摘要
目的探讨血红素加氧酶-1(heme oxygenase-1,HO-1)调节巨噬细胞极化在海水淹溺引起的急性肺损伤中的作用及意义。方法用人工海水(seawater,SW)分别刺激Raw264.7细胞8、24和72 h,将这些细胞分为对照组、SW 8 h组、SW 24 h组和SW 72 h组,观察细胞形态变化,检测细胞凋亡、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和HO-1蛋白含量。肺泡巨噬细胞条件性Hmox1基因敲除Hmox1_(flox/flox)Cre^(+/-)Hmox1^(-/-)(HO-1M-KO)小鼠随机分为HO-1^(flox/flox)对照组、HO-1^(flox/flox) SW 24 h组、HO-1M-KO对照组和HO-1M-KO SW 24 h组(每组n=6)。小鼠置于镂空的容器中,置于水深6 cm、温度(25±2)℃的人工海水中28 s,建立小鼠海水淹溺性急性肺损伤(acute lung injury,ALI)模型。淹溺24 h后取材进行肺泡灌洗,检测支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中细胞总数和蛋白浓度,观察肺组织的病理变化和肺组织中iNOS的蛋白含量。结果海水刺激Raw264.7细胞后不规则细胞增多,细胞数量减少;对照组、SW 8 h组、SW 24 h组和SW 72 h组的凋亡率分别为(5.99±0.23)%、(16.71±1.16)%、(41.80±2.50)%和(77.84±1.59)%,凋亡率随淹溺时间增加(P<0.01)。对照组、SW 8 h组、SW 24 h组和SW 72 h组细胞中HO-1蛋白含量分别为(1.07±0.06)、(1.42±0.01)、(2.77±0.22)和(0.99±0.10),SW 8 h组和SW 24 h组的HO-1蛋白含量高于其他2组(P<0.05)。对照组、SW 8 h组、SW 24 h组和SW 72 h组细胞中iNOS蛋白表达量分别为(0.94±0.10)、(3.44±0.32)、(1.52±0.09)和(2.26±0.11),SW 8 h组、SW 24 h组和SW 72 h组iNOS蛋白表达量均高于对照组(P<0.01)。但是SW 24 h组相较于SW 8 h组,HO-1蛋白表达量显著上升,iNOS表达量下降(P<0.01)。HO-1M-KO小鼠淹溺后肺组织损伤明显加重,肺泡腔塌陷、缩小,肺泡腔内出血,肺泡间隔明显增厚,炎性细胞浸润,BALF中的细胞数量和蛋白浓度显著升高(P<0.01),肺组织中iNOS含量显著上升(P<0.01)。结论HO-1可通过抑制M1巨噬细胞极化,减轻海水淹溺引起的小鼠ALI。
Objective To investigate the role and significance of heme oxygenase-1(HO-1)in regulating macrophage polarization on seawater-drowning-induced acute lung injury(ALI)in mice.Methods Raw 264.7 cells were divided into four groups.The control group was not stimulated with artificial seawater(SW),while the SW 8 h group,SW 24 h group,and SW 72 h group stimulated with artificial seawater for 8 h,24 h,and 72 h,respectively.The morphological changes of cells were observed,the apoptosis,the contents of inducible nitric oxide synthase(iNOS),and HO-1 protein were detected.Hmox1^(flox/flox)Cre^(+/-)and Hmox1^(-/-)(HO-1M-KO)mice with conditional knockout of HO-1 gene in alveolar macrophages were produced and randomly divided into HO-1flox/flox Control group,HO-1^(flox/flox) SW 24 h group,HO-1M-KO Control group,and HO-1M-KO SW 24 h Group,with 6 mice in each group.The mouse model of seawater-drowning-induced ALI was established by placing mice in a hollow container and then immersing them in 6 cm deep and(25±2)℃artificial seawater for 28 s.Samples were taken 24 hours after seawater drowning to carry out alveolar lavage,and then the total cell counts and protein concentrations in bronchoalveolar lavage fluid(BALF),and the pathological changes and the iNOS protein content in lung tissues were observed.Results Abnormally shaped Raw 264.7 cells increased while the total number of Raw 264.7 cells decreased after seawater stimulation.The apoptosis rates of the control group,SW 8 h group,SW 24 h group,and SW 72 h group were increased to(5.99±0.23)%,(16.71±1.16)%,(41.80±2.50)%,and(77.84±1.59)%,respectively,with statistically significant differences(P<0.01).The contents of HO-1 protein in the control group,SW 8 h group,SW 24 h group,and SW 72 h group were(1.07±0.06),(1.42±0.01),(2.77±0.22),and(0.99±0.10),respectively,and the contents of HO-1 protein of both SW 8 h group and SW 24 h group increased significantly(P<0.05).The contents of iNOS protein in the cells of the control group,SW 8 h group,SW 24 h group,and SW 72 h group were(0.94±0.10),(3.44±0.32),(1.52±0.09),and(2.26±0.11),respectively,and the contents of iNOS protein of SW 8 h group,SW 24 h group,and SW 72 h group increased significantly(P<0.05);compared with the SW 8 h group,the HO-1 protein content increased significantly,while the iNOS protein content decreased in the SW 24 h group significantly(P<0.01).Lung tissue injury in HO-1M-KO mice was significantly aggravated after drowning.The cavity of pulmonary alveoli collapsed and shrunk,intra-alveolar hemorrhage occurred,alveolar septum thickened,and inflammatory cell infiltration aggravated.The cell number and protein concentration in BALF significantly increased(P<0.01),and the iNOS content in lung tissue significantly increased(P<0.01).Conclusion HO-1 can alleviate the seawater-drowning-induced ALI in mice by inhibiting the M1 macrophage polarization.
作者
万斌斌
刘钢
袁佳宁
胡珺婕
王敏英
丁铎
吴亚先
陈丹
庞庆丰
Wan Binbin;Liu Gang;Yuan Jianing;Hu Junjie;Wang Minying;Ding Duo;Wu Yaxian;Chen Dan;Pang Qingfeng(Wuxi School of Medicine,Jiangnan University,Wuxi 214122,China)
出处
《中华航海医学与高气压医学杂志》
CAS
CSCD
2021年第3期350-355,共6页
Chinese Journal of Nautical Medicine and Hyperbaric Medicine
基金
国家自然科学基金(81871518)。
