丙酮酸乙酯对THP-1源性泡沫细胞自噬和凋亡的影响
发布时间：2019-05-23 06:22
【摘要】：目的：巨噬细胞吞噬氧化低密度脂蛋白(oxygenized low density lipoproteins, ox-LDL)并转化为泡沫细胞。研究发现,泡沫细胞大量死亡可导致局部堆积大量脂质,斑块进展甚至破裂。细胞死亡方式包括：自噬性死亡,凋亡和坏死。丙酮酸乙酯(Ethyl pyruvate,EP)是一种丙酮酸衍生物,多个研究显示该药物能通过抑制细胞自噬和凋亡,减少细胞死亡,从而发挥对脏器的保护作用。本研究拟观察EP对泡沫细胞自噬和凋亡的影响,并初步探讨可能的机制。方法：1.THP-1源性泡沫细胞模型的构建：用不同浓度ox-LDL(0mg/L～150mg/L)诱导构建THP-1源性泡沫细胞模型。之后用油红O染色鉴定造模情况,用MTT法测细胞生存率。2. ox-LDL刺激对泡沫细胞自噬和凋亡的影响：THP-1细胞经PMA诱导贴壁后,给予不同浓度的ox-LDL(0mg/L～150mg/L)孵育24小时,用流式细胞术检测细胞自噬和凋亡水平,以筛选最佳模型构建条件。3.EP对THP-1源性泡沫细胞自噬及凋亡的影响：用上述研究筛选的最适ox-LDL浓度刺激泡沫细胞生成,应用不同浓度EP(5mM-20mM)进行干预,观察其对泡沫细胞自噬和细胞凋亡的影响。分组如下：①对照组：不加任何干预因素：②泡沫细胞组：加入100mg/L的ox-LDL;③低浓度EP组：加入100mg/L的ox-LDL和5mM的EP；④中浓度EP组：加入100mg/L的ox-LDL和10mM的EP；⑤高浓度EP组：加入100mg/L的ox-LDL和20mmM的EP。共同孵育24小时后,用western blot、流式细胞术、免疫荧光、PCR技术观察细胞自噬水平、细胞凋亡率。评估不同浓度EP对THP-1源性泡沫细胞脂质蓄积、细胞自噬和细胞凋亡的影响。用免疫荧光、PCR技术评估各组HMGB1水平表达的差异。4.EP对泡沫细胞自噬和凋亡影响可能的分子机制：用免疫荧光、PCR技术评估ox-LDL组与ox-LDL+20mMEP组HMGB1、Beclin1、Rage等基因水平表达的差异,探讨EP作用于泡沫细胞自噬和凋亡水平可能的机制。结果：1.ox-LDL为25mg/L时,细胞生存率即下降至87.18%±5.34%(与对照组比较P0.05)。随着ox-LDL浓度升高,细胞活力逐渐下降,油红O染色可见泡沫细胞内大量脂滴。2.THP-1源性泡沫细胞自噬率及凋亡率水平随着ox-LDL浓度增高增加,0mgL组,25mg/L组,50mg/L组,100mg/L组,150mg/L组的自噬率分别为0.05%±0.01%,1.28%±0.32%,2.09%±0.17%,5.01%±0.33%,5.41%±0.27%,凋亡率分别为10.01%±0.19%,20.36%±3.42%,29.83±6.27%,49.29%±10.66%,62.70%±1.95%。但100mg/L组与150mg/L组间差异均无统计学意义(P0.05)。3.流式细胞术、]eal-time PCR. Western blot等检测手段证实,随着EP浓度增高,自噬率、自噬相关基因的表达水平逐渐下降。泡沫细胞组凋亡率较对照组明显增加(48.77±7.22 VS 5.87±1.03)(P0.05)。不同浓度EP组(5mM,10mM,20mM)细胞凋亡率分别为26.12%±3.99%,12.92%±1.32%,9.46%±0.53%,均较泡沫细胞组明显下降(P0.05),凋亡标志性蛋白caspase-3表达随着EP浓度增加而减少。即随着EP浓度升高,凋亡率下降明显。4.免疫荧光染色、PCR显示ox-LDL+20mMEP组HMGB1、Beclin1、Rage和TNF-a基因mRNA表达水平较ox-LDL组显著下降。结论：1.以PMA诱导THP-1细胞24小时可以促使该细胞贴壁分化为巨噬细胞；之后再以ox-LDL诱导泡沫细胞形成。方法简单可重复性好。2.在ox-LDL诱导THP-1源性泡沫细胞中自噬和凋亡水平较对照组均增加。3.不同浓度EP可以抑制泡沫细胞中的自噬和凋亡,呈浓度依赖性。4.EP可能是通过抑制HMGB1减少了HMGB1-Beclin1互动及抑制HMGB1-RAGE通路来减少泡沫细胞中的自噬；可能是通过HMGB1-TLR4-TNF-α轴通路来抑制凋亡现象。
[Abstract]:Objective: To investigate the effect of macrophages on oxidized low density lipoprotein (ox-LDL) and to convert them into foam cells. The study found that a large number of deaths in the foam cells could result in a significant accumulation of lipid, the progression of the plaque, or even the rupture. The mode of cell death includes autophagic death, apoptosis and necrosis. Ethyl Pyruvate (EP) is a pyruvic acid derivative, and a number of studies show that the drug can play a protective role in organs by inhibiting autophagy and apoptosis of cells and reducing cell death. This study is to observe the effect of EP on autophagy and apoptosis of foam cells and to explore possible mechanisms. Methods:1. Construction of THP-1-derived foam cell model: The model of THP-1-derived foam cells was constructed by different concentration of ox-LDL (0 mg/ L ~ 150 mg/ L). The cell survival rate was measured by MTT method. The effect of ox-LDL on the autophagy and apoptosis of the foam cells was: after the THP-1 cells were induced by PMA, different concentrations of ox-LDL (0 mg/ L-150 mg/ L) were given for 24 hours, and the autophagy and the level of apoptosis were detected by flow cytometry to screen the best model construction conditions. The optimal ox-LDL concentration was used to stimulate the generation of the foam cells and to use different concentrations of EP (5 mM-20 mM) to interfere with the autophagy and apoptosis of the foam cells. The group was as follows: control group: non-foam cell group: add 100 mg/ L of ox-LDL; low-concentration EP group: add 100 mg/ L of ox-LDL and 5 mM of EP; concentration of EP group: add 100 mg/ L of ox-LDL and 10 mM of EP; high-concentration EP group: add 100 mg/ L of ox-LDL and 20 mmM of EP. After 24 hours of incubation, the autophagy level and the cell apoptosis rate were observed by western blot, flow cytometry, immunofluorescence and PCR. The effects of different concentrations of EP on lipid accumulation, autophagy and apoptosis in THP-1-derived foam cells were evaluated. The difference of the expression of HMGB1 in each group was assessed by means of immunofluorescence and PCR.4. The possible molecular mechanism of the effect of EP on autophagy and apoptosis of the foam cells: the difference of the level of HMGB1, Beclin 1, and Rage of the ox-LDL group and the ox-LDL + 20 mMEP group was assessed by immunofluorescence and PCR. The possible mechanisms of EP on autophagy and apoptosis were discussed. Results: The survival rate of the cells decreased to 87.18% and 5.34% when the concentration of 1. oxen-LDL was 25 mg/ L (P0.05). As the concentration of ox-LDL increased, the cell viability gradually decreased, and the oil red O staining showed a large number of lipid droplets in the foam cells.2. The autophagocytosis and the apoptosis rate of the THP-1-derived foam cells increased with the increase of the ox-LDL concentration, the self-phagocytosis of the group of 0 mg/ L,25 mg/ L,50 mg/ L,100 mg/ L and 150 mg/ L was 0.05% and 0.01%, respectively. 1.28%, 0.32%, 2.09%, 0.17%, 5.01%, 0.33%, 5.41%, 0.27%, and the apoptosis rate was 10.01%, 0.19%, 20.36%, 3.42%, 29.83% 6.27%, 49.29%, 10.66%, 62.70% and 1.95%, respectively. However, there was no significant difference between 100 mg/ L and 150 mg/ L (P0.05). Flow cytometry,] eal-time PCR. Western blot and other methods of detection confirmed that with the increase of EP, the self-phagocytosis and autophagy-related genes decreased gradually. The apoptosis rate of the foam cells increased significantly (48.77% 7.22 VS 5.87-1.03) (P0.05). The apoptosis rates of different concentration EP groups (5 mM,10 mM,20 mM) were 26.12%, 3.99%, 12.92%, 1.32%, 9.46% and 0.53%, respectively. In other words, with the increase of EP, the rate of apoptosis was decreased. The expression of HMGB1, Bechin1, Rage and TNF-a gene in the ox-LDL + 20 mMEP group was significantly lower than that of the ox-LDL group. Conclusion:1. The induction of THP-1 cells by PMA for 24 hours can cause the cell to adhere to the macrophages and then induce the formation of the foam cells by ox-LDL. The method is simple and repeatable. The level of autophagy and apoptosis in the THP-1-derived foam cells induced by ox-LDL increased. Different concentrations of EP can inhibit autophagy and apoptosis in the foam cells, in a concentration-dependent manner.4. EP may be to reduce autophagy in the foam cells by inhibiting the HMGB1-Beclo1 interaction and to inhibit the HMGB1-RAGE pathway; it may be the inhibition of apoptosis by the HMGB1-TLR4-TNF-necrosis axis pathway.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543.5
[Abstract]:Objective: To investigate the effect of macrophages on oxidized low density lipoprotein (ox-LDL) and to convert them into foam cells. The study found that a large number of deaths in the foam cells could result in a significant accumulation of lipid, the progression of the plaque, or even the rupture. The mode of cell death includes autophagic death, apoptosis and necrosis. Ethyl Pyruvate (EP) is a pyruvic acid derivative, and a number of studies show that the drug can play a protective role in organs by inhibiting autophagy and apoptosis of cells and reducing cell death. This study is to observe the effect of EP on autophagy and apoptosis of foam cells and to explore possible mechanisms. Methods:1. Construction of THP-1-derived foam cell model: The model of THP-1-derived foam cells was constructed by different concentration of ox-LDL (0 mg/ L ~ 150 mg/ L). The cell survival rate was measured by MTT method. The effect of ox-LDL on the autophagy and apoptosis of the foam cells was: after the THP-1 cells were induced by PMA, different concentrations of ox-LDL (0 mg/ L-150 mg/ L) were given for 24 hours, and the autophagy and the level of apoptosis were detected by flow cytometry to screen the best model construction conditions. The optimal ox-LDL concentration was used to stimulate the generation of the foam cells and to use different concentrations of EP (5 mM-20 mM) to interfere with the autophagy and apoptosis of the foam cells. The group was as follows: control group: non-foam cell group: add 100 mg/ L of ox-LDL; low-concentration EP group: add 100 mg/ L of ox-LDL and 5 mM of EP; concentration of EP group: add 100 mg/ L of ox-LDL and 10 mM of EP; high-concentration EP group: add 100 mg/ L of ox-LDL and 20 mmM of EP. After 24 hours of incubation, the autophagy level and the cell apoptosis rate were observed by western blot, flow cytometry, immunofluorescence and PCR. The effects of different concentrations of EP on lipid accumulation, autophagy and apoptosis in THP-1-derived foam cells were evaluated. The difference of the expression of HMGB1 in each group was assessed by means of immunofluorescence and PCR.4. The possible molecular mechanism of the effect of EP on autophagy and apoptosis of the foam cells: the difference of the level of HMGB1, Beclin 1, and Rage of the ox-LDL group and the ox-LDL + 20 mMEP group was assessed by immunofluorescence and PCR. The possible mechanisms of EP on autophagy and apoptosis were discussed. Results: The survival rate of the cells decreased to 87.18% and 5.34% when the concentration of 1. oxen-LDL was 25 mg/ L (P0.05). As the concentration of ox-LDL increased, the cell viability gradually decreased, and the oil red O staining showed a large number of lipid droplets in the foam cells.2. The autophagocytosis and the apoptosis rate of the THP-1-derived foam cells increased with the increase of the ox-LDL concentration, the self-phagocytosis of the group of 0 mg/ L,25 mg/ L,50 mg/ L,100 mg/ L and 150 mg/ L was 0.05% and 0.01%, respectively. 1.28%, 0.32%, 2.09%, 0.17%, 5.01%, 0.33%, 5.41%, 0.27%, and the apoptosis rate was 10.01%, 0.19%, 20.36%, 3.42%, 29.83% 6.27%, 49.29%, 10.66%, 62.70% and 1.95%, respectively. However, there was no significant difference between 100 mg/ L and 150 mg/ L (P0.05). Flow cytometry,] eal-time PCR. Western blot and other methods of detection confirmed that with the increase of EP, the self-phagocytosis and autophagy-related genes decreased gradually. The apoptosis rate of the foam cells increased significantly (48.77% 7.22 VS 5.87-1.03) (P0.05). The apoptosis rates of different concentration EP groups (5 mM,10 mM,20 mM) were 26.12%, 3.99%, 12.92%, 1.32%, 9.46% and 0.53%, respectively. In other words, with the increase of EP, the rate of apoptosis was decreased. The expression of HMGB1, Bechin1, Rage and TNF-a gene in the ox-LDL + 20 mMEP group was significantly lower than that of the ox-LDL group. Conclusion:1. The induction of THP-1 cells by PMA for 24 hours can cause the cell to adhere to the macrophages and then induce the formation of the foam cells by ox-LDL. The method is simple and repeatable. The level of autophagy and apoptosis in the THP-1-derived foam cells induced by ox-LDL increased. Different concentrations of EP can inhibit autophagy and apoptosis in the foam cells, in a concentration-dependent manner.4. EP may be to reduce autophagy in the foam cells by inhibiting the HMGB1-Beclo1 interaction and to inhibit the HMGB1-RAGE pathway; it may be the inhibition of apoptosis by the HMGB1-TLR4-TNF-necrosis axis pathway.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543.5
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