This study investigated the effects of hyperoxia on dynamic changes of thioredoxin-1 （Trx1） and thioredoxin reductase-1 （TrxR1） in alveolar type Ⅱ epithelial cells （AECⅡ） of premature rats. Pregnant Sprague-Da...This study investigated the effects of hyperoxia on dynamic changes of thioredoxin-1 （Trx1） and thioredoxin reductase-1 （TrxR1） in alveolar type Ⅱ epithelial cells （AECⅡ） of premature rats. Pregnant Sprague-Dawley rats were sacrificed on day 19 of gestation. AECⅡ were isolated and purified from the lungs of premature rats. When cultured to 80% confluence, in vitro cells were randomly divided into air group and hyperoxia group. Cells in the hyperoxia group were continuously exposed to 95% O2/5% CO2 and those in the air group to 95% air/5% CO2. After 12, 24 and 48 h, cells in the two groups were harvested to detect their reactive oxygen species （ROS）, apoptosis, TrxR1 activity and the expressions of Trx1 and TrxR1 by corresponding protocols, respectively. The results showed that AECⅡ exposed to hyperoxia generated excessive ROS and the apoptosis percentage in the hyperoxia group was increased significantly at each time points as compared with that in the air group （P0.001）. Moreover, TrxR1 activity was found to be markedly depressed in the hyperoxia group in comparison to that in the air group （P0.001）. RT-PCR showed the expressions of both Trx1 and TrxR1 mRNA were significantly increased in AECⅡ exposed to hyperoxia for 12 and 24 h （P0.01）, respectively. At 48 h, the level of Trx1 mRNA as well as that of TrxR1 mRNA in the hyperoxia group was reduced and showed no significant difference from that in the air group （P0.05）. Western blotting showed the changes of Trx1 protein expressions in the hyperoxia group paralleled those of Trx1 mRNA expressions revealed by RT-PCR. It was concluded that hyperoxia can up-regulate the protective Trx1/TrxR1 expressed by AECⅡ in a certain period, however, also cause dysfunction of the cytoplasmic thioredoxin system by decreasing TrxR1 activity, which may contribute to the progression of oxidative stress and cell apoptosis and finally result in lung injury.展开更多
Purpose:This study aims to elucidate the electrotaxis response of alveolar epithelial cells(AECs)in direct-current electric fields(EFs),explore the impact of EFs on the cell fate of AECs,and lay the foundation for fut...Purpose:This study aims to elucidate the electrotaxis response of alveolar epithelial cells(AECs)in direct-current electric fields(EFs),explore the impact of EFs on the cell fate of AECs,and lay the foundation for future exploitation of EFs for the treatment of acute lung injury.Methods:AECs were extracted from rat lung tissues using magnetic-activated cell sorting.To elucidate the electrotaxis responses of AECs,different voltages of EFs(0,50,100,and 200 mV/mm)were applied to two types of AECs,respectively.Cell migrations were recorded and trajectories were pooled to better demonstrate cellular activities through graphs.Cell directionality was calculated as the cosine value of the angle formed by the EF vector and cell migration.To further demonstrate the impact of EFs on the pulmonary tissue,the human bronchial epithelial cells transformed with Ad12-SV402B(BEAS-2B cells)were obtained and experimented under the same conditions as AECs.To determine the influence on cell fate,cells underwent electric stimulation were collected to perform Western blot analysis.Results:The successful separation and culturing of AECs were confirmed through immunofluorescence staining.Compared with the control,AECs in EFs demonstrated a significant directionality in a voltage-dependent way.In general,type I alveolar epithelial cells migrated faster than type II alveolar epithelial cells,and under EFs,these two types of cells exhibited different response threshold.For type II alveolar epithelial cells,only EFs at 200 mV/mm resulted a significant difference to the velocity,whereas for,EFs at both 100 mV/mm and 200 mV/mm gave rise to a significant difference.Western blotting suggested that EFs led to an increased expression of a AKT and myeloid leukemia 1 and a decreased expression of Bcl-2-associated X protein and Bcl-2-like protein 11.Conclusion:EFs could guide and accelerate the directional migration of AECs and exert antiapoptotic effects,which indicated that EFs are important biophysical signals in the re-epithelialization of alveolar epithelium in lung injury.展开更多
目的构建急性肺损伤体外细胞模型。方法以TNF-α10ng/mL刺激肺泡Ⅱ型上皮A549细胞,采用酶联免疫吸附法检测细胞上清液中IL-4、IL-1β浓度,比色法检测丙二醛浓度、总超氧化物歧化酶活力,RT-PCR及免疫印迹法分别检测NF-κB m RNA及蛋白的...目的构建急性肺损伤体外细胞模型。方法以TNF-α10ng/mL刺激肺泡Ⅱ型上皮A549细胞,采用酶联免疫吸附法检测细胞上清液中IL-4、IL-1β浓度,比色法检测丙二醛浓度、总超氧化物歧化酶活力,RT-PCR及免疫印迹法分别检测NF-κB m RNA及蛋白的表达水平,流式细胞术检测细胞凋亡率。结果以TNF-α刺激A549细胞,细胞上清液IL-1β、IL-4浓度上升;细胞内丙二醛增多,超氧化物歧化酶活力下降;NF-κB在基因及蛋白水平表达上调(P<0.05)。结论以TNF-α刺激肺泡Ⅱ型上皮A549细胞可诱导炎症反应放大、氧化应激失衡、NF-κB通路活化、细胞凋亡增加,符合急性肺损伤的主要特点,可作为ALI体外细胞模型。展开更多
基金supported by a grant from the National Natural Sciences Foundation of China (No. 30770944)
文摘This study investigated the effects of hyperoxia on dynamic changes of thioredoxin-1 （Trx1） and thioredoxin reductase-1 （TrxR1） in alveolar type Ⅱ epithelial cells （AECⅡ） of premature rats. Pregnant Sprague-Dawley rats were sacrificed on day 19 of gestation. AECⅡ were isolated and purified from the lungs of premature rats. When cultured to 80% confluence, in vitro cells were randomly divided into air group and hyperoxia group. Cells in the hyperoxia group were continuously exposed to 95% O2/5% CO2 and those in the air group to 95% air/5% CO2. After 12, 24 and 48 h, cells in the two groups were harvested to detect their reactive oxygen species （ROS）, apoptosis, TrxR1 activity and the expressions of Trx1 and TrxR1 by corresponding protocols, respectively. The results showed that AECⅡ exposed to hyperoxia generated excessive ROS and the apoptosis percentage in the hyperoxia group was increased significantly at each time points as compared with that in the air group （P0.001）. Moreover, TrxR1 activity was found to be markedly depressed in the hyperoxia group in comparison to that in the air group （P0.001）. RT-PCR showed the expressions of both Trx1 and TrxR1 mRNA were significantly increased in AECⅡ exposed to hyperoxia for 12 and 24 h （P0.01）, respectively. At 48 h, the level of Trx1 mRNA as well as that of TrxR1 mRNA in the hyperoxia group was reduced and showed no significant difference from that in the air group （P0.05）. Western blotting showed the changes of Trx1 protein expressions in the hyperoxia group paralleled those of Trx1 mRNA expressions revealed by RT-PCR. It was concluded that hyperoxia can up-regulate the protective Trx1/TrxR1 expressed by AECⅡ in a certain period, however, also cause dysfunction of the cytoplasmic thioredoxin system by decreasing TrxR1 activity, which may contribute to the progression of oxidative stress and cell apoptosis and finally result in lung injury.
基金National Natural Science Foundation of China(82272908,81672287,82222038)Natural Science Foundation of Chongqing(CSTB2022NSCQ-MSX1110)+1 种基金Open Project Program of the State Key Laboratory of Trauma,Burn and Combined Injury(SKLYQ202102,SKLKF2022011)Daping Hospital of Army Medical University(2019CXJSB004,2019CXJSC024)。
文摘Purpose:This study aims to elucidate the electrotaxis response of alveolar epithelial cells(AECs)in direct-current electric fields(EFs),explore the impact of EFs on the cell fate of AECs,and lay the foundation for future exploitation of EFs for the treatment of acute lung injury.Methods:AECs were extracted from rat lung tissues using magnetic-activated cell sorting.To elucidate the electrotaxis responses of AECs,different voltages of EFs(0,50,100,and 200 mV/mm)were applied to two types of AECs,respectively.Cell migrations were recorded and trajectories were pooled to better demonstrate cellular activities through graphs.Cell directionality was calculated as the cosine value of the angle formed by the EF vector and cell migration.To further demonstrate the impact of EFs on the pulmonary tissue,the human bronchial epithelial cells transformed with Ad12-SV402B(BEAS-2B cells)were obtained and experimented under the same conditions as AECs.To determine the influence on cell fate,cells underwent electric stimulation were collected to perform Western blot analysis.Results:The successful separation and culturing of AECs were confirmed through immunofluorescence staining.Compared with the control,AECs in EFs demonstrated a significant directionality in a voltage-dependent way.In general,type I alveolar epithelial cells migrated faster than type II alveolar epithelial cells,and under EFs,these two types of cells exhibited different response threshold.For type II alveolar epithelial cells,only EFs at 200 mV/mm resulted a significant difference to the velocity,whereas for,EFs at both 100 mV/mm and 200 mV/mm gave rise to a significant difference.Western blotting suggested that EFs led to an increased expression of a AKT and myeloid leukemia 1 and a decreased expression of Bcl-2-associated X protein and Bcl-2-like protein 11.Conclusion:EFs could guide and accelerate the directional migration of AECs and exert antiapoptotic effects,which indicated that EFs are important biophysical signals in the re-epithelialization of alveolar epithelium in lung injury.
文摘目的构建急性肺损伤体外细胞模型。方法以TNF-α10ng/mL刺激肺泡Ⅱ型上皮A549细胞,采用酶联免疫吸附法检测细胞上清液中IL-4、IL-1β浓度,比色法检测丙二醛浓度、总超氧化物歧化酶活力,RT-PCR及免疫印迹法分别检测NF-κB m RNA及蛋白的表达水平,流式细胞术检测细胞凋亡率。结果以TNF-α刺激A549细胞,细胞上清液IL-1β、IL-4浓度上升;细胞内丙二醛增多,超氧化物歧化酶活力下降;NF-κB在基因及蛋白水平表达上调(P<0.05)。结论以TNF-α刺激肺泡Ⅱ型上皮A549细胞可诱导炎症反应放大、氧化应激失衡、NF-κB通路活化、细胞凋亡增加,符合急性肺损伤的主要特点,可作为ALI体外细胞模型。
