甘草酸二铵脂质配位体抑制内毒素引起的急性肺损伤中炎性细胞浸润及细胞间连接蛋白的降解
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摘要
目的探讨甘草有效成分甘草酸二铵脂质配位体(diammonium glycyrrhizinate lipid ligand,DGLL)对内毒素(lipopolysaccharide,LPS)诱发的肺损伤和肺水肿的改善作用及其机制。方法本研究利用腹腔给入LPS(10mg/kg)建立大鼠急性肺损伤(acute lung injury,ALI)模型,在LPS诱导前1小时灌胃给入DGLL(30、60、120mg/k g)。在LPS诱导6小时后,用HE组织学染色方法评价肺损伤,用肺湿干重比、肺泡灌洗液蛋白含量检测和肺组织伊文思蓝渗出法综合评价肺水肿,用ELISA方法检测肺组织中炎性因子和粘附分子含量,用免疫组织化学染色方法检测肺组织白细胞浸润标志物髓过氧化物酶(myeloperoxidase,MPO)的表达,用Western blot方法检测炎症相关蛋白以及肺微血管通透性相关蛋白水平。结果 DGLL前给药可以显著抑制LPS诱导的肺组织损伤,降低LPS诱导的MPO免疫反应性增强,降低肺组织中炎性因子TNF-α、IL-6和粘附分子ICAM-1的表达;同时,DGLL可以抑制LPS诱导的肺水肿、降低肺泡灌洗液中的蛋白含量以及肺组织伊文思蓝的渗出;抑制血管内皮钙黏蛋白(vascular endothelial cadherin,VE-cadherin)和ZO-1、Occludin、JAM-1等紧密连接蛋白的降解。结论 DGLL对LPS诱导的大鼠ALI具有明显的抑制作用,其作用机制与抑制炎性细胞的浸润以及细胞间连接蛋白的降解相关。该结果为D GLL在临床治疗ALI提供了新的理论依据。
Objective To explore the mechanism of diammonium glycyrrhizinate lipid ligand(DGLL) in improving lipopolysaccharide(LPS) induced acute lung injury and pulmonary edema. Methods In the study, rat acute lung injury(ALI) model was induced by intraperitonreal injection of LPS(10 mg/kg). DGLL(30, 60, 120 mg/kg) was administrated by gastric gavage one hour prior. Six hours later, pathological changes of lung injury were evaluated by HE staining, while the degree of pulmonary edema was assessed by tissue wet-to-dry weight ratio, protein content in bronchoalveolar lavage(BAL) fluid and the accumulation of Evans blue dye in alveolar space. Inflammatory cytokines and adhesion molecules in injured lung tissues were quantified by ELISA or Western blot. In addition, immunohistochemistry was used to identify leukocyte infiltration marker myeloperoxidase(MPO). Result DGLL significantly reduced the number of MPO positive cells and the expressions of TNF-α, IL-6 I and CAM-1 in injured lung tissues. Meanwhile, DGLL appeared to relieve pulmonary edema indicated by decreased protein content in BAL fluid and Evans blue in alveolar space. DGLL also inhibited the degradation of vascular endothelial cadherin(VE-Cadherin) and tight junction proteins ZO-1, Occludin, and JAM-1. Conclusion DGLL demonstrated a significant inhibitory effect on LPS-induced rat ALI. The mechanism was likely related to its inhibition of inflammatory cell infiltration and cell junctional protein degradation. The results provided a new theoretical basis for the clinical treatment of ALI by DGLL.
Objective To explore the mechanism of diammonium glycyrrhizinate lipid ligand(DGLL) in improving lipopolysaccharide(LPS) induced acute lung injury and pulmonary edema. Methods In the study, rat acute lung injury(ALI) model was induced by intraperitonreal injection of LPS(10 mg/kg). DGLL(30, 60, 120 mg/kg) was administrated by gastric gavage one hour prior. Six hours later, pathological changes of lung injury were evaluated by HE staining, while the degree of pulmonary edema was assessed by tissue wet-to-dry weight ratio, protein content in bronchoalveolar lavage(BAL) fluid and the accumulation of Evans blue dye in alveolar space. Inflammatory cytokines and adhesion molecules in injured lung tissues were quantified by ELISA or Western blot. In addition, immunohistochemistry was used to identify leukocyte infiltration marker myeloperoxidase(MPO). Result DGLL significantly reduced the number of MPO positive cells and the expressions of TNF-α, IL-6 I and CAM-1 in injured lung tissues. Meanwhile, DGLL appeared to relieve pulmonary edema indicated by decreased protein content in BAL fluid and Evans blue in alveolar space. DGLL also inhibited the degradation of vascular endothelial cadherin(VE-Cadherin) and tight junction proteins ZO-1, Occludin, and JAM-1. Conclusion DGLL demonstrated a significant inhibitory effect on LPS-induced rat ALI. The mechanism was likely related to its inhibition of inflammatory cell infiltration and cell junctional protein degradation. The results provided a new theoretical basis for the clinical treatment of ALI by DGLL.
引文
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[9]李铮,黄华,陈凤坤,等.甘草酸二铵与黄芪注射液对急性肺损伤肺保护作用的研究.世界中西医结合杂志,2013,10(7):10 25-1027.
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[12]宋丽君,王朝霞,迟宝荣,等.甘草酸二铵对支气管哮喘辅助T细胞1/2偏移调节作用的实验研究.中华医学杂志,2007,87(40):2865-2867.
[13]赵晓琴,张剑峰,邝晓聪,等.甘草酸二铵对急性肺损伤大鼠肺泡巨噬细胞介素10及18表达的影响.中华结核和呼吸杂志,2004,27(2):126-127.
[14]张剑峰,李超乾,磨静佳,等.甘草酸二铵对急性肺损伤大鼠糖皮质激素受体及核因子κB表达的影响.中华急诊医学杂志,2010,19(3):245-249.
[15]Daniela D,Christian M,Tanja K,et al.Lung endothelial ADAM17 regulates the acuteinfl ammatory response to lipopolysaccharide.EMBO Mol Med,2012,4(5):412–423.
[16]Veszelka S,Pasztoi M,Farkas AE,et al.Pentosanpolysulfate protects brain endothelial cells against bacterial lipopolysaccharide-induced damages.Neurochem Int,2007,1(50):219–228.
[17]Mehta D,Malik AB.Signaling mechanisms regulating endothelial permeability.Physiol Rev,2006,86(1):279-367.
[2]Meduri GU,Headley AS,Golden E,et al.Effect of prolonged methylprednisolone therapy in unresolving acute respiratory distress syndrome:a randomized controlled trial.JAMA,1998,280(2):159-165.
[3]Matthay MA,Zimmerman GA.Acute lung injury and the acute respiratory distress syndrome:four decades of inquiry into pathogenesis and rational management.Am J Respir Cell Mol Biol,2005,33(4):319-327.
[4]Johnson ER,Matthay MA.Acute lung injury:epidemiology,pathogenesis,and treatment.J Aerosol Med Pulm Drug Deliv,2010,23(4):243-252.
[5]刘梅梅,王栋,陈晓蓉,等.甘草酸二铵脂质配位体对非酒精性脂肪肝大鼠的影响.安徽医科大学学报,2010,45(6):770-773.
[6]刘梅梅.甘草酸二铵脂质配位体对非酒精性脂肪肝大鼠肿瘤坏死因子-α表达的影响及机制.安徽医科大学学报,2011,46(8):725-727.
[7]刘梅梅,王齐,刘晓利,等.甘草酸二铵脂质配位体对非酒精性脂肪肝大鼠白细胞浸润的影响.中国组织化学与细胞化学杂志,2013,22(5):426-431.
[8]张剑峰,曾光,李超乾,等.甘草酸二铵治疗急性肺损伤的临床观察.中国急救医学,2011,31(7):646-648.
[9]李铮,黄华,陈凤坤,等.甘草酸二铵与黄芪注射液对急性肺损伤肺保护作用的研究.世界中西医结合杂志,2013,10(7):10 25-1027.
[10]Chen H,Bai CX,Wang XD.The value of the lipopolysaccharide-induced acute lung injury model in respiratory medicine.Expert Rev Resp Med,2010,4(6):773-783.
[11]Welbourn CR,Young Y.Endotoxin,septic shock and acute lung injury:neutrophils,macrophages and inflammatory mediators.Br J Surg,1992,79(10):998–1003.
[12]宋丽君,王朝霞,迟宝荣,等.甘草酸二铵对支气管哮喘辅助T细胞1/2偏移调节作用的实验研究.中华医学杂志,2007,87(40):2865-2867.
[13]赵晓琴,张剑峰,邝晓聪,等.甘草酸二铵对急性肺损伤大鼠肺泡巨噬细胞介素10及18表达的影响.中华结核和呼吸杂志,2004,27(2):126-127.
[14]张剑峰,李超乾,磨静佳,等.甘草酸二铵对急性肺损伤大鼠糖皮质激素受体及核因子κB表达的影响.中华急诊医学杂志,2010,19(3):245-249.
[15]Daniela D,Christian M,Tanja K,et al.Lung endothelial ADAM17 regulates the acuteinfl ammatory response to lipopolysaccharide.EMBO Mol Med,2012,4(5):412–423.
[16]Veszelka S,Pasztoi M,Farkas AE,et al.Pentosanpolysulfate protects brain endothelial cells against bacterial lipopolysaccharide-induced damages.Neurochem Int,2007,1(50):219–228.
[17]Mehta D,Malik AB.Signaling mechanisms regulating endothelial permeability.Physiol Rev,2006,86(1):279-367.