调控Hippo信号通路促进间充质干细胞修复急性呼吸窘迫综合征肺损伤

Modulation of Hippo signaling pathway facilitates mesenchymal stem cells to repair lung injury in murine lipopolysaccharide induced acute respiratory distress syndrome

目的探讨通过调控Hippo信号通路促进间充质干细胞(MSC)修复脂多糖(LPS)诱导急性呼吸窘迫综合征(ARDS)肺损伤的作用。方法通过慢病毒载体转染构建低表达大肿瘤抑制因子1(LATS1)基因的C57BL/6小鼠骨髓来源的MSC(mMSCs)细胞系,气道内给予2 mg/mL LPS 50μL复制小鼠ARDS模型,SPF级C57BL/6雄性小鼠随机(随机数字法)分为4组(n=36):正常对照组、ARDS组、ARDS+空干扰病毒转染的mMSCs(MSC-shcontrol)治疗组、ARDS+转染干扰LATS1病毒的mMSCs(MSC-shLATS1)治疗组。成模后3、7、14 d后处死小鼠并收集肺组织及支气管肺泡灌洗液(BALF)样本。近红外荧光成像、免疫荧光染色及Western blot评估mMSCs在肺组织的存留及向肺泡Ⅱ型肺泡上皮(ATⅡ)细胞的分化;测定肺组织湿质量/体质量比(LWW/BW)、BALF中总蛋白(TP)和白蛋白(ALB)水平及评价mMSCs对肺水肿的改善效应;Western blot检测上皮紧密连接蛋白Occludin反映mMSCs对肺泡上皮通透性影响;ELISA检测BALF中IL-1β、IL-6及IL-10含量评估mMSCs对肺组织炎症反应的影响;肺组织行HE染色及肺损伤评分评估mMSCs对肺损伤的修复作用;肺组织行Masson染色及肺纤维化评分评估mMSCs对肺纤维化的影响。结果MSC-shLATS1治疗组3、7、14 d mMSCs在ARDS肺组织内存留数量(个/HP)[(26.25±4.58)、(20.49±3.86)、(13.77±3.55)]均较MSC-shcontrol治疗组[(12.13±3.75)、(9.97±2.76)、(6.89±2.10)]更多(均P<0.05),14 d向ATⅡ细胞的分化比例[(64.12±15.29)%]也较MSC-shcontrol治疗组[(19.64±3.71)%]更高(P<0.05);MSC-shLATS1治疗组3 d及14 d LWW/BW、BALF中TP和ALB水平[(9.85±1.51)及(6.11±0.83)mg/g、(5.12±0.87)及(3.05±0.87)(mg/mL)、(0.44±0.18)及(0.33±0.04)mg/mL]均较MSC-shcontrol治疗组[(14.88±1.74)及(8.04±1.70)mg/g、(8.08±1.72)及(5.94±1.20)(mg/mL)、(0.71±0.21)及(1.07±0.29)(mg/mL)]下降(均P<0.05),其14 d Occludin蛋白相对表达[(0.79±0.11)]较MSC-shcontrol治疗组[(0.49±0.19)]升高(P<0.05);MSC-shLATS1治疗组BALF中IL-1β、IL-6浓度[(60.11±8.58)、(101.74±11.55)(pg/mL)]较MSC-shcontrol治疗组[(99.26±14.32)、(145.54±13.29)(pg/mL)]更低(均P<0.05),但IL-10浓度[(316.65±37.88)(pg/mL)]较MSC-shcontrol治疗组[(190.83±22.61)(pg/mL)]更高(P<0.05);MSC-shLATS1治疗组3 d、14 d肺损伤评分及14 d肺纤维化评分[(7.18±1.12)、(3.33±0.49)及(0.68±0.12)]均较MSC-shcontrol治疗组[(9.72±1.45)、(5.11±0.86)及(1.47±0.18)]降低(均P<0.05)。结论通过低表达LATS1基因抑制Hippo信号通路能促进mMSCs对ARDS肺损伤的修复效应。

Objectives To determine the effect of Hippo signaling pathway on lung injury repair of bone-marrow derived mesenchymal stem cells (mMSCs) in murine lipopolysaccharide (LPS) induced acute respiratory distress syndrome (ARDS). Methods C57BL/6 mouse bone marrow-derived MSC (mMSCs) cell lines with low expression of large tumor suppressor 1 (LATS1) were constructed by lentiviral vector transfection. ARDS was modeled by intratracheally injection of 2 mg/mL lipopolysaccharide (LPS) 50 μL. C57BL/6 mice were randomly(random number) divided into four groups (n=36): normal control group, ARDS group, ARDS mice transplanted with mMSCs transfected with blank lentivirus vector (MSC-shcontrol group) or sh-LATS1 lentivirus vector (MSC-shLATS1 group). Mice were sacrifi ced at 3, 7, and 14 d after modeling, and lung tissue and bronchoalveolar lavage fl uid (BALF) were collected. Near-infrared fl uorescence imaging, immunofl uorescence staining and Western blot were used to evaluate retention and differentiation of mMSCs in lung tissue. Lung tissue wet weight/body weight ratio (LWW/ BW) and total protein (TP) and albumin (ALB) in BALF were determined to refl ect pulmonary edema. The expression of Occludin protein in lung epithelium was tested by Western blot. The levels of interleukins (IL-1β, IL-6, and IL-10) in BALF were assessed by enzyme-linked immunosorbent assay (ELISA). Lung injury score and pulmonary fi brosis score in lung tissue were assessed. Results The retention of mMSCs at 3, 7 and 14 d in the MSC-shLATS1 group were signifi cantly higher than those in the MSC-shcontrol group [(26.25±4.58) vs (12.13±3.75) cells/HP,(20.49±3.86) vs (9.97±2.76) cells/HP, and (13.77±3.55) vs (6.89±2.10) cells/HP, all P<0.05], so was the differentiation of mMSCs into type Ⅱ alveolar epithelial cells at 14 d [(64.12±15.29)% vs (19.64±3.71)%, P<0.05]. LWW/BW and TP and ALB in BALF at 3 and 14 d in the MSC-shLATS1 group [(9.85±1.51),(6.11±0.83)(mg/g) and (5.12±0.87),(3.05±0.87)(mg/mL) and (0.44±0.18),(0.33±0.04)(mg/mL)] were higher than those in the MSC-shcontrol group [(14.88±1.74),(8.04±1.70)(mg/g) and (8.08±1.72),(5.94±1.20)(mg/mL) and (0.71±0.21),(1.07±0.29)(mg/mL)](all P<0.05), so was the relative expression of Occludin protein[(0.79±0.11) vs (0.49±0.19),(P<0.05)]. The levels of IL-1β and IL-6(pg/mL) in BALF in the MSC-shLATS1 group [(60.11±8.58),(101.74±11.55)] was lower than those in the MSC-shcontrol group [(99.26±14.32),(145.54±13.29)](all P<0.05), but the levels of IL-10 in BALF in the MSC-shLATS1 group (316.65±37.88)pg/mL was higher than those in the MSC-shcontrol group (190.83±22.61)pg/mL (P<0.05). Lung injury scores at 3 and 14 d in the MSC-shLATS1 group [(7.18±1.12),(3.33±0.49)] was lower than those in the MSC-shcontrol group [(9.72±1.45),(5.11±0.86)](all P<0.05), so was pulmonary fi brosis score at 14 d [(0.68±0.12) vs (1.47±0.18), P<0.05]. Conclusion Inhibition of Hippo signaling pathway through underexpression of LATS1 could improve the therapeutic effects of mMSCs in murine LPS-induced ARDS.