转化生长因子-β/Smad信号通路对 急性肺损伤小鼠免疫平衡的影响

Regulatory role of transforming growth factor-β/Smad pathway on immune imbalance in a mouse model of acute lung injury

目的：探讨转化生长因子-β（TGF-β）/Smad信号通路对急性肺损伤（ALI）小鼠肺组织辅助性T细胞17/调节性T细胞（Th17/Treg）平衡的影响及机制。方法按随机数字表法将60只雄性C57BL/6小鼠分为3组，每组20只。白细胞介素-17（IL-17）预处理组先经腹腔注射抗小鼠IL-17单克隆抗体200μg，ALI模型组则注射200μg同型对照抗体IgG2a；24h后两组均经气管内注入脂多糖（LPS）4mg/kg〔溶于30μL磷酸盐缓冲液（PBS）中〕诱导ALI小鼠模型，对照组经气管内滴注30μLPBS。各组于制模后24h处死小鼠，取肺组织和支气管肺泡灌洗液（BALF）。光镜下观察肺组织病理学改变；测定肺湿/干重（W/D）比值和BALF中蛋白含量；用酶联免疫吸附试验（ELISA）检测肺组织匀浆中IL-6、IL-10、IL-17A和TGF-β1水平；流式细胞仪检测肺组织中Treg和Th17细胞量；实时定量聚合酶链反应（PCR）检测肺组织维甲酸相关孤核受体γt（ROR-γt）、TGF-β1、叉头蛋白P3（Foxp3）、Smad2、Smad3、Smad7的mRNA表达；蛋白质免疫印迹试验（WesternBlot）检测肺组织磷酸化Smad2/3、Smad7（p-Smad2/3、p-Smad7）表达。结果与对照组比较，ALI模型组肺组织可见大量炎性细胞浸润，伴有肺泡壁间隔增厚、充血水肿/塌陷和点状出血；肺W/D比值、BALF中蛋白含量及肺组织IL-6、IL-17A、TGF-β1水平、Th17/Treg细胞比例、ROR-γt、TGF-β1、Foxp3、Smad2、Smad3的mRNA表达明显升高，Smad7mRNA表达明显下降。与ALI模型组比较，IL-17预处理组镜下显示，肺损伤明显减轻；肺W/D比值明显减小（5.84±0.65比8.27±1.32），BALF中蛋白含量明显降低（g/L：0.09±0.02比0.18±0.05）；肺组织IL-6（pg/mg：465±105比1098±145）、IL-17A（pg/mg：32±12比876±178）、TGF-β1（pg/mg：45±10比96±26）明显下降；肺组织Th17细胞量〔（6.78±0.35）%比（11.32±1.25）%〕和Th17/Treg比值（1.24±0.29比1.98±0.21）明显降低；肺组织ROR-γt、TGF-β1、Smad2、Smad3的mRNA表达（2-ΔΔCt：1.04±0.05比2.32±0.34，2.38±0.12比3.48±0.31，2.44±0.12比3.25±0.34，2.36±0.09比2.98±0.13）和p-Smad2/3（A值：1.78±0.25比2.58±0.32）明显下降，Smad7mRNA表达（2-ΔΔCt：0.84±0.09比0.69±0.03）和p-Smad7（A值：0.76±0.12比0.53±0.05）明显升高，差异均有统计学意义（均P＜0.05），而Foxp3mRNA表达相当（2-ΔΔCt：1.74±0.05比1.62±0.13，P＞0.05）。各组肺组织IL-10水平差异无统计学意义。结论 Th17/Treg失衡可能参与了ALI的发生，TGF-β/Smad信号通路调控了Th17/Treg的免疫失衡。

Objective To investigate the regulatory role and its mechanism of transforming growth factor-β （TGF-β）/Smad pathway on the balance of helper T cell 17/T-regulatory cells （Th17/Treg） in a mouse model of acute lung injury （ALI）. Methods Sixty C57BL/6 mice were divided into three groups according to randomize number table method： control group （n = 20）, ALI model group （n = 20）, and interleukin-17 （IL-17） antibody （Ab） pretreatment group （n = 20）. The mice in IL-17 Ab pretreatment group were delivered 200 μg IL-17 Ab intraperitoneally, while the ALI model group were given 200 μg Iso-IgG2a. Both groups were challenged by 4 mg/kg lipopolysaccharides （LPS） [dissolved in 30 μL phosphate buffer saline （PBS）] intratracheally to induce ALI. The mice in control group were given 30 μL PBS. Twenty-four hours later, all mice were sacrificed and lung tissues and bronchoalveolar lavage fluid （BALF） were harvested. The pathological images were observed under light microscope, the lung wet weight/dry weight （W/D） ratio and the protein content in BALF were measured to assess the severity of lung injury. The levels of IL-6, IL-10, IL-17A and TGF-β1 in lung homogenates were assessed by enzyme-linked immunosorbent assay （ELISA）. The ratio of Treg and Th17 was examined by flow cytometer. The mRNA expression levels of retinoid-related orphan nuclear receptor γt （ROR-γt）, TGF-β1, Foxp3, Smad2, Smad3 and Smad7 were quantitatively measured by real-time polymerase chain reaction （PCR）. The phosphates Smad2/3 and Smad7 （p-Smad2/3, p-Smad7） of lung tissues were detected by Western Blot. Results Compared with the control group, a large number of inflammatory cells infiltration could be observed and accompanied by alveolar septal thickening, alveolar congestion and edcma/collapse and dot bleeding; the ratio of W/D, the protein levels in BALF, the concentrations of IL-6, IL-17A and TGF-β1 in lung tissue homogenates, the ratio of Th17/Treg and the mRNA expressions of ROR-γt, TGF-β1, Foxp3, Smad2, and Smad3 were significantly elevated, the mRNA expression of Smad7 was significantly decreased in the ALI model group （all P 〈 0.05）. Interestingly, compared with the ALI model group, the obvious reduction in the disruption of lung tissue structure and congestion were seen under light microscope; the ratio of W/D （5.84±0.65 vs. 8.27±1.32）, the protein levels in BALF （g/L： 0.09±0.02 vs. 0.18±0.05）, the concentrations of IL-6 （pg/mg： 465±105 vs. 1 098±145）, IL-17A （pg/mg： 32±12 vs. 876±178） and TGF-β1 （pg/mg： 45±10 vs. 96±26） in lung tissue homogenates were significantly decreased in IL-17 Ab pretreatment group （all P 〈 0.05）. In addition, all mice with pretreatment of IL-17 Ab resulted in significantly reduced percentage of Th17 [（6.78±0.35）% vs. （11.32±1.25）%] and ratio of Th17/Treg （1.24±0.29 vs. 1.98±0.21）, mRNA expressions of ROR-γt, TGF-β1, Smad2, and Smad 3 （2-ΔΔCt： 1.04±0.05 vs. 2.32±0.34, 2.38±0.12 vs. 3.48±0.31, 2.44±0.12 vs. 3.25±0.34, 2.36±0.09 vs. 2.98±0.13）, and the p-Smad2/3 （A value： 1.78±0.25 vs. 2.58±0.32） was significantly decreased, while the mRNA （2-ΔΔCt： 0.84±0.09 vs. 0.69±0.03） and protein expression of p-Smad7 （A value： 0.76±0.12 vs. 0.53±0.05） was significantly up-regulated in lung tissue homogenates, the differences were statistically significant （all P 〈 0.05）. The mRNA expression of Foxp3 （2-ΔΔCt： 1.74±0.05 vs. 1.62±0.13, P 〉 0.05） was similar. There were no significant differences in protein levels of IL-10 of lung tissue among all the groups. Conclusion The imbalance of Th17/Treg may play an important role in the immunological mechanism of ALI, which may be partially modulated by TGF-β/Smad pathway.