mTORC2介导的信号通路影响肺泡上皮钠通道对急性肺损伤肺组织的保护作用  

mTORC2-mediated signaling pathway affects protective role of alveolar epithelial sodium channel in mice of acute lung injury

作　　者：钟曦[1] 赵燕[1] 王导新[1] 

机构地区：[1]重庆医科大学附属第二医院呼吸与危重症医学科,重庆400010

出　　处：《第三军医大学学报》2015年第24期2438-2443,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81270141)~~

摘　　要：目的通过调控肺泡上皮细胞α钠离子通道(epithelial sodium channelα,ENa C-α)上游信号通路,探讨m TORC2/SGK1途径在急性肺损伤(acute lung injury,ALI)情况下对肺水清除方面的作用机制。方法将50只BALB/c雄性小鼠按随机数表法分成对照组、模型组、胰岛素组、雷帕霉素组及PP242抑制剂组,每组10只。滴鼻给予5 mg/kg LPS构建ALI动物模型。建模2 h后,分别腹腔注射0.8 U/kg胰岛素、4 mg/kg雷帕霉素、60 mg/kg PP242干预,8 h后处死小鼠。A549细胞分别以培养基、10 mmol/m L胰岛素、5 mmol/m L雷帕霉素、5 mmol/m L PP242处理。采用ELISA检测各组肺泡灌洗液(BALF)中IL-6及TNF-α浓度,并测量各组右上肺组织的湿/干质量比(W/D)评估肺水清除(alveolar fluid clearance,AFC),HE染色观察各组肺组织病理改变。采用Real-time PCR、Western blot、免疫荧光法检测A549细胞rictor、糖皮质激素调节蛋白激酶1(serum and glucocorticoid-regulated protein kinase1,SGK1)、磷酸泛素化连接酶(E3 ubiquitin protein ligase,Nedd4-2)及ENa C-α的表达。结果体内实验发现,与模型组比较,胰岛素组小鼠BALF中IL-6[(77.48±6.04)vs(49.17±8.49)pg/m L]和TNF-α[(116.2±13.5)vs(92.4±8.37)pg/m L]水平显著降低,并伴有湿/干质量比减低,病检示肺损伤程度减轻(P<0.05)。PP242抑制剂组BALF中IL-6[(77.48±6.04)vs(100.6±14.69)pg/m L]和TNF-α[(116.2±13.5)vs(192.5±16.01)pg/m L]水平上升,湿/干质量比显著升高,肺组织损伤明显,程度较模型组更重(P<0.05)。而雷帕霉素组与模型组中各项指标及病理学改变差异无统计学意义(P>0.05)。体外实验中,A549细胞中rictor、SGK1、Nedd4-2及ENa C-αmRNA和蛋白表达在胰岛素组明显增高,在PP242抑制剂组中明显降低(P<0.05),而在雷帕霉素组中无明显改变(P>0.05)。结论调控m TORC2/SGK1信号通路可影响ENa C-α的表达,从而发挥其在ALI情况下对肺组织的保护作用。Objective To explore the role and mechanism of mTORC2/SGK1 signaling pathway in alveolar fluid clearance （AFC） of acute lung injury （ALI） by regulating alveolar epithelial alpha sodium channel （ENaC-α） upstream signaling pathway. Methods Fifty male BALB/c mice were randomly divided into blank group, lipopolysaccharide （LPS） group, insulin group, LPS＋rapamycin （mTORC1 inhibitor） group, and LPS＋PP242 （mTORC1 and mTORC2 double inhibitor） group （n=10）. ALI animal models were constructed by intranasal administration of 5 mg/kg LPS. After 2 h, the models were treated by intraperitoneal injection of 0.8 U/kg insulin, 4 mg/kg rapamycin, and 60 mg/kg PP242, respectively, and the mice were put to death after 8 h. A549 cells were divided into control group, insulin group, rapamycin group and PP242 group, which were treated with culture medium, 10 mmol/L insulin, 5 mmol/L rapamycin, and 5 mmol/L PP242, respectively. The bronchoalveolar lavage fluid （BALF） IL-6 and TNF-α were measured by ELISA, and wet/dry （W/D） weight ratio was determined to evaluate AFC. HE staining was adopted to observe the lung tissue pathological changes. Real-time PCR, Western blotting and immunofluorescence assay were used to detect the expressions of rictor, SGK1, Nedd4-2 and ENaC-α in A549 cells. Results In vivo experiment showed that, compared with the LPS group, BALF IL-6 （77.48±6.04 vs 49.17±8.49）, TNF-α （116.2±13.5 vs 92.4±8.37） and W/D weight ratio were significantly decreased, AFC was increased, and lung injury was alleviated in the insulin group （all P〈0.05）. Compared with the LPS group, BALF IL-6 （77.48±6.04 vs 100.6±14.69）, TNF-α （116.2±13.5 vs 192.5±16.01） and W/D weight ratio were significantly increased, AFC was decreased, and lung injury was significantly worse in the LPS＋PP242 group（all P〈0.05）. The inflammation indexes and pathological change had no significantly statistical differences between the LPS group and LPS＋rapamycin group（all P〈0.05）

关 键 词：肺泡上皮钠通道 mTORC2/SGK1 急性肺损伤 肺水清除率 

分 类 号：R322.35[医药卫生—人体解剖和组织胚胎学] R363[医药卫生—基础医学]