HSP22在PPARγ激动剂抗动脉粥样硬化中表达的研究

发布时间：2018-10-08 21:08

【摘要】：目的:研究过氧化物酶体增殖物激活受体-γ(peroxisome proliferator-activated receptor-γ,PPARγ)激动剂吡格列酮(pioglitazone,P io)对动脉粥样硬化模型载脂蛋白E基因敲除(Apo E-/-)小鼠主动脉中热休克蛋白22(heat shock protein 22,HSP22)表达的影响,并探讨HSP22在其中所起的作用。方法:1、36只8~9周龄Apo E-/-雄性小鼠,随机分为4组:对照组(n=9,普通饲料),对照干预组(n=9,普通饲料+Pio),模型组(n=9,高脂饲料)与模型干预组(n=9,高脂饲料+Pio);对照组、对照干预组喂食普通饲料12周,模型组、模型干预组喂食高脂饲料12周,即高脂饮食(high-fat diet,HFD),其中干预组从第五周开始给予Pio(20mg/kg/d)干预处理,共干预8周。2、称量各组小鼠每周体重;检测各组小鼠基线及干预结束的血脂水平,包括:甘油三脂(triglyceride,TG)、总胆固醇(total cholesterol,TC)、高密度脂蛋白-胆固醇(high-density lipoprotein cholesterol,HDL-C)、低密度脂蛋白-胆固醇(low-density lipoprotein cholesterol,LDL-C);检测干预结束时小鼠的空腹血糖。3、主动脉整体油红O染色及苏木素-伊红(hematoxylin and eosin,HE)染色法观察各组小鼠动脉粥样硬化斑块形成情况及病理学变化。4、免疫组化法检测小鼠主动脉HSP22表达,Western Blot法检测PPARγ、HSP22、细胞间粘附分子1(intercellular adhesio n molecule-1,ICAM-1)、内皮型一氧化氮合酶(endothelial nitric oxide synthase,e NOS)、p-e NOS(ser1177)表达,ELISA法检测血清白细胞介素6(interleukin-6,IL-6)浓度。结果:1、血脂、血糖变化(1)基线血脂:各组Apo E-/-小鼠的基线血清TG、TC、HDL-C、LDL-C的水平无差异(P0.05);(2)干预结束时血脂:模型组和模型干预组小鼠血清TC、LDL-C较对照组及对照干预组明显升高(P0.01),HDL-C显著降低(P0.01),TG的变化无统计学意义。经Pio干预后,模型干预组小鼠血清TC、LDL-C较模型组明显降低(TC:P0.05,LDL-C:P0.01),HDL-C明显升高(P0.01),TG的变化无统计学意义;对照干预组小鼠血清LDL-C较对照组显著降低(P0.01),TG、TC、HDL-C无明显变化。(3)干预结束时血糖:经高脂饲料饲养后,模型组和模型干预组小鼠血糖较对照组及对照干预组明显升高(P0.01);模型干预组较模型组无明显变化。2、组织病理学观察及分析(1)主动脉整体油红O染色:模型组和模型干预组小鼠主动脉红染脂质斑块多,其斑块相对面积明显高于对照组、对照干预组(P0.01),而模型干预组较模型组减少(P0.01);对照干预组较对照组有减少,但差异无统计学意义(P0.05)。(2)主动脉根部HE染色:与对照组、对照干预组比较,模型组及模型干预组主动脉根部可见大量动脉粥样硬化斑块形成,其斑块相对面积显著增加(P0.01),与模型组比较,模型干预组斑块面积减少(P0.01)。3、Western Blot法检测主动脉PPARγ蛋白的表达模型组主动脉PPARγ的表达较对照组显著升高(P0.01);而经Pio治疗后,模型干预组PPARγ的表达较模型组进一步升高(P0.01),对照干预组PPARγ的表达较对照组进一步升高(P0.01)。4、主动脉HSP22蛋白的表达(1)Western Blot法:模型组及模型干预组主动脉HSP22的表达较对照组、对照干预组显著升高(P0.01);而模型干预组经Pio治疗后HSP22的表达较模型组降低(P0.01);(2)免疫组织化学法:对照组及对照干预组主动脉内棕色着色浅,HSP22表达量少;模型组主动脉斑块及血管壁棕色着色深,HSP22大量表达;模型干预组与对照组相比,HSP22表达量也增加,但较模型组明显较少;5、Western Blot法检测主动脉e NOS、p-e NOS蛋白的表达模型组主动脉e NOS蛋白的表达较对照组、对照干预组显著降低(P0.01),而模型干预组经Pio干预后,e NOS的表达较模型组升高(P=0.027)。模型组主动脉p-e NOS蛋白的表达较对照组降低(P=0.028)、较对照干预组显著降低(P0.01);而模型干预组p-e NOS的表达较模型组显著升高(P0.01)。6、Western Blot法检测ICAM-1蛋白的表达模型组及模型干预组主动脉ICAM-1的表达较对照组、对照干预组显著升高(P0.01),给予Pio治疗后模型干预组主动脉ICAM-1的表达较高脂模型组降低(P0.01)。7、血清IL-6水平模型组血清IL-6的浓度较对照组、对照干预组显著升高(P0.01),而给予Pio干预后,模型干预组血清IL-6的浓度较模型组降低(P0.01)。结论:1、高脂饮食可上调主动脉PPARγ、HSP22的表达,下调e NOS、p-e NOS的表达,上调ICAM-1的表达,促进动脉粥样硬化形成。2、吡格列酮可激活PPARγ,下调主动脉内HSP22的表达,上调e NOS、p-e NOS的表达,下调ICAM-1的表达,抑制动脉粥样硬化形成。
[Abstract]:AIM: To study the effects of pioglitazone (Pio) on the proliferation of peroxidases in the aorta of apolipoprotein E knockout mice (ApoE-/-) mice. HSP22 expression, and explore the role of HSP22 in it. Methods: 1, 36 male mice aged 8 to 9 weeks were randomly divided into 4 groups: control group (n = 9, normal feed), control intervention group (n = 9, normal feed + PIo), model group (n = 9, high fat feed) and model intervention group (n = 9, high fat feed + Pio); control group. In the control intervention group, 12 weeks of normal feed were fed, the model group and the model intervention group were fed with the high fat diet for 12 weeks, that is, the high-fat diet (HFD), among which the intervention group began to give PIo (20mg/ kg/ d) intervention treatment from the fifth week to intervene for 8 weeks. Serum lipids were detected at the end of baseline and intervention in each group, including triglyceride (TG), total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C); To detect the formation and pathological changes of atherosclerotic plaques in mice at the end of intervention, the formation and pathological changes of atherosclerotic plaques in mice were observed by the staining of whole oil red O and hematoxylin and eosin (HE) staining. The levels of interleukin-6 (IL-6) and interleukin-6 (IL-6) in serum were measured by Western blot. The expression of interleukin-6 (IL-6) was detected by ELISA. Results: There was no difference in serum TG, TC, HDL-C and LDL-C in the baseline serum TG, TC, HDL-C and LDL-C in all groups of Aso E-/-mice (P0.05). LDL-C was significantly higher than that in control group (P0.01), HDL-C decreased significantly (P <0.01), and TG had no statistical significance. After PIO intervention, the serum TC and LDL-C of the model intervention group were significantly lower than that in the model group (TC: P0.05, LDL-C: P0.01), HDL-C increased significantly (P <0.01), TG did not change significantly, and the serum LDL-C of the control group was significantly lower than that in the control group (P0.01), TG, TC and HDL-C had no obvious change. (3) At the end of the intervention, the blood glucose in the model group and the model intervention group was significantly higher than that in the control group (P0.01). Histopathological observation and analysis (1) The whole aorta oil red O staining: the model group and the model intervention group were significantly higher than the control group and the control intervention group (P0.01), while the model intervention group was lower than that in the model group (P0.01). There was no significant difference between control intervention group and control group (P0.05). (2) HE staining of the aortic root: Compared with the control group and the control intervention group, the model group and the model intervention group showed a large number of atherosclerotic plaques in the aortic root, and the relative area of the plaque increased significantly (P0.01). Compared with the model group, the plaque area of the model intervention group decreased (P0.01). Compared with the control group (P0.01), the expression of CD44v6 in the model intervention group was higher than that in the control group (P0.01). The expression of HSP22 in aorta and the expression of HSP22 in the model group and the model intervention group were significantly higher than that in the control group (P0.01). The expression of HSP22 in the model intervention group was lower than that of the model group (P0.01). In the control group and the control intervention group, the brown coloration of the aorta was shallow and the expression of HSP22 was small; the model group aortic plaque and blood vessel wall brown staining depth, HSP22 were expressed in large numbers; the model intervention group also increased the expression level of HSP22 compared with the control group, but the model group was less obvious; 5, The expression of NOS, p-e NOS protein in aorta was lower than that in control group (P = 0.01), and the expression of e NOS in the model group was higher than that in control group (P = 0. 01). The expression of p-e NOS protein in the model group was lower than that in the control group (P = 0.028), but the expression of p-e NOS in the model group was significantly higher than that in the control group (P0.01). The expression model of ICAM-1 and the expression of ICAM-1 in the model intervention group were higher than that in the control group (P0.01). The expression of ICAM-1 in the model intervention group was lower than that in the control group (P0.01). The serum IL-6 concentration in serum IL-6 group was higher than that in control group (P0.01), and the concentration of IL-6 in serum IL-6 group was lower than that in control group (P0.01). Conclusion: 1. High fat diet can upregulate the expression of ENOS, p-e NOS, regulate the expression of ICAM-1, regulate the expression of ICAM-1 and promote the formation of atherosclerosis. Down-regulates the expression of ICAM-1 and inhibits the formation of atherosclerosis.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R543.5

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