布地奈德对宫内感染致支气管肺发育不良新生大鼠肺部血管发育及血管内皮生长因子、核苷酸结合寡聚化结构域样蛋白3表达的影响

Effects of Budesonide on pulmonary vascular development and the expressions of vascular endothelial growth factor,nucleotide binding oligomerization domain-like receptor protein 3 in newborn rats with bronchopulmonary dysplasia caused by intrauterine infection

目的探究布地奈德(BUD)对宫内感染致支气管肺发育不良(BPD)新生大鼠肺部血管发育及血管内皮生长因子(VEGF)和核苷酸结合寡聚化结构域样蛋白3(NLRP3)表达的影响。方法将孕15 d的SD大鼠分为对照组和感染组[0.35 mg/(kg·d)脂多糖腹腔注射],并将各组所产的新生大鼠分为BUD组(0.5 mg BUD悬液)、正常对照组(NC组,等量9 g/L盐水)、BPD组(等量9 g/L盐水),每组40只,均采用雾化吸入,2次/d,持续14 d。分别于出生时及给药后3 d、7 d、14 d选取10只新生大鼠,HE染色后观察肺组织病理变化和放射状肺泡计数(RAC),并测量肺泡呼吸膜厚度;免疫组织化学法检测肺组织血小板-内皮细胞黏附分子(PECAM-1/CD_(31)),并计算肺微血管密度;免疫印迹法检测肺组织VEGF、NLRP3及半胱天冬氨酸蛋白酶-1(Caspase-1)蛋白表达水平;酶联免疫吸附试验检测血清白细胞介素(IL)-1β、IL-18水平。结果随日龄增加,NC组新生大鼠肺组织逐渐发育成熟,肺泡结构清晰、大小均匀、计数明显增加,未见明显病理变化;BPD组新生大鼠肺组织结构紊乱,肺泡大小不一、计数少,肺泡腔或肺泡间隔有炎症细胞渗出;与BPD组相比,BUD组新生大鼠肺组织病理变化显著减轻,给药后3 d、7 d、14 d,与NC组比较,BPD组和BUD组新生大鼠RAC减少,CD31阳性细胞平均积分光密度值、肺微血管密度、肺组织VEGF蛋白水平降低,差异均有统计学意义(均P<0.05),呼吸膜厚度增大,肺组织NLRP3、Caspase-1蛋白水平及血清IL-1β、IL-18水平升高,差异均有统计学意义(均P<0.05);与BPD组比较,BUD组新生大鼠RAC增多,CD31阳性细胞平均积分光密度值、肺微血管密度、肺组织VEGF蛋白水平明显升高,差异均有统计学意义(均P<0.05),呼吸膜厚度减小,肺组织NLRP3、Caspase-1蛋白水平及血清IL-1β、IL-18水平明显降低,差异均有统计学意义(均P<0.05)。结论宫内感染致BPD新生大鼠病理变化的发生发展可通过肺组织炎症反应影响肺血管发育。BUD可通过降低炎症反应及上调VEGF表达,促进肺组织血管重塑,增加肺组织微血管密度,缓解BPD新生大鼠肺组织病理变化。

Objective To investigate the effect of Budesonide(BUD)on pulmonary vascular development and the expression of vascular endothelial growth factor(VEGF)and nucleotide binding oligomerization domain-like receptor protein 3(NLRP3)in newborn rats with bronchopulmonary dysplasia(BPD)caused by intrauterine infection.Methods The 15-day-pregnant SD rats were divided into control group and infection group[intraperitoneal injection of 0.35 mg/(kg·d)lipopolysaccharide],and the newborn rats born by the above groups were divided into 3 groups:BUD group(0.5 mg of BUD suspension),normal control group(NC group,equal amount of 9 g/L saline),BPD group(equal amount of 9 g/L saline),with 40 rats in each group,all of them were inhaled twice a day for 14 days.Ten newborn rats were selected at birth,on the 3^(rd),7^(th) and 14^(th) day after administration.Pulmonary histopathological changes and radial alveolar counts(RAC)were observed after HE staining,and the thickness of alveolar respiratory membrane was measured;the platelet-endothelial cell adhesion molecule(PECAM-1/CD31)in lung tissue was detected by immunohistochemistry,and the density of pulmonary microvessels was calculated;the expressions of VEGF,NLRP3 and Caspase-1 were detected by Western blot;and the levels of serum interleukin(IL)-1βand IL-18 were measured by enzyme-linked immunosorbent assay.Results With the increase of day-old,the lung tissue of newborn rats in NC group was gradually developed and matured,the structure of alveoli was clear,the size was uniform,the count was significantly increased,and no obvious pathological changes were observed.In BPD group,the lung tissue structure was disordered,the alveoli were different in size and few in count,and inflammatory cells were exuded from the alveoli or the alveoli space.Compared with BPD group,the pathological changes of lung tissue in BUD group were significantly reduced.On the 3^(rd),7^(th) and 14^(th) day after administration,compared with NC group,the RAC,average integral optical density of CD31 positive cells,density of pulmonary microvessel and level of VEGF protein in lung tissue of BPD group and BUD group were lower,and the differences were statistically significant(all P<0.05);while the thickness of respiratory membrane,level of NLRP3,Caspase-1 proteins in lung tissue and serum levels of IL-1β,IL-18 were significantly higher,and the differences were statistically significant(all P<0.05).Compared with BPD group,the RAC,average integral optical density of CD31 positive cells,density of pulmonary microvessel and level of VEGF protein in lung tissue of BPD group and BUD group were significantly higher,and the differences were statistically significant(all P<0.05);while the thickness of respiratory membrane,level of NLRP3,Caspase-1 proteins in lung tissue and serum levels of IL-1β,IL-18 were significantly lower,and the differences were statistically significant(all P<0.05).Conclusions The occurrence and development of pathological changes of BPD newborn rats caused by intrauterine infection can affect the development of pulmonary vessels through the inflammatory response of lung tissue.BUD can alleviate pathological changes in lung tissues of BPD newborn rats by reducing inflammatory reaction and up-regulating VEGF expression,promoting pulmonary vascular remodeling,and increasing pulmonary microvascular density.