谷氨酰胺通过内质网应激途径对新生大鼠高氧肺损伤的保护作用

doi:10.13481/j.1671-587x.20200102

摘要/Abstract

摘要： 目的：探讨谷氨酰胺（GLN）通过内质网应激（ERS）途径对高氧诱导新生大鼠肺损伤的保护作用，并阐明其作用机制。方法：足月新生Wistar大鼠90只，随机分为对照组（吸入氧浓度为21%）、高氧组（吸入氧浓度>85%）和高氧+GLN组（吸入氧浓度>85%且腹腔内注射GLN，剂量为0.75g·kg^-1·d^-1），每组30只。实验开始第3、7和14天测定大鼠体质量和肺组织含水量，采用HE染色法检测大鼠肺组织形态表现，氯化硝基四氮唑蓝（NBT）法测定肺组织中超氧化物歧化酶（SOD）活性，硫代巴比妥酸（TBA）法测定大鼠肺组织中丙二醛（MDA）水平，蛋白免疫印记（Western blotting）法测定大鼠肺组织中半胱氨酸蛋白酶12（Caspase-12）、葡萄糖调节蛋白78（GRP78）、生长抑制DNA损害基因153（GADD153）、B细胞淋巴瘤-2（Bcl-2）和Bcl-2相关X蛋白（Bax）蛋白表达水平。结果：实验第3、7和14天，与同时间对照组比较，高氧组大鼠体质量明显降低（P<0.05），肺组织含水量明显升高（P<0.05），肺组织中SOD活性明显降低（P<0.05），MDA水平明显升高（P<0.05），肺组织中Caspase-12、GRP78、GADD153和Bax蛋白表达水平明显升高（P<0.05），Bcl-2蛋白表达水平和Bcl-2/Bax比值明显降低（P<0.05）；与同时间高氧组比较，高氧+GLN组大鼠体质量明显升高（P<0.05），肺组织含水量明显降低（P<0.05），肺组织中SOD活性明显升高（P<0.05），MDA水平明显降低（P<0.05），肺组织中Caspase-12、GRP78、GADD153和Bax蛋白表达水平明显降低（P<0.05），Bcl-2蛋白表达水平和Bcl-2/Bax比值明显升高（P<0.05）。对照组大鼠肺组织结构规则，未见肺泡水肿，肺泡大小及肺泡间隔基本一致，无炎性细胞浸润；高氧组大鼠肺组织中支气管及肺泡上皮细胞肿胀，肺泡管腔增大，间质细胞水肿，可见炎性细胞浸润及纤维渗出；高氧+GLN组大鼠肺组织中肺泡损伤程度、炎性渗出和纤维组织增生程度均减轻，介于高氧组与对照组之间。结论：GLN可减轻高氧诱导新生大鼠肺组织水肿及炎症反应，其机制之一是通过ERS途径，降低GADD153、GRP78、Caspase-12和Bax蛋白表达水平，上调Bcl-2蛋白表达水平而发挥保护作用。

关键词: 高氧肺损伤, 新生大鼠, 谷氨酰胺, 内质网应激, 半胱氨酸蛋白酶12, 生长抑制DNA损害基因153, 葡萄糖调节蛋白78, B细胞淋巴瘤-2, Bcl-2相关X蛋白

Abstract: Objective: To explore the protective effect of glutamine(GLN) on the hyperoxia-induced lung injuryof the neonatal rats through endoplasmic reticulum stress (ERS) pathway, and to elucidate its mechanisms. Methods: A total of 90 Wistar rats were randomly divided into control group (FiO₂=21%), hyperoxia group(FiO₂>85%), and hyperoxia+GLN group (FiO₂>85%,the concentration of intraperitoneal injection of GLN was 0.75 g·kg^-1·d^-1);there were 30 rats in each group. The body weights and water contents in the lung tissue of the neonatal rats were measured on the 3rd, 7th and 14th days of the experiment. HE staining was used to determine the morphology of lung tissue of the rats. The superoxide dismutase (SOD) activity in lung tissue of the rats was detected by nitro blue tetrazolium chloride(NBT), and the malondialdehyde (MDA) level was determined by thiobarbital acid(TBA). The expression levels of Caspase-12, GADD153, GRP78, Bcl-2,and Bax in lung tissue of the rats were detected by Western blotting method. Results: Compared with control group at the same time, the body weights of the neonatal rats in hyperoxia group on the 3rd,7th and 14th days were significantly decreased(P<0.05), the water contents in lung tissue of the neonatal rats were increased(P<0.05), the SOD activities were significantly decreased(P<0.05), the levels of MDA in the lung tissue of the neonatal rats were increased(P<0.05), the expressions levels of Caspase-12, GADD153, GRP78 and Bax proteins were significantly increased(P<0.05),and the expression levels of Bcl-2 protein and the Bcl-2/Bax ratios were significantly decreased(P<0.05).Compared with hyperoxia group at the same time, the body weights of the neonatal rats in hyperoxia + GLN group on the 3rd,7th and 14th days were significantly increased(P<0.05), the water contents in lung tissue of the neonatal rats were decreased(P<0.05), the SOD activities were significantly increased(P<0.05), the levels of MDA in lung tissue of the neonatal rats were decreased(P<0.05), the expression levels of Caspase-12, GADD153, GRP78 and Bax proteins were significantly decreased(P<0.05), the expression levels of Bcl-2 protein and the Bcl-2/Bax ratios were increased(P<0.05).The pathological sections of lung tissue of the rats in control group showed that lung tissue structure was regular, no alveolar edema was found,the alveolar size and alveolar septum were approximately the same, and no inflammatory cell infiltration was found; the histopathological sections of lung tissue of the rats in hyperoxia group showed swelling of brochial and alveolar epithelial cells, enlargement of alveolar lumen, edema of interstitial cells, inflammatory cell infiltration and fibrous exudation;the degrees of alveolar damage, the inflammatory exudation and the proliferation of fibrons tissue in hyperoxia+GLN group were alleviated which was between hyperoxia group and control group. Conclusion: GLN can alleviate the hyperoxia-induced lung tissue edema and inflammatory response of the neonatal rats, and one of mechanisms is that GLN can down-regulate the expression levels of Caspase-12, GADD153, GRP78 and Bax proteins and up-regulate the expression level of Bcl-2 protein through ERS pathway to protect hypoxic lung injury.

Key words: hyperoxic lung injury, neonatal rats, glutamine, endoplasmic reticulum stress, Caspase-12, growth arrest-and DNA damage-inducible gene153, glucose regulation protein 78, B-cell lymphoma-2, Bcl-2 associated X protein

中图分类号:

R563

王叶, 王红, 张书剑, 姬华祎, 金正勇. 谷氨酰胺通过内质网应激途径对新生大鼠高氧肺损伤的保护作用[J]. 吉林大学学报(医学版), 2020, 46(01): 7-13.

WANG Ye, WANG Hong, ZHANG Shujian, JI Huayi, JING Zhengyong. Protective effect of glutamine on hyperoxic lung injury of neonatal rats through endoplasmic reticulum stress pathway[J]. Journal of Jilin University(Medicine Edition), 2020, 46(01): 7-13.

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