热休克蛋白70调控Bcl-2/Bax对缺氧性肺高压新生大鼠肺血管内皮细胞凋亡及肺动脉压力的作用

Study of heat shock protein 70 regulating Bcl-2/Bax on apoptosis of pulmonary vascular endothelial cells and pulmonary arterial pressure of hypoxic pulmonary hypertension in neonatal rats

目的探讨热休克蛋白70(HSP70)调控Bcl-2和Bax对缺氧性肺动脉高压(hypoxia pulmonary hypertension,HPH)新生大鼠肺血管内皮细胞凋亡和肺动脉压力的影响。方法128只Wistar新生大鼠随机分为对照组和缺氧组,缺氧组建立HPH模型,按干预方法分为HPH组、HSP70+HPH组和KNK437+HSP70+HPH组。HSP70+HPH组和KNK437+HSP70+HPH组经口气管插管注入Ad-HSP70-EGFP(增强型绿色荧光蛋白标记的HSP70腺病毒载体),HPH组和对照组经口气管插管注入Ad-CON-EGFP(增强型绿色荧光蛋白标记的空腺病毒载体),KNK437+HSP70+HPH组每日腹腔注射HSP70抑制剂KNK437,对照组常氧饲养,各组分别于缺氧3、7、10、14 d检测平均右心室收缩压(RVSP),观察肺血管内皮细胞凋亡变化,并检测肺组织中HSP70、Bcl-2和Bax的表达水平。结果(1)平均RVSP:各观察时间点HPH组平均RVSP均高于对照组(P<0.05);缺氧3、7、10 d,HSP70+HPH组平均RVSP低于HPH组和KNK437+HSP70+HPH组(P<0.05),和对照组对比差异无统计学意义。(2)HSP70的表达:缺氧3、7、10 d HPH组HSP70表达高于对照组(P<0.05),缺氧3、7 d HSP70+HPH组HSP70表达高于HPH组(P<0.05),缺氧10、14 d与HPH组比较差异无统计学意义(P>0.05);缺氧3、7、10 d KNK437+HSP70+HPH组HSP70表达均低于HSP70+HPH组和HPH组(P<0.05)。(3)Bcl-2和Bax的表达:HPH组缺氧3 d Bcl-2表达低于对照组(P<0.05),缺氧3、7 d Bax表达高于对照组(P<0.05);HSP70+HPH组在各观察时间点Bcl-2表达均高于HPH组(P<0.05),缺氧3 d Bax表达低于HPH组(P<0.05);KNK437+HSP70+HPH组Bcl-2表达缺氧3、7、10 d低于HSP70+HPH组(P<0.05),缺氧3 d Bax表达高于HSP70+HPH组(P<0.05)。结论腺病毒介导外源性HSP70在新生大鼠肺组织中表达升高,可能通过上调Bcl-2和下调Bax,在缺氧早期抑制了肺血管内皮细胞凋亡,降低了肺动脉压力,在新生大鼠HPH中发挥保护作用。

Objective To study the effect of Heat shock protein 70(HSP70)regulating Bcl-2 and Bax on pulmonary vascular endothelial cell apoptosis and pulmonary artery pressure in neonatal rats with hypoxia pulmonary hypertension(HPH).Methods A total of 128 Wistar neonatal rats were randomly divided into control group and hypoxia group,and HPH model was established in hypoxia group which were subsequently divided into HPH group,HSP70+HPH group and KNK437+HSP70+HPH group based on different intervention method.In HSP70+HPH and KNK437+HSP70+HPH group,Ad-HSP70-EGFP(enhanced green fluorescent protein labeled HSP70 adenoviral vector)and Ad CON-EGFP(enhanced green fluorescent protein labeled empty adenovirus vector)were injected through oral tracheal intubation,and KNK437+HSP70+HPH group was intraperitoneally injected with the HSP70 inhibitor KNK437 daily,and the control group was fed under normoxia condition.Average right ventricular systolic pressure(RVSP)was tested at 3,7,10,14 days in each group,changes in pulmonary vascular endothelial cell apoptosis were observed and the expression levels of HSP70,Bcl-2 and Bax in lung tissue were measured.Results(1)Average RVSP:The average RVSP in HPH group was higher than that of the control group at each observation time point(P<0.05),indicating that the model establishment was successful;the average RVSP in HSP70+HPH group was lower than that of the HPH group and KNK437+HSP70 at 3,7,and 10 days of hypoxia(P<0.05),while there was no significant difference compared with the control group.(2)The expression of HSP70:the expression of HSP70 in the HPH group was higher than that of the control group at 3,7 and 10 days of hypoxia(P<0.05),the expression of HSP70 in the HSP70+HPH group was higher than that of the HPH group at 3 and 7 days of hypoxia(P<0.05),and there was no statistical difference in 10 and 14 days of hypoxia(P>0.05).The expression of HSP70 in KNK437+HSP70+HPH group was lower than HSP70+HPH group and HPH group at 3,7,and 10 days of hypoxia(P<0.05).(3)The expression of Bcl-2 and Bax:The expression of Bcl-2 in the HPH group was lower than that of the control group(P<0.05),and the expression of Bax was higher than that of the control group(P<0.05).The expression of Bcl-2 at the different observation time point in the HSP70+HPH group was higher than that of the HPH group(P<0.05),and the expression of Bax in 3 days of hypoxia was lower than that of the HPH group(P<0.05);the expression of Bcl-2 in the KNK437+HSP70+HPH group was lower than that in the HSP70+HPH group at 3,7,10 days of hypoxia(P<0.05),and the expression of Bax was higher than that in the HSP70+HPH group at 3 days of hypoxia(P<0.05).Conclusions Adenovirus-mediated increase in the expression of exogenous HSP70 in the lung tissue of neonatal rats may be through up-regulation of Bcl-2 and down-regulation of Bax,then inhibit pulmonary endothelial cell apoptosis in the early stage of hypoxia and reducing pulmonary artery pressure,which plays a protective role in development of HPH in neonatal rat.