TAZ蛋白在血管紧张素Ⅱ诱导的高血压小鼠主动脉纤维化中的作用及其机制

Role and mechanism of TAZ in aortic fibrosis of angiotensinⅡ-induced hypertensive mice

目的探讨PDZ结合域的转录共刺激因子(TAZ)蛋白在血管紧张素Ⅱ(AngⅡ)诱导的高血压小鼠主动脉纤维化中的作用及其机制。方法将18只雄性C57BL/6小鼠随机分为正常对照组、AngⅡ组和AngⅡ+维替泊芬(Ve)组,每组6只。AngⅡ组和AngⅡ+Ve组小鼠皮下植入注满AngⅡ的渗透压微量泵,持续14 d释放AngⅡ(1.1 mg·kg^(-1)·d^(-1))诱导小鼠高血压模型;正常对照组小鼠不进行AngⅡ干预。AngⅡ+Ve组小鼠隔日腹腔注射1次Ve(60 mg·kg^(-1))至实验结束;AngⅡ组和正常对照组小鼠隔日腹腔注射等量生理盐水。造模结束后,采用尾动脉测压法测定小鼠收缩压(SBP)、舒张压(DBP)和心率;使用水合氯醛麻醉小鼠,打开胸腔,分离主动脉,采用苏木精-伊红染色法检测小鼠主动脉厚度,Masson染色法检测小鼠主动脉纤维化情况,Western blot法检测TAZ、转化生长因子-β(TGF-β)、母亲信号蛋白同源物3(Smad3)、磷酸化母亲信号蛋白同源物3(p-Smad3)和Ⅰ型胶原蛋白(collagenⅠ)表达。结果3组小鼠SBP、DBP比较差异有统计学意义(F=79.900、40.650,P<0.05)。AngⅡ组和AngⅡ+Ve组小鼠SBP、DBP显著高于正常对照组,AngⅡ+Ve组小鼠SBP、DBP显著低于AngⅡ组(P<0.05)。3组小鼠心率比较差异无统计学意义(F=0.090,P>0.05)。3组小鼠主动脉壁厚度比较差异有统计学意义(F=6.791,P<0.05);AngⅡ组小鼠主动脉壁厚度显著高于正常对照组(t=3.435,P<0.05),AngⅡ+Ve组小鼠主动脉壁厚度显著低于AngⅡ组(t=2.598,P<0.05),AngⅡ+Ve组与正常对照组小鼠主动脉壁厚度比较差异无统计学意义(t=0.361,P>0.05)。3组小鼠主动脉壁胶原纤维面积百分比比较差异有统计学意义(F=357.700,P<0.05)。AngⅡ组和AngⅡ+Ve组小鼠主动脉壁胶原纤维面积百分比显著高于正常对照组(t=25.810、4.882,P<0.05),AngⅡ+Ve组小鼠主动脉壁胶原纤维面积百分比显著低于AngⅡ组(t=20.580,P<0.05)。AngⅡ组小鼠主动脉中TAZ、TGF-β、p-Smad3、collagenⅠ蛋白相对表达量显著高于正常对照组(P<0.05);AngⅡ+Ve组小鼠主动脉中TAZ、TGF-β、p-Smad3、collagenⅠ蛋白相对表达量显著低于AngⅡ组(P<0.05);AngⅡ+Ve组小鼠主动脉中TGF-β蛋白相对表达量显著高于正常对照组(P<0.05),TAZ、p-Smad3、collagenⅠ蛋白相对表达量与正常对照组比较差异无统计学意义(P>0.05)。3组小鼠主动脉中Smad3蛋白相对表达量比较差异无统计学意义(F=46.010,P>0.05)。结论TAZ可通过增加纤维化相关蛋白p-Smad3及collagenⅠ表达,激活Hippo通路,促进AngⅡ诱导的高血压小鼠主动脉增生和纤维化。

Objective To investigate the role and mechanism of transcriptional co-activator with PDZ-binding motif(TAZ)in angiotensinⅡ(AngⅡ)-induced hypertensive mice.Methods Eighteen male C57BL/6 mice were randomly divided into normal control group,AngⅡgroup,AngⅡ+verteporfin(Ve)group,with six mice in each group.The mice in the AngⅡgroup and AngⅡ+Ve group were subcutaneously implanted with osmotic pressure micropump filled with AngⅡ,and AngⅡ(1.1 mg·kg^(-1)·d^(-1))was released for 14 days to induce hypertension models.The mice in the normal control group were not treated with AngⅡ.The mice in the AngⅡ+Ve group were intraperitoneally injected with Ve(60 mg·kg^(-1)),every other day,until the end of the experiment.The mice in the AngⅡgroup and normal control group were intraperitoneally injected with same amount of saline,every other day.After modeling,the systolic blood pressure(SBP),diastolic blood pressure(DBP)and heart rate of mice were measured by tail artery method;the mice were anesthetized with chloral hydrate,the thoracic cavity was opened,the aorta was isolated,and the thickness of aorta of the mice was measured by hematoxylin-eosin staining,the condition of the fibrosis of aorta of mice was detected by Masson staining,and the expressions of TAZ,transforming growth factor-β(TGF-β),mothers against decapentaplegic homolog 3(Smad3),phosphorylated mothers against decapentaplegic homolog 3(p-Smad3)and collagenⅠ(collagenⅠ)were detected by Western blot.Results There were significant differences in SBP and DBP of mice among the three groups(F=79.900,40.650;P<0.05).The SBP and DBP of mice in the AngⅡgroup and AngⅡ+Ve group were significantly higher than those in the normal control group,the SBP and DBP of mice in the AngⅡ+Ve group were significantly lower than those in the AngⅡgroup(P<0.05).There was no significant difference in heart rate of mice among the three groups(F=0.090,P>0.05).There was significant difference in the thickness of aortic wall of mice among the three groups(F=6.791,P<0.05);the thickness of aortic wall of mice in the AngⅡgroup was significantly higher than that in the normal control group(t=3.435,P<0.05),and the thickness of aortic wall of mice in the AngⅡ+Ve group was significantly lower than that in the AngⅡgroup(t=2.598,P<0.05),there was no significant difference in the thickness of aortic wall of mice between the AngⅡ+Ve group and normal control group(t=0.361,P>0.05).There was significant difference in the percentage of collagen fiber area in the aortic wall of mice among the three groups(F=357.700,P<0.05).The percentage of collagen fibers area in the aortic wall of mice in the AngⅡgroup and AngⅡ+Ve group was significantly higher than that in the normal control group(t=25.810,4.882;P<0.05),and the percentage of collagen fibers area in the aortic wall of mice in the AngⅡ+Ve group was significantly lower than that in the AngⅡgroup(t=20.580,P<0.05).The relative expressions of TAZ,TGF-β,p-Smad3 and collagenⅠprotein in aorta of mice in the AngⅡgroup were significantly higher than those in the normal control group(P<0.05);the relative expressions of TAZ,TGF-β,p-Smad3 and collagenⅠprotein in aorta of mice in the AngⅡ+Ve group were significantly lower than those in the AngⅡgroup(P<0.05);the relative expression of TGF-βprotein in aorta of mice in the AngⅡ+Ve group was significantly higher than that in the normal control group(P<0.05);there was no significant difference in the relative expressions of TAZ,p-Smad3,collagenⅠprotein in aorta of mice between the AngⅡ+Ve group and normal control group(P>0.05).There was no significant difference in the relative expression of Smad3 protein in aorta of mice among the three groups(F=46.010,P>0.05).Conclusion TAZ can activate Hippo pathway by increasing the expression of fibrosis-associated protein p-Smad3 and collagenⅠ,and promote aortic proliferation and fibrosis in AngⅡ-induced hypertensive mice.