【作者】 王小燕；

【导师】 李勋；

【作者基本信息】 苏州大学 ， 心血管内科， 2011， 硕士

【摘要】 目的:探讨阿托伐他汀对大鼠急性心肌梗死后心肌纤维化的作用及其机制。方法:选择健康雄性SD(Sprague Dawley)大鼠,称重后用10%水合氯醛腹腔注射麻醉并行气管插管,开胸结扎左冠状动脉前降支建立心肌梗死模型,假手术组仅将缝线穿过前降支而不结扎。将心肌梗死大鼠随机分为两个亚组,一组接受阿托伐他汀(10mg/kg/d)灌胃给药5周,设为心梗药物干预组(MI干预组),另一组给予等体积生理盐水,设为心肌梗死组(MI组)。第6周时存活的大鼠共20只,其中假手术组(sham组)6只,MI组8只,MI干预组6只。将大鼠称重后麻醉,开胸取出心脏,称左心室重量并取左心室非梗死区心肌组织液氮冻藏备用。采用WST-1法测定心肌组织超氧阴离子(O2-)浓度及NADPH氧化酶活性,实时荧光定量聚合酶链反应法检测NADPH氧化酶亚单位gp91phox和p22phox mRNA及基质金属蛋白酶-2和-9 mRNA的表达,用考马斯亮蓝法检测心肌胶原蛋白含量。结果: 1. MI组大鼠左室重量指数为(3.18±0.33)mg/g,sham组为(2.69±0.21)mg/g,两组间差异有统计学意义(P<0.01),MI干预组为(2.94±0.17)mg/g,较MI组显著降低(P<0.01),但与sham组无统计学差异(P>0.05)。2.⑴MI组大鼠非梗死区心肌组织O2-浓度及NADPH氧化酶活性分别为(12550.47±7726.33)RLU/mg和(4.98±0.65)RLU/mg,均较sham组[分别为(2181.01±236.8)RLU/mg和(2.62±0.29)RLU/mg]显著升高(P<0.01),MI干预组O2-浓度为(4145.96±1229.83)RLU/mg,NADPH氧化酶活性为(3.33±0.44)RLU/mg,与MI组比较差异有统计学意义(P<0.01),但仍较sham组高,其中O2-浓度与sham组间无统计学差异(P>0.05),但NADPH氧化酶活性与sham组比较仍有统计学意义(P<0.01)。⑵MI组大鼠NADPH氧化酶亚单位gp91phox mRNA的表达水平是sham组的(2.73±0.78)倍,两者比较有统计学意义(P<0.01),是MI干预组的(1.37±0.39)倍,二者比较亦有统计学差异(P<0.05),而MI干预组gp91phox mRNA表达水平是sham组的(2.13±0.90)倍,两者比较有统计学差异(P<0.01);MI组p22phox mRNA的表达水平是sham组的(23.42±7.06)倍,两者比较有统计学意义(P<0.01),是MI干预组的(4.16±2.10)倍,二者比较亦有统计学差异(P<0.01),而MI干预组p22phox mRNA的表达水平是sham组的(6.68±3.49)倍,两者比较有统计学差异(P<0.01)。(3)O2-浓度与NADPH氧化酶活性显著相关(R=0.865,P<0.01);gp91phox和p22phox mRNA表达与O2-浓度亦显著相关(分别为R=0.656,P<0.01;R=0.449,P<0.05)。3.(1)MI组MMP-2 mRNA的表达水平是sham组的(29.14±9.33)倍,是MI干预组的(3.78±1.24)倍,而MI干预组则是sham组的(8.37±3.18)倍,三组间两两比较均有统计学差异(P<0.01);MI组MMP-9 mRNA的表达水平是sham组的(4.94±0.66)倍,是MI干预组的(2.52±0.50)倍,而MI干预组则是sham组的(1.99±0.28)倍,三组间两两比较亦均有统计学差异(P<0.01)。(2)MMP-2和MMP-9 mRNA表达与O2-浓度显著相关(分别为R=0.676,P<0.01;R=0.700,P<0.01)。4. sham组非梗死区羟脯氨酸含量和胶原蛋白含量分别为(1.97±1.03)μg/mg和(14.72±7.67)μg/mg,MI组分别为(9.82±1.52)μg/mg和(73.26±11.37)μg/mg,二组间差异有统计学意义(P<0.01),MI干预组分别为(6.23±1.38)μg/mg和(46.46±10.3)μg/mg,较MI组显著下降(P<0.01),但仍高于sham组,且亦有显著差异(P<0.01)。O2-浓度与心肌胶原蛋白含量显著相关(R=0.648,P<0.01)。结论: 1. MI组大鼠非梗死区心肌组织O2-浓度、NADPH氧化酶活性及其亚单位gp91phox和p22phox mRNA表达水平较sham组显著升高,且O2-浓度与NADPH氧化酶活性及其亚单位表达水平显著相关,表明心肌梗死后NADPH氧化酶系统被激活,阿托伐他汀干预使非梗死区心肌组织中O2-浓度、NADPH氧化酶活性及其亚单位gp91phox和p22phox mRNA表达水平显著下降,说明阿托伐他汀通过抑制NADPH氧化酶活性及其表达减少O2-产生。2. MI组大鼠非梗死区心肌组织MMP-2和MMP-9 mRNA的表达均较sham组显著升高,且与O2-浓度显著相关,阿托伐他汀干预后MMP-2和MMP-9 mRNA的表达显著下降,提示阿托伐他汀通过抑制O2-浓度,进而抑制MMP-2和MMP-9 mRNA的表达。3. MI组大鼠左室重量指数及非梗死区羟脯氨酸和胶原蛋白含量均较sham组明显增高,且胶原蛋白含量与O2-浓度显著相关,阿托伐他汀干预后上述指标均显著下降,表明阿托伐他汀通过减少O2-产生可减轻心肌梗死后心肌纤维化及左室重构。更多还原

【Abstract】 Objective: To investigate the effect and mechanism of atorvastatin on myocardial fibrosis in rats after acute myocardial infarction.Methods: Healthy male Sprague Dawley rats were used and anesthesiaed by intraperitoneal injection of chloral hydrate(10%). After endotracheal intubation, thoracotomy was performed, MI group was produced by ligation of anterior descending coronary artery, the sham group underwent the same procedure without coronary artery ligation. They were assigned randomly to intragastrically receive atorvastatin (10 mg/ kg/d), named MI intervention group, or saline, named MI group, 24 hours after surgery for 5 weeks. A total of 20 rats survived after 5 weeks, 6 rats were in sham group, 6 rats were in MI intervention group and 8 rats were in MI group. The rats were killed and left ventricle weight weighed. Non-infarcted left ventricular myocardium was taken and frozed in liquid nitrogen. The concentration of O2- and the activity of NADPH oxidase were measured by WST-1, the expression level of gp91phox mRNA, p22phox mRNA, MMP-2 mRNA and MMP-9 mRNA were detected with RT-qPCR, and the content of myocardial collagen was measured with Coomassie brilliant blue.Results: 1. Left ventricular mass index (LVMI)was (3.18±0.33) mg/g in MI group, sham group was (2.69±0.21) mg/g, there was significant difference between the two groups(P<0.01). MI intervention group was (2.94±0.17) mg/g, reduced significantly than MI group(P<0.01), but without significance compared with sham group (P>0.05). Left ventricular weight and body weight were not statistically significant among MI group, sham group and MI intervention group (P>0.05).2. (1)The concentration of superoxide anion(O2-) and the activity of NADPH oxidase were (12550.47±7726.33)RLU/mg and (4.98±0.65)RLU/mg respectively in MI group, significantly increased compared to sham group [ ( 2181.01±236.8 ) RLU/mg and (2.62±0.29)RLU/mg respectively] (P<0. 01). The concentration of superoxide anion and the activity of NADPH oxidase in MI intervention group were (4145.96±1229.83)RLU/mg and (3.33±0.44)RLU/mg respectively, significantly decreased than MI group(P<0.01), increased than sham group,but there was no significant difference in the concentration of superoxide anion between the MI intervention group and sham group. (2) The expression level of gp91phox mRNA in MI group was [2.73±0.78] times than sham group, significantly different between the two groups (P<0.01), the expression level of gp91phox mRNA in MI group was [1.37±0.39] times than MI intervention group, significantly decreased(P < 0.05). The expression level of gp91phox mRNA in MI intervention group was [2.13±0.90] times than sham group, there was statistical difference also(P<0.01). The expression level of p22phox mRNA in MI group was [23.42±7.06] times than sham group, significantly different between the two groups(P<0.01), the expression level of p22phox mRNA in MI group was [4.16±2.10] times than MI intervention group, the latter group decreased significantly(P<0.01). The expression level of p22phox mRNA in MI intervention group was [6.68±3.49] times than sham group, there was statistical difference also(P<0.01). (3)The concentration of O2- positively correlated to the activity of NADPH oxidase(R=0. 865,P<0.01), the expression level of gp91phox mRNA(R=0.656,P<0.01) and p22phox mRNA (R=0.449,P<0.05).3. The expression level of MMP-2 mRNA in MI group was [29.14±9.33] times than sham group, significantly different between the two groups(P<0. 01), the expression level of MMP-2 mRNA in MI group was [3.78±1.24] times than MI intervention group, the latter group decreased significantly(P<0.01). The expression level of MMP-2 mRNA in MI intervention group was [8.37±3.18] times than sham group, there was statistical difference also(P<0.01). The expression level of MMP-9 mRNA in MI group was [4.94±0.66] times than sham group, significantly different between the two groups(P<0. 01). The expression level of MMP-9 mRNA in MI group was [2.52±0.50] times than MI intervention group, the latter group decreased significantly(P<0.01). The expression level of MMP-9 mRNA in MI intervention group was [1.99±0.28] times than sham group, there was statistical difference also(P<0.01). (2) The expression level of MMP-2 mRNA and MMP-9 mRNA positively correlated to the concentration of O2- (R=0.676,P<0.01;R=0.700,P<0.01).4. The content of hydroxyproline and collagen content were (1.97±1.03)μg/mg and (14.72±7.67)μg/mg respectively in non-infarcted myocardium in sham group, and (9.82±1.52)μg/mg and (73.26±11.37)μg/mg respectively in MI group, there was significant difference between the two groups(P < 0.01), while the content of hydroxyproline and collagen content were (6.23±1.38)μg/mg and(46.46±10.3)μg/mg respectively in MI intervention group, significantly increased than sham group(P<0.01) and significantly decreased than MI group(P<0.01). The concentration of O2- positively correlated to the content of collagen content(R=0.648,P<0.01).Conclusion: 1. The concentration of superoxide anion, the activity of the NADPH oxidase, the expression level of gp91phox and p22phox mRNA in MI group increased significantly compared to sham group, and the concentration of O2- positively correlated to the activity of NADPH oxidase, the expression level of gp91phox and p22phox mRNA. The concentration of the superoxide anion, the activity of NADPH oxidase, the expression level of gp91phox and p22phox mRNA in non-infarcted left ventricular myocardium in MI intervention group decreased significantly than MI group. This illustrates that atorvastatin reduces the production of O2- by inhibiting NADPH oxidase activity and its expression.2. The expression level of MMP-2 and MMP-9 mRNA in MI group were significantly higher than sham group, while the expression level of MMP-2 and MMP-9 mRNA in MI intervention group significantly decreased than MI group. Besides they positively correlated to the concentration of O2-. This indicates that atorvastatin decreases the expression of MMP-2 and MMP-9 mRNA by inhibiting O2-.3. The content of hydroxyproline, collagen content and LVMI in MI group were significantly higher than sham group, while MI intervention group was significantly decreased than MI group, which demonstrates that atorvastatin decreases the concentration of O2- ,and thereby relieves myocardial fibrosis and left ventricular remodeling after MI.更多还原