双调蛋白对小鼠损伤肺组织的修复作用及机制研究

The repair effect and mechanism of amphiregulin on injured lung tissue in mice

目的探讨双调蛋白(amphiregulin, Areg)对急性呼吸窘迫综合征(acute respiratory distress syndrome, ARDS)小鼠损伤肺组织的修复作用及机制。方法使用脂多糖(lipopolysaccharide, LPS)气管滴注制作小鼠ARDS模型, 连续7 d提取支气管肺泡灌洗液(bronchoalveolar lavage fluid, BALF)。将成年雄性C57BL/6小鼠用随机数字法分为5组(n=4/组):①空白组;②Areg组(腹腔注射重组Areg蛋白);③LPS+PBS组;④LPS+Areg组;⑤LPS+Anti-Areg组(③④⑤组小鼠气管滴注LPS, 30 min后腹腔注射PBS、重组Areg或Areg中和抗体)。于ARDS后1、3、5、7 d提取肺组织与BALF, HE染色评估肺组织病理变化, BCA法检测BALF中总蛋白含量, ELISA法检测肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、白介素-1β(IL-1β)与免疫球蛋白M(IgM)浓度, Western Blot检测表皮生长因子受体(epidermal growth factor receptor, EGFR)、增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)、表面活性蛋白-C(surface proteins-C, SP-C)的表达情况, 免疫荧光检测肺组织PCNA与SP-C共表达情况。符合正态分布的计量资料多组间比较采用方差分析, 两组间比较采用LSD-t检验。结果与建模前相比[(51.05±2.47) pg/mL], ARDS后小鼠肺组织中的Areg含量在第1天[(71.97±6.51) pg/mL, P<0.01]与第3天[(147.58±7.56) pg/mL, P<0.01]显著升高。在ARDS后第1天, LPS+PBS组和LPS+Areg组出现明显肺组织间质水肿、中性粒细胞浸润和肺泡结构塌陷, 且损伤程度无明显差异。在第3、5、7天, 与LPS+PBS组相比, LPS+Areg组的肺组织病理损伤情况均有明显好转, 而LPS+Anti-Areg组的损伤情况却更为严重。与空白组相比, LPS+PBS组小鼠BALF中总蛋白、IgM、中性粒细胞数量、TNF-α、IL-1β、IL-6均有明显升高, Areg处理显著降低了这些指标的水平。LPS+Areg组PCNA(1.34±0.10)、SP-C(1.48±0.10)、p-EGFR(0.92±0.032)的表达水平较LPS+PBS组[(0.88±0.03), (1.06±0.15), (0.68±0.03), P<0.05]上调, 且LPS+Areg组PCNA及SP-C双阳性细胞较LPS+PBS组明显增多, 而LPS+Anti-Areg组则有所下降。结论 Areg可以通过激活EGFR通路增加肺泡Ⅱ型上皮细胞的增殖, 从而促进ARDS损伤肺组织的修复。

Objective To investigate the repair effect of amphiregulin(Areg)on injured lung tissue in mice with acute respiratory distress syndrome(ARDS)and its underlying mechanism.Methods The ARDS mouse model was made by tracheal infusion of lipopolysaccharide(LPS),and bronchoalveolar lavage fluid(BALF)was extracted for 7 consecutive days.Adult male C57BL/6 mice were randomly(random number)divided into 5 groups(n=4 per group):(1)Control group;(2)Areg group:mice were treated intraperitoneally(i.p.)with recombinant Areg;(3)LPS+PBS group;(4)LPS+Areg group;and(5)LPS+Anti-Areg group;mice were instilled with LPS,then were injected i.p.with PBS,Areg or Areg neutralization antibody(Anti-Areg)30 min later.Lung tissue and BALF were extracted at day 1,3,5 and 7 after ARDS.HE staining was used to evaluate the pathological changes of lung tissues.The total protein content in BALF was detected by BCA method,and the concentrations of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),IL-1β and immunoglobulin M(IgM)were determined by ELISA method.The phosphorylated levels of epidermal growth factor receptor(EGFR)and expressions of proliferating cell nuclear antigen(PCNA)and surface proteins-C(SP-C)were tested by Western blot.The immunofluorescence was used to detect the co-expression of PCNA and SP-C in lung tissues.Oneway analysis of variance was used to compare the mean values of normally distributed measurement data between groups.Comparisons between groups were performed using the least significant difference/-test.Results Compared with that at before modeling[(51.05±2.47)pg/mL],Areg concentrations were increased significantly at day 1[(71.97±6.51)pg/mL;P<0.01]and day 3[(147.58±7.56)pg/mL,P<0.01]in the BALF after ARDS.At day 1 after ARDS,there were significant interstitial edema,neutrophil infiltration and alveolar collapse in the LPS+PBS group and LPS+Areg group.Compared with the LPS+PBS group at day 3,5 and 7,the pathological changes of lung tissues were notably improved in the LPS+Areg group,while were more serious in the LPS+Anti-Areg group.Compared with the control group,the LPS+PBS group had higher levels of neutrophil number,total protein,IgM,TNF-α,IL-1β,and IL-6.However,Areg treatment significantly reduced the levels of these indicators.Moreover,the expressions of PCNA(1.34±0.10),SP-C(1.48±0.10)and p-EGFR(0.92±0.032)in the LPS+Areg group were significantly up-regulated compared with those in the LPS+PBS group(0.88±0.03,1.06±0.15,and 0.68±0.03,all p<0.01).And compared with the LPS+PBS group,PCNA and SP-C double positive cells were significantly increased in the LPS+Areg group,but decreased in the LPS+Anti-Areg group.Conclusions Areg enhances the proliferation of alveolar type Ⅱ epithelial cells by activating EGFR pathway,therefore promotes the repair of lung tissues during ARDS development.