不同浓度氧气对脂多糖诱导的肺泡Ⅱ型上皮细胞炎性反应的影响

Effect of different concentrations of oxygen on inflammatory response in lipopolysaccharide-induced alveolar type Ⅱ cells

目的 观察不同浓度氧气对脂多糖（LPS）诱导的大鼠肺泡Ⅱ型上皮细胞（AT-Ⅱ）分泌炎性因子的影响并探讨其机制.方法 原代培养的AT-Ⅱ随机分为5组（n=6）：A、B、C、D组,E组分别置于无菌培养箱：A箱37℃、21％O2、5％ CO2,B箱37℃、21％O2、5％ CO2,C箱37℃、40％O2、55％N2、5％ CO2,D箱37℃、60％O2、35％N2、5％ CO2,E箱37℃、90％O2、5％ N2、5％CO2;24 h后酶联免疫吸附试验（ELISA）检测各组细胞白细胞介素-8（IL-8）含量,反转录-聚合酶链反应（PT-PCR）及Western blot检测各组细胞p38丝裂原活化蛋白激酶（p38MAPK） mRNA及蛋白表达水平.结果 A、B、C、D、E组IL-8含量分别为（40.22 ±7.25）、（109.35±10.19）、（213.98±30.51）、（369.71 ±21.73）、（489.32 ±42.35） ng/L;p38MAPK mRNA表达水平分别为0.066± 0.010、0.163±0.008、0.228 ±0.027、0.268 ±0.016、0.292±0.010;p38 MAPK蛋白表达水平分别为0.055±0.005、0.124 ±0.006、0.182±0.009、0.227±0.013、0.303±0.010.与A组比较B、C、D、E组IL-8含量、p38MAPK mRNA和蛋白表达水平明显升高,差异有统计学意义（P＜0.05）;与B组比较C、D、E组IL-8含量、p38MAPK mRNA和蛋白表达水平逐渐升高,差异有统计学意义（P＜0.05）;C、D、E组间两两比较IL-8含量、p38MAPK mRNA和蛋白表达水平逐渐升高,差异有统计学意义（P＜0.05）.结论 40％～90％的氧气可使LPS诱导的AT-Ⅱ分泌IL-8呈浓度依赖性增加,其机制可能与胞内p38MAPK表达上调有关.

Objective To investigate the secretion of inflammatory cytokines in lipopolysaccharide （LPS）-induced aveolar epithelium type Ⅱ cells （AT-Ⅱ） cells exposed to various oxygen concentrations,and the possible mechanism.Methods Primary cultured AT-Ⅱ cells were randomly divided into five groups：A,B,C,D,and E,and then placed in sterilized incubators：37 ℃,21% O2,5% CO2 ;37 ℃,21% O2,5% CO2; 37 ℃,40% O2,55% N2,5% CO2; 37 ℃,60% O2,35% N2,5% CO2;37 ℃,90% O2,5% N2,5% CO2.Towenty-four h later,the supernatant fluid was used to assay the content of interleukin-8 （IL-8） by enzyme linked immunosorbent assay （ELISA）.The expression of p38 mitogen-activated protein kinase （p38MAPK） mRNA and protein was detected in AT-Ⅱ cells by reverse transcription-polymerase chain reaction and Western blotting.Results ELISA,reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting showed that the content of IL-8 was （40.22±7.25）,（109.35 ±10.19）,（213.98±30.51）,（369.71 ±21.73） and （489.32± 42.35） ng/L,p38MAPK mRNA expression level was 0.066 ± 0.010,0.163 ± 0.008,0.228 ± 0.027,0.268 ±0.016 and 0.292 ±0.010,and p38MAPK protein expression level was 0.055 ±0.005,0.124 ± 0.006,0.182 ±0.009,0.227 ±0.013 and 0.303 ±0.010 in groups A,B,C,D and E respectively.As compared with group A,the content of IL-8 in the supernatant fluid,and the expression of p38MAPK mRNA and protein were significantly increased in groups B,C,D and E （P 〈 0.05）.As compared with groups B,the content of IL-8 in the supernatant fluid,and the p38MAPK mRNA and protein expression level in groups C,D and E were gradually increased with the difference being statistically significant （P 〈 0.05）.In different oxygen concentration groups,with the increases in oxygen concentrations,the content of IL-8 in the supematant fluid,and the expression of p38MAPK mRNA and protein expression level were gradually increased with the difference being statistically significant （P 〈 0.05）.Condusion 40%-90% oxygen could concentrasion-dependently increase the LPS-induced secretion of IL-8 in AT-Ⅱ cells probably by up-regulating the intracellular p38MAPK expression.