益生菌衍生的可溶性因子对于宿主防御的调节机制：对消化系统的健康影响课件

1、Fang Yan, M.D., Ph.D. Department of PediatricsDivision of Gastroenterology, Hepatology and NutritionVanderbilt University School of MedicineNashville, TNSupport: National Institute of Diabetes and Digestive Diseases and Kidney Diseases, Crohns and Colitis Foundation of America, Vanderbilt Digestive

2、Disease Research Center, the Nestle Nutrition Grant, and Atticus-Brown Family TrustProbiotic-derived soluble factors regulate host defense mechanisms: impact on digestive health糖尿病、消化系统疾病和肾病国家研究所、美国克洛病和结肠炎基金会、范德比特消化系统疾病研究中心、雀巢营养公司的授权、布朗家族信托益生菌衍生的可溶性因子对于宿主防御的调节机制：对消化系统的健康影响范德比特大学医学院肠道学、肝胆学和营养学部门儿科田纳西

3、州纳什维尔Research projects:1. Prevention and treatment of intestinal inflammation - Probiotic (Lactobacillus GG)-derived soluble proteins- Berberine (an alkaloid isolated from plants) 2. The roles of EGF and TNF signaling pathways in regulation of injury and repair mechanisms in intestinal inflammation

4、and carcinogenesis3. The mechanisms of H.pylori-induced gastric inflammation and cancer development 预防和治疗肠道炎症-LGG的分泌蛋白-黄连素（一种从植物中分离的生物碱）-可治疗腹泻EGF和TNF的信号通路在肠道炎症及癌变中对损伤和修复机制的调节作用。 幽门螺杆菌诱导胃炎症和癌变的机制Intestinal epithelial cell homeostasis and functions肠上皮细胞的动态平衡和功能 Digestion and absorption Water and elect

5、rolyte transport Protective defense mechanisms Barrier function (tight junctions) Anti-bacterial substancesInnate and adaptive immunity 肠细胞保护防御机制屏障功能腺管小肠绒毛杯状细胞潘氏细胞内分泌细胞干细胞先天免疫后天免疫抗菌物质The gastrointestinal microbiota1014 GI bacteria1013 cells in the body1012 skin bacteriaOral cavity200 species Stomach

6、H. PyloriSmall intestine108 bacteria/mlColon1010-1011 bacteria/g400-500 speciesBacteriodesEubacteriumBifidobacteriumRuminococcusBacillusClostridiumLactobacillusEnterococcusEnterobacter杆菌属真菌属双歧杆菌属瘤胃球菌属芽孢杆菌属梭菌属乳杆菌属肠球菌属肠杆菌属胃肠道细菌幽门螺杆菌Commensal bacteria in the gastrointestinal tract胃肠道中的共生细菌Beneficial ef

7、fects: Promote intestinal development. Benefit host health by enhancing polysaccharide digestion, uptake of nutrients, and antimicrobial activity. Modulate intestinal immune responses. Regulate intestinal epithelial cells.Yan and Polk, Curr Opin Gastroenterol 20: 565-571, 2004扁平细胞树突状细胞基底膜上皮紧密连接粘液促进肠

8、道发育提高多糖的消化和营养物质的吸收，提高抗菌活性，从而有益于宿主健康调节肠道免疫反应调节肠道上皮细胞Lactobacillus rhamnosus GG (LGG) Isolated from a healthy human One of the best-studied probiotic bacteria in clinical trial to prevent and/or treat: Diarrhea, Clostridium difficile, Rotavirus and Enterococci infections Ulcerative colitis, Atopic der

9、matitisLGG-derived soluble factors:Intestinal epithelial cells: Survival and proliferation Barrier function Synthesis of cytoprotective proteinsMacrophages: Decrease LPS-induced TNF production Increase G-CSF LGG巨噬细胞肠上皮细胞鼠李糖乳杆菌GG株在预防和治疗轮状病毒腹泻、梭状芽孢杆菌、肠球菌感染的溃疡性结肠炎，过敏性皮炎的临床试验中，它是研究的最多的益生菌之一。细胞保护蛋白的合成细菌脂

10、多糖粒细胞集落刺激因子肿瘤坏死因子LGG and factors recovered from LGG broth culture supernatant regulate intestinal epithelial cell signaling pathways to block apoptosis LGG和从LGG肉汤培养基的上清液分离的因子通过调节肠上皮细胞的信号转导途径来阻止细胞凋亡Yan and Polk, J. Biol. Chem., 52, 50959, 2002. (pro-apoptotic)(Anti-apoptotic)ApoptosisTNFIFNIL-1Colon

11、epithelial cellCytokine ReceptorLGGLGG soluble proteinsAktp38Cytokine receptor 细胞因子受体Colon epithelial cell 结肠上皮细胞Apoptosis 细胞凋亡Pro-apoptotic 促凋亡Anti-apoptotic 抗凋亡Apoptosis 细胞凋亡To define mechanisms through which LGG-secreted soluble proteins regulate intestinal epithelial cell function 确定LGG分泌的哪种可溶性蛋

12、白调节肠道上皮细胞功能的机制Aim 1Purification of p40 and p75 from LGG broth culture supernatant (LGG-s)p40p75Yan et al., Gastroenterology, 132, 562, 2007p40 and p75 activation of Akt颗粒过滤器LGG分泌蛋白阳离子交换色谱NaCl洗脱p40 and p75 prevent TNF-induced apoptosisin colon epithelial cells.在结肠上皮细胞p40和p75阻止TNF-诱导细胞凋亡Yan et al., Ga

13、stroenterology, 132, 562, 2007 * p 0.01 compared to TNF treatmentEGF receptor (EGFR) signaling pathway regulates Akt activation and cellular responses.表皮生长因子受体（EGFR）信号通路的调节Akt的活化和细胞的反应 EGFR: Type 1 receptor tyrosine kinase Expressed basolaterally in the intestinal epitheliumcolon epithelial cell结肠上皮

14、细胞proliferation增殖migration迁移differentiation区别survival生存RTKsGPCR G蛋白欧联受体Cytokine细胞因子Receptor接收器srcMMPs基质金属蛋白酶Ligands配体trans-membrane ligands 跨膜配体EGFR表皮生长因子受体 丝裂原活化蛋白激酶胞内磷脂酰肌醇激酶，与vsrc和vras等癌基因的产物相关，且PI3K本身具有丝氨酸/苏氨酸(Ser/Thr)激酶的活性，也具有磷脂酰肌醇激酶的活性 EGFR为I型跨膜酪氨酸激酶生长因子受体定位于细胞膜 与H ER2ErbB2Neup l 85、HER3ErbB一3、

15、HER4ErbB-4等同归入HEREerb家族。由胞外区、跨膜区及胞内区三部分组成。其配体包括表皮生长因子(epidermal growth factor。EGF)、转化生长因子Of(transforming growth factor-or，TGFd)、8一细胞素(betacellulin，BTC)、双调蛋白(amphiregulin)、表皮素(epiregulin)、肝素结合的表皮生长因子(heparinbinding EGF，HBEGF)等，以EGF和TGFd最为重要。EGFR与其配体胞外区结合后相互作用形成同源二聚体或异源二聚体。与同源二聚体相比，异源二聚体在介导细胞增殖、分化、迁移等

16、信号传递中起着更为重要的作用。二聚体的形成导致胞内酪氨酸激酶区的激活，进而通过转磷酸化和磷酸化作用，促使受体酪氨酸残基磷酸化启动rasMAPK、P13K、PLCyPKC、STAT等一系列级联反应将信号传到细胞核内最终引起一系列相关基因活化促进细胞从G，期过渡到S期141从而对核内基因表达和细胞生长分化产生调节作用。EGFR is required for p40 prevention of cytokine-induced apoptosis in intestinal epithelial cells在肠上皮细胞中预防细胞因子诱导的凋亡，EGFR对P40来说是必需的p40 or p75 pr

17、event H2O2 disruption of tight junction in Caco2 cells.p40或p75可以防止H2O2中断Caco2细胞紧密连接H2O2 H2O2/p40 H2O2/p40/AG1478 H2O2/p75 H2O2/p75/AG1478ZO-1 OccludinAG1478: EGF receptor kinase inhibitorAG1478：表皮生长因子受体激酶抑制剂Seth et al. Am J Phys Gastrointest Liver Physiol 2008p40 requires EGFR kinase activity to sti

18、mulate Akt activation in mouse colon explants.在小鼠结肠外植体中，p40需要EGFR激酶活性刺激Akt的活性wt (C57BL/6)EGFRwa5p40 (10 ng/ml)Colon explantDMEM 0.5% FBSColonic mucosal lysatesEGFR kinase inhibitorAG1478 (10 nM)结肠黏膜裂解p40 inhibition of TNF-induced apoptosis requires EGFR activity in colonic organ culture.在结肠器官培养物中，p4

19、0抑制TNF-诱导的细胞凋亡需要EGFR的活性EGFR mediates p40 prevention of TNF-induced redistribution of occludin in intestinal epithelial cells.在肠上皮细胞中，EGFR介导p40预防TNF-诱导的密封蛋白的再分配Red: Occludin, Blue: DAPIControl TNF TNF + p40wtEGFRwa5红色代表Occludin 密封蛋白 蓝色：DAPIp40 prevents cytokine-induced apoptosis and disruption of int

20、egrity through activation of EGF receptor (EGFR) and Akt in intestinal epithelial cells.在肠上皮细胞p40通过激活表皮生长因子受体（EGFR）和Akt，阻止细胞因子诱导的细胞凋亡和破坏其完整性。Yan et al. Gastroenterology 32:562-75, 2007, J Clin Invest 121:2242-53, 2011. Colon epithelial cellAktActivationEGFRTNFIFNIL-1Cytokine ReceptorApoptosisDisrupt

21、ion of barrierp40?p40 is a novel proteinp40是一种新的蛋白Full-length p40 protein sequence (412 aa) MKFNKAMITLVAAVTLAGSVSALTPVFADTSASIASNKSETNDLLKQIEAANTEVINLNKQIDAKNGEISDATAKISATDAKIASLSGEITAAQKNVAARKNNLKDQLISLQKKAGSSVSGNVYIDFVLNSQSLSDLIARTMTVGKLSQASKDALDAVTVAKDKLAALKSEQETARQTLVSTKASLETQKSQLETLQKTASDKQDALN

22、KEIADHKDELVALQSQFAQEQSEAAKATQAALKTAAASTASSSTSSTSNKSANSSVLSTGTSSTNTSSNSGASSTVISSNTASGSGSHADYSGSGNTYPWGQCTWYVKSVASWAGNGWGNGAEWGASAAAAGFTVNHTPAAGSIIVFAAGQSVGGQWTADGSYGHVAYVQSVSGDSVTITQGGMGFSSPTGPNTQTISGASSYVGreen: N-terminal sequence detected by Edman degradation.Orange: internal sequence detected by M

23、ALDI-TOF/MS/MS and LC/MS/MS analysis.Purple: Leader sequence for protein secretion.绿：N-末端序列由Edman降解法检测。橙：由MALDI-TOF/MS/MS和LC/ MS / MS分析检测到的内部序列。紫色：前导序列的蛋白质分泌。Yan et al., Gastroenterology, 132, 562, 2007Prediction of p40 tertiary structure for structure-function analysisp40的三级结构的结构与功能分析预测p40 amino ac

24、id sequence homology analysisHigh-confidence templates for p40 domainsUncharacterized domain-sheetcoiled-coilNH2 1 75 150 225 300 375 412 COOHp40的氨基酸序列的同源性分析高信任度的p40域模板p40 stimulates HB-EGF release, but not TGF or amphiregulin, in mice在小鼠实验中，P40刺激HB-EGF的释放，而不是TGF或双调蛋白Gavage p40-pectin/zein beadsBloo

25、dELISAKnock-down HB-EGF suppresses activation of EGFR and inhibition of apoptosis by p40 in YAMC cells.在YAMC细胞中去除HB-EGF通过p40抑制EGFR的活性并抑制细胞凋亡Metalloproteinase inhibitors block p40-stimulated EGFR activation in YAMC cells.在YAMC细胞中，金属蛋白酶抑制剂阻止 p40 刺激表皮生长因子的活性ADAM: A Disintegrin And Metalloproteinasep40:

26、 10 ng/ml, 1 hrEGF: 10 ng/ml, 5 minMetalloproteinase inhibitors: GM6001 (10 nM) and TAPI-1 (10 nM) Anti-EGFR antibody: C225, 1:1000 dilutionInactive Membranebound ligandactivePropeptideMetalloproteaseDisintegrinCystein-richTransmembranecytoplasmic A Disintegrin And Metalloproteinase 去整合素和金属蛋白酶inacti

27、ve 无活性propeptide肽Metalloprotease 金属蛋白酶Disintegrin 整合素cystein-rich 富含半胱氨酸Transmembrane 跨膜Cytoplasmic 细胞质membrane bound ligand 膜结合配体p40 stimulates TACE (ADAM17) activity in colon epithelial cells in vitro and in vivo.在体内或体外的结肠上皮细胞中，p40刺激TACE（肝动脉栓塞？）（ADAM17）的活性YAMCp40Lysates: TACE activity assayGavage

28、p40-pectin/zein beadsColonic epithelial cellsTACE activity assayHour after gavageTACE mediates p40 regulated anti-apoptotic response in colon epithelial cells.在结肠上皮细胞上，TACE介导P40的抗凋亡性反应p40 transactivation of EGFR depends on TACE.TACE mediates inhibition of apoptosis by p40.P40的表皮生长因子受体的转录依赖于TACE通过p40

29、，TACE介导抑制细胞凋亡Summaryp40-stimulated TACE activity, leading to HB-EGF release, serves as a mechanism underlying EGF receptor transactivation in intestinal epithelial cells在肠上皮细胞，P40-刺激TACE的活性，导致HB-EGF释放，从而使其在表皮生长因子受体下转录Aim 3To test the hypothesis that p40 prevents experimental colitis in mice in an EG

30、FR-dependent manner.为了检验这个假设，p40依赖表皮生长因子受体来抑制小鼠的实验性结肠炎Preparation of pectin/zein beads for specific delivery of p40 to the colon in vivo. 制备果胶/玉米醇溶蛋白的药丸（包埋有p40），能够使其在体内到达结肠位置。 Gavaged with pectin/zein beads (2 beads/mouse) containing FITC-His-p40 or His-p40- Sacrificed after 4 hours. FITC-labeled Hi

31、s-p40 beads His-p40 beadsGreen: FITC-His-p40 Red: E-cadherin Blue: DAPIStomach Small intestine Colon Colonpectin/zein beadHis-p40: 5 g/bead1 cmYan et al., JCI, 121, 2242, 2011-灌胃果胶/玉米醇溶蛋白的药丸（每只小鼠两颗），药丸里面包含有FITC-His-p40（标记的）或His-p40（未标记的）-药丸在4h后完全消化 上表皮钙粘着蛋白p40 activates EGFR and Akt in colon epithel

32、ial cells in vivo.在体内的结肠上皮细胞中，p40激活EGFR和Akt Yan et al., JCI, 121, 2242, 2011磷酸化表皮生长因子受体p40 prevents DSS-induced colitis in wt, but not EGFRwa2 mice.在wt小鼠模型中p40防止DSS诱导结肠炎，但在EGFRwa2小鼠模型中不起作用Yan et al., JCI, 121, 2242, 2011损伤或炎症得分牺牲/处理P40 treats DSS-induced colitis in wt, but not EGFRwa2 mice.在wt小鼠模型中p

33、40可以治疗DSS诱导的结肠炎，但在EGFRwa2小鼠模型中不起作用Yan et al., JCI, 121, 2242, 2011p40 blocks DSS-induced colon epithelial cell apoptosis in an EGFR-dependent manner.p40需要依赖EGFR来抑制DSS诱导结肠上皮细胞凋亡Yan et al., JCI, 121, 2242, 2011Water DSS-7D DSS+p40半胱氨酸天冬氨酸蛋白酶-3：被激活后可以通过对caspase靶蛋白的水解，导致程序性细胞死亡Bcl-2基因（即B细胞淋巴瘤/白血病-2基因）是一

34、种原癌基因，它具有抑制凋亡的作用p40 prevents DSS-induced disruption of intestinal epithelial tight junctions in an EGFR-dependent manner. p40需要依赖EGFR来抑制DSS诱导肠上皮细胞紧密连接的中断Yan et al., JCI, 121, 2242, 2011体内渗透检测血清FITC-葡聚糖紧密连接蛋白ZO-1Generation of transgenic mice with EGF receptor specifically deleted in the intestinal ep

35、ithelium.新一代的转基因小鼠的肠上皮细胞中专门去除EGFRYan et al., JCI, 121, 2242, 2011上皮基质EGF receptor expression in the colon epitheliumis required for p40 treating DSS-induced colitis in mice.p40治疗DSS诱导的小鼠结肠炎需要EGFR在结肠上皮细胞的表达Day: 0 4 73% DSS Water10 g of p40/day/mouseYan et al., JCI, 121, 2242, 2011外伤/炎症评分血清中FITC标记的聚葡萄

36、糖凋亡细胞所占百分比p40 ameliorating oxazolone-induced colitis requires EGF receptor expression in the colon epithelium.p40改善恶唑酮诱导的小鼠结肠炎需要EGFR在结肠上皮细胞的表达乙醇p40 activation of EGF receptor in intestinal epithelial cells mediates amelioration of intestinal inflammation in mice.在肠上皮细胞中p40激活EGFR的活性，间接地改善了小鼠肠道炎症Yan e

37、t al., J Clin Invest 121:2242-53, 2011破坏屏障炎症Aim 4To detect immune responses regulated by p40 during colitis in mice.检测小鼠结肠炎期间由p40调控的免疫反应p40 treatment decrease macrophage and neutrophil infiltration in DSS-induced colitis in mice.在具有DSS诱导的结肠炎的小鼠中，p40处理后可以减少巨噬细胞和嗜中性白血球的渗透活动嗜中性白血球巨噬细胞p40 treatment decr

38、eases TNF, IFN-, IL-6, KC, and IL-17 mRNA levels in DSS treated mouse colon mucosa.在DSS处理的结肠粘膜的小鼠中，p40处理后可以减少TNF, IFN-, IL-6, KC, 和IL-17 mRNA巨噬细胞产生TNF, IL-6和KC，TH1细胞产生IFN-p40 treatment does not affect IL-10, IL-1 mRNA levels in DSS treated mouse colon mucosa.在DSS处理的结肠粘膜的小鼠中，p40处理后不影响IL-10，IL-1mRNA水平

39、IL-10、IL-13主要由th2细胞产生p40 does not affect IL-13 production in oxazolone-induced colitis in mice.在具有恶唑酮诱导的结肠炎的小鼠中，p40处理后不影响IL-13的量Colon mucosal IL-13 mRNA levelReal time PCR Colon mucosal IL-13 protein levelFlow cytometry 实时定量PCR流式细胞仪结肠黏膜IL-13蛋白水平结肠黏膜IL-13 mRNA水平IL-13由th2细胞产生SummaryBased on the cytoki

40、ne profiles that are regulated by p40, it is possible that p40 plays a role in regulation of innate immunity and a Th1 immune response in colitis, such as regulation of TNF, IL-6 and KC production by macrophages and IFN- production by Th1 cells. 根据上述资料，p40可能在结肠炎中发挥先天免疫和Th1型的免疫应答的调节作用，如调节巨噬细胞产生TNF, I

41、L-6和KC，TH1细胞产生IFN-。Our findings support development of probiotic-derived proteins as novel reagents for more convenient and direct therapeutic intervention in preventing and/or treating ulcerative intestinal inflammatory disorders. 我们的研究结果表明益生菌的衍生蛋白质可以可以作为一种新型的试剂更方便、更直接的治疗和预防溃疡性肠道炎症疾病。ConclusionLactobacillus GG-fermented milk prevents DSS-induced colitis and regulates intestinal epithelial homeostasis through activation of epidermal growth factor receptorLGG发酵乳可以通过调节EGFR的活性防止DSS诱导的