脑肠肽Ghrelin对脓毒症大鼠小肠上皮短肽载体-1调节作用研究
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摘要
目的探讨外源性脑肠肽Ghrelin对脓毒症大鼠小肠上皮短肽载体-1表达及功能的影响。
方法雄性SD大鼠80只,随机分为4组,正常组、假手术组、脓毒症组和Ghrelin干预组,每组20只；采用盲肠结扎穿刺术模型,制模后立即给予Ghrelin静脉干预；模型建立后20h每组随机处理10只大鼠,Elisa方法检测TNF-α、IL-1β和Ghrelin水平,免疫组化、实时定量PCR和蛋白印迹分别检测PepTl分布、mRNA和蛋白表达,高效液相色谱法检测PepTl摄取功能；每组剩余10只大鼠观察生存时间(7d)。
结果(1)与正常组和假手术组相比,脓毒症时小肠黏膜受损明显,血清及小肠黏膜TNF-α、IL-1β明显升高(P<0.05)；小肠黏膜PepTl分布减少,PepTl mRNA表达、蛋白表达以及对底物摄取能力明显降低(P<0.05)；(2)与脓毒症组比较,Ghrelin干预组大鼠小肠黏膜损伤较轻,血清及肠黏膜TNF-α、IL-1β(P<0.05);小肠黏膜PepTl分布量增多,生存率、PepTl mRNA表达蛋白表达及对底物摄取能力显著升高(P<0.05)；(3)正常组与假手术组各指标比较无显著差异(P>0.05)。
结论脓毒症时大鼠小肠上皮PepTl mRNA、蛋白表达以及PepTl在肠黏膜分布明显下降,机体在基因及蛋白水平下调了小肠上皮PepTl生物学功能；Ghrelin干预可降低炎性反应,对脓毒症小肠上皮PepTl mRNA、蛋白表达以及摄取能力均有上调作用。
Objective To investigate the effect of Ghrelin on PepT1of the small intestinal epithelial in sepsis rats.
Methods Eighty male SD rats were divided into four groups:normal group, sham operation, sepsis group and Ghrelin group, and there twenty rats in every group, by random number table.The model of sepsis was made with cecal ligation and puncture (CLP).After the procedure Ghrelin was injected via vein in the Ghrelin group. Ten rats by random in every group were used to investigate the intestinal histological. Serum and intestinal mucosas TNF-α, IL-1β and Ghrelin were detected with Elisa. In addition, Immunohistochemical, Realtime PCR and Western-blot were used to detect PepT1distribution, PepT1mRNA expression and PepT1protein expression levels respectively in every group. And the uptake of PepT1of small intestinal epithelial cells was measured with high performance liquid chromatography. The others were recorded the time of the survival of the rats in the treatment period (7d)
Results (1) The levels of serum and intestinal mucosas TNF-α and IL-1β in the sepsis group were significantly higher than in normal and sham operation groups (P<0.05). The PepT1distribution, PepT1mRNA expression levels, PepT1protein expression levels and the uptake of PepT1in the sepsis group were significantly lower than in normal and sham operation groups (P<0.05).And the damage to the small intestine mucosa was more serious in sepsis group compared to normal and sham operation groups.(2)Comparing with the sepsis group, thhe levels of serum and intestinal mucosas TNF-α and IL-1β in the Ghrelin group were significantly decreased (P<0.05). The survival rate of rats, PepT1distribution, PepT1mRNA expression levels, PepT1protein expression levels and the uptake of PepT1in the Ghrelin group were significantly improved than in sepsis group (P<0.05), and a lighter damage to the small intestine mucosa.(3) There were no differences in various indicators between sham operation and normal groups (P>0.05)
Conclusion The PepT1mRNA and protein expression levels with sepsis were significantly decreased, which could effect the uptake of PepT1.Ghrelin could decline the inflammatory response and greatly upregulate the physiological function of PepT1of small intestinal epithelial cells.
方法雄性SD大鼠80只,随机分为4组,正常组、假手术组、脓毒症组和Ghrelin干预组,每组20只；采用盲肠结扎穿刺术模型,制模后立即给予Ghrelin静脉干预；模型建立后20h每组随机处理10只大鼠,Elisa方法检测TNF-α、IL-1β和Ghrelin水平,免疫组化、实时定量PCR和蛋白印迹分别检测PepTl分布、mRNA和蛋白表达,高效液相色谱法检测PepTl摄取功能；每组剩余10只大鼠观察生存时间(7d)。
结果(1)与正常组和假手术组相比,脓毒症时小肠黏膜受损明显,血清及小肠黏膜TNF-α、IL-1β明显升高(P<0.05)；小肠黏膜PepTl分布减少,PepTl mRNA表达、蛋白表达以及对底物摄取能力明显降低(P<0.05)；(2)与脓毒症组比较,Ghrelin干预组大鼠小肠黏膜损伤较轻,血清及肠黏膜TNF-α、IL-1β(P<0.05);小肠黏膜PepTl分布量增多,生存率、PepTl mRNA表达蛋白表达及对底物摄取能力显著升高(P<0.05)；(3)正常组与假手术组各指标比较无显著差异(P>0.05)。
结论脓毒症时大鼠小肠上皮PepTl mRNA、蛋白表达以及PepTl在肠黏膜分布明显下降,机体在基因及蛋白水平下调了小肠上皮PepTl生物学功能；Ghrelin干预可降低炎性反应,对脓毒症小肠上皮PepTl mRNA、蛋白表达以及摄取能力均有上调作用。
Objective To investigate the effect of Ghrelin on PepT1of the small intestinal epithelial in sepsis rats.
Methods Eighty male SD rats were divided into four groups:normal group, sham operation, sepsis group and Ghrelin group, and there twenty rats in every group, by random number table.The model of sepsis was made with cecal ligation and puncture (CLP).After the procedure Ghrelin was injected via vein in the Ghrelin group. Ten rats by random in every group were used to investigate the intestinal histological. Serum and intestinal mucosas TNF-α, IL-1β and Ghrelin were detected with Elisa. In addition, Immunohistochemical, Realtime PCR and Western-blot were used to detect PepT1distribution, PepT1mRNA expression and PepT1protein expression levels respectively in every group. And the uptake of PepT1of small intestinal epithelial cells was measured with high performance liquid chromatography. The others were recorded the time of the survival of the rats in the treatment period (7d)
Results (1) The levels of serum and intestinal mucosas TNF-α and IL-1β in the sepsis group were significantly higher than in normal and sham operation groups (P<0.05). The PepT1distribution, PepT1mRNA expression levels, PepT1protein expression levels and the uptake of PepT1in the sepsis group were significantly lower than in normal and sham operation groups (P<0.05).And the damage to the small intestine mucosa was more serious in sepsis group compared to normal and sham operation groups.(2)Comparing with the sepsis group, thhe levels of serum and intestinal mucosas TNF-α and IL-1β in the Ghrelin group were significantly decreased (P<0.05). The survival rate of rats, PepT1distribution, PepT1mRNA expression levels, PepT1protein expression levels and the uptake of PepT1in the Ghrelin group were significantly improved than in sepsis group (P<0.05), and a lighter damage to the small intestine mucosa.(3) There were no differences in various indicators between sham operation and normal groups (P>0.05)
Conclusion The PepT1mRNA and protein expression levels with sepsis were significantly decreased, which could effect the uptake of PepT1.Ghrelin could decline the inflammatory response and greatly upregulate the physiological function of PepT1of small intestinal epithelial cells.
引文
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[2]Thompson JS. The intestinal response to critical illness[J]. Am J Gastroenterol 1995; 90(2):190-200
[3]Swank GM, Deitch EA. Role of the gut in multiple organ failure:bacterial translocation and permeability changes[J]. World J Surg 1996; 20(4):411-417
[4]Magnotti LJ, Deitch EA. Burns, bacterial translocation, gut barrier function, and failure[J]. J Burn Care Rehabil 2005; 26(5):383-391
[5]王俊健,张寿熙.肠道营养因子与肠黏膜屏障保护[J].实用医学进修杂志1999；27(4)：249-252
[6]康白,主编.双歧杆菌[M].大连：大连海事大学出版社1998：76-120
[7]李兰娟.肝功能衰竭并发感染与肠道细菌易位[J].中国微生态学杂志2001；13(2)：63-65
[8]Fei YJ, Kanai Y, Nussberger S. et al. Expression cloning of a mammalian proton-coupled oligopeptide transporter[J]. Nature 1994; 368(6471):563-566
[9]Ferraris RP, Diamond J, Kwan WW. Dietary regulation of intestinal transport of the dipeptide carnosine[J]. Am J Physiol 1988; 255(2 Pt 1):G 143-150
[10]Sun BW, Zhao XC, Wang GJ, et al. Hormonal regulation of dipeptide transporter (PepTl) in Caco-2 cells with normal and anoxia/reoxygenation management[J]. World J Gastroenterol 2003;9(4):808-812
[11]Shu HJ, Takeda H, Shinzawa H, et al. Effect of lipopolysaccharide on peptide transporter 1 expression in rat small intestine and its attenuation by dexamethasone[J]. Digestion 2002; 65(1):21-29
[12]Vavricka SR, Musch MW, Fujiya M, et al. Tumor necrosis factor-alpha and interferon-gamma increase PepT1 expression and activity in the human colon carcinoma cell line Caco-2/bbe and in mouse intestine[J]. Pflugers Arch 2006; 452(1):71-80
[13]Kojima M, Hosoda H. Date Y, et al. Ghrelin is a growth-hormone-releasing acylated peptide from stomach[J]. Nature 1999; 402(6762):656-660
[14]Krsek M, Rosicka M, Haluzik M, et al. Plasma ghrelin levels in patients with short bowel syndrome[J]. Endocr Res 2002; 28(1-2):27-33
[15]Ariyasu H. Takaya K, Tagami T, et al. Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelin-like immunoreactivity levels in humans[J]. J Clin Endocrinol Metab 2001; 86(10):4753-4758
[16]Ariga H, Tsukamoto K, Chen C. et al. Endogenous acyl ghrelin is involved in mediating spontaneous phase Ⅲ-like contractions of the rat stomach[J]. Neurogastroenterol Motil 2007; 19:675-680
[17]Ariga H, Nakade Y, Tsukamoto K, et al. Ghrelin accelerates gastric emptying via early manifestation of antro-pyloric coordination in conscious rats[J]. Regul Pept 2008; 146:112-116
[18]Asakawa A, Inui A, Kaga T, et al. Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin[J]. Gastroenterology 2001; 120(2):337-345
[19]Masuda Y, Tanaka T, Inomata N, et al. Ghrelin stimulates gastric acid secretion and motility in rats[J]. Biochem Biophys Res Commun 2000; 276(3):905-908
[20]Sibilia V, Rindi G, Pagani F, et al. Ghrelin protects against ethanol-induced gastric ulcers in rats:studies on the mechanisms of action[J]. Endocrinology 2003; 144(1):353-359
[21]Brzozowski T, Konturek PC, Konturek SJ, et al. Exogenous and endogenous ghrelin in gastroprotection against stress-induced gastric damage[J]. Regul Pept 2004; 120(1-3):39-51
[22]Brzozowski T, Konturek PC, Drozdowicz D, et al. Role of central and peripheral ghrelin in the mechanism of gastric mucosal defence[J]. Inflammopharmacology 2005; 13(1-3):45-62
[23]Konturek PC, Brzozowski T, Walter B, et al. Ghrelin-induced gastroprotection against ischemia-reperfusion injury involves an activation of sensory afferent nerves and hyperemia mediated by nitric oxide[J]. Eur J Pharmacol 2006; 536:171-181
[24]Brzozowski T, Konturek PC, Sliwowski Z, et al. Neural aspects of ghrelin-induced gastroprotection against mucosal injury induced by noxious agents[J]. J Physiol Pharmacol 2006; 57 Suppl 6:63-76
[25]褚万立等ghrelin对失血性休克大鼠胃黏膜的保护作用[J].中国危重病急救医学杂志2009；21(5)：263-265
[26]Wu R, Dong W, Qiang X, et al. Orexigenic hormone ghrelin ameliorates gut barrier dysfunction in sepsis in rats[J]. Crit Care Med 2009; 37:2421-2426
[27]Vila G, Maier C, Riedl M, et al. Bacterial endotoxin induces biphasic changes in plasma ghrelin in healthy humans[J]. J Clin Endocrinol Metab 2007; 92:3930-3934
[28]Das UN. Relationship between gut and sepsis:Role of ghrelin[J]. World Journal of Diabetes 2011; 2:1
[29]Wu R, Dong W, Qiang X, et al. Orexigenic hormone ghrelin ameliorates gut barrier dysfunction in sepsis in rats[J]. Critical Care Medicine 2009; 37(8):2421-2426
[30]何晓童,樊晓明Ghrelin的抗凋亡作用[J].国际内分泌学杂志2009；29(sup)：29-31
[31]Wu R, Zhou M, Cui X, et al. Upregulation of cardiovascular ghrelin receptor occurs in the hyperdynamic phase of sepsis[J]. Am J Physiol Heart Circ Physiol 2004; 287(3):H1296-1302
[32]Chang L, Du JB, Gao LR, et al. Effect of ghrelin on septic shock in rats[J]. Acta Pharmacol Sin 2003; 24(1):45-49
[33]Chang L. Zhao J, Yang J, et al. Therapeutic effects of ghrelin on endotoxic shock in rats[J]. Eur J Pharmacol 2003; 473(2-3):171-176
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