大黄牡丹汤对脓毒症急性肠功能障碍大鼠肠道髓系细胞触发受体-1表达的影响
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摘要
目的:研究大黄牡丹汤对脓毒症大鼠急性肠功能障碍的治疗作用是否与调控肠道髓系细胞触发受体-1(TREM-1)表达有关。方法:雄性SD大鼠100只,腹腔注射4. 5 mg·kg~(-1)的脂多糖(LPS)进行造模,造模成功后随机分为5组:模型组,谷氨酰胺组(3. 75 g·kg~(-1)),大黄牡丹汤低、中、高剂量组(7. 5,15,30 g·kg~(-1)),另设正常大鼠10只作为正常组。每日1次。7 d后所有大鼠灌胃混悬液2 m L(乳果糖100 mg,甘露醇50 mg)。留取24 h尿液,运用带电化学检测器的高压液相色谱仪分析尿中乳果糖(lactulose)与甘露醇(mannitol)比值(L/M)。取血和回肠组织,运用高效液相色谱法检测血清瓜氨酸浓度;酶联免疫吸附法检测血清髓系细胞触发受体-1(TREM-1),肿瘤坏死因子-α(TNF-α),肠型脂肪酸结合蛋白(iFABP),D-乳酸浓度;实时荧光定量PCR检测回肠组织TREM-1,Toll样受体2(TLR2),Toll样受体4(TLR4),髓样细胞分化蛋白(MyD88),核转录因子-κB(NF-κB) mRNA的表达水平;电镜观察肠黏膜损伤的病理变化。结果:与正常组比较,模型组血清TREM-1,TNF-α浓度明显升高(P <0. 05,P <0. 01);回肠组织TREM-1,TLR2,TLR4,MyD88,NF-κB mRNA表达水平明显升高(P <0. 05,P <0. 01); i FABP,D-乳酸浓度,尿L/M明显升高(P <0. 05);血清瓜氨酸浓度明显降低(P <0. 05);回肠黏膜厚度、绒毛长度明显下降(P <0. 05)。与模型组比较,各给药组血清TREM-1,TNF-α浓度明显下降(P <0. 05,P <0. 01);回肠组织TREM-1,TLR2,TLR4,MyD88,NF-κB mRNA表达水平明显下降(P <0. 05,P <0. 01),4组之间无差异;谷氨酰胺组,大黄牡丹汤中、高剂量组血清i FABP,D-乳酸浓度,尿L/M明显下降(P <0. 05,P <0. 01);血清瓜氨酸浓度明显升高(P <0. 05,P <0. 01);绒毛长度及黏膜层厚度明显升高(P <0. 05);肠黏膜Chiu氏评分明显下降(P <0. 05),3组之间无差异。结论:大黄牡丹汤能有效治疗脓毒症大鼠急性肠功能障碍,其作用机制可能与通过调控肠道髓系细胞触发受体-1表达,减轻肠道炎症反应有关。
Objective: To investigate whether the therapeutic effect of Dahuang Mudan Tang on septic acute intestinal dysfunction in sepsis ratsis related to the regulation of expression of triggering receptor expressed on myeloid cells-1( TREM-1). Method: Totally 100 male SD rats were injected intraperitoneally with lipopolysaccharide( LPS) at a dose of 4. 5 mg·kg~(-1) to build sepsis model. The sepsis model rats were randomly divided into five groups: model group,glutamine group( 3. 75 g·kg~(-1)),low,medium,high-dose Dahuang Mudan Tang group( 7. 5,15,30 g·kg~(-1)),and another 10 normal rats were selected as normal group. Seven days later,2 m L suspension( 100 mg lactulose and 50 mg mannitol) was orally administrated by gavage,and 24 h urinewas collected. The ratio of lactulose to mannitol in urine( L/M) was detected by HPLC with pulsed electrochemical detection( HPLC-PED). Serum citrulline concentrationsin blood and ileum were determined by HPLC. Enzyme linked immunesorbent assay( ELISA) was used to detect the concentrations of triggering receptor expressed on myeloid cells-1( TREM-1),tumor necrosis factor-α( TNF-α),intestinal fatty acid binding protein( i FABP) and D-lactic acid. Real-time PCR was used to detect the mRNA expressions of TREM-1,Toll-like receptors2( TLR2), Toll-like receptors 4( TLR4), myeloid cell differentiation protein( MyD88), nuclear transcription factor-κB( NF-κB). Electron microscopy was used to observe the pathological changes of intestinal mucosa injury. Result: Compared with normal group,the serum concentrations of TREM-1,TNF-α,i FABP,D-lactate; the ratio of lactulose to mannitol in urine( L/M) and the expressions of TREM-1,TLR2,TLR4,MyD88,NF-κB mRNA in model group were increased obviously( P < 0. 05,P < 0. 01); citrulline concentration was decreased obviously( P < 0. 05); the lengths of the villus and thicknesses of the mucosal layer were decreased obviously( P < 0. 05); the Chiu score was increased obviously( P < 0. 01). Compared with model group,the expressions of TREM-1,TLR2,TLR4,My D88,NF-κB mRNA,and the serum concentrations of TREM-1 and TNF-α in all medication administration groups were decreased obviously( P < 0. 05),with no difference between these groups. Compared with model group,the serum concentrations of i FABP,D-lactate,L/M,the Chiu scorein glutamine group,medium-dose Dahuang Mudan Tang group and high-dose Dahuang Mudan Tang group were decreased obviously( P < 0. 05),lengths of villus and thicknesses of mucosal layers were increased obviously( P <0. 05); and the citrulline concentrations were increased obviously( P < 0. 05). There was no difference between the three groups. Conclusion: Dahuang Mudan Tang can effectively treat SAID in rats,and its mechanism may be realized by regulating the expression of TREM-1 and relieving intestinal inflammation of intestinal tract.
Objective: To investigate whether the therapeutic effect of Dahuang Mudan Tang on septic acute intestinal dysfunction in sepsis ratsis related to the regulation of expression of triggering receptor expressed on myeloid cells-1( TREM-1). Method: Totally 100 male SD rats were injected intraperitoneally with lipopolysaccharide( LPS) at a dose of 4. 5 mg·kg~(-1) to build sepsis model. The sepsis model rats were randomly divided into five groups: model group,glutamine group( 3. 75 g·kg~(-1)),low,medium,high-dose Dahuang Mudan Tang group( 7. 5,15,30 g·kg~(-1)),and another 10 normal rats were selected as normal group. Seven days later,2 m L suspension( 100 mg lactulose and 50 mg mannitol) was orally administrated by gavage,and 24 h urinewas collected. The ratio of lactulose to mannitol in urine( L/M) was detected by HPLC with pulsed electrochemical detection( HPLC-PED). Serum citrulline concentrationsin blood and ileum were determined by HPLC. Enzyme linked immunesorbent assay( ELISA) was used to detect the concentrations of triggering receptor expressed on myeloid cells-1( TREM-1),tumor necrosis factor-α( TNF-α),intestinal fatty acid binding protein( i FABP) and D-lactic acid. Real-time PCR was used to detect the mRNA expressions of TREM-1,Toll-like receptors2( TLR2), Toll-like receptors 4( TLR4), myeloid cell differentiation protein( MyD88), nuclear transcription factor-κB( NF-κB). Electron microscopy was used to observe the pathological changes of intestinal mucosa injury. Result: Compared with normal group,the serum concentrations of TREM-1,TNF-α,i FABP,D-lactate; the ratio of lactulose to mannitol in urine( L/M) and the expressions of TREM-1,TLR2,TLR4,MyD88,NF-κB mRNA in model group were increased obviously( P < 0. 05,P < 0. 01); citrulline concentration was decreased obviously( P < 0. 05); the lengths of the villus and thicknesses of the mucosal layer were decreased obviously( P < 0. 05); the Chiu score was increased obviously( P < 0. 01). Compared with model group,the expressions of TREM-1,TLR2,TLR4,My D88,NF-κB mRNA,and the serum concentrations of TREM-1 and TNF-α in all medication administration groups were decreased obviously( P < 0. 05),with no difference between these groups. Compared with model group,the serum concentrations of i FABP,D-lactate,L/M,the Chiu scorein glutamine group,medium-dose Dahuang Mudan Tang group and high-dose Dahuang Mudan Tang group were decreased obviously( P < 0. 05),lengths of villus and thicknesses of mucosal layers were increased obviously( P <0. 05); and the citrulline concentrations were increased obviously( P < 0. 05). There was no difference between the three groups. Conclusion: Dahuang Mudan Tang can effectively treat SAID in rats,and its mechanism may be realized by regulating the expression of TREM-1 and relieving intestinal inflammation of intestinal tract.
引文
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[26]张保国,刘庆芳.大黄牡丹汤现代药效学研究与临床应用[J].中国药学杂志,2009,44(21):1601-1604.
[27]邓芳芳,曹淼,张文兴.大黄牡丹汤恢复急性阑尾炎术后肠功能的Meta分析[J].中国中西医结合外科杂志,2015,21(2):124-127.
[28]周健,高淳,唐学典.大黄牡丹汤加减治疗急性胰腺炎对机体炎症和应激反应的影响[J].中医药信息,2017,34(1):62-66.
[29]关云艳,沈丽娟,吴锡平,等.血清I-FABP在诊断脓毒症大鼠急性肠功能障碍中的作用及谷氨酰胺对其表达的影响[J].中国生化药物杂志,2014,34(4):44-46.
[30]舒逍,董京文,杨海峰,等.通腑清胰方辅助治疗重症胰腺炎的疗效及对肠黏膜屏障功能的保护作用[J].中国实验方剂学杂志,2013,19(21):280-284.
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[33]刘桂伟,任维聃,姜国胜.谷氨酰胺强化肠内营养对结肠癌患者左半结肠切除术后肠通透性及感染并发症的作用[J].中国全科医学,2018,21(5):526-530.
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[2] Rowlands B J,Soong C V,Gardiner K R,et al.Thegastrointestinal tract as a barrier in sepsis[J]. Br Med Bull,1999,55(1):196-211.
[3]马晓春,尹晓晗,栾正刚.血红素加氧酶-1对脓毒症患者胃肠道保护作用研究进展[J].创伤与急危重病医学,2013,1(1):17-20.
[4]官永海,陈福刚.中药灌肠结合针刺疗法治疗ICU重症感染合并胃肠功能障碍疗效观察[J].现代中西医结合杂志,2017,26(9):967-969.
[5]孙燕妮,承解静,杨晓燕,等.大黄牡丹汤含药血清对LPS刺激的小鼠肺巨噬细胞TLR4和MyD88表达的影响[J].山东医药,2014,54(10):10-13.
[6] Bouchon A,Dietrich J,Colonna M. Cutting edge:inflammatory responses can be triggered by TREM-1,a novel re-ceptor expressed on neutrophils and monocytes[J]. J Immunol,2000,164(10):4991-4995.
[7]关云艳,王倩,吴海荣.髓样细胞表达的触发受体-1研究进展[J].国际免疫学杂志,2010,33(1):49-52.
[8]苗大兴,肖天保,梁宛伶.大黄牡丹汤含药血清对巨噬细胞释放炎症因子的影响[J].时珍国医国药,2014,25(4):843-845.
[9] Stahl O,Loffler B,Haier J,et al. Mimicry of human sepsis in a rat model-prospects and limitations[J]. J Surg Res,2013,179(1):e167-175.
[10]何艳,秦雪梅,鲁卫华,等.脓毒症大鼠肠道瞬时感受器电位香草酸受体1及降钙素基因相关肽的表达及作用[J].中国临床药理学与治疗学,2016,21(1):38-41.
[11]沈丽娟,王倩,吴锡平,等.谷氨酰胺对脓毒症大鼠急性肠黏膜损伤的保护作用[J].内科急危重症杂志,2018,24(3):253-255.
[12]苗大兴,肖天保,梁宛伶,等.大黄牡丹汤含药血清对小鼠骨髓源巨噬细胞吞噬功能及Toll样受体mRNA表达的影响[J].中国老年学杂志,2017,37(8):1891-1893.
[13] Chiu C J,Mcardle A H,Brown R,et a1. Intestinal mucosa lesion in low flow states[J]. Arch Surg,1970,101(4):478-483.
[14] SHEN L J,GUAN Y Y,WU X P,etal. Serumcitrulline as a diagnostic marker of sepsis-induced intestinal dysfunction[J]. Clin Res Hepatol Gastroenterol,2015,39(2):230-236.
[15] Livak K J,Schmittgen T D. Analysis of relative gene expression data using Real-time quantitative PCR and the(2-Delta Delta C(T))method[J]. Methods,2001,25(4):402-408.
[16]刘洪斌,吴咸中,李东华,等.清热解毒方对脓毒症大鼠的治疗作用[J].中国中西医结合外科杂志,2008,14(6):580-584.
[17] Singer M,Deutschman C S,Seymour C W,et al. The third international consensus definitions for sepsis and septic shock(Sepsis-3)[J]. JAMA,2016,315(8):801-810.
[18] Gaieski D F, Edwards J M, Kallan M J, et al.Benchmarking the incidence and mortality of severe sepsis in the United States[J]. Crit Care Med,2013,41(5):1167-74.
[19] Shankar-Hari M, Phillips G S,Levy M L, et al.Developing a new definition and assessing new clinical criteria for septic shock:for the third international consensus definitions for sepsis and septic shock(sepsis-3)[J]. JAMA,2016,315(8):775-787.
[20] Sertaridou E,Papaioannou V,Kolios G,et al. Gut failure in critical care:old school versus new school[J]. Ann Gastroenterol,2015,28(3):309-322.
[21] Clark J A,Coopersmith C M. Intestinal crosstalk:a new paradigm for understanding the gut as the “motor”of critical illness[J]. Shock,2007,28(4):384-393.
[22] Klingensmith N J,Coopersmith C M. The gut as the motor of multiple organ dysfunction in critical illness[J]. Crit Care Clin,2016,32(2):203-212.
[23] Shimizu K,Ogura H,Hamasaki T,et al. Altered gut flora are associated with septic complications and death in critically ill patients with systemic inflammatory response syndrome[J]. Dig Dis Sci,2011,56(4):1171-1177.
[24]周秀红,张灵恩,陆铸今,等.危重患儿急性胃肠功能衰竭的病因、死亡危险因素分析和干预措施[J].中国小儿急救医学,2006,8(4):339-342.
[25]刘洋,江宇泳,王宪波,等.肠功能障碍的中医药治疗进展[J].中医药学报,2013,41(6):87-89.
[26]张保国,刘庆芳.大黄牡丹汤现代药效学研究与临床应用[J].中国药学杂志,2009,44(21):1601-1604.
[27]邓芳芳,曹淼,张文兴.大黄牡丹汤恢复急性阑尾炎术后肠功能的Meta分析[J].中国中西医结合外科杂志,2015,21(2):124-127.
[28]周健,高淳,唐学典.大黄牡丹汤加减治疗急性胰腺炎对机体炎症和应激反应的影响[J].中医药信息,2017,34(1):62-66.
[29]关云艳,沈丽娟,吴锡平,等.血清I-FABP在诊断脓毒症大鼠急性肠功能障碍中的作用及谷氨酰胺对其表达的影响[J].中国生化药物杂志,2014,34(4):44-46.
[30]舒逍,董京文,杨海峰,等.通腑清胰方辅助治疗重症胰腺炎的疗效及对肠黏膜屏障功能的保护作用[J].中国实验方剂学杂志,2013,19(21):280-284.
[31] Glatz J F,Van D V G J. Cellular fatty acid-binding proteins:their function and physiological significance[J]. Prog Lipid Res,1996,35(3):243-282.
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