葶苈大枣泻肺汤对急性肺损伤小鼠PTEN/PI3K/AKT通路的影响

Effect of Tingli Dazao Xiefei Decoction on PTEN/PI3K/AKTSignaling Pathway in Acute Lung Injury Mice

目的:探讨葶苈大枣泻肺汤对急性肺损伤小鼠磷酸酶与张力蛋白同源物(phosphatase and tension homology,PTEN)/磷脂酰肌醇-3-激酶(phosphatidylinositol-3-hydroxykinase,PI3K)/蛋白激酶B(protein kinase B,AKT)信号通路的影响。方法:将50只C57BL/6J小鼠随机分为正常组、模型组、葶苈大枣泻肺汤低剂量组(5.85 g·kg^(-1))、葶苈大枣泻肺汤高剂量组(11.7 g·kg^(-1))、地塞米松组(5 mg·kg^(-1)),每组10只。各药物组按照相应剂量灌胃给药,正常组和模型组灌胃等体积生理盐水,每日1次,连续7 d。末次灌胃后,除正常组外,其余小鼠腹腔注射脂多糖(lipopolysaccharide,LPS)溶液建立急性肺损伤模型。采用HE染色观察肺组织病理学改变,ELISA检测肺泡灌洗液中白细胞介素-1β(interleukin 1β,IL-1β)、白细胞介素-6(interleukin 6,IL-6)、肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)含量,RT-qPCR检测肺组织IL-6、TNF-α、信号转导和转录激活因子3(signal transducer and activator of transcription 3,STAT3)、细胞因子信号转导抑制因子3(suppressors of cytokine signaling 3,SOCS3)基因表达水平;Western Blot检测肺组织PI3K、p-AKT/AKT、AKT、PTEN表达水平。结果:正常组小鼠肺组织未见明显病理性改变;模型组小鼠可见间质性肺炎和出血;与模型组比较,葶苈大枣泻肺汤低剂量组、高剂量组小鼠的间质性肺炎、出血情况减轻;地塞米松组小鼠肺组织未见明显病理性改变。与正常组比较,模型组小鼠肺泡灌洗液IL-1β、IL-6、TNF-α含量升高(P<0.01);与模型组比较,葶苈大枣泻肺汤低剂量组、高剂量组和地塞米松组小鼠肺泡灌洗液IL-1β、IL-6、TNF-α含量降低(P<0.01)。与正常组比较,模型组小鼠肺组织IL-6 mRNA、TNF-αmRNA、STAT3 mRNA、SOCS3 mRNA水平升高(P<0.01);与模型组比较,葶苈大枣泻肺汤低剂量组、高剂量组和地塞米松组小鼠肺组织IL-6 mRNA、TNF-αmRNA、STAT3 mRNA、SOCS3 mRNA水平降低(P<0.05)。与正常组比较,模型组小鼠肺组织PI3K、p-AKT/AKT蛋白表达水平升高(P<0.01);与模型组比较,葶苈大枣泻肺汤低剂量组、高剂量组和地塞米松组小鼠肺组织PI3K、p-AKT/AKT蛋白表达水平降低(P<0.01)。与正常组比较,模型组小鼠肺组织PTEN蛋白表达水平降低(P<0.01);与模型组比较,葶苈大枣泻肺汤低剂量组、高剂量组和地塞米松组小鼠肺组织PTEN蛋白表达水平升高(P<0.05)。结论:葶苈大枣泻肺汤可减轻急性肺损伤小鼠肺组织病变,其机制可能与调控PTEN/PI3K/AKT信号通路,抑制炎症反应有关。

Objective:To investigate the effects of Tingli Dazao Xiefei Decoction on phosphatase and tension homolog(PTEN)/phosphatidylinositol-3-hydroxykinase(PI3K)/protein kinase B(AKT)signaling pathway in mice with acute lung injury.Methods:Thirty C57BL/6J mice were randomly divided into a normal group,a model group,a low-dose group of Tingli Dazao Xiefei Decoction(5.85 g·kg^(-1)),a high-dose group of Tingli Dazao Xiefei Decoction(11.7 g·kg^(-1)),and a dexamethasone group(5 mg·kg^(-1)),with 6 mice in each group.Each drug group was administered by gavage at the corresponding dose,while the normal group and model group were given an equal volume of physiological saline by gavage once a day for 7 consecutive days.After the last gavage,except for the normal group,other mice were injected intraperitoneally with lipopolysaccharide(LPS)solution to establish an acute lung injury model.HE staining was used to observe pathological changes in lung tissue,and ELISA was used to detect interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)content.Detect the gene expression levels of IL-6,TNF-α,signal transducer and activator of transcription 3(STAT3)and suppressors of cytokine signaling 3(SOCS3)in lung tissue with RT-qPCR.Western blot was used to detect the expression levels of PI3K,p-AKT/AKT,AKT and PTEN in lung tissue.Results:No significant pathological changes were observed in the lung tissue of the normal group mice.Interstitial pneumonia and bleeding were observed in the model group mice.Compared with that of the model group,a reduction in interstitial pneumonia and bleeding in mice was found in the low-dose and high-dose groups of Tingli Dazao Xiefei Decoction.No significant pathological changes were observed in the lung tissue of mice treated with dexamethasone.Compared with that of the normal group,IL-1β,IL-6 and TNF-αcontent increased significantly in the alveolar lavage fluid of the model group mice(P<0.01).Compared with that of the model group,IL-1β,IL-6 and TNF-αcontent in alveolar lavage fluid decreased significantly in the low-dose group,high-dose group with Tingli Dazao Xiefei Decoction and dexamethasone group(P<0.01).Compared with that of the normal group,the levels of IL-6 mRNA,TNF-αmRNA,STAT3 mRNA and SOCS3 mRNA significantly increased in the lung tissue of the model group mice(P<0.01).Compared with that of the model group,the levels of IL-6 mRNA,TNF-αmRNA,STAT3 mRNA and SOCS3 mRNA significantly decreased in the low-dose and high-dose group of Tingli Dazao Xiefei Decoction as well as dexamethasone group(P<0.05).Compared with the normal group,the expression levels of PI3K and p-AKT/AKT proteins increased significantly in the lung tissue of the model group mice(P<0.01);Compared with that of the model group,the expression levels of PI3K and p-AKT/AKT proteins reduced in the lung tissue of the low-dose and high-dose group of Tingli Dazao Xiefei Decoction as well as dexamethasone group(P<0.01).Compared with that of the normal group,the expression level of PTEN protein significantly decreased in the lung tissue of the model group mice(P<0.01);Compared with that of the model group,the expression levels of PTEN protein significantly increased in the lung tissue of mice in the low-dose group,high-dose group of Tingli Dazao Xiefei Decoction and dexamethasone group(P<0.05).Conclusion:Tingli Dazao Xiefei Decoction can alleviate lung tissue lesions in acute lung injury mice,the mechanism of which may be related to regulating the PTEN/PI3K/AKT signaling pathway and inhibiting inflammatory response.