【作者】 石磊；

【导师】 蔡尚郎；

【作者基本信息】 青岛大学 ， 心血管内科（专业学位）， 2019， 博士

【摘要】 目的(1)观察白黎芦醇对动脉粥样硬化模型小鼠肝脏及血清指标的影响。(2)研究不同浓度白藜芦醇干预对动脉粥样硬化小鼠模型巨噬细胞自噬和凋亡的影响及可能机制。方法(1)动脉粥样硬化模型小鼠的建立:将50只小鼠随机分为5个小组,各组分别给予不同的干预,观察不同浓度白藜芦醇干预对动脉粥样硬化模型小鼠肝脏和血脂等指标的影响;(2)分别用0、12.5μM、25μM、50μM、100μM、200μM共6个浓度梯度的白藜芦醇干预培养的鼠巨噬细胞24 h后,采用显微镜观察细胞形态变化、MTT染色法分析细胞存活率大小,Western-Blot检测小鼠巨噬细胞自噬(LC3、Beclin1、P62、ATG5、Sirt1、p-P70 s6 k、AMPK、P-AMPK)、凋亡(P53、cle-Caspase3、cle-PARP、Bcl-2/Bax)相关蛋白以及脂代谢相关分子PPARα、CYP7 A1的表达。结果(1)分为基础组、动脉粥样硬化(AS)组、RSVI组、RSVII组、RSVIII组5组小鼠。各组小鼠饮食、精神状态良好,生长发状况育未见明显异常。5组小鼠体重增加量以RSVI组最高,但与其他各组比较,差异无统计学意义(P>0.05)。(2)5组小鼠在实验期间平均摄食量均随饲养周期延长而增加,6周后小鼠活动量减少,摄食量增幅有所降低。基础组、干预III组小鼠平均摄食量高于其余3组,但组间比较差异无统计学意义(P>0.05)。(3)5组小鼠肝脏指数组间比较结果示差异无统计学意义(P>0.05)。(4)小鼠肝脏的病理学变化:基础组、RSVII组和RSVIII组小鼠肝脏颜色鲜红,边沿锐利,质地软;而AS组和RSVI组小鼠肝脏边沿变钝,质地韧,剖面呈油腻感。小鼠肝脏HE染色切片显示:基础组小鼠肝脏细胞形态大致正常,脂肪变性肝细胞极少见;模型组小鼠的肝脏切片中可见部分脂肪变性肝细胞;RSV干预的三组小鼠肝脏切片中可见少数脂肪变性肝细胞。(5)与基础组比较,AS组血清TC、TG、LDL-C水平显著升高(P<0.01),HDL-C水平无显著性差异;与AS组比较,用白藜芦醇干预的3组中,TC、TG、LDL-C含量均明显减少(P<0.01,P<0.05),HDL-C含量均明显升高(P<0.01);RSVI组、RSVII组和RSVIII组组间两两比较发现,TC、TG、LDLC水平逐渐降低,HDL-C水平逐渐增加,差异有统计学意义(P<0.05)。(6)与基础组比较,AS组动脉粥样硬化指数AI1、AI2显著上升(P<0.01);与AS组比较,白藜芦醇干预的三组中,AI1、AI2均降低(P<0.01,P<0.05);RSVI组、RSVII组和RSVIII组组间两两比较发现,AI1、AI2水平逐渐降低,差异有统计学意义(P<0.05)。结果显示,白藜芦醇对动脉粥样硬化指数AI1、AI2有一定的影响。(7)小鼠血清抗氧化水平:与基础组比较,AS组血清T-SOD、CAT、GSH-Px水平降低(P<0.01),MDA水平升高(P<0.01);与AS组比较,用白藜芦醇干预的3组中,T-SOD、CAT、GSH-Px含量均显著升高(P<0.01,<0.05),MDA含量均明显降低(P<0.01);RSVI组、RSVII组和RSVIII组组间两两比较发现,T-SOD、CAT、GSH-Px水平逐渐升高,MDA含量逐渐下降,差异有统计学意义(P<0.05)。(8)小鼠肝组织抗氧化水平:与基础组比较,AS组肝脏中T-SOD、CAT、GSH-Px水平降低(P<0.01),MDA水平升高(P<0.01);与AS组比较,用白藜芦醇干预的3组中,T-SOD、CAT、GSH-Px含量均显著升高(P<0.01,<0.05),MDA含量均明显降低(P<0.01);RSVI组、RSVII组和RSVIII组组间两两比较发现,T-SOD、CAT、GSH-Px水平逐渐升高,MDA水平逐渐降低,差异有统计学意义(P<0.05)。结果显示,白藜芦醇对于肝脏中抗氧化酶T-SOD、CAT、GSH-Px及MDA水平的影响呈现出一定的剂量依赖性。(9)随着处理浓度增加,细胞间距变大、细胞折光性变差、死细胞数量增多,活细胞状态也变差,细胞活力都呈剂量依赖性的负相关。MTT法检测显示,不同浓度梯度的RSV干预可一定程度抑制小鼠巨噬细胞的增殖,随着RSV浓度升高,巨噬细胞生存活力下降。(10)Western-Blot检测结果显示,经RSV干预后,AS模型小鼠巨噬细胞中自噬相关分子LC3-Ⅱ、ATG5、Beclin1、p62、AMPK、P-AMPK和Sirt1表达均上调,p-P70 s6k、LC3-1的表达下调。(11)Western-Blot检测结果提示,经RSV干预后,AS模型小鼠巨噬细胞中凋亡相关因子P53、cle-Caspase3、cle-PARP、Bax表达上调,凋亡因子Bcl-2的表达下降。(12)Western-Blot检测结果显示,经RSV干预后,脂代谢相关分子PPARα和CYP7A1在AS模型小鼠巨噬细胞中表达增强。结论(1)白藜芦醇可以减轻小鼠肝脏的脂肪变性、降低小鼠外周血的血脂水平,降低小鼠的动脉粥样硬化指数,还可以使其肝组织和血清中抗氧化分子的水平升高,从而影响小鼠的动脉粥样硬化进展。(2)白藜芦醇可以通过影响小鼠巨噬细胞的自噬和凋亡,调节其脂代谢相关因子,达到抗动脉粥样硬化的作用。更多还原

【Abstract】 Objectives(1)To observe the effects of resveratrol on hepatic and serum indexes in atherosclerosis model mice.(2)To study the effects of different resveratrol concentrations on autophagy and apoptosis of macrophages in atherosclerosis model mice and its possible mechanism.Methods(1)To establish atherosclerosis model mice,50 mice were divided into 5 groups and given different interventions to observe the effect of resveratrol on liver and serum lipids metabolism of atherosclerosis model mice.(2)Rat macrophages cultured with resveratrol at different concentrations of 0,12.5 μM,25μM,50μM,100μM,and 200μM were treated for 24 hours respectively.The morphological changes of the cells were observed under microscope and the survival rate was analyzed by MTT staining.Western-Blot was used to detect protein expression of autophagy(LC3,Beclin1,P62,ATG5,Sirt1,p-P70s6 k,AMPK,P-AMPK),apoptosis(P53 、 cle-Caspase3 、 cle-PARP 、 Bcl-2/Bax)and lipid metabolism related molecules PPARa and CYP7A1.Results(1)Mice were divided into five groups: basic group,atherosclerosis model group,RSVI group,RSVII group and RSVIII group.The diet and mental status of mice in each group were good,and there was no obvious abnormality in growth and development.The weight increasement of RSVI group was the highest in the five groups,but there was no significant difference compared with other groups(P > 0.05).(2)During the experiment,the average food intake of the five groups of rats increased with the extension of the feeding cycle.After 6 weeks,the activity of the mice decreased and the food intake increasement decreased.The average food intake of the basic group and the RSVIII group was higher than that of the other three groups and the difference between groups was not statistically significant(P > 0.05).(3)There was no significant difference in liver index between groups(P>0.05).(4)Pathological changes of mice liver: The liver of the basic group,RSVIII group and RSVII group was bright red with sharp edges and soft texture,while that of the AS model group and RSVI group was blunt,tough and greasy.HE staining sections of mice liver showed that the morphology of hepatocytes in the basic group was normal,and steatotic hepatocytes were rare;some steatotic hepatocytes could be seen in the liver sections of AS model group mice;a few steatotic hepatocytes could be seen in the liver sections of three groups of mice intervened by RSV.(5)Compared with the basic group,the serum levels of TC,TG and LDL-C in the atherosclerosis model group were significantly increased(P < 0.01),while the levels of HDL-C were not significantly different.Compared with the AS model group,the levels of TC,TG and LDL-C in the three groups with resveratrol intervention were significantly reduced(P <0.01,P < 0.05),and the levels of HDL-C were significantly increased(P < 0.01).Comparison between RSVI group,RSVII group and RSVIII group showed that the levels of TC,TG and LDL-C decreased gradually,while the levels of HDL-C increased gradually,and the difference between groups was statistically significant(P < 0.05).(6)Compared with the basic group,the atherosclerosis index AI1 and AI2 in the AS model group were significantly increased(P < 0.01).Compared with the model group,AI1 and AI2 in the three groups treated with resveratrol decreased gradually(P <0.01,P < 0.05).Pairwise comparison between RSVI group,RSVII group and RSVIII group showed that AI1 and AI2 levels decreased gradually,and the difference was statistically significant(P < 0.05).The results showed that resveratrol had certain effect on the atherosclerosis index AI1 and AI2.(7)Antioxidant levels in mice serum: Compared with the basic group,serum T-SOD,CAT and GSH-px levels of in the AS model group were decreased(P < 0.01),while MDA levels were increased(P < 0.01).Compared with the AS model group,the levels of T-SOD,CAT and GSH-px in the three groups with resveratrol intervention were significantly increased(P< 0.01,< 0.05),and the levels of MDA were significantly decreased(P < 0.01).Pairwise comparison between RSVI group,RSVII group and RSVIII group showed that the levels of T-SOD,CAT and GSH-px increased gradually,while the levels of MDA decreased gradually,and the difference was statistically significant(P < 0.05).(8)Antioxidant levels in mice liver tissue: compared with the basic group,T-SOD,CAT and GSH-px levels of mice liver in the AS model group were decreased(P < 0.01),while MDA levels were increased(P < 0.01).Compared with the AS model group,the levels of T-SOD,CAT and GSH-px in the three groups with resveratrol intervention were significantly increased(P < 0.01,< 0.05),and the levels of MDA were significantly decreased(P < 0.01).Pairwise comparison between RSVI group,RSVII group and RSVIII group showed that the levels of T-SOD,CAT and GSH-px increased gradually,while the levels of MDA decreased gradually,and the difference had statistically significant(P<0.05).The results showed that resveratrol had a dose-dependent effect on the levels of antioxidant enzymes T-SOD,CAT,GSH-px and MDA in the liver.(9)With the increasement of treatment concentration,cell spacing increased,cell refraction decreased,the number of dead cells increased,and the state of living cells also deteriorated.Cell viability showed a dose-dependent negative correlation.MTT assay showed that RSV intervention with different concentration gradients could inhibit the proliferation of mouse macrophages to a certain extent.With the increasement of RSV concentration,the viability of macrophages decreased.(10)Western-Blot assay showed that after RSV intervention,autophagy related molecules LC3-II,ATG5,Beclin1,p62,AMPK,P-AMPK and Sirt-1 in macrophages of AS mice were up-regulated,while the expression of p-P70S6 K and LC3-1 was down-regulated.(11)Western-Blot results showed that after RSV intervention,the expressions of apoptotic related factors P53,cle-Caspase 3,cle-PARP and Bax in macrophages of AS mice were up-regulated,while the expression of apoptotic factor Bcl-2 was down-regulated.(13)Western-Blot assay showed that the expression of PPARa and CYP7A1 in macrophages of AS mice increased after RSV intervention.Conclusions(1)Resveratrol could alleviate the steatosis of liver,reduce the level of blood lipid in peripheral blood,reduce the atherosclerosis index of mice,and increase the level of antioxidant molecules in serum,thus could affect the progress of atherosclerosis in mice.(2)Resveratrol could inhibit atherosclerosis by affecting autophagy and apoptosis of macrophages and regulating lipid metabolism related factors in AS model mice.更多还原