Role and mechanism of FIRRE in LPS-induced acute lung injury

doi:10.3760/cma.j.issn.1007-1245.2024.20.002

Abstract

Abstract:

Objective　To explore the role and mechanism of FIRRE in lipopolysaccharide (LPS) induced acute lung injury (ALI). Methods　The study was conducted at Binzhou Medical University Hospital from June 2020 to May 2022. Part I: The role of FIRRE in LPS-induced A549 cell injury. A549 cells were divided into a control group, a low molecular weight heparin calcium (LMWH) group, a LPS group, and a LPS+LMWH group, which were treat by normal saline, 5 IU/ml LMWH, 10 mg/L LPS, and 10 mg/L LPS+5 IU/ml LMWH for 24 h. The cell activity of each group was detected by CCK-8, the expressions of interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α were detected by enzyme-linked immunosorbent assay (ELISA), and the relative expression of FIRRE gene was detected by real-time quantitative PCR. Part Ⅱ: The role of FIRRE in LPS-induced ALI in C57BL/6J mice. Twenty-four healthy and clean SPF grade male C57BL/6J mice (body weight 25-30 g, 6-8 weeks old) were randomly divided into 3 groups. The control group was injected with equal volume of normal saline, the ALI group was injected with 5 mg/kg LPS to induce ALI, and the LMWH group was intraperitoneally injected with 5 mg/kg LPS + 5 AXa IU/kg LMWH. The changes of hair color, reaction ability, and resistance to external environment were evaluated at 6 h and 12 h after LPS injection. HE staining was used to observe the degree of lung tissue injury, and the lung injury score was calculated. Blood samples were collected from the abdominal main vein, and the expressions of IL-6 and IL-10 in serum were detected by ELISA. The levels of IL-6 and IL-10 in bronchoalveolar lavage fluid (BALF) were detected by ELISA. The relative expression of FIRRE gene was detected by RT-PCR. One-way analysis of variance, LSD-t test, and Dunnett-t test were used. Results　Part I: The activity of A549 cells in the LPS group was lower than that in the control group, and the activity of A549 cells in the LPS+LMWH group was higher than that in the LPS group, with a statistically significant difference among multiple groups (F=44.25, P<0.05). The levels of IL-6, IL-10, and TNF-α in the LPS group and LPS+LMWH group were higher than those in the control group and LMWH group, and the levels of IL-6, IL-10, and TNF-α in the LPS+LMWH group were lower than those in the LPS group (all P<0.05). The expression levels of FIRRE in A549 cells of the LPS group and LPS+LMWH group were higher than that of the control group, and that of the LPS+LMWH group was lower than that of the LPS group, with a statistically significant difference among multiple groups (F=242.23, P<0.001). Part II: The reaction ability and resistance to external environment of the mice in the ALI group and LMWH group were lower than those in the control group. At 6 h and 12 h after LPS injection, in the ALI group, HE staining of lung tissue sections showed a large number of inflammatory cell infiltration in the alveolar cavity, alveolar collapse, The lung injury score was 4. The inflammatory injury of lung tissue sections showed by HE staining in the LMWH group was lower than that in the ALI group. The levels of IL-6 and IL-10 in serum and BALF in the ALI group were higher than those in the control group (all P<0.05), and the levels of IL-6 and IL-10 in serum and BALF in the LMWH group were lower than those in the ALI group (all P<0.05).The FIRRE expressions in the ALI group and LMWH group were higher than that in the control group, and the expression in the LMWH group was lower than that in the ALI group, with statistically significant differences among multiple groups (F=7.23 and 6.51, both P<0.05). Conclusions　Part I: LMWH may reduce LPS-induced injury of A549 cells by inhibiting the expression of FIRRE gene and down-regulating the levels of inflammatory factors. Part II: LMWH may reduce LPS-induced ALI in C57BL/6J mice by inhibiting the expression of FIRRE gene and down-regulating the levels of inflammatory factors.

Key words:

Acute lung injury, Lipopolysaccharide, FIRRE, Low molecular weight heparin calcium, Cell injury, Animal experiment

摘要：

目的　探讨FIRRE在脂多糖（LPS）诱导急性肺损伤（ALI）中的作用及机制。方法　研究时间为2020年6月至2022年5月，地点为滨州医学院附属医院。第一部分：FIRRE在LPS诱导A549细胞损伤中的作用研究。A549细胞分为4组，对照组采用生理盐水，低分子肝素钙组采用5 IU/ml低分子肝素钙，LPS组采用10 mg/L LPS，LPS+低分子肝素钙组采用10 mg/L LPS+5 IU/ml低分子肝素钙；处理24 h。采用CCK-8检测各组细胞活性；采用酶联免疫吸附试验（ELISA）检测白细胞介素（IL）-6、IL-10和肿瘤坏死因子（TNF-α）的表达；采用实时定量PCR检测FIRRE基因的相对表达量。第二部分：FIRRE在LPS诱导C57BL/6J小鼠ALI中的作用研究。将24只健康清洁SPF级雄性C57BL/6J小鼠（体质量25～30 g，6～8周）随机分为3组，对照组腹腔注射等体积生理盐水，ALI组注射5 mg/kg LPS诱发ALI，低分子肝素钙组腹腔注射5 mg/kg LPS诱发ALI+低分子肝素钙5 AXa IU/kg。分别于6 h、12 h评估各组小鼠毛色、反应能力、对抗外界阻力等变化；肺组织切片HE染色观察肺组织损伤程度，并进行肺损伤评分；腹主静脉取血，采用ELISA检测血清IL-6、IL-10的表达情况；采用ELISA检测肺泡灌洗液IL-6、IL-10含量；采用RT-PCR检测FIRRE基因的相对表达量。采用单因素方差分析、LSD-t检验、Dunnett-t检验。结果　第一部分：LPS组A549细胞活性低于对照组，LPS+低分子肝素钙组A549细胞活力高于LPS组，多组间比较差异有统计学意义（F=44.25，P<0.05）。LPS组、LPS+低分子肝素钙组IL-6、IL-10、TNF-α水平均高于对照组、低分子肝素钙组，LPS+低分子肝素钙组IL-6、IL-10、TNF-α水平均低于LPS组（均P<0.05）。LPS组、LPS+低分子肝素钙组A549细胞FIRRE表达水平均高于对照组，LPS+低分子肝素钙组低于LPS组，多组间比较差异有统计学意义（F=242.23，P<0.001）。第二部分：ALI组及低分子肝素钙组小鼠反应能力、对抗外界阻力能力均低于对照组。LPS注射后6 h、12 h，ALI组HE染色肺组织切片显示，肺泡腔内大量炎症细胞浸润，肺泡塌陷肺损伤评分为4分；低分子肝素钙组HE染色肺组织切片炎性损伤低于ALI组；ALI组血清及肺泡灌洗液中IL-6、IL-10水平均高于对照组（均P<0.05）；低分子肝素钙组血清及肺泡灌洗液中IL-6、IL-10水平均低于ALI组（均P<0.05）；ALI组、低分子肝素钙组FIRRE表达量均高于对照组，低分子肝素钙组低于ALI组，多组间比较差异均有统计学意义（F=7.23、6.51，均P<0.05）。结论　第一部分：低分子肝素钙可能通过抑制FIRRE基因的表达，下调炎症因子水平，减轻LPS诱导A549细胞损伤。第二部分：低分子肝素钙可能通过抑制FIRRE基因的表达，下调炎症因子水平，减轻LPS诱导C57BL/6J小鼠ALI。

关键词:

急性肺损伤, 脂多糖, FIRRE, 低分子肝素钙, 细胞损伤, 动物实验

Zhao Xiaoxia, Li Xiaomei, Sang Yi, Xu Conglei, Li Zhaolun, Sun Wenjing, Liu Xiaoming.

Role and mechanism of FIRRE in LPS-induced acute lung injury [J]. International Medicine and Health Guidance News, 2024, 30(20): 3351-3356.

赵晓霞李晓梅桑义许从磊李昭伦孙文静刘晓明.

FIRRE在脂多糖诱导急性肺损伤中的作用及机制研究 [J]. 国际医药卫生导报, 2024, 30(20): 3351-3356.

References

[1] Thompson BT, Chambers RC, Liu KD. Acute respiratory distress syndrome[J]. N Engl J Med, 2017,377(6):562-572.DOI: 10.1056/NEJMra1608077.
[2] Butt Y, Kurdowska A, Allen TC. Acute lung injury: a clinical and molecular review[J]. Arch Pathol Lab Med, 2016,140(4):345-350.DOI: 10.5858/arpa.2015-0519-RA.
[3] Matthay MA, Zemans RL. The acute respiratory distress syndrome: pathogenesis and treatment[J]. Annu Rev Pathol, 2011,6:147-163.DOI: 10.1146/annurev- pathol-011110-130158.
[4] Bao H, Gao F, Xie G,et al. Angiotensin-converting enzyme 2 inhibits apoptosis of pulmonary endothelial cells during acute lung injury through suppressing MiR-4262[J]. Cell Physiol Biochem, 2015,37(2):759-767.DOI: 10.1159/000430393.
[5] Liu S, Tang J, Huang L,et al. Cordyceps militaris alleviates severity of murine acute lung injury through miRNAs-mediated CXCR2 inhibition[J]. Cell Physiol Biochem,2015,36(5):2003-2011.DOI: 10.1159/000430168.
[6] Deng X, Jin K, Li Y,et al. Platelet-derived growth factor and transforming growth factor β1 regulate ARDS-associated lung fibrosis through distinct signaling pathways[J]. Cell Physiol Biochem, 2015,36(3):937-946.DOI: 10.1159/000430268.
[7] Ding S, Hou X, Yuan J,et al. Wedelolactone protects human bronchial epithelial cell injury against cigarette smoke extract-induced oxidant stress and inflammation responses through Nrf2 pathway[J]. Int Immunopharmacol, 2015,29(2):648-655.DOI: 10.1016/j.intimp.2015.09.015.
[8] Kawamoto Y, Morinaga Y, Kimura Y,et al. TNF-α inhibits the growth of legionella pneumophila in airway epithelial cells by inducing apoptosis[J]. J Infect Chemother, 2017,23(1):51-55.DOI: 10.1016/j.jiac.2016.09.010.
[9] Shi X, Cui Z, Liu X,et al. LncRNA FIRRE is activated by MYC and promotes the development of diffuse large B-cell lymphoma via Wnt/β-catenin signaling pathway[J]. Biochem Biophys Res Commun,2019,510(4):594-600.DOI: 10.1016/j.bbrc.2019.01.105.
[10] Lu Y, Liu X, Xie M,et al. The NF-κB-responsive long noncoding RNA FIRRE regulates posttranscriptional regulation of inflammatory gene expression through interacting with hnRNPU[J]. J Immunol, 2017,199(10):3571-3582.DOI: 10.4049/jimmunol.1700091.
[11] 李银娇.PKR分子在脂多糖诱导的小鼠急性肺损伤中的作用及机制研究[D].上海:上海交通大学,2017.
[12] Confalonieri M, Salton F, Fabiano F. Acute respiratory distress syndrome[J]. Eur Respir Rev, 2017,26(144):160116.DOI: 10.1183/16000617.0116-2016.
[13] 王胜超,吴敏,黄尤光. 长链非编码 RNA在结直肠癌中的研究进展[J]. 国际遗传学杂志,2019,42(4):290-294. DOI:10.3760/cma.j.issn.1673-4386.2019.04.006.
[14] Yi L, Huang X, Guo F,et al. Yes-associated protein (YAP) signaling regulates lipopolysaccharide-induced tissue factor expression in human endothelial cells[J]. Surgery, 2016 ,159(5):1436-1448. DOI: 10.1016/j.surg.2015. 12.008.
[15] Luhr OR, Antonsen K, Karlsson M,et al. Incidence and mortality after acute respiratory failure and acute respiratory distress syndrome in Sweden, Denmark, and Iceland. The ARF study group[J]. Am J Respir Crit Care Med, 1999,159(6):1849-1861. DOI: 10.1164/ajrccm.159.6.9808136.