【作者】 朱敏；

【导师】 吴延庆；

【作者基本信息】 南昌大学 ， 内科学（心血管）， 2017， 硕士

【摘要】 目的:研究热休克蛋白22（heat shock protein 22,HSP22）在高脂诱导动脉粥样硬化（atherosclerosis,AS）病变中的作用,以及HSP22对阿托伐他汀（atorvastatin,ator）干预的影响。方法:1、8～9周龄Apo E^-/-、HSP22^-/-//Apo E^-/-、HSP22⁺//Apo E^-/-雄性小鼠各18只,适应性喂养1周后,按体重分层随机分为2亚组,即对照组与他汀干预组（ator组）,HSP22^-/-对照组（KO组）与HSP22^-/-他汀干预组（KO+ator组）,HSP22⁺对照组（Tg组）与HSP22⁺他汀干预组（Tg+ator组）,各组小鼠均高脂饮食12周,其中ator组、KO+ator组及Tg+ator组从第5周开始给予ator(10mg/kg^-1·d^-1)干预,所有对照组给予等量生理盐水干预,共8周。2、每周称小鼠体重,检测小鼠基线及干预结束时血脂。3、主动脉整体油红O染色及HE染色法观察小鼠AS斑块情况。4、免疫组化法或Western Blot法检测主动脉HSP22、NF-κB、e NOS及ICAM-1蛋白的表达,ELISA法检测血清HSP22和IL-6浓度。结果:1、血脂变化（1）基线血脂:各组小鼠基线血脂水平无差异（P>0.05）;（2）干预结束时血脂:各对照组小鼠血清中TC、TG及HDL-C间的差异无统计学显著性（P>0.05）。Tg组小鼠血清LDL-C较KO组降低（13.59±0.84vs 15.41±0.39,P<0.01）。给予ator干预8周后,各干预组之间血脂水平无统计学显著性（P>0.05）。2、组织病理学观察主动脉整体油红O染色结果可见Tg组主动脉斑块相对面积低于KO组（（12.68±0.75）%vs（13.75±0.56）%,P<0.05）,经ator干预8周后,Tg+ator组主动脉斑块面积较KO+ator组减少（（3.78±0.20）%vs（4.90±0.65）%,P<0.01）。3、Western Blotting或免疫组化法检测主动脉HSP22、e NOS、NF-κB及ICAM-1蛋白表达小鼠主动脉HSP22及e NOS蛋白表达检测结果显示Tg组高于KO组（HSP22:1.08±0.06 vs 0.06±0.04,P<0.01;e NOS:0.40±0.03 vs 0.21±0.01,P<0.01）及对照组（HSP22:1.08±0.06 vs 0.31±0.14,P<0.01）,对照组高于KO组（HSP22:0.31±0.14 vs 0.06±0.04,P<0.01;e NOS:0.39±0.02 vs 0.21±0.01,P<0.01）。对照组小鼠主动脉NF-κB及ICAM-1蛋白表达较KO组降低（NF-κB:0.46±0.02 vs0.52±0.02,P<0.01;ICAM-1:0.34±0.01 vs 0.74±0.02,P<0.01）、较Tg组升高（NF-κB:0.46±0.02 vs 0.29±0.01,P<0.01;ICAM-1:0.34±0.01 vs 0.18±0.01,P<0.01）。经ator干预8周后,主动脉HSP22及e NOS蛋白表达结果显示Tg+ator组高于ator组（HSP22:0.70±0.03 vs 0.13±0.02,P<0.01）及KO+ator组（HSP22:0.70±0.03 vs 0.03±0.02,P<0.01;e NOS:0.47±0.02 vs 0.35±0.03,P<0.01）,ator组高于KO+ator组（HSP22:0.13±0.02 vs 0.03±0.02,P<0.01;e NOS:0.46±0.02vs 0.35±0.03,P<0.01）。主动脉ICAM-1及NF-κB蛋白表达结果显示KO+ator组较ator组及Tg+ator组升高（ICAM-1:0.70±0.03 vs 0.23±0.02 or 0.04±0.01,P<0.01;NF-κB:0.39±0.02 vs 0.17±0.01 or 0.15±0.01,P<0.01）,Tg+ator组较ator组降低（ICAM-1:0.04±0.01 vs 0.23±0.02,P<0.01）。4、ELISA法检测血清HSP22和IL-6的水平小鼠血清中HSP22水平结果显示KO组低于对照组和Tg组（45.84±1.18 vs181.37±21.21 or 248.17±36.78,P<0.01）,对照组低于Tg组（181.37±21.21 vs248.17±36.78,P<0.01）;对照组、KO组及Tg组间比较血清IL-6水平差异无统计学显著性（271.43±12.13 vs 263.62±11.58 vs 251.29±39.98,P>0.05）。经ator干预8周后,KO+ator组HSP22浓度低于ator组及Tg+ator组（38.97±8.36 vs145.83±6.95 or 203.28±18.82,P<0.01）,Tg+ator组高于ator组（203.28±18.82 vs145.83±6.95,P<0.01）;而ator组、KO+ator组及Tg+ator组间比较血清IL-6水平差异无统计学显著性（160.16±6.27 vs 172.44±9.48 vs 144.95±11.98,P>0.05）。结论:1、高脂饮食上调NF-κB、ICAM-1、IL-6的表达,下调e NOS表达,诱导AS形成,他汀可上调e NOS的表达,下调NF-κB、IL-6、ICAM-1的表达,改善AS。2、HSP22基因缺失可上调NF-κB、ICAM-1的表达,降低e NOS的表达,加速AS的进展,HSP22基因过表达可降低NF-κB、ICAM-1的表达,从而改善AS。3、HSP22基因缺失部分限制了他汀下调NF-κB及ICAM-1、上调e NOS表达的作用,其过表达可促进他汀下调ICAM-1表达,进一步改善AS。更多还原

【Abstract】 Objective:To evaluate the role of heat shock protein 22（HSP22）in atherosclerosis（AS）induced by high-fat diet and in intervention with atorvastatin（ator）.Methods:1.Total 3 groups of 8-9 weeks-old Apo E^-/-,HSP22^-/-//Apo E^-/-,HSP22⁺//Apo E^-/-male mice were used,sheach group were 18 mice.After one week of adaptive feeding,the mice in each group was randomly divided into 2 subgroups:control group and ator group,HSP22 knockout group（KO group）and HSP22 knockout ator group（KO+ator group）,HSP22 overexpression group（Tg group）and HSP22 overexpression ator group（Tg+ator group）were set up.All these mice were fed with high fat diet for 12weeks to establish atherosclerosis models.Atro(ator 10mg/kg^-1-d^-1)was administered to mice of ator group,KO+ator group and Tg+ator group from the 5th week.Other groups were administered by saline.2.The weight of all mice was recorded every week.The lipid levels were measured at baseline and the end of intervention.3.The Oil Red O staining and HE staining of the aortic wall of the mice were used to measure the atherosclerotic lesion burdens.4.The protein levels of HSP22,NF-κB,e NOS,ICAM-1 and IL-6 in the aortas or serum were examined by Western blotting or immunohist ochemistry（IHC）or ELISA.Results:1.The changes of lipid level（1）The baseline lipid shows no difference in all groups（P>0.05）;（2）The lipid at the end of intervention:there were no significant difference in serum TC,TG and HDL-C of mice among the control group,KO group and Tg group（P>0.05）.LDL-C in Tg group was significantly less than KO group（13.59±0.84vs15.41±0.39,P<0.01）.After 8 weeks of ator intervention,there were no difference in serum TC,TG,LDL-C and HDL-C of mice among the ator group,KO+ator group and Tg+ator group（P>0.05）.2.Histopathological observationAortic Oil Red O staining showed the relative area of aorta plaque in Tg group was less than that in KO group（（12.68±0.75）%vs（13.75±0.56）%,P<0.05）.After 8weeks of ator intervention,the relative area of aorta plaque in Tg+ator group was significantly less than that in KO+ator group（（3.78±0.20）%vs（4.90±0.65）%,P<0.01）.3.The protein expression of HSP22,e NOS,NF-κB and ICAM-1 were measured by WB or IHCThe protein expression of HSP22 and e NOS in Tg group was significantly higher than that in control group（HSP22:1.08±0.06vs0.31±0.14,P<0.01）and KO group（HSP22:1.08±0.06vs0.06±0.04,P<0.01;e NOS:0.40±0.03vs0.21±0.01,P<0.01）,and their expression in control group was significantly higher than that in KO group（HSP22:0.31±0.14vs0.06±0.04,P<0.01;e NOS:0.39±0.02vs0.21±0.01,P<0.01）.The pro tein expression of NF-κB and ICAM-1 in control group was significantly less compared with KO group（NF-κB:0.46±0.02vs0.52±0.02,P<0.01;ICAM-1:0.34±0.01vs0.74±0.02,P<0.01）,and their expression was significantly higher than that in Tg gro up（NF-κB:0.46±0.02vs0.29±0.01,P<0.01;ICAM-1:0.34±0.01vs0.18±0.01,P<0.01）.After 8 weeks of ator intervention,the protein expression of HSP22 and e NOS in Tg+ator group was significantly higher than that in ator group（HSP22:0.70±0.03vs0.13±0.02,P<0.01）and KO+ator group（HSP22:0.70±0.03vs0.03±0.02,P<0.01;e NOS:0.47±0.02vs0.35±0.03,P<0.01）,and their expressions in ator group were significantly higher than KO+ator group（HSP22:0.13±0.02vs0.03±0.02,P<0.01;e NOS:0.46±0.02vs0.35±0.03,P<0.01）.The expressions of NF-κB and ICAM-1 protein in KO+ator group were significantly higher than ator group and Tg+ator group（ICAM-1:0.70±0.03vs0.23±0.02or0.04±0.01,P<0.01;NF-κB:0.39±0.02vs0.17±0.01or0.15±0.01,P<0.01）,and the protein expression of ICAM-1 in Tg+ator group was significantly less than that in ator group（ICAM-1:0.04±0.01vs0.23±0.02,P<0.01）.4.The secretions of HSP22 and IL-6 from serum samples of all groups were measured by ELISAThe concentration of serum HSP22 in KO group was significantly less than that in control group and Tg group（45.84±1.18vs181.37±21.21or248.17±36.78,P<0.01）,and its expression in control group was significantly less than that in Tg group（181.37±21.21vs 248.17±36.78,P<0.01）.No difference in concentration of serum IL-6 was found between Tg group,KO group and control group（271.43±12.13vs263.62±11.58or 251.29±39.98,P>0.05）.After 8 weeks of ator intervention,the concentration of serum HSP22 in KO+ator group was significantly less than that in ator group and Tg+ator group（38.97±8.36vs145.83±6.95or203.28±18.82,P<0.01）,and its expression in Tg+ator group was significantly higher than that in ator group（203.28±18.82vs145.83±6.95,P<0.01）.The concentration of serum IL-6 between Tg+ator group,KO+ator group and ator group was not different（160.16±6.27vs172.44±9.48or144.95±11.98,P>0.05）.Conclusion:1.High fat feeding induces the expression of NF-κB,ICAM-1 and IL-6,and inhibits the expression of e NOS,accelerates AS.The intervention with ator up-regulates the expression of e NOS and down-regulates the expression of NF-κB,IL-6 and ICAM-1,thus attenuating AS development.2.HSP22 gene deletion up-regulates the expression of NF-κB and ICAM-1,and down-regulates the expression of e NOS,accelerates AS.HSP22 overexpression decreases the expression of NF-κB and ICAM-1,thus attenuating AS development.3.HSP22 gene deletion partial limits the role of ator on the expression of NF-κB,ICAM-1 and e NOS.HSP22 overexpression amplifies the reduced expression of ICAM-1 by the intervention with ator,further improveattenuates AS development.Key words:atherosclerosis;heat shock protein 22;atorvastatin;inflammation.更多还原