分子氢减轻脂多糖诱导急性肺损伤的机制探究

Mechanism of molecular hydrogen attenuating acute lung injury induced by lipopolysaccharid

目的探讨分子氢对脂多糖(lipopolysaccharide,LPS)诱导急性肺损伤(acute lung injury,ALI)的作用机制。方法将24只Balb/c小鼠随机(随机数字法)分为4组:control组、control+H_(2)组、LPS组、LPS+H_(2)组,6只/组。观察各组肺组织形态,检测肺组织丙二醛(malondialdehyde,MDA)、Fe^(2+)水平,对肺组织进行免疫荧光染色检测F4/80阳性巨噬细胞浸润水平。将A549细胞分为control、control+H_(2)、erastin、erastin+H_(2)组,检测各组活性氧自由基(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutathione,GSH)、细胞死亡数量、乳酸脱氢酶(lactate dehydrogenase,LDH)释放量,荧光定量PCR定量核因子红细胞2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、血红素氧化酶1(heme oxygenase-1,HO-1)mRNA表达水平,蛋白质免疫印迹检测Nrf2蛋白表达水平,免疫荧光法检测Nrf2核转位水平。计量资料统计前进行方差齐性检验,多组间差异比较采用单因素方差分析。结果与control组比较,LPS组肺组织损伤明显加重,F4/80阳性巨噬细胞浸润程度、脂质过氧化水平增高(均P<0.05);与LPS组比较,LPS+H_(2)组肺损伤程度明显减轻(均P<0.05);体外实验中,与control组比较,erastin组ROS、MDA水平、细胞死亡数量、LDH释放量增多(均P<0.05),GSH、GPX4 mRNA水平下降,HO-1 mRNA、Nrf2核转位水平增高(均P<0.05),与erastin组相比,earstin+H_(2)组ROS、MDA水平、细胞死亡数量、LDH释放量下降(均P<0.05),GSH、GPX4 mRNA、Nrf2 mRNA、HO-1 mRNA水平提高,Nrf2核转位水平升高(均P<0.05)。结论分子氢促进Nrf2核转位,抑制肺泡上皮细胞铁死亡减轻LPS诱导的ALI。

Objective To investigate the role and mechanism of molecular hydrogen in lipopolysaccharide(LPS)-induced acute lung injury(ALI).Methods Balb/c male mice were randomly(random number)divided into control group,control+H_(2),LPS and LPS+H_(2) group with 6 mice in each group.The levels of malondialdehyde(MDA)and Fe^(2+)in lung tissue were detected by kits.The lung tissue morphology was observed.The infi ltration levels of F4/80 positive macrophages in lung tissue were detected by immunofl uorescence staining.A549 cells were divided into control,control+H_(2),erastin and erastin+H_(2) group.The reactive oxygen species(ROS),malondialdehyde,(MDA),lactate dehydrogenase(GSH),number of cell death and lactate dehydrogenase(LDH)release in each group were detected by kits.Nrf2,GPX4,and HO-1mRNA were quantifi ed by real-time PCR,the protein expression level of Nrf2 was detected by western blot,and the nuclear translocation level of Nrf2 was observed by immunofl uorescence.The chi-square test was performed before the measurement data were counted.One-way analysis of variance was used to compare differences between multiple groups.Results Compared with the control group,the histopathological damage was aggravated,and the levels of MDA,Fe^(2+)signifi cantly increased in the LPS group,and F4/80 positive immune cells infiltration significantly increased(all P<0.05).Compared with LPS group,the degree of lung injury in LPS+H_(2) group significantly reduced(all P<0.05).In vitro experiments,compared with the control group,the ROS,MDA levels,number of cell death and LDH release signifi cantly increased in erastin group(all P<0.05),while GSH,and GPX4 mRNA levels decreased(all P<0.05).HO-1mRNA and Nrf2 nuclear translocation levels increased(all P<0.05).Compared with erastin group,ROS,MDA levels,cell death number and LDH release decreased in earstin+H_(2) group(all P<0.05).The levels of GSH,GPX4 mRNA,Nrf2 mRNA,HO-1 mRNA and Nrf2 nuclear translocation levels increased(all P<0.05).Conclusions Molecular hydrogen attenuates LPSinduced ALI by promoting Nrf2 nuclear translocation to inhibit ferroptosis of alveolar epithelial cells.