谷氨酰胺诱导热休克蛋白70在缺氧性肺动脉高压新生大鼠中的保护作用研究

Protection of glutamine-induced heat shock protein 70 on neonatal rats from hypoxic pulmonary hypertension

目的探讨谷氨酰胺对缺氧性肺动脉高压新生大鼠肺损伤的保护作用。方法将84只Wistar新生大鼠随机分为缺氧性肺动脉高压(HPH)组和对照组。HPH组包括模型组、盐水组、谷氨酰胺(Gln)组,后两组新生大鼠通过尾静脉注射无菌盐水、Gln注射液,1次/d,连续3 d,3组同时建立HPH模型,并分别于缺氧3、7、14 d和对照组同步观察不同时间点肺动脉压力(mPAP)变化;用光学显微镜观察肺血管结构变化,肺小血管重塑指标(MT%、MA%)变化;用Western blot法检测各组新生大鼠肺组织中热休克蛋白70(HSP70)、缺氧诱导因子-1α(HIF-1α)、内皮素-1(ET-1)、诱导型一氧化氮合酶(iNOS)的蛋白质表达。结果(1)模型组及盐水组缺氧3 d、7 d、14 d的mPAP水平与同日龄对照组比较显著增高(P<0.05),表明造模成功。Gln组缺氧3 d、7 d、14 d的mPAP水平与同日龄模型组及盐水组比较,显著降低(P<0.05);(2)缺氧3 d,各组间测量的肺血管重塑指标中层横截面积占血管总横截面积的百分比(MA%)、肺动脉血管壁中层壁厚占外径的百分比(MT%),差异无统计学意义[F(MA%)=0.312、F(MT%)=0.252,P>0.05];缺氧7 d、14 d各组间测量的肺血管MA%、MT%比较,差异有统计学意义[F(MA%7 d)=5.367,F(MT%7 d)=6.102,P<0.01;F(MA%14 d)=8.672,P<0.01,F(MT%14 d)=5.132、P<0.05)],其中对照组及Gln组分别与模型组、盐水组比较,差异具有统计学意义(P<0.05),Gln组与对照组比较,差异具有统计学意义(P>0.05);(3)在缺氧3 d、7 d、14 d各组间HSP70的蛋白质表达比较差异有统计学意义(F=13.547,P<0.01;F=3.546,P<0.05;F=15.625,P<0.01);HPH各组表达增强,Gln组与模型组、盐水组比较,差异具有统计学意义(P<0.01),HSP70在Gln组表达增强;在缺氧3 d、7 d、14 d各组间HIF-1α、ET-1、iNOS蛋白质表达比较,差异具有统计学意义(P<0.05),其中对照组与模型组、盐水组比较,差异具有统计学意义(P<0.05),HPH各组表达增强,Gln组与模型组、盐水组比较,差异具有统计学意义(P<0.05),对照组与Gln组比较差异无统计学意义(P>0.05)。结论谷氨酰胺可以诱导缺氧性肺动脉高压新生大鼠肺组织HSP70的表达,降低HIF-1α、ET-1、iNOS的表达,减轻缺氧性肺损伤,可能成为治疗新生儿HPH的一种新策略。

ObjectiveTo investigate the protective effect of glutamine on lung injury in newborn rats with hypoxic pulmonary hypertension(HPH).Methods84 Wistar newborn rats were randomly divided into HPH and control group.HPH group was further divided into model group,saline group and Gln group.The latter two groups of neonatal rats were injected sterile saline and Gln injection through tail vein once a day for 3 consecutive days.HPH models were established in three groups at the same time,and the changes were observed of pulmonary artery pressure(mPAP)at different observation time points in hypoxia 3,7,14 days and control group,respectively.The expression of HSP70,HIF-1a,ET-1 and iNOS in lung tissue of neonatal rats were detected by Western blot.Results(1)The levels of mPAP in the model group and saline group were significantly higher than those in the control group(P<0.05),indicating that the model was successfully established.Compared with the model group and saline group,the levels of mPAP in the Gln group on the 3rd,7th and 14th days of hypoxia were significantly reduced(P<0.05).(2)No significant difference was observed in MA%and MT%of pulmonary vascular remodeling between groups after 3 days of hypoxia[F(MA%)=0.312,F(MT%)=0.252,P>0.05)];whreas there was significant difference in MA%and MT%of pulmonary vessels between groups after 7 days and 14 days of hypoxia[F(MA%7d)=5.367,F(MT%7d)=6.102,P<0.01;F(MA%14d)=8.672,P<0.01,F(MT%14d)=5.132,P<0.05)].Among them,the control group and Gln group was compared with model group and saline group(P<0.05),and group Gln was compared with control group(P>0.05).(3)There were significant differences of HSP70 protein expression among groups on 3 d,7 d and 14 d of hypoxia(F=13.547,P<0.01;F=3.546,P<0.05;F=15.625,P<0.01).The expression of HPH was significantly increased in each group,compared with model group and saline group(P<0.01),HSP70 increased in Gln group.HIF-1a,ET-1 and iNOS protein expression in 3 d,7 d and 14d of hypoxia(P<0.05).The expression of HPH was increased in the control group and model group,saline group(P<0.05).No significant difference was identified between the Gln group and model group,saline group(P<0.05).No significant difference was observed between the control group and the Gln group(P>0.05),either.ConclusionGlutamine was shown to be able to induce the HSP70 expression in pulmonary tissue of newborn rats with HPH,reducing the manifestation of HIF-1a,ET-1 and iNOS,whereby reducing the hypoxic injury,which may be used for the treatment of HPH in newborn baby.