维甲酸X受体对缺氧/复氧诱导的大鼠肺泡Ⅱ型上皮细胞自噬的调控作用

Role of retinoic acid X receptor in regulation of autophagy in rat alveo⁃lar typeⅡepithelial cells induced by hypoxia/reoxygenation

目的:探讨维甲酸X受体(RXR)对缺氧/复氧(H/R)诱导的大鼠肺泡Ⅱ型上皮细胞(AECⅡ)自噬的调控作用及分子机制。方法:常氧培养大鼠肺泡Ⅱ型上皮细胞,将生长至对数生长期的细胞随机分为5组:(1)对照组:细胞于正常条件下培养30 h;(2)缺氧/复氧组:细胞缺氧6 h,复氧24 h;(3)溶剂DMSO组:细胞于造模前1.5 h用浓度低于0.1%的DMSO进行预处理,其余处理同H/R组;(4)9-顺式维甲酸(9-RA)组:细胞于缺氧前1 h用9-RA(100 nmol/L)预处理;(5)HX531组:细胞先用9-RA处理0.5 h,然后用HX531(2.5μmol/L)处理1 h。采用CCK-8法检测各组细胞活力;免疫荧光染色法观察各组细胞RXRα表达变化;用透射电镜观察细胞内部超微结构的改变;RT-PCR检测腺苷酸活化蛋白激酶(AMPK)、自噬相关蛋白beclin 1及LC3、哺乳动物雷帕霉素靶蛋白(mTOR)和P62 mRNA水平的表达。Western blot检测p-AMPK、beclin 1、LC3-Ⅱ、p-mTOR和P62蛋白的表达。结果:与对照组相比,其余4组细胞的细胞活力显著降低,AMPK、beclin 1和LC3 mRNA表达显著增多,p-AMPK、beclin 1和LC3-Ⅱ蛋白表达显著增多,而mTOR和P62 mRNA表达降低,p-mTOR和P62蛋白表达降低(P<0.05);与缺氧/复氧和HX531组相比,9-RA组细胞损伤减轻,自噬水平也显著降低(P<0.05)。结论:(1)缺氧/复氧可诱导AECⅡ自噬水平升高;(2)激活RXR可一定程度上减轻缺氧/复氧引起的AECⅡ损伤,其机制可能与抑制细胞自噬有关。

AIM:To investigate the regulatory effect of retinoic acid X receptor(RXR)on autophagy induced by hypoxia/reoxygenation(H/R)in rat alveolar typeⅡepithelial cells(AECⅡ)and its molecular mechanism.METHODS:AECⅡwere cultured in normoxia.The cells growing to logarithmic growth phase were randomly divided in⁃to 5 groups:(1)control(Con)group:cells were cultured for 30 h under normal operation;(2)H/R group:cells were cul⁃tured in hypoxia condition for 6 h and then in reoxygenation condition for 24 h;(3)DMSO group:cells were pretreated 1.5 h with medium containing less than 0.1%DMSO before modeling,and the rest were treated the same as the H/R group;(4)9-cis-retinoic acid(9-RA)group:cells were pretreated for 1 h with 9-RA(100 nmol/L)before hypoxia;(5)HX531 group:cells were treated with 9-RA(100 nmol/L)for 0.5 h,then treatment with HX531(2.5μmol/L)for 1 h.CCK-8 assay was used to detect the cell viability.Immunofluorescence staining was used to observe the expression of RXRα.Transmission electron microscope was used to observe the changes of intracellular ultrastructure,and the mRNA expression of adenosine AMP-activated protein kinase(AMPK),beclin 1,LC3,mammalian target of rapamycin(mTOR)and P62 was detected by RT-PCR.Western blot was used to detected the protein levels of p-AMPK,beclin 1,LC3-Ⅱ,pmTOR and P62.RESULTS:Compared with Con group,the cell viability in H/R,DMSO,9-RA and HX531 groups were significantly decreased.The mRNA expression of AMPK,beclin 1 and LC3 was significantly increased,and the protein levels of p-AMPK,beclin 1 and LC3-Ⅱwere also increased.The mRNA expression of mTOR and P62 was decreased,and the protein levels of p-mTOR and P62 were also decreased(P<0.05).The cell injury in 9-RA group was alleviated and autophagy level was significantly lower than that in H/R,DMSO and HX531 groups(P<0.05),and no significant dif⁃ference among H/R,DMSO and HX531 groups was observed(P>0.05).CONCLUSION:H/R induces autophagy of AECⅡ.Activating RXR reduce the damage of AECⅡcells induced by H/R,and its mechanism may be related to the in⁃hibition of autophagy.