基于网络药理学和整体动物实验探讨凉膈散通过ERK1/2与PI3K/AKT通路治疗脓毒症急性肺损伤

Exploring the treatment of sepsis-associated acute lung injury with Liangge Powder via ERK1/2 and PI3K/AKT pathways:based on network pharmacology and whole animal experimentation

目的探讨凉膈散对脓毒症大鼠急性肺损伤(ALI)的保护作用及其机制。方法于2021年4至12月,采用网络药理学方法对凉膈散成分及其抗脓毒症ALI的靶点进行分析,富集相关信号通路。将90只雄性SD大鼠随机分为假手术组,脓毒症ALI模型组(模型组),凉膈散低、中、高剂量组。其中,假手术组10只,其他4组每组20只。采用盲肠穿孔结扎法(CLP)建立大鼠脓毒症ALI模型。假手术组:灌胃生理盐水2 ml,不进行盲肠结扎穿孔手术;模型组:进行手术且灌胃生理盐水2 ml;凉膈散低、中、高剂量组:进行手术且分别灌胃凉膈散3.9、7.8、15.6 g/kg。检测大鼠肺组织湿/干质量比,评价肺泡毛细血管屏障通透性。HE染色观察大鼠肺组织病理形态表现,ELISA法检测肺泡灌洗液中肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-6和IL-1β浓度,蛋白免疫印迹法检测p-磷脂酰肌醇3-激酶(PI3K)、p-蛋白激酶B(AKT)、p-细胞外调节蛋白激酶(ERK)相对蛋白表达水平。结果通过网络药理学筛选出凉膈散177个活性成分,抗脓毒症ALI靶点88个。GO功能富集到354个条目,KEGG富集到基因通路108条。PI3K/AKT信号通路在凉膈散抗脓毒症ALI中起重要作用。与假手术组比较,模型组大鼠肺组织湿/干质量比(6.35±0.95)增加(P<0.001),HE染色可见模型组大鼠肺组织正常结构破坏,肺泡灌洗液IL-6[(392.36±66.83)pg/ml]、IL-1β[(137.11±26.83)pg/ml]、TNF-α[(238.34±59.36)pg/ml]含量升高(P<0.001、=0.001、<0.001),肺组织中p-PI3K、p-AKT、p-ERK1/2蛋白表达水平(1.04±0.15、0.51±0.04、2.31±0.41)升高(P=0.002、0.003、0.005)。与模型组比较,凉膈散各剂量组肺组织病理改变较轻,凉膈散中剂量组肺组织湿/干质量比(4.29±1.26)降低(P=0.019),TNF-α含量[(147.85±39.05)pg/ml]降低(P=0.022),p-PI3K(0.37±0.18)、p-ERK1/2(1.36±0.07)相对蛋白表达水平降低(P=0.008、0.017);高剂量组肺组织湿/干质量比(4.16±0.66)降低(P=0.003),IL-6、IL-1β、TNF-α含量[(187.98±53.28)pg/ml、(92.45±25.39)pg/ml、(129.77±55.94)pg/ml]降低(P=0.001、0.027、0.018),p-PI3K、p-AKT、p-ERK1/2相对蛋白表达水平(0.65±0.05、0.31±0.08、1.30±0.12)降低(P=0.013、0.018、0.015)。结论凉膈散对脓毒症ALI大鼠具有治疗作用,其机制可能与抑制肺组织ERK1/2与PI3K/AKT通路激活有关。

Objective To investigate the therapeutic effect and mechanism of Liangge Powder against sepsis-induced acute lung injury(ALI).Methods From April to December 2021,the key components of Liangge Powder and its targets against sepsis-induced ALI were analyzed by network pharmacology,and to enrich for relevant signaling pathways.A total of 90 male Sprague-Dawley rats were randomly assigned to sham-operated group,sepsis-induced ALI model group(model group),Liangge Powder low,medium and high dose group,ten rats in the sham-operated group and 20 rats in each of the remaining four groups.Sepsis-induced ALI model was established by cecal ligation and puncture.Sham-operated group:gavage with 2 ml saline and no surgical treatment.Model group:surgery was performed and 2 ml saline was gavaged.Liangge Powder low,medium and high dose groups:surgery and gavage of Liangge Powder 3.9,7.8 and 15.6 g/kg,respectively.To measure the wet/dry mass ratio of rats lung tissue and evaluate the permeability of alveolar capillary barrier.Lung tissue were stained with hematoxylin and eosin for histomorphological analysis.The levels of tumor necrosis factor-alpha(TNF-α),interleukin(IL)-6 and IL-1βin bronchoalveolar lavage fluid(BALF)were measured by enzyme-linked immunosorbent assay.The relative protein expression levels of p-phosphatidylinositol 3-kinase(PI3K),p-protein kinase B(AKT),and p-ertracellular regulated protein kinases(ERK)were detected via Western blot analysis.Results Network pharmacology analysis indicated that 177 active compounds of Liangge Powder were selected.A total of 88 potential targets of Liangge Powder on sepsis-induced ALI were identified.354 GO terms of Liangge Powder on sepsis-induced ALI and 108 pathways were identified using GO and KEGG analysis.PI3K/AKT signaling pathway was recognized to play an important role for Liangge Powder against sepsis-induced ALI.Compared with the sham-operated group,the lung tissue wet/dry weight ratio of rats in the model group(6.35±0.95)was increased(P<0.001).HE staining showed the destruction of normal structure of lung tissue.The levels of IL-6[(392.36±66.83)pg/ml],IL-1β[(137.11±26.83)pg/ml]and TNF-α[(238.34±59.36)pg/ml]were increased in the BALF(P<0.001,=0.001,<0.001),and the expression levels of p-PI3K,p-AKT and p-ERK1/2 proteins(1.04±0.15,0.51±0.04,2.31±0.41)were increased in lung tissue(P=0.002,0.003,0.005).The lung histopathological changes were reduced in each dose group of Liangge Powder compared with the model group.Compared with the model group,the wet/dry weight ratio of lung tissue(4.29±1.26)was reduced in the Liangge Powder medium dose group(P=0.019).TNF-αlevel[(147.85±39.05)pg/ml]was reduced(P=0.022),and the relative protein expression levels of p-PI3K(0.37±0.18)and p-ERK1/2(1.36±0.07)were reduced(P=0.008,0.017).The wet/dry weight ratio of lung tissue(4.16±0.66)was reduced in the high-dose group(P=0.003).Levels of IL-6,IL-1βand TNF-α[(187.98±53.28)pg/ml,(92.45±25.39)pg/ml,(129.77±55.94)pg/ml]were reduced(P=0.001,0.027,0.018),and relative protein expression levels of p-PI3K,p-AKT and p-ERK1/2(0.65±0.05,0.31±0.08,1.30±0.12)were reduced(P=0.013,0.018,0.015).Conclusion Liangge Powder has therapeutic effects in rats with sepsis-induced ALI,and the mechanism may be related to the inhibition of ERK1/2 and PI3K/AKT pathway activation in lung tissue.