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

Ferroptosis of bronchial epithelial cells induced by pseudomonas aeruginosa infection and the immunomodulatory mechanism of sodium butyrate

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,Westernblot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Fe^(2+)水平;加入NaB预处理后,BODIPY581/591C11荧光探针检测细胞内脂质过氧化水平,Westernblot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,Westernblot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2.5mmol/L以下,2.5mmol/LNaB处理细胞36h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Fe^(2+)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Fe^(2+)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。

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 Fe^(2+)level was detected by Fe^(2+)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 Fe^(2+)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-1βand TNF-αwere 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(P>0.05).Conclusion:PA infection leads to the accumulation of Fe^(2+)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.