四氢生物蝶呤通过增加磷酸化蛋白激酶B的表达改善高血压小鼠左心室舒张功能

Tetrahydrobiopterin improves left ventricular diastolic function possibly through upregulating phosphorylated protein kinase B expression in hypertensive mice induced by deoxycorticosterone acetate

目的 探讨四氢生物蝶呤（BH4）改善高血压小鼠左心室舒张功能的作用与磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（Akt）信号通路及Akt磷酸化水平的关系.方法 采用10周龄雄性C57BL/6小鼠建立DOCA-盐型高血压小鼠模型,同周龄假手术小鼠作为对照.将小鼠分为4组,分别为假手术组（n=20）、假手术＋ BH4组（n=20）、DOCA组（n=22）和DOCA ＋BH4组（n=22）,各组行相应的手术后14 d,予以假手术＋BH4组和DOCA ＋BH4组小鼠2.5％的BH4溶液0.1 ml/10 g灌胃,同时予以假手术组和DOCA组小鼠等体积生理盐水灌胃.干预7d后,测定鼠尾动脉压;彩色多普勒超声心动图和导管法血液动力学评价小鼠心脏结构与功能;高效液相色谱法检测小鼠左心室心肌组织BH4和二氢生物蝶呤（BH2）含量;免疫组织化学法检测磷酸化受磷蛋白（p-PLB）的表达;ELISA法检测小鼠左心室心肌组织中环磷鸟嘌呤核苷（cGMP）、丙二醛（MDA）、一氧化氮（NO）含量和超氧化物歧化酶（SOD）活性;Western blot检测小鼠左心室心肌组织中PI3K、Akt和磷酸化（p）-Akt的表达.结果 （1）各组小鼠收缩压和舒张压：DOCA组小鼠收缩压和舒张压均明显高于假手术组（P均＜0.05）.DOCA＋BH4组小鼠收缩压明显低于DOCA组（P=0.027）,舒张压两组间差异无统计学意义.（2）各组小鼠左心室功能：DOCA组小鼠左心室舒张功能指标二尖瓣环舒张早期组织运动速度（E＇）、E＇/二尖瓣环舒张晚期组织运动速度（A＇）均明显低于假手术组,二尖瓣口舒张早期血流峰值速度（E）/E＇明显高于假手术组（P均＜0.05）,DOCA＋ BH4组E ＇明显高于DOCA组,E/E＇明显低于DOCA组（P均＜0.05）.DOCA组小鼠左心室舒张末压（LVEDP）、左心室舒张末压力容积系数（EDPVR）和左心室松弛时间常数Tau指数均明显高于假手术组（P分别为0.002、0.011和0.016）.DOCA ＋BH4组小鼠EDPVR和Tau指数则明显低于DOCA组（P均＜0.05）.（3）各组小鼠心肌组织中BH4和BH2含量：DOCA组小鼠心肌细胞BH4和BH2含量均明显低于假手术组（P均＜0.05）.假手术＋ BH4组和DOCA＋ BH4组小鼠心肌细胞BH4含量和BH4/BH2比值均明显高于DOCA组（P均＜0.05）,但BH2含量组间比较差异均无统计学意义.（4）各组小鼠左心室心肌组织中cGMP、MDA和NO含量以及SOD活性：DOCA组小鼠左心室心肌组织中cGMP、SOD和NO含量均明显低于假手术组（P均＜0.05）,而MDA含量则明显高于假手术组（P＜0.05）.DOCA＋ BH4组小鼠左心室心肌组织中NO含量和SOD活性均明显高于DOCA组（P均＜0.05）.（5）各组小鼠左心室心肌组织中p-PLB蛋白表达：DOCA组小鼠左心室心肌组织中p-PLB蛋白表达明显高于假手术组（P＜0.05）,DOCA＋BH4组明显低于DOCA组（P＜0.05）.（6）各组小鼠左心室心肌组织中PI3K、Akt和p-Akt蛋白表达：DOCA组小鼠左心室心肌组织中PI3K和p-Akt（Ser473和Thr308）蛋白表达水平均明显低于假手术组（P均＜0.05）,DOCA＋BH4组PI3K和p-Akt（Ser473）均明显高于DOCA组（P均＜0.05）,而p-Akt（Thr308）蛋白表达水平在DOCA＋ BH4组和DOCA组间差异无统计学意义（P＞0.05）.结论 BH4可改善高血压小鼠左心室舒张功能,其可能通过影响Akt和PLB磷酸化水平而降低心肌氧化应激水平.

Objective To investigate whether tetrahydrobiopterin （BH4） could improve left ventricular diastolic function through phosphoinositide-3 kinase/protein kinase B （PI3K/Akt） signaling pathway in hypertensive mice.Methods Ten-week-old male C57BL/6 mice were used to establish the deoxycorticosterone acetate （DOCA）-salt hypertensive model,age matched Sham mice serve as the controls.Mice were divided into four groups：Sham （n =20）,Sham ＋ BH4 （n =20）,DOCA （n =22）,and DOCA ＋ BH4 （n =22）.On the t4 days after surgery,mice in Sham ＋ BH4 and DOCA ＋ BH4 groups received BH4 （0.1 ml/10 g） supplement for 7 days,while mice in Sham and DOCA groups were given equal volume of normal saline.The blood pressure measurements were performed 7 days later.Hemodynamic and echocardiographic parameters were used to assess left ventricular functions.High performance liquid chromatography （HPLC） analysis was used to measure cardiac biopterins BH4 and BH2.The phosphorylated phospholamban （p-PLB） was detected by immunohistochemical staining.PI3 K,Akt and phosphorylated Akt were assayed with Western blot analysis.Results （1） The systolic and diastolic blood pressure of DOCA group were significantly higher than control group （P ＜ 0.05）.Compared with DOCA group,the systolic blood pressure was lower in DOCA ＋ BH4 mice （P =0.027）.Diastolic blood pressure was similar between the groups.（2） Compared with Sham group,the left ventricular diastolic function indexes such as mitral annulus velocity （E＇） and E＇/A＇ratio were significantly lower,while the E/E＇ratio was significantly higher （P ＜ 0.05） in DOCA mice.The E/E＇ratio of DOCA ＋ BH4 group was significantly lower than that of DOCA group （P ＜ 0.05）.Compared with Sham group,the lefi ventricular end-diastolic pressure （LVEDP）,left ventricular end-diastolic pressure volumetric coefficient （EDPVR） and lefi ventricular relaxation time constant Tau index were significantly higher in DOCA mice （P =0.002,0.011 and 0.016,respectively）.The EDPVR and Tau index were significantly lower in DOCA ＋ BH4 group than in DOCA group （P ＜ 0.05）.（3） Compared with Sham group,the myocardial contents of BH4 and BH2 were significantly lower in DOCA mice （P ＜ 0.05）.The BH4 levels and BH4/BH2 ratio were significantly higher in Sham ＋ BH4 and DOCA ＋ BH4 groups than in the DOCA group （P ＜0.05）,but the BH2 levels were similar between groups.（4） The cGMP content,SOD activity and NO content in the lefi ventricular myocardial tissue were significantly lower （P ＜ 0.05）,while the MDA content was significantly higher in DOCA mice than in Sham mice.The NO content and SOD activity in DOCA ＋ BH4 groups were significantly higher than in the DOCA group （P ＜ 0.05）.（5） Compared with DOCA group,the expression of p-PLB was significantly higher in Sham mice and lower in DOCA ＋ BH4 mice （P ＜ 0.05）.（6） The expression of PI3 K,Akt and p-Akt （Ser473 and Thr308） in DOCA mice were significantly lower than in Sham group （P ＜ O.05）.The expression of PI3 K,Akt and p-Akt （Ser473） was significantly higher in DOCA ＋ BH4 group than in DOCA group （P ＜ 0.05）.p-Akt （Thr308） expression was similar between DOCA ＋ BH4 group and DOCA group （all P ＞ 0.05）.Conclusion Our results suggest that BH4 could improve left ventricular diastolic function in hypertensive mice,this effect might be mediated by reducing the oxidative stress in ventricular myocardium through modlating the expression of Akt and PLB phosphorylation.