Background
Ventilator-induced lung injury (VILI) is commonly associated with barrier dysfunction and inflammation reaction. Glutamine could ameliorate VILI, but its role has not been fully elucidated. This study examined the relationship between inflammatory cytokines (interleukin [IL]-6, tumor necrosis factor [TNF]-α, and IL-10) and adherens junctions (E-cadherin, p120-catenin), which were ameliorated by glutamine in VILI, both in vitro and in vivo.
Conclusions
High tidal volume mechanical ventilation and 20% cyclic stretching could cause VILI. Glutamine regulates VILI by improving cytokines and increasing the adherens junctions, protein E-cadherin and p120-catenin, to enhance the epithelial barrier function. 谷氨酰胺在机械通气肺损伤中调节E-钙黏素、p120连环蛋白及炎症的作用摘要背景：机械通气相关肺损伤与屏障功能障碍和炎症反应有关，谷氨酰胺能够改善机械通气造成的肺损伤，但是其作用机制尚不十分清楚。本研究通过体内实验和体外实验两方面，探讨谷氨酰胺改善机械通气造成肺损伤的机制。 方法：30只体重为25~30g的健康C57BL/6小鼠，利用随机数字表法随机分为5组，每组6只小鼠：对照组（C组）、小潮气量组（L组）、小潮气量+谷氨酰胺组（L+G组）、大潮气量组（H组）、大潮气量+谷氨酰胺组（H+G组）。C组小鼠自主呼吸，其余四组小鼠进行机械通气4小时。肺泡上皮细胞（MLE-12）和谷氨酰胺预处理过的MLE-12细胞均以20%的牵张幅度进行机械牵张4小时。收集细胞和肺组织，用免疫印迹法、细胞因子测定、苏木精-伊红（HE）染色和免疫荧光来检测黏连蛋白（E-cadherin、p120-catenin）、炎症细胞因子（IL-6、TNF-α、IL-10）和肺病理变化。 结果：体内实验，与C组比较，H组肺泡灌洗液中的细胞总数（t= -28.182，P<0.01）、中性粒细胞（t= −28.095，P<0.01）、IL-6（t= −28.296，P<0.01）、TNF-α（t= -19.812，P<0.01）、肺损伤评分（t= −6.708，P<0.01）和湿干比（t= −15.595， P<0.01）均增加，肺泡灌洗液中IL-10（t=9.093，P<0.01）、E-cadherin（t=10.044，P<0.01）和p120-catenin（t=13.218，P<0.01）表达均下降。与H组比较，H+G组的肺泡灌洗液中的细胞总数（t=14.844，P<0.01）、中性粒细胞（t=18.077， P<0.01）、肺泡灌洗液中IL-6（t=18.007，P<0.01）和TNF-α（t=10.171，P<0.01）表达均降低，而肺泡灌洗液中IL-10（t= −7.531，P<0.01）、E-cadherin（t= −14.814，P<0.01）、p120-catenin（t= −9.114，P<0.01）表达均增加。体外实验，与对照组比较，机械牵张组IL-6（t= −21.111，P<0.01）和TNF-α（t= −15.270，P<0.01）表达增加，IL-10（t=5.450，P<0.01）和E-cadherin（t=17.736，P<0.01）、p120-catenin（t=16.136，P<0.01）表达下降。与机械牵张组比较，谷氨酰胺预处理组的IL-6（t=11.818，P<0.01）、TNF-α（t=8.631，P<0.01）表达下降，IL-10（t= −3.203，P<0.05）、E-cadhrein（t= −13.567，P<0.01）、p120-catenin（t= −10.013，P<0.01）表达增加。 结论：大潮气量机械通气和幅度为20%的机械牵张可能导致机械通气相关肺损伤。谷氨酰胺通过改善细胞因子和增加黏附连接蛋白E-cadherin和p120-catenin来改善机械通气肺损伤，增强屏障功能。.
Methods
For the in vivo study, 30 healthy C57BL/6 mice weighing 25-30 g were randomly divided into five groups with random number table (n = 6 in each group): control (Group C); low tidal volume (Group L); low tidal volume + glutamine (Group L + G); high tidal volume (Group H); and high tidal volume + glutamine (Group H + G). Mice in all groups, except Group C, underwent mechanical ventilation for 4 h. For the in vitro study, mouse lung epithelial 12 (MLE-12) cells pretreated with glutamine underwent cyclic stretching at 20% for 4 h. Cell lysate and lung tissue were obtained to detect the junction proteins, inflammatory cytokines, and lung pathological changes by the Western blotting, cytokine assay, hematoxylin and eosin staining, and immunofluorescence.
Results
In vivo, compared with Group C, total cell counts (t = -28.182, P < 0.01), the percentage of neutrophils (t = -28.095, P < 0.01), IL-6 (t = -28.296, P < 0.01), and TNF-α (t = -19.812, P < 0.01) in bronchoalveolar lavage (BAL) fluid, lung injury scores (t = -6.708, P < 0.01), and the wet-to-dry ratio (t = -15.595, P < 0.01) were increased in Group H; IL-10 in BAL fluid (t = 9.093, P < 0.01) and the expression of E-cadherin (t = 10.044, P < 0.01) and p120-catenin (t = 13.218, P < 0.01) were decreased in Group H. Compared with Group H, total cell counts (t = 14.844, P < 0.01), the percentage of neutrophils (t = 18.077, P < 0.01), IL-6 (t = 18.007, P < 0.01), and TNF-α (t = 10.171, P < 0.01) in BAL fluid were decreased in Group H + G; IL-10 in BAL fluid (t = -7.531, P < 0.01) and the expression of E-cadherin (t = -14.814, P < 0.01) and p120-catenin (t = -9.114, P < 0.01) were increased in Group H + G. In vitro, compared with the nonstretching group, the levels of IL-6 (t = -21.111, P < 0.01) and TNF-α (t = -15.270, P < 0.01) were increased in the 20% cyclic stretching group; the levels of IL-10 (t = 5.450, P < 0.01) and the expression of E-cadherin (t = 17.736, P < 0.01) and p120-catenin (t = 16.136, P < 0.01) were decreased in the 20% cyclic stretching group. Compared with the stretching group, the levels of IL-6 (t = 11.818, P < 0.01) and TNF-α (t = 8.631, P < 0.01) decreased in the glutamine group; the levels of IL-10 (t = -3.203, P < 0.05) and the expression of E-cadherin (t = -13.567, P < 0.01) and p120-catenin (t = -10.013, P < 0.01) were increased in the glutamine group. Conclusions: High tidal volume mechanical ventilation and 20% cyclic stretching could cause VILI. Glutamine regulates VILI by improving cytokines and increasing the adherens junctions, protein E-cadherin and p120-catenin, to enhance the epithelial barrier function. 谷氨酰胺在机械通气肺损伤中调节E-钙黏素、p120连环蛋白及炎症的作用摘要背景：机械通气相关肺损伤与屏障功能障碍和炎症反应有关，谷氨酰胺能够改善机械通气造成的肺损伤，但是其作用机制尚不十分清楚。本研究通过体内实验和体外实验两方面，探讨谷氨酰胺改善机械通气造成肺损伤的机制。 方法：30只体重为25~30g的健康C57BL/6小鼠，利用随机数字表法随机分为5组，每组6只小鼠：对照组（C组）、小潮气量组（L组）、小潮气量+谷氨酰胺组（L+G组）、大潮气量组（H组）、大潮气量+谷氨酰胺组（H+G组）。C组小鼠自主呼吸，其余四组小鼠进行机械通气4小时。肺泡上皮细胞（MLE-12）和谷氨酰胺预处理过的MLE-12细胞均以20%的牵张幅度进行机械牵张4小时。收集细胞和肺组织，用免疫印迹法、细胞因子测定、苏木精-伊红（HE）染色和免疫荧光来检测黏连蛋白（E-cadherin、p120-catenin）、炎症细胞因子（IL-6、TNF-α、IL-10）和肺病理变化。 结果：体内实验，与C组比较，H组肺泡灌洗液中的细胞总数（t= -28.182，P<0.01）、中性粒细胞（t= −28.095，P<0.01）、IL-6（t= −28.296，P<0.01）、TNF-α（t= -19.812，P<0.01）、肺损伤评分（t= −6.708，P<0.01）和湿干比（t= −15.595， P<0.01）均增加，肺泡灌洗液中IL-10（t=9.093，P<0.01）、E-cadherin（t=10.044，P<0.01）和p120-catenin（t=13.218，P<0.01）表达均下降。与H组比较，H+G组的肺泡灌洗液中的细胞总数（t=14.844，P<0.01）、中性粒细胞（t=18.077， P<0.01）、肺泡灌洗液中IL-6（t=18.007，P<0.01）和TNF-α（t=10.171，P<0.01）表达均降低，而肺泡灌洗液中IL-10（t= −7.531，P<0.01）、E-cadherin（t= −14.814，P<0.01）、p120-catenin（t= −9.114，P<0.01）表达均增加。体外实验，与对照组比较，机械牵张组IL-6（t= −21.111，P<0.01）和TNF-α（t= −15.270，P<0.01）表达增加，IL-10（t=5.450，P<0.01）和E-cadherin（t=17.736，P<0.01）、p120-catenin（t=16.136，P<0.01）表达下降。与机械牵张组比较，谷氨酰胺预处理组的IL-6（t=11.818，P<0.01）、TNF-α（t=8.631，P<0.01）表达下降，IL-10（t= −3.203，P<0.05）、E-cadhrein（t= −13.567，P<0.01）、p120-catenin（t= −10.013，P<0.01）表达增加。 结论：大潮气量机械通气和幅度为20%的机械牵张可能导致机械通气相关肺损伤。谷氨酰胺通过改善细胞因子和增加黏附连接蛋白E-cadherin和p120-catenin来改善机械通气肺损伤，增强屏障功能。.