谷氨酰胺对急性肺损伤小鼠MUC5AC的调节及其对肺脏的保护作用
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摘要
旨在探讨谷氨酰胺(GLN)对脂多糖(LPS)诱导急性肺损伤小鼠黏蛋白MUC5AC表达的调节作用及其对肺损伤的保护作用。选用雌性ICR小鼠32只随机分成4组:正常对照组(PBS组)、谷氨酰胺组(GLN)、内毒素组(LPS)、谷氨酰胺+内毒素组(GLN+LPS)。采用气管滴注LPS的方法刺激小鼠建立急性肺损伤(ALI)模型,在滴注LPS12 h后采集肺组织进行湿干重比值,IL-6、TNF-α、IL-1β、IL-8、HSP70、MUC5AC的mRNA水平,IL-6、TNF-α含量以及HSP70、MUC5AC蛋白含量的测定,通过HE染色观察肺组织形态学变化。结果显示:与对照组相比,LPS组MUC5AC、IL-6、TNF-α、IL-1β、IL-8 mRNA表达量极显著升高,HSP70 mRNA表达量极显著降低;MUC5AC蛋白的表达量极显著增加,HSP70蛋白的表达量极显著降低;IL-6、TNF-α含量极显著升高,肺组织湿干比值极显著增加;肺泡破裂较严重,细支气管黏液分泌增加且可见杯状细胞部分脱落;与LPS组相比,GLN+LPS组IL-6、TNF-α、IL-1βmRNA表达量极显著降低,MUC5AC、IL-8 mRNA表达量显著降低,HSP70 mRNA表达量显著升高;MUC5AC蛋白的表达量显著减少,HSP70蛋白的表达量显著升高,IL-6、TNF-α含量极显著降低;肺组织湿干比值显著减少;部分肺泡破裂,杯状细胞排列整齐,细支气管黏液分泌量减少。结果表明:静脉注射谷氨酰胺能减少LPS诱导的小鼠肺组织中MUC5AC、IL-6、TNF-α、IL-1β、IL-8 mRNA和MUC5AC蛋白的表达,减少IL-6、TNF-α的含量,增加HSP70 mRNA和HSP70蛋白的表达,减轻肺组织损伤;谷氨酰胺可能通过增加HSP70的表达来对LPS诱导的急性肺损伤小鼠起到保护作用。
To study the regulatory effect of glutamine(GLN) on the expression of MUC5AC in mice with acute lung injury induced by LPS and the protective effect GLN on the injured lung, thirty-two female ICR mice were used and randomly divided into four groups: the normal control group(PBS), the glutamine treated group(GLN), the LPS treated group(LPS), the LPS and glutamine treated group(GLN+LPS). An acute lung injury(ALI) model was established in the mice stimulated by lipopolysaccharide(LPS). After 12 hours of LPS instillation, the lung tissue samples were collected from the treated mice to determine the ratio of wet-dry weight, the expressions of IL-6, TNF-α, IL-1β, IL-8, HSP70 and MUC5AC mRNA, the concentration of IL-6 and TNF-α, and the expressions of HSP70 and MUC5AC proteins. Morphological changes of the lung tissues were observed after HE staining. The results showed that: compared with the PBS group, the expressions of IL-6, TNF-α, IL-1β, IL-8 and MUC5AC mRNA and the concentrations of IL-6 and TNF-α in the LPS group were extremely significantly increased, while the expression of HSP70 mRNA was highly significantly decreased. And the expression of MUC5AC was extremely significantly upregulated but the expression of HSP70 was highly significantly down-regulated. The wet-dry ratio of the lung tissue increased significantly. Many alveoli collapsed, mucus secretion increased and the goblet cells were partially exfoliated. Compared with the LPS group,the expressions of IL-6, TNF-α and IL-1β mRNA in the GLN+LPS group were extremely significantly decreased, the expressions of MUC5AC and IL-8 mRNA were significantly decreased, too, the expression of HSP70 mRNA was significantly upregulated, that of
To study the regulatory effect of glutamine(GLN) on the expression of MUC5AC in mice with acute lung injury induced by LPS and the protective effect GLN on the injured lung, thirty-two female ICR mice were used and randomly divided into four groups: the normal control group(PBS), the glutamine treated group(GLN), the LPS treated group(LPS), the LPS and glutamine treated group(GLN+LPS). An acute lung injury(ALI) model was established in the mice stimulated by lipopolysaccharide(LPS). After 12 hours of LPS instillation, the lung tissue samples were collected from the treated mice to determine the ratio of wet-dry weight, the expressions of IL-6, TNF-α, IL-1β, IL-8, HSP70 and MUC5AC mRNA, the concentration of IL-6 and TNF-α, and the expressions of HSP70 and MUC5AC proteins. Morphological changes of the lung tissues were observed after HE staining. The results showed that: compared with the PBS group, the expressions of IL-6, TNF-α, IL-1β, IL-8 and MUC5AC mRNA and the concentrations of IL-6 and TNF-α in the LPS group were extremely significantly increased, while the expression of HSP70 mRNA was highly significantly decreased. And the expression of MUC5AC was extremely significantly upregulated but the expression of HSP70 was highly significantly down-regulated. The wet-dry ratio of the lung tissue increased significantly. Many alveoli collapsed, mucus secretion increased and the goblet cells were partially exfoliated. Compared with the LPS group,the expressions of IL-6, TNF-α and IL-1β mRNA in the GLN+LPS group were extremely significantly decreased, the expressions of MUC5AC and IL-8 mRNA were significantly decreased, too, the expression of HSP70 mRNA was significantly upregulated, that of
引文
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[18] 崔艳茹,刘海云,屈飞.贝母辛对LPS致黏液高分泌小鼠MUC5AC和EGFR表达的影响[J].中药药理与临床,2015,31(3):17-21.
[19] 荆科,孙梅.谷氨酰胺对肠组织NF-κB及TNF-α的调节与肠损伤保护作用的关系[J].中国当代儿科杂志,2011,13(8):661-664.
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[22] 万宁,张建勇. TLR4在慢性支气管炎大鼠模型中对炎症损伤与黏液分泌的调控作用[J].中国老年学杂志,2015,(19):5398-5400.
[23] Hou Y C,Pai M H,Chiu W C,et al. Effects of dietary glutamine supplementation on lung injury induced by lipopolysaccharide administration[J].Am J Physiol-Lung Cell Mol Physiol, 2009,296(3):288-295.
[24] Zapata-Sirvent R L,Hansbrough J F,Ohara M M,et al. Bacterial translocation in burned mice after administration of various diets including fiber and glutamine-enriched enteral formulas[J].Crit Care Med,1994,22(4):690-696.
[25] Gianotti L,Alexander J W,Gennari R,et al. Oral glutamine decreases bacterial translocation and improves survival in experimental gut-origin sepsis[J].J Parenter Enteral Nutr,1995,19(1):69-74.
[26] Hugh R B. Pelham. Hsp70 accelerates the recovery of nucleolar morphology after heat shock[J].Embo J,1984,13(3):3095-30100.
[27] Flynn G C,Rohl J,Flocco M T,et al. Peptide-binding specificity of the molecular chaperone BiP[J].Nature 1991,(353):726-730.
[28] Blond E S,Cwirla S E,Dower W J,et.al. Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP[J].Cell,1993,(75):717-729.
[29] 张科东,朱彩侠,刘小科.羧甲司坦对气道黏蛋白MUC5AC的抑制作用研究[J].宁夏医学杂志,2017,39(7):594-596.
[30] 王砚,唐法娣,赵春贞,等.QTY06对LPS引起的大鼠慢性气道炎症及MUC5AC表达的影响[J].浙江大学学报(医学版),2008,37(4):345-350.
[31] 万宁,张建勇,唐凤鸣,等.TLR4在慢性支气管炎大鼠气道黏液高分泌中的作用[J].四川医学,2015,36(11):1521-1524.
[32] Smirnova M G,Guo L,Birchall J P,et al. LPS up-regulates mucin and cytokine mRNA expression and stimulates mucin and cytokine secretion in goblet cells[J].Cell Immunol,2003,221(1):42-49.
[33] Magez S,Radwanska M,Drennan M,et al. Tumor necrosis factor (TNF) receptor-1 (TNFp55) signal transduction and macrophage-derived soluble TNF are crucial for nitric oxide-mediated Trypanosoma congolense parasite killing[J].J Infect Dis,2007,196(6):954-962.
[34] Salkowski C A, Detore G, McNally R,et al. Regulation of inducible nitric oxide synthase messenger RNA expression and nitric oxide production by lipopolysaccharide in vivo: the roles of macrophages,endogenous IFN-gamma,and TNF receptor-1-mediated signaling[J].J Immunol. 1997,158(2):905-912.
[35] Levitt J E,Gould M K,Ware L B,et al. The pathogenetic and prognostic value of biologic markers in acute lung injury[J].J Intensive Care Med,2009,24(3):151-167.
[36] Pithon-Curi T C,Trezena A G,Tavares-Lima W,et al. Evidence that glutamine is involved in neutrophil function[J].Cell Biochem Funct,2002,20(2):81-86.
[37] Lagranha C J,Levada-Pires A C,Sellitti D F,et al. The effect of glutamine supplementation and physical exercise on neutrophil function[J].Amin Acid,2008,34(3):337-346.
[38] Singleton K D,Beckey V E,Wischmeyer P E. Glutamine prevents activation of NF-κb and stress kinase pathways,attenuates inflammatory cytokine release,and prevents acute respiratory distress syndrome (ARDS) following sepsis[J].Shock,2005,24(6):583-389.
[2] Rubi B,Ishihara H,Hegardt F G,et al. GAD65-mediated glutamate decarboxylation reduces glucose stimulated insulin secretion in pancreatic β-cells[J].J Biol Chem,2001,276(39):36391-36396.
[3] Gleeson M,Bishop N C. Modification of immune responses to exercise by carbohydrate,glutamine and anti-oxidant supplements[J].Immunol Cell Biol,2000,5(78):554-561.
[4] Renee E F,Irene B H,Erwin T C,et al. The role of glutamine and other alternate substrates as energy sources in the fetal rat lung type II cell[J].Pediatr Res,1996,40(1):135-141.
[5] Ehrensvard G,fischer A,stjernholm R. Protein metabolism of tissue cells in vitro; the chemical nature of some obligate factors of tissue cell nutrition[J].Acta Physiol Scand,1949,18(2-3):218-230.
[6] Askanazi J,Elwyn D H,Kinney J M,et al. Muscle and plasma amino acids after injury: the role of inactivity[J].Ann Surg,1978,188(6):797-803.
[7] Smith R J,Wilmore D W. Glutamine nutrition and requirements[J].J Parenter Enter Nutr,1990,14(4):94-99.
[8] Rogeri P S,Costa R L. Plasma glutamine concentration in spinal cord injured patients[J].Life Sci,2005,77(19):2351-2360.
[9] Wischmeyer P E,Kahana M,Wolfson R,et al. Glutamine reduces cytokine release,organ damage,and mortality in a rat model of endotoxemia[J].Shock,2001,16(5):398-402.
[10] McComb M,Spurlock M. Expression of stress proteins in porcine tissues: developmental changes and effect of immunological challenge[J].J Anim Sci,1997,75(1):195-201.
[11] Singleton K D,Wischmeyer P E. Glutamine’s protection against sepsis and lung injury is dependent on heat shock protein 70 expression[J].Am J Physiol Regul Intergr Comp Physiol,2007,292(5):1839-1845.
[12] Singleton K D,Serkova N,Banerjee A,et al. Glutamine attenuates endotoxin-induced lung metabolic dysfunction: Potential role of enhanced heat shock protein 70[J].Nutrition,2005,21(2):214-223.
[13] Perng W C,Huang K L,Li M H,et al. Glutamine attenuates hyperoxia-induced acute lung injury in mice[J].Clin Exp Pharmacol Physiol,2010,37(1):56-61.
[14] Zhang F,Wang X,Pan L,et al. Glutamine attenuates lipopolysaccharide-induced acute lung injury[J].Nutrition,2009,25(6):692-698.
[15] Singleton K D,Serkova N,Beckey V E,et al. Glutamine attenu-ates lung injury and improves survival after sepsis: role of enhanced heat shock protein expression[J].Crit Care Med,2005,33(6):1206-1213.
[16] Liu J C,Wang H T,Wang W. Protective effects of alanyl-glutamine on acute lung injury induced by lipopolysaccharide in rats[J]. J Cent South Univ(Med Sic),2008,33(12):1095-1100.
[17] 张媛莉,陈秋萍,姚华国.谷氨酰胺对急性肺损伤大鼠肺泡上皮屏障功能的影响[J].实用医学杂志,2008(12):2053-2055.
[18] 崔艳茹,刘海云,屈飞.贝母辛对LPS致黏液高分泌小鼠MUC5AC和EGFR表达的影响[J].中药药理与临床,2015,31(3):17-21.
[19] 荆科,孙梅.谷氨酰胺对肠组织NF-κB及TNF-α的调节与肠损伤保护作用的关系[J].中国当代儿科杂志,2011,13(8):661-664.
[20] 周昌妮,韩德恩,徐如冰,等.舒肺贴对COPD大鼠肺组织IL-8、TNF-α及黏蛋白MUC5AC表达的影响[J].中成药,2016,38(11):2391-2324.
[21] Hou Y C,Pai M H,Liu J J,et al. Alanyl-glutamine resolves lipopolysaccharide-induced lung injury in mice by modulating the polarization of regulatory T cells and T helper 17 cells[J].J Nutr Biochem,2013,24(9):1555-1563.
[22] 万宁,张建勇. TLR4在慢性支气管炎大鼠模型中对炎症损伤与黏液分泌的调控作用[J].中国老年学杂志,2015,(19):5398-5400.
[23] Hou Y C,Pai M H,Chiu W C,et al. Effects of dietary glutamine supplementation on lung injury induced by lipopolysaccharide administration[J].Am J Physiol-Lung Cell Mol Physiol, 2009,296(3):288-295.
[24] Zapata-Sirvent R L,Hansbrough J F,Ohara M M,et al. Bacterial translocation in burned mice after administration of various diets including fiber and glutamine-enriched enteral formulas[J].Crit Care Med,1994,22(4):690-696.
[25] Gianotti L,Alexander J W,Gennari R,et al. Oral glutamine decreases bacterial translocation and improves survival in experimental gut-origin sepsis[J].J Parenter Enteral Nutr,1995,19(1):69-74.
[26] Hugh R B. Pelham. Hsp70 accelerates the recovery of nucleolar morphology after heat shock[J].Embo J,1984,13(3):3095-30100.
[27] Flynn G C,Rohl J,Flocco M T,et al. Peptide-binding specificity of the molecular chaperone BiP[J].Nature 1991,(353):726-730.
[28] Blond E S,Cwirla S E,Dower W J,et.al. Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP[J].Cell,1993,(75):717-729.
[29] 张科东,朱彩侠,刘小科.羧甲司坦对气道黏蛋白MUC5AC的抑制作用研究[J].宁夏医学杂志,2017,39(7):594-596.
[30] 王砚,唐法娣,赵春贞,等.QTY06对LPS引起的大鼠慢性气道炎症及MUC5AC表达的影响[J].浙江大学学报(医学版),2008,37(4):345-350.
[31] 万宁,张建勇,唐凤鸣,等.TLR4在慢性支气管炎大鼠气道黏液高分泌中的作用[J].四川医学,2015,36(11):1521-1524.
[32] Smirnova M G,Guo L,Birchall J P,et al. LPS up-regulates mucin and cytokine mRNA expression and stimulates mucin and cytokine secretion in goblet cells[J].Cell Immunol,2003,221(1):42-49.
[33] Magez S,Radwanska M,Drennan M,et al. Tumor necrosis factor (TNF) receptor-1 (TNFp55) signal transduction and macrophage-derived soluble TNF are crucial for nitric oxide-mediated Trypanosoma congolense parasite killing[J].J Infect Dis,2007,196(6):954-962.
[34] Salkowski C A, Detore G, McNally R,et al. Regulation of inducible nitric oxide synthase messenger RNA expression and nitric oxide production by lipopolysaccharide in vivo: the roles of macrophages,endogenous IFN-gamma,and TNF receptor-1-mediated signaling[J].J Immunol. 1997,158(2):905-912.
[35] Levitt J E,Gould M K,Ware L B,et al. The pathogenetic and prognostic value of biologic markers in acute lung injury[J].J Intensive Care Med,2009,24(3):151-167.
[36] Pithon-Curi T C,Trezena A G,Tavares-Lima W,et al. Evidence that glutamine is involved in neutrophil function[J].Cell Biochem Funct,2002,20(2):81-86.
[37] Lagranha C J,Levada-Pires A C,Sellitti D F,et al. The effect of glutamine supplementation and physical exercise on neutrophil function[J].Amin Acid,2008,34(3):337-346.
[38] Singleton K D,Beckey V E,Wischmeyer P E. Glutamine prevents activation of NF-κb and stress kinase pathways,attenuates inflammatory cytokine release,and prevents acute respiratory distress syndrome (ARDS) following sepsis[J].Shock,2005,24(6):583-389.