肠道必需氨基酸代谢及其功能的研究进展

在肠道代谢过程中,食物必需氨基酸可不同程度地被肠道组织利用。这些必需氨基酸,除了用于合成肠黏膜蛋白质外,还可通过不同途径在肠上皮细胞内代谢。它们不仅是小肠黏膜的能量物质,同时还参与肠道内氨基酸、谷胱甘肽和多胺等多种生物活性物质的合成,对维持肠黏膜完整性和肠道功能有重要意义,对整个机体的代谢产生重要的影响。     

Dephosphorylation of myo-inositol phosphates in the in vitro intestinal Caco-2 cell model

Plant and microbial phytases present in raw materials can cause a dephosphorylation of phytate (myo-inositol hexakisphosphate) (InsP₆)) during food processing resulting in a broad range of different myo-inositol phosphates such as pentakisphosphate (InsP₅) and tetrakisphosphate (InsP₄) in foods. Here, we investigated whether the human intestinal epithelium is able to dephosphorylate myo-inositol phosphates (InsP₆, InsP₅-, InsP₄-, InsP₃-isomers) using an in vitro model with differentiated human Caco-2 cells cultured on semipermeable inserts. Incubation of InsP₆ and an InsP₅-isomer with cells for 3?h showed no dephosphorylation of both InsPs. Treatment of cells with a mixture of different InsP₄-isomers, however, caused a formation of about 3.5% of an InsP₃-isomer (Ins(1,5,6)P₃) and treatment with a mixture of different InsP₃-isomers caused about 20% formation of InsP₂-isomers, respectively. Thus, human intestinal cells can contribute to the dephosphorylation of myo-inositol phosphates of partly dephosphorylated forms such as InsP₃ and InsP₄.     

铜对Caco-2细胞单层屏障功能的影响

目的研究铜对Caco2细胞单层细胞间通透性、P糖蛋白(Pgp)活性的影响。方法应用Caco2细胞单层模型,通过测定铜暴露后细胞单层的跨上皮细胞电阻(TEER)来反映通透性的改变;通过免疫荧光和荧光染色观察铜对紧密连接蛋白ZO1和F微丝的影响;通过测定细胞单层对罗丹明123的跨膜转运和细胞内罗丹明123的蓄积来反映P糖蛋白活性的改变。结果细胞单层顶面铜暴露(30～100μmol/L,Hanks缓冲溶液,0～3h)可导致TEER值呈浓度和时间依赖性降低,同时伴随着F微丝的解聚,但对紧密连接蛋白ZO1无显著影响;在不影响细胞活性和细胞单层通透性的剂量下,顶面铜暴露(300μmol/L,完全培养基,24h)后细胞单层对罗丹明123的表观通透系数(Papp)BL→AP从对照组的(737±020)10-6cm/s降低为(643±027)×10-6cm/s,PappAP→BL从对照组(123±005)×10-7cm/s增加为(341±008)×10-7cm/s,同时细胞内罗丹明-123的蓄积量从对照组的每膜(031±001)nmol增加为每膜(050±003)nmol。结论铜可显著增加Caco2细胞单层的通透性和抑制Pgp的活性,从而推测可能影响肠道上皮细胞的正常屏障功能。     

NF-kappaB activation as a key mechanism in ethanol-induced disruption of the F-actin cytoskeleton and monolayer barrier integrity in intestinal epithelium.

Intestinal barrier disruption has been implicated in several intestinal and systemic disorders including alcoholic liver disease (ALD). Using monolayers of intestinal (Caco-2) cells, we showed that ethanol (EtOH) disrupts the barrier integrity via destabilization of the cytoskeleton. Because proinflammatory conditions are associated with activation of NF-kappa B (NF-kappaB), we hypothesized that EtOH induces disruption of cytoskeletal assembly and barrier integrity by activating NF-kappaB. Parental cells were pretreated with pharmacological modulators of NF-kappaB. Other cells were stably transfected with a dominant negative mutant for the NF-kappaB inhibitor, I-kappaBalpha. Monolayers of each cell type were exposed to EtOH and we then monitored monolayer barrier integrity (permeability); cytoskeletal stability and molecular dynamics (confocal microscopy and immunoblotting); intracellular levels of the I-kappaBalpha (immunoblotting); subcellular distribution and activity of NF-kappaB (immunoblotting and sensitive ELISA); and intracellular alterations in the 43kDa protein of the actin cytoskeleton, polymerized F-actin, and monomeric G-actin (SDS-PAGE fractionation). EtOH caused destabilizing alterations, including I-kappaBalpha degradation, NF-kappaB nuclear translocation, NF-kappaB subunit (p50 and p65) activation, actin disassembly (upward arrow G-, downward arrow F-), actin cytoskeleton instability, and barrier disruption. Inhibitors of NF-kappaB and stabilizers of I-kappaBalpha (e.g., MG-132, lactacystin, etc) prevented NF-kappaB activation while protecting against EtOH-induced injury. In transfected I-kappaBalpha mutant clones, stabilization of I-kappaBalpha to inactivate NF-kappaB protected against all measures of EtOH-induced injury. Our data support several novel mechanisms where NF-kappaB can affect the molecular dynamics of the F-actin cytoskeleton and intestinal barrier integrity under conditions of EtOH injury. (1) EtOH induces disruption of the F-actin cytoskeleton and of intestinal barrier integrity, in part, through I-kappaBalpha degradation and NF-kappaB activation; (2) The mechanism underlying this pathophysiological effect of the NF-kappaB appears to involve instability of the assembly of the subunit components of actin network.     

铜对人类肠道上皮Caco-2细胞的毒性研究

目的　了解铜对人类肠道上皮Caco 2细胞的毒性影响。方法　应用噻唑蓝 (MTT)实验、P 糖蛋白(P gp)活性检测实验、活性氧检测及克隆形成实验研究铜对肠道上皮Caco 2细胞的毒性及可能作用机制 ,同时利用Caco 2细胞和肠炎沙门氏菌为模型 ,研究铜对Caco 2细胞对细菌侵入和存活易感性的影响。结果　在一定的暴露场景下 ,铜可明显地降低细胞的活力 ,抑制细胞膜表面P gp的活性 ,促进细胞内活性氧自由基的产生 ,降低细胞的克隆形成能力。此外 ,细胞经铜暴露后 ,肠炎沙门氏菌侵入细胞的数量增加 ,但细胞内存活的细菌数量却下降。结论　过量的铜可导致Caco 2细胞氧化损伤 ,从而引起更广泛的细胞毒性效应 ,但其对细胞对细菌侵入和存活易感性的影响有待进一步研究。     

Profile and bioavailability analysis of myo-inositol phosphates in rye bread supplemented with phytases: a study using an in vitro method and Caco-2 monolayers

Commercial preparations of 6-phytase A alone and in combination with phytase B were used in rye breadmaking. Determination of bioavailability of myo-inositol phosphates from bread was performed by an in vitro digestion method followed by the measurement of an uptake by Caco-2 cells in culture. In bread supplemented with a combination of 6-phytase A and phytase B, a significant reduction in phytate content was observed from 3.62?μmol/g in the control to 0.7?μmol/g. Bioavailability of phytate estimated by an in vitro method simulating digestion in the human alimentary tract was 9% in the bread supplemented with phytase B, 7% (6-phytase A) and 50% in the control bread. In cell culture, the bioaccessibilities of inositol triphosphates from bread baked with the addition of 6-phytase A was higher by 36% as compared to the samples baked with phytase B and by 32% in breads baked with combination of both phytases.     

基因重组肠三叶因子对肠上皮细胞修复和保护作用的研究

目的:探讨基因重组肠三叶因子(ITF)对肠上皮细胞(IEC-6)是否具有促进修复和保护的作用.方法:制备不同浓度的重组ITF.首先,采用MTT法观察ITF对IEC-6细胞增殖活性的影响;其次,制作IEC-6细胞机械损伤模型,观察ITF对IEC-6细胞移行速率的影响;最后,采用烧伤血清致IEC-6细胞损伤模型,观察ITF对IEC-6细胞的保护作用. 结果:各种浓度的ITF对IEC-6细胞具有一定的促增殖作用,但差异无统计学意义(P＝0.19).ITF可加快IEC-6细胞移行速度,是对照组的2～3倍(P＜0.01).ITF可明显降低烧伤血清致IEC-6细胞损伤程度,反映细胞损伤的谷草转氨酶(AST)、乳酸脱氢酶(LDH)活性显著下降,且作用持续12 h.联合使用黏蛋白(Mucin)可显著加强ITF的细胞保护作用,谷丙转氨酶(ALT)、AST、LDH活性显著降低,且保护作用延长至48 h(P＜0.01). 结论:ITF可加快细胞移行速度,促进受损肠黏膜屏障的重建,对肠上皮细胞具有明显的保护作用.单独使用ITF对细胞增殖影响不明显,与Mucin联合使用,其保护作用明显增强.     

神经导向因子在缺血肠道及缺氧细胞中的表达及作用

目的:探讨神经导向因子(Netrin-1)在急性肠系膜缺血(AMI)病人肠道组织及低氧条件下人结直肠腺癌(Caco-2)细胞中的表达及其对肠道功能恢复和微血管新生的作用. 方法:采用免疫印迹、免疫组化技术分别检测46例肠系膜上静脉血栓病人缺血肠道Netrin-1表达情况,并分析其表达量与病人主要临床结果之间的关系;培养Caco-2细胞,应用免疫印迹、Real-time PCR和免疫荧光技术检测不同低氧时间下Caco-2细胞Netrin-1 mRNA和蛋白的表达. 结果:预后较好的病人肠道局部Netrin-1表达量明显高于有严重并发症(30 d内死亡、二次手术探查、全肠外营养(TPN)时间＜14 d、脓毒症、需机械通气的ARDS、MODS、SBS)的病人,差异有显著性统计学意义(P＜0.05).同时,Caco-2细胞低氧时间与细胞Nertin-1存在一定相关性,且随着低氧时间逐渐延长,Netrin-1 mRNA及其蛋白表达量逐渐增加. 结论:AMI病人肠道局部的Netrin-1可能参与微血管新生及血管网络的构建而保护已缺血肠道,进而减少肠坏死等并发症,促进肠道功能恢复.     

大、小鼠吸收和代谢双酚A的差异

目的 探讨相同剂量双酚A(bisphenol A,BPA)经口染毒后,引起大、小鼠血清BPA浓度差异的机制.方法 无特定病原体(specic pathogen free,SPF)级雄性SD大鼠和ICR小鼠各18只,一次性经口给予300 mg/kg的BPA后,在第0.5、1.0、12.0小时时间点各采集6只大、小鼠血液,用荧光-高效液相色谱法(fluorescence-high performance liquid chromatography,FL-HPLC)检测血清中BPA浓度;大、小鼠各6只采用原位小肠吸收模型循环灌流100 ml 0.1 mmol/L BPA灌流液,用FL-HPLC法分别检测第0.5、1.0、2.0小时时间点灌注液中BPA浓度和灌流后2.0 h血清中的浓度;采用逆转录PCR(RT-PCR)方法检测大、小鼠肝脏尿苷二磷酸葡萄糖醛酸转移酶2B1(UDP-glucuronosyltransferase 2B1,UGT2B1)mRNA的表达水平,并采用FL-HPLC法检测UGT2B1的酶活性;大、小鼠各6只禁食24 h后,一次性经口给予300 ms/kg BPA,收集24 h粪便,用FL-HPLC法检测粪便中BPA含量.结果 300 ms/kg BPA经口染毒后第0.5、1.0、12.0小时时间点小鼠血清中BPA浓度分别为(66.57±14.95)、(51.16±16.06)、(22.73±5.00)μg/ml,大鼠血清中BPA浓度分别为(15.63±5.65)、(18.34±5.02)、(7.65±2.58)μg/ml,各时间点小鼠均高于大鼠,差异有统计学意义(F值分别为50.660,17.957,8.420,P值均＜0.05);0～、0.5～、1.0～2.0 h小鼠小肠各时间段吸收速率分别为(10.20±4.20)、(1.49±0.67)、(1.31±0.55)μg·cm-2·min-1均高于大鼠相应时间段吸收率[(1.87 ± 0.69)、(0.47±0.13)、(0.36 ± 0.08)μg·cm-2·min-1],差异均有统计学意义(F值分别为14.954、8.877、11.536,P值均＜0.05).0.1 mmol/L BPA灌肠2 h后小鼠血清中的BPA浓度为(22.64±4.35)μg/ml,高于大鼠的(4.13±0.83)μg/ml,差异有统计学意义(F=74.643,P=0.000);大鼠肝脏中BPA代谢酶UGT2B1 mRNA的表达水平和酶活性明显高于小鼠;300 mg/kg BPA经口染毒24 h后,大鼠经粪便排出的BPA量为(1.50±0.32)mg/g,高于小鼠的(0.57±0.35)ms/g,差异有统计学意义(F=21.215,P=0.001).结论 大、小鼠经口给予300 mg/kg BPA染毒后,由于小鼠肠吸收BPA的能力高于大鼠,而大鼠代谢及排出BPA能力高于小鼠,引起小鼠血清中BPA的浓度明显高于大鼠.     

促炎细胞因子对肠上皮细胞紧密连接调控的分子机制

促炎细胞因子是由细胞分泌的、具有促进和调节炎性反应作用的小分子多肽,主要有IL-1、IL-6、肿瘤坏死因子-α(TNF-α)、γ-干扰素(IFN-γ)等.炎症性肠病病人血清中促炎细胞因子水平显著增高,并且能引起肠上皮细胞紧密连接的改变.现就促炎细胞因子对肠上皮细胞紧密连接调控的分子机制作一综述,旨在为开展基础研究和临床提供参考.     

In vitro intestinal transport and antihypertensive activity of ACE inhibitory pea and whey digests

Angiotensin I converting enzyme (ACE) inhibitory peptides cause an antihypertensive effect if they reach the systemic circulation. This was investigated for the high ACE inhibitory activity present in peas and whey in vitro gastrointestinal digests. The samples retained high ACE inhibitory activity when incubated in Caco-2 homogenates or rat intestinal acetone powder, both sources of small intestine peptidases. Only little ACE inhibitory activity was transported through Caco-2 cell monolayers in 1?h. As the Caco-2 model is tighter than intestinal mammalian tissue, sufficient absorption of these peptides might occur in vivo. After intravenous administration of 50?mg protein?kg^?1 BW in spontaneously hypertensive rats (SHR), pea digest exerted a transient, but strong antihypertensive effect of 44.4?mmHg. Whey digest exerted no effect at this dose. These results suggest that pea digest could be a promising source of ACE inhibitory peptides for use in the prevention and treatment of hypertension.     

内毒素对大鼠肝巨噬细胞谷氨酰胺代谢的影响

目的 :体外培养条件下观察内毒素对大鼠肝巨噬细胞 (Kupffer cells,KC)谷氨酰胺代谢的影响。　方法 :分离、纯化获得的大鼠 KC体外培养 ,观察在不同浓度内毒素时 ,KC对谷氨酰胺 (Gln)消耗、利用率和细胞内 Gln浓度的影响。　结果 :培养液中不含内毒素时 ,KC对 Gln的消耗、利用率和细胞内含量分别为 (2 2 5± 2 4)μmol/ L、(9.33± 0 .99) %和 (142 5± 2 0 1)μmol/ 1.5× 10 6 KC。内毒素浓度为 10 mg/ L ,其 Gln的消耗、利用率和细胞内含量最大值分别为 (4937± 2 89)μmol/ L、(73.2 9± 4.72 ) %和 (5 117± 389)μmol/ 1.5× 10 6 KC。　结论 :Gln是静息状态和内毒素激活时 KC的重要代谢底物。     

肠上皮细胞紧密连接基因及信号调控的研究进展

肠道屏障功能对于临床多种疾病的发生发展发挥着重要的作用,紧密连接结构对维持肠道屏障功能及调控细胞分化具有重要作用,细胞通过与周边细胞和胞外基质相互作用调节紧密连接蛋白的基因表达,进一步影响细胞的屏障功能、细胞增殖、细胞极性和细胞凋亡等.紧密连接作为一种重要的细胞连接,其调节与一些信号转导通路有关,包括经典的环磷酸腺苷-蛋白激酶A-cAMP反应元件结合蛋白通路、二磷酸肌醇通路、丝裂原活化蛋白激酶通路、磷脂酰肌醇-3-激酶通路及特殊的闭锁小带蛋白1相关核酸连接蛋白通路、细胞周期蛋白、磷酸化和甲基化调控途径等,其基因及蛋白质表达的调控较复杂.紧密连接蛋白表达及调控紊乱与临床疾病的发生密切相关,如屏障破坏、顽固性感染、消化系统疾病、肿瘤.本文主要综述紧密连接蛋白如何参与基因表达、相关基因的调控,及其参与基因表达和细胞分化的自身调节相关机制.     

肠道菌群参与宿主代谢对医疗个性化的影响

肠道菌群的代谢流程与宿主的代谢流程存在交汇与互补的情况,即交互式代谢(metabolicexchange)和共同代谢(co_metabolism)。这种相互作用与许多人类疾病的发生、发展有着重要联系,但我们现在对此却知之甚少。由于这种代谢上的关联,肠道菌群对药物在宿主体内的吸收、代谢、毒理存在显著影响。这提示我们在设计个性化医疗时需充分考虑个体间肠道菌群的差异性。     

Bioactivity and cell metabolism of in vitro digested sweet cherry (Prunus avium) phenolic compounds

In this study, the bioaccessibility of phenolic compounds after in vitro gastrointestinal digestion of two cherry cultivars was assessed. The phenolic profile was modified during in vitro digestion, with a considerable decrease of total and individual phenolic compounds. Hydroxycinnamic acids and especially coumaroylquinic acids showed the highest bioaccessibility. Isomerisation of caffeoylquinic and coumaroylquinic acids was observed after in vitro digestion. Modification of the phenolic profile after digestion resulted in an increased or decreased scavenging activity depending on the assay. In vitro digested phenolic-rich fractions also showed antiproliferative activity against SW480 but no effect against Caco-2 cell lines. Both Caco-2 and SW480 cell lines were able to metabolise cherry phenolic compounds with remarkable differences. An accumulation of glycosylated flavonols was observed in SW480 medium. In conclusion, phenolic compounds from cherries and especially hydroxycinnamic acids were efficiently released and remained bioaccessible after in vitro digestion, resulting in antioxidant and antiproliferative activities.     

Particle size of milled chokeberry pomace did not influence in vitro cellular absorption and transport efficiencies of anthocyanins,phenolic acids and flavonols

Abstract

Industrial chokeberry pomace is very rich in polyphenols. The main focus here lies on the possible relationship between the particle size of chokeberry milled pomace and an enhanced absorption and transport of polyphenols by Caco-2 cells. Wet milling was used to produce materials with particle size distributions in the micrometre and in the sub-micrometre to nanometre ranges starting from chokeberry pomace. Milled materials with about 50% of the particles with a mean size (x_50,3) of 223?±?13?µm (coarse milling) and about 90% of the particles with x_50,3 of 160?±?40?nm (fine milling, sonication) were obtained. None of the milled materials exhibited cytotoxic effects within the tested concentration-ranges. The polyphenol absorption and the transport efficiencies from the fine and the coarse milled materials were similar. Thus, no effect of the particle size upon cellular uptake and transport could be established, but agglomeration of particle during incubation cannot be excluded as the cause. Furthermore, based on polyphenol stability we postulate that direct milling may be applied to valorise the processing by-product from commercial fruit juice production.     

Genotoxic risk assessment in white blood cells of occupationally exposed workers before and after alteration of the polycyclic aromatic hydrocarbon (PAH) profile in the production material: comparison with PAH air and urinary metabolite levels

Objective: Workers in various industries can be exposed to polycyclic aromatic hydrocarbons (PAHs). The relationship between biomarkers of genotoxic risk, PAH compounds in air (ambient monitoring) and PAH metabolites in urine (internal exposure) were studied in 17 workers exposed to PAHs in a fireproof-material producing plant before and 3 months after the PAH profile was altered in the binding pitch. Methods: Two biomarkers of exposure, specific DNA adducts of (±)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) and non-specific DNA adduct of 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodGuo) were determined in white blood cells (WBCs). In addition, DNA strand breaks were analysed in lymphocytes by single-cell gel electrophoresis in a genotoxic risk assessment. Sixteen PAH compounds in air were determined by personal air sampling, and hydroxylated metabolites of phenanthrene, pyrene and naphthalene were determined in urine. Results: After substitution of the binding pitch the concentrations of benzo[a]pyrene in air decreased (P<0.01). No changes could be observed for pyrene, while levels of phenanthrene (P=0.0013) and naphthalene (P=0.0346) in air increased. Consequently, median DNA adduct rates of anti-BPDE decreased after alteration of the production material (from 0.9 to <0.5 adducts/10⁸ nucleotides). No changes in the excretion of 1-hydroxypyrene in urine could be determined, whereas increased levels of 1-, 2+9-, 3- and 4-hydroxyphenanthrene (P<0.0001) and 1-naphthol and 2-naphthol (P=0.0072) were found in urine. In addition, a statistically significant increase in DNA strand break frequencies (P<0.01) and elevated 8-oxodGuo adduct levels (P=0.7819, not statistically significant) were found in the WBCs of exposed workers 3 months after the PAH profile in the binding pitch had been altered. Conclusion: The results presented here show that the increased concentration of naphthalene and/or phenanthrene in the air at the work place could induce the formation of DNA strand breaks and alkali-labile sites in WBCs of exposed workers.