铜绿假单胞菌感染支气管上皮细胞诱导铁死亡及丁酸钠的免疫调控作用

doi:10.3969/j.issn.2095-9400.2024.07.005

科研成果详情

题名	铜绿假单胞菌感染支气管上皮细胞诱导铁死亡及丁酸钠的免疫调控作用
其他题名	Ferroptosis of bronchial epithelial cells induced by pseudomonas aeruginosa infection and the immunomodulatory mechanism of sodium butyrate
作者	周娈1; 何一帆2; 赵伊敏 3; 任锡凯1; 耿轩1; 黄楠 1; 苏苗赏1
发表日期	2024-07-28
发表期刊	温州医科大学学报影响因子和分区
语种	中文
原始文献类型	Periodical
关键词	铜绿假单胞菌支气管上皮细胞铁死亡丁酸钠炎症因子
其他关键词	pseudomonas aeruginosa ; bronchial epithelial cells ; ferroptosis ; sodium butyrate ; inflammatory factor
摘要	目的:探究铜绿假单胞菌(PA)感染对人支气管上皮细胞(BEAS-2B)铁死亡与炎症反应的诱导作用及丁酸钠(NaB)对炎症的调节作用.方法:体外培养BEAS-2B细胞,加入不同浓度NaB后通过CCK-8筛选其安全浓度和作用时间,Western blot检测酰基辅酶A合成酶长链家族成员4(ACSL4)、磷酸化蛋白激酶B(P-AKT)蛋白表达水平,确定NaB的用药条件.PA感染BEAS-2B细胞(MOI=1)不同时间后,CCK-8检测细胞存活率,倒置显微镜观察细胞形态变化,透射电镜观察细胞线粒体形态变化,二价铁离子探针检测细胞内Fe2+水平;加入NaB预处理后,BODIPY 581/591 C11荧光探针检测细胞内脂质过氧化水平,Western blot检测ACSL4、P-AKT、谷胱甘肽过氧化物酶4(GPX4)蛋白表达水平;加入ACSL4抑制剂吡格列酮(PIO)后,Western blot验证其抑制效果,ELISA检测各组细胞上清液中炎症因子水平变化.结果:NaB对BEAS-2B细胞的安全浓度在2.5 mmol/L以下,2.5 mmol/L NaB处理细胞36 h后,ACSL4和P-AKT蛋白表达水平显著下降(P＜0.05).PA感染BEAS-2B细胞后细胞存活率显著降低(P＜0.05);细胞形态变为圆形;线粒体体积变小、膜密度增高,嵴减少甚至消失;胞内Fe2+和脂质过氧化水平显著升高(P＜0.05);细胞ACSL4和P-AKT蛋白表达水平上升(P＜0.05),GPX4蛋白表达水平显著下降(P＜0.05).NaB预处理细胞后,与PA组相比ACSL4和P-AKT蛋白的表达水平下降(P＜0.05),对GPX4蛋白的降低及胞内脂质过氧化水平为促进作用.ELISA检测结果显示,与对照组相比,PA感染后IL-6、IL-8、IL-1β和TNF-α水平显著上升(P＜0.05),IL-10水平轻微下降(P＜0.05);NaB或PIO预处理后,IL-6、IL-8、IL-1β和TNF-α水平均显著下降(P＜0.05),IL-10水平差异无统计学意义(P＞0.05).结论:PA感染导致支气管上皮细胞内Fe2+及脂质过氧化物的积累从而发生铁死亡并引起炎症反应,NaB可能通过抑制ACSL4和AKT磷酸化途径起到免疫调节作用.
其他摘要	Objective:To investigate the inductive effect of pseudomonas aeruginosa(PA)infection on ferroptosis and inflammation in human bronchial epithelial cells(BEAS-2B)and the immunomodulatory mechanism of sodium butyrate(NaB).Methods:In the in vitro BEAS-2B cell culture system,different concentrations of NaB were added to screen the safe concentration and the suitable time by CCK8 assay.Changes in the expression of acyl-CoA synthetase long-chain family member 4(ACSL4)and phosphorylated protein kinase B(P-AKT)were detected by Western blot to determine the concentration of NaB.BEAS-2B cells infected with PA(MOI=1),and after different period of time,cell viability was measured using CCK-8 assay;changes in cell morphology were observed by inverted microscopy and mitochondrial morphology were observed by transmission electron microscopy,and intracellular Fe2+level was detected by Fe2+fluorescence probe.After the pretreatment with NaB,the intracellular lipid peroxidation level was detected by BODIPY 581/591 C11 fluorescence probe and changes in the expression of ACSL4,P-AKT and glutathione peroxidase 4(GPX4)were detected by Western blot.After the pretreatment with pioglitazone(PIO),the inhibitory effect on ACSL4 was verified by Western blot,and the inflammatory factors in different groups were detected by ELISA.Results:The safe concentration of NaB for BEAS-2B cells was below 2.5 mmol/L,and with the concentration of 2.5 mmol/L treated for 36 hours,the protein levels of ACSL4 and P-AKT protein were decreased significantly(P<0.05).After PA infection,cell viability was reduced in BEAS-2B cells(P<0.05).The cell became round in shape,mitochondria were the smaller and mitochondrial crista decreased or even disappeared in the ferroptotic cells;the intracellular Fe2+and lipid peroxidation levels were significantly increased(P<0.05)and the protein levels of ACSL4 and P-AKT were increased(P<0.05),while the protein level of GPX4 was significantly decreased(P<0.05).Compared with the PA group,the protein levels of ACSL4 and P-AKT were decreased with the pretreatment of NaB(P<0.05),which,however,promoted the decreased levels of GPX4 and the accumulation of lipid peroxides.Compared with the control group,the ELISA results showed that the levels of IL-6,IL-8,IL-1β and TNF-α were increased significantly after PA infection(P<0.05),and the level of IL-10 was slightly decreased(P<0.05).After the pretreatment with NaB or PIO,the level of proinflammatory factors was significantly decreased(P<0.05),while the level of IL-10 showed no statistical significance(P0.05).Conclusion:PA infection leads to the accumulation of Fe2+and lipid peroxides in bronchial epithelial cells,which induces ferroptosis and inflammation,and NaB may play an immunomodulatory role by inhibiting the pathways of ACSL4 and AKT.
资助项目	Y202352557:浙江省教育厅一般科研项目
ISSN	2095-9400
卷号	54 期号:7 页码:554-562
DOI	10.3969/j.issn.2095-9400.2024.07.005
页数	9
收录类别	万方 ; 维普 ; CNKI ; ISTIC
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引用统计
文献类型	期刊论文
条目标识符	https://kms.wmu.edu.cn/handle/3ETUA0LF/217266
专题	附属第二医院附属第二医院_内分泌科第二临床医学院、附属第二医院、育英儿童医院附属第二医院_儿童呼吸科
通讯作者	苏苗赏
作者单位	1.温州医科大学附属第二医院育英儿童医院儿童呼吸科,浙江温州 325027; 2.温州医科大学附属第二医院育英儿童医院儿童呼吸科,浙江温州 325027;龙湾区第一人民医院内分泌科,浙江温州 325024; 3.温州医科大学第二临床医学院,浙江温州 325035
第一作者单位	附属第二医院; 第二临床医学院，附属第二医院、育英儿童医院; 儿童呼吸科
第一作者的第一单位	附属第二医院
推荐引用方式 GB/T 7714	周娈,何一帆,赵伊敏,等. 铜绿假单胞菌感染支气管上皮细胞诱导铁死亡及丁酸钠的免疫调控作用[J]. 温州医科大学学报,2024,54(7):554-562.
APA	周娈., 何一帆., 赵伊敏., 任锡凯., 耿轩., ... & 苏苗赏. (2024). 铜绿假单胞菌感染支气管上皮细胞诱导铁死亡及丁酸钠的免疫调控作用. 温州医科大学学报, 54(7), 554-562.
MLA	周娈,et al."铜绿假单胞菌感染支气管上皮细胞诱导铁死亡及丁酸钠的免疫调控作用".温州医科大学学报 54.7(2024):554-562.