自噬对BPD大鼠NLRP3炎症小体的调控作用

Regulation of autophagy on NLRP3 inflammasome in neonatal rats with BPD

目的:探讨在高氧诱导的新生大鼠支气管肺发育不良(BPD)中自噬和核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体之间的关系及其对肺发育结局的影响。方法:用85%O 2制造BPD新生大鼠模型,与常氧(21%O 2)新生大鼠对比,选择生后第3、7、14天的新生大鼠肺组织做病理切片,使用苏木精-伊红染色观察肺组织形态变化(RAC、MLI),Western blot测肺组织LC3、P62蛋白,确定自噬流变化。雷帕霉素组新生大鼠与高氧组在同一高氧环境中,于生后第2、4、6天予以腹腔注射雷帕霉素。高氧组与常氧组注射同等量生理盐水。选择生后第7天,Western blot测肺组织MTOR、LC3、P62、NLRP3、ASC、cleaved-caspase-1、cleaved-IL-1β,PCR测LC3、NLRP3、cleaved-caspase-1的mRNA,确定其自噬与炎症小体活性之间的关系。结果:肺组织HE染色示生后第7、14天RAC(P<0.05)常氧组均高于高氧组;MLI(P<0.05)高氧组均高于常氧组。Western blot示生后第3、7天LC3-Ⅱ/LC3-Ⅰ(P<0.01),P62(P<0.05)高氧组均高于常氧组。生后第7天肺组织HE染色示高氧组RAC低于常氧组和雷帕霉素组(P<0.05),MLI高于常氧组和雷帕霉素组(P<0.05),生后第7天Western blot示高氧组的MTOR、LC3-Ⅱ/LC3-Ⅰ、P62、NLRP3、ASC、cleaved-caspase-1、cleaved-IL-1β均高于正常组和雷帕霉素组(P<0.05)。qPCR示高氧组的LC3mRNA低于正常组和雷帕霉素组(P<0.05),而NLRP3、caspase-1的mRNA均高于正常组和雷帕霉素组(P<0.05)。结论:自噬流与炎症小体之间的相互作用是BPD发生发展的重要因素,通过增加自噬流可以减少炎症小体的活性,减少cleaved-IL-1β的生成,进而改善BPD,为进一步治疗BPD提供依据。

Objective:To investigate the relationship between autophagy and nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome in hyperoxy-induced bronchopulmonary dysplasia(BPD)in neonatal rats,and its impact on the developmental outcome of the lungs.Methods:Neonatal rat model of BPD was initially established using 85%oxygen,and then compared with neonatal rats kept in normoxia(21%oxygen).Pathological sections were obtained from lung tissues of rats on postnatal days 3,7,and 14.Hematoxylin-eosin staining was used to observe morphological changes in lung tissue(RAC and MLI).The changes of autophagy flux were determined by Western blot analysis of LC3 and P62 proteins in lung tissue.Rapamycin group(Drug+Hyperoxia,DO group)of neonatal rats was exposed to the same hyperoxia environment as hyperoxia group,and intraperitoneal injections of rapamycin were administered on postnatal days 2,4,and 6.The hyperoxia group and normoxia group(HO and NO group)were injected with an equivalent volume of saline.On the 7 th day after birth,Western blot was performed to measure the expression of MTOR,LC3,P62,NLRP3,ASC,cleaved caspase-1 and cleaved IL-1βin lung tissues.mRNA levels of LC3,NLRP3,cleaved caspase-1 were as sessed by PCR to determine the relationship between autophagy and inflammasome activity.Results:HE staining showed that the RAC at postnatal days 7 and 14 were higher in the NO group than in the HO group(P<0.05),while the MLI was higher in the HO group(P<0.05).Western blot showed that the HO group had higher expression of LC3-Ⅱ/LC3-Ⅰ(P<0.01)and P62(P<0.05)at postnatal days 3 and 7 than the NO group.On postnatal day 7,HE staining revealed that the RAC in the HO group was lower than that in the NO and DO groups(P<0.05),while the MLI in the HO group was higher(P<0.05).On postnatal day 7,Western blot demonstrated that MTOR,LC3-Ⅱ/LC3-Ⅰ,P62,NLRP3,ASC,cleaved caspase-1,and cleaved IL-1βin the HO group were higher than those in the NO and DO groups(P<0.05).qPCR showed that LC3 mRNA in the HO group was lower than that in the NO and DO groups(P<0.05),while the mRNA levels of NLRP3 and caspase-1 in the HO group were higher(P<0.05).Conclusion:The interaction between autophagy flux and inflammasome is an important factor in its occurrence and development.Increased autophagy flux can reduce the activity of inflammasome,decrease the production of cleaved IL-1βand improve BPD,providing a basis for further treatment of BPD.