糖皮质激素减轻烟雾吸入性大鼠急性肺损伤后肺纤维化

Glucocorticoid attenuating pulmonary fibrosis in rats with acute lung injury induced by smoke inhalation

目的探究早期应用不同时程甲泼尼龙(MP)对大鼠烟雾吸入性肺损伤后期纤维化的改善作用。方法 178只成年雄性SD大鼠随机分为对照组(n=18)、单纯烟雾吸入组(smoke组,n=40)、烟雾吸入+MP1 d治疗组[smoke+MP(1D)组,n=40]、烟雾吸入+MP 3 d治疗组[smoke+MP(3D)组,n=40]、烟雾吸入+MP 7 d治疗组[smoke+MP(7D)组,n=40]。烟雾吸入大鼠在烟雾箱中烟雾暴露30 min以达到急性肺损伤标准,之后分别于烟雾吸入的1 d、1～3 d、1～7 d给予腹腔注射MP(每天4 mg/kg),计算大鼠28 d生存率;烟雾吸入后的第7、14、28天取大鼠肺组织行马松三色染色、天狼星红染色,并进行肺纤维化评分;通过实时荧光定量PCR测定大鼠肺组织中转化生长因子β1(TGF-β1)和α-平滑肌肌动蛋白(α-SMA)的mRNA表达;通过蛋白质印迹法测定大鼠肺组织中髓过氧化物酶(MPO)、结缔组织生长因子(CTGF)、高迁移率族蛋白1(HMGB1)、白细胞介素6(IL-6)的表达情况。结果 Smoke组大鼠烟雾吸入28 d生存率为47.50%,给予不同时程MP治疗后,生存率均提高至80%以上(P均<0.01)。马松三色染色及天狼星红染色结果显示smoke组大鼠肺纤维化程度加重,而smoke+MP(3D)组和smoke+MP(7D)组较smoke组和smoke+MP(1D)组纤维化程度改善,胶原沉积减轻(P<0.05或P<0.01)。烟雾吸入后smoke组和smoke+MP(1D)组大鼠肺组织中TGF-β1和α-SMA的mRNA表达水平逐步升高,28 d时smoke组和smoke+MP(1D)组高于smoke+MP(3D)和smoke+MP(7D)组(P<0.05或P<0.01)。烟雾吸入后28 d时smoke+MP(3D)组和smoke+MP(7D)组大鼠肺组织中MPO、CTGF、HMGB1、IL-6的表达水平低于smoke组和smoke+MP(1D)组(P<0.05,P<0.01)。但smoke+MP(3D)组和smoke+MP(7D)组上述指标差异均无统计学意义(P均>0.05)。结论大鼠烟雾吸入后给予MP治疗可以提高其生存率,3 d或7 d的MP治疗可以改善大鼠28 d肺纤维化程度,但这两种治疗方案之间差异无统计学意义。

Objective To explore the effect of methylprednisolone(MP) with different durations on pulmonary fibrosis in rats with acute lung injury induced by smoke inhalation.Methods A total of 178 male SD rats were randomized into 5 groups:control group(n=18),simple smoke inhalation group(smoke group,n=40),smoke inhalation+ MP treatment for 1d,3d and 7d groups,naming smoke+MP(1D) group(n=40),smoke+MP(3D) group(n=40) and smoke+MP(7D) group(n=40),respectively.The rats were exposed to smoke for 30 min in the smoke box to meet the criteria of acute lung injury.MP(4mg/kg per day) was injected intraperitoneally at 1d,1-3d and 1-7d after smoke inhalation.Survival rates were calculated at 28 d after smoke inhalation.At 7d,14d and 28d after smoke inhalation,the lung tissues were stained with Masson’s trichrome staining and Sirius Red staining to evaluate pulmonary fibrosis.The mRNA expression levels of transforming growth factor β1(TGF-β1) and α-smooth muscle actin(α-SMA) were detected by real-time fluorescent quantitative PCR,and the expression levels of myeloperoxidase(MPO),connective tissue growth factor(CTGF),high mobility group box 1(HMGB1) and interleukin 6(IL-6) were detected by Western blotting.Results The survival rate of the rats in the smoke group was 47.50%,and the survival rates were significantly improved to more than 80% after MP treatment with different durations(all P<0.01).Masson’s trichrome staining and Sirius Red staining staining showed that the lung fibrosis of the rats in the smoke group was aggravated,and the fibrosis and collagen deposition in the smoke+MP(3D) and smoke+ MP(7D) groups were significantly attenuated compared with the smoke and smoke+MP(1D) groups(P<0.05 or P<0.01).As time progressed,the mRNA expression levels of TGF-β1 and α-SMA after smoke inhalation were increased in the smoke and smoke+MP(1D) groups.At 28d after smoke inhalation,the TGF-β1 and α-SMA mRNA levels were significantly higher in the smoke and smoke+MP(1D) groups compared with the smoke+MP(3D) and smoke+MP(7D) groups(P<0.05 or P<0.01).At 28d after smoke inhalation,the protein expression levels of MPO,CTGF,HMGB1 and IL-6 were significantly reduced in smoke+MP(3D) and smoke+MP(7D) groups compared with the smoke and smoke+MP(1D) groups(P<0.05,P<0.01).However,the above indicators have no significant difference between smoke+MP(3D) group and smoke+MP (7D) group(all P>0.05).Conclusion MP can significantly improve survival rate of rats with smoke inhalation injury.MP treatment for 3d or 7d can significantly attenuate smoke inhalation induced pulmonary fibrosis at 28d after smoke inhalation,but there is no significant difference between these two treatment protocols.