病的預(yù)防和治療起到有益作用。SIRT1參與細胞在CR時的代謝反應(yīng)和調(diào)控。SIRT1是一種NAD+依賴的脫乙酰酶,能夠?qū)M蛋白和很多轉(zhuǎn)錄因子脫乙�；{(diào)節(jié),包括NF-κB、p53、Fox Os、PGC-1α,并從而調(diào)節(jié)一系列細胞功能如細胞生存、衰老、自噬和代謝。小分子化合物ZLN005[2-(4-叔丁基苯基)苯并咪唑]是一種新發(fā)現(xiàn)的PGC-1α轉(zhuǎn)錄調(diào)節(jié)劑,在小鼠糖尿病模型中能夠調(diào)節(jié)代謝水平起到保護作用,但是對于ZLN005是否能在心血管疾病中發(fā)揮作用未見研究報道�！狙芯磕康摹�1.研究ZLN005是否能夠保護心肌對抗高糖產(chǎn)生的損害;2.研究ZLN005是否能夠調(diào)節(jié)巨噬細胞極化和膽固醇代謝;3.探討ZLN005發(fā)揮作用的機制,初步做出新藥開發(fā)的可行性評價�！狙芯糠椒ā�1.培養(yǎng)原代乳鼠心肌細胞,給予高糖刺激并使用ZLN005干預(yù),觀察細胞形態(tài),檢測細胞活性、細胞氧化應(yīng)激水平、細胞自噬水平和凋亡比率;2.培養(yǎng)巨噬細胞,給予高膽固醇環(huán)境并用ZLN005干預(yù),觀察泡沫細胞形成情況、檢測細胞內(nèi)膽固醇含量;3.培養(yǎng)巨噬細胞并給予LPS刺激,檢測ZLN005作用下細胞活性變化及M1和M2標(biāo)志表達的情況;4.研究ZLN005對細胞內(nèi)SIRT1表達的影響,及在ZLN005作用時特異性抑制SIRT1功能對細胞活力、細胞自噬和凋亡及膽固醇代謝、巨噬細胞極化的影響;5.通過分子對接分析ZLN005激活SIRT1的方式和結(jié)合能力并與其它SIRT1激動劑對比�！狙芯拷Y(jié)果】1.在33m M高糖刺激下,培養(yǎng)的原代乳鼠心肌細胞活力下降、細胞氧化應(yīng)激水平增高、細胞自噬下調(diào),凋亡比率上升,給予ZLN005干預(yù)能夠上調(diào)細胞自噬水平,減少應(yīng)激,部分對抗高糖引起的細胞損害;2.在ox LDL-c作用下,Thp-1細胞來源的巨噬細胞形成泡沫細胞,同時給予ZLN005可見其減少了泡沫細胞內(nèi)脂質(zhì)成分比例,經(jīng)檢測證實ZLN005干預(yù)減少細胞內(nèi)膽固醇含量;3.LPS引起Thp-1細胞來源的M2巨噬細胞向M1分化并降低細胞活力,添加ZLN005減少細胞活力下降的程度并減少M1標(biāo)志即炎性因子的表達。4.ZLN005能夠上調(diào)心肌細胞內(nèi)SIRT1的m RNA和蛋白表達。特異性抑制SIRT1可消除ZLN005對自噬相關(guān)蛋白表達的影響,抑制ZLN005對高糖致細胞凋亡的保護作用。5.在Thp-1來源的巨噬細胞中,特異性抑制SIRT1導(dǎo)致ZLN005對LPS損害細胞活力和高脂促進泡沫細胞形成中的保護作用減少,增加了ZLN005作用組細胞內(nèi)膽固醇水平。抑制SIRT1還對抗了ZLN005對巨噬細胞極化的影響,增加了M1標(biāo)志的表達。ZLN005在對巨噬細胞中通過SIRT1調(diào)節(jié)ABCA1和ABCG1的表達;6.通過分子對接得出了ZLN005與SIRT1結(jié)合的可能方式和具體結(jié)合位置以及與ZLN005分子結(jié)構(gòu)的具體關(guān)系,其結(jié)合能力強于多種SIRT1激動劑,并有以其結(jié)構(gòu)為基礎(chǔ)進行改進的可能,初步判斷有進一步進行藥物開發(fā)的可行性�！窘Y(jié)論】1.ZLN005能夠通過SIRT1途徑對抗高糖造成的心肌細胞損傷,其機制可能與調(diào)節(jié)自噬抑制凋亡有關(guān);2.ZLN005能夠通過SIRT1途徑調(diào)節(jié)Thp-1來源巨噬細胞膽固醇代謝,可能與增加ABCA1和ABCG1表達有關(guān),并進一步減少巨噬細胞源性泡沫細胞內(nèi)脂滴含量;3.ZLN005能夠通過SIRT1途徑調(diào)控Thp-1來源巨噬細胞極化,抑制其向M1分化;4.ZLN005增加心肌細胞內(nèi)SIRT1轉(zhuǎn)錄和翻譯,通過分子對接分析其可直接結(jié)合SIRT1起到變構(gòu)激活效應(yīng),推測其結(jié)合能力強于多種SIRT1激動劑。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R54
，
本文編號：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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R54
，
本文編號：2363358
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