左金丸对IEC6、HCT116细胞共培养模型肠上皮屏障功能的保护及机制研究

目的 研究左金丸对IEC6、HCT116细胞共培养模型肠上皮屏障功能的影响,并初步探讨其作用机制。方法 采用左金丸醇提物低、中、高剂量(5、10、20μg·mL^(-1))处理HCT116、IEC6细胞,采用MTT法检测细胞增殖情况。通过体外构建IEC6、HCT116细胞共培养体系模拟结肠癌微环境,以左金丸(5、10、20μg·mL^(-1))进行干预;检测IEC6细胞跨膜电阻(TEER)值及细胞通透性;采用高效液相色谱(HPLC)法检测HCT116、IEC6细胞多胺含量;微量法检测HCT116细胞的葡萄糖消耗量、乳酸及ATP含量;Western Blot法检测IEC6细胞Zonula occluden 1(ZO-1)、Occludin蛋白和HCT116细胞鸟氨酸脱羧酶(ODC)、精眯/精胺N1-乙酰基转移酶(SSAT)、鸟氨酸脱羧酶抗酶1(OAZ1)、SLC22A1、缺氧诱导因子1(HIF1α)、C-Myc、己糖激酶2(HK2)、丙酮酸激酶M2亚型(PKM2)、丙酮酸脱氢酶激酶1(PDK1)、葡萄糖转运体1(GLUT1)、乳酸脱氢酶(LDHA)蛋白表达情况。结果 (1)与对照组比较,左金丸低、中、高剂量(5、10、20μg·mL^(-1))处理HCT116细胞24、48、72 h后,细胞增殖抑制率显著升高(P<0.01);左金丸低、中、高剂量(5、10、20μg·mL^(-1))处理IEC6细胞24 h后,细胞增殖抑制率无明显变化(P>0.05)。(2)与IEC6细胞单培养组比较,共培养模型组细胞的TEER及FD4荧光强度显著降低(P<0.01);紧密连接蛋白ZO-1、Occludin表达量显著降低(P<0.01);IEC6细胞的多胺含量显著降低(P<0.01)。与HCT116细胞单培养组比较,共培养模型组HCT116细胞的多胺含量显著增加(P<0.01)。与共培养模型组比较,左金丸中、高剂量组细胞的TEER及FD4荧光强度显著升高(P<0.01);左金丸高剂量组IEC6细胞ZO-1、Occludin蛋白表达明显上调(P<0.05,P<0.01);左金丸低、中、高剂量组的IEC6细胞多胺含量显著增加(P<0.01),而左金丸中、高剂量组HCT116细胞的多胺含量显著降低(P<0.01)。(3)与对照组比较,左金丸能明显下调HCT116细胞多胺合成酶ODC、多胺转运体SLC22A1蛋白的表达(P<0.05,P<0.01),明显上调SSAT、OAZ1蛋白表达(P<0.05,P<0.01);能够明显抑制HCT116细胞的葡萄糖消耗量、乳酸生产量及ATP含量(P<0.05,P<0.01);明显下调糖酵解通路HIF1α、C-Myc、HK2、PKM2、PDK1、GLUT1、LDHA蛋白表达(P<0.05,P<0.01)。结论 左金丸对IEC6、HCT116细胞共培养模型肠上皮屏障功能具有保护作用,对结肠癌HCT116细胞具有增殖抑制作用,可能与其调节HCT116细胞多胺代谢、转运以及抑制肿瘤糖酵解水平有关。

Effects of Echinacea purpurea Polysaccharide on IEC-6 Cell Proliferation

[Objective] This study was conducted to investigate the effects of Echi-nacea purpurea polysaccharides （EPS） on proliferation of rat intestinal epithelial cel IEC-6. [Method] The proliferation rate of IEC-6 cel s cultured in EPS at different concentrations and for different time was measured by MTT assay and analyzed by statistic methods. [Result] The proliferation rate of IEC-6 cel s cultured in EPS at al the concentrations and for different time was improved by different extents in com-parison with the control. In detail, 50 and 200 μg/ml EPS greatly improved the IEC-6 cel proliferation after 24 h of culture; then, the cel proliferation rate in the two treatments increased from 24 to 48 h, and declined from 48 to 72 h. The cel pro-liferation was also significantly improved by culturing in 100 μg/ml EPS for 72 h and in 500 μg/ml EPS for 48 h. After 48 h of culture, the proliferation rate of IEC-6 cel increased in a EPS dose-dependent manner. [Conclusion] EPS can promote IEC-6 cel proliferation, and thus improve the intestinal mucosal absorption and immune function of rat.

Ephrin-B reverse signaling induces expression of wound healing associated genes in IEC-6 intestinal epithelial cells

AIM： Eph receptors and ephrin ligands play a pivotal role in development and tissue maintenance. Since previous data have indicated an involvement of ephrin-B2 in epithelial healing, we investigated the gene expression and downstream signaling pathways induced by ephrin-B mediated cell-cell signaling in intestinal epithelial cells. METHODS： Upon stimulation of ephrin-B pathways in IEC-6 cells with recombinant rat EphB1-Fc, gene expression was analyzed by Affymetrix rat genome 230 high density arrays at different time points. Differentially expressed genes were confirmed by real-time RT-PCR. In addition, MAP kinase pathways and focal adhesion kinase （FAK） activation downstream of ephrin-B were investigated by immunoblotting and fluorescence microscopy. RESULTS： Stimulation of the ephrin-B reverse signaling pathway in IEC-6 cells induces predominant expression of genes known to be involved into wound healing/cell migration, antiapoptotic pathways, host defense and inflammation. Cox-2, c-Fos, Egr-1, Egr-2, and MCP-1 were found among the most significantly regulated genes. Furthermore, we show that the expression of repair- related genes is also accompanied by activation of the ERKI/2 MAP kinase pathway and FAK, two key regulators of epithelial restitution. CONCLUSION： Stimulation of the ephrin-B reverse signaling pathway induces a phenotype characterized by upregulation of repair-related genes, which may partially be mediated by ERK1/2 pathways.

Differential activation of mitogen-activated protein kinases by γ-irradi-ation in IEC-6 cells: Role of intracellular Ca^(2+)

Objective: To explore the effects of γ-irradiation on mitogen-activated protein kinases (MAPKs) and role of intracellular calcium in this event in intestinal epithelial cell line 6 (IEC-6 cells). Methods: After cultured rat IIEC-6 cells with or without the pretreatment of intracellular Ca2+ chelator were exposed to Y-ir-radiation of 6 Gy, the total and phosphorylated MAPKs in the cells were determined with Western blotting and apoptosis was examined with flow cytometry. Activities of Extracellular signal-regulated protein kinase (ERK) and p38 MAPK were determined by using immuoprecipitation followed by Western blotting. Results: In response to γ-irradiation, phosphorylation of ERK was not significantly observed, while the levels of phosphorylated c-Jun NH2-terminal kinase (JNK) and p38 MAPK were increased in 30 min and reached the peak 2 h after exposure to 6 Gy γ-irradiation, though the cell viability was significantly lowered 12 h. On the other hand, no obvious changes were seen in the total protein levels of ERK, JNK and p38 MAPK. Chelation of intracellular Ca2+ almost completely suppressed the JNK and p38 MAPK phosphorylation induced by γ-irradia-tion, but removal of external Ca2+ had no such effect. Activation of p38 MAPK, but not of ERK, was seen to have a correlation with γ-irradiation induced apoptosis. Conclusion: The results suggest that γ-irradiation is a potent activator for JNK and p38 MAPK, and Ca2+ mobilized from intracellular stores plays an important role in the activation of MAPKs and the induction of apoptosis in IEC-6 cells.

金荞麦提取物对猪肠道上皮屏障因子和炎症因子的影响

为了解金荞麦提取物对猪肠道上皮屏障因子和炎症因子的影响,试验用60%乙醇提取金荞麦茎叶有效成分,培养猪小肠上皮细胞(IEC),用不同浓度金荞麦茎叶提取物(10、50、100、250μg/mL)处理IEC,48 h后刮取IEC,提取RNA,实时荧光定量RT-PCR测定肠道炎症因子TNF-α、IL-6、IL-8、IL-22、IL-10和肠道屏障相关因子ZO-1、Occludin的表达。结果:金荞麦茎叶提取物为黑色粉末或块状物,提取率为13.1%;不同浓度金荞麦提取物均可下调炎症相关基因的表达并显著升高抗炎因子IL-10的表达,肠道屏障相关因子ZO-1、Occludin的表达升高。结果表明:金荞麦茎叶乙醇提取物浓度为250μg/mL时可显著抑制肠道炎症因子,并强化肠道上皮细胞的紧密连接屏障。

穿心莲内酯在镉致IEC6细胞线粒体自噬中的作用

为探讨穿心莲内酯(Ad)在镉诱导肠上皮细胞线粒体自噬中的作用,体外培养IEC6细胞,以5μmol/L镉处理不同时间(0、6、12 h),利用荧光显微镜观察细胞中外源性GFP-RFP-LC3聚点的变化。用7μmol/L Ad预处理IEC6细胞16 h后,5μmol/L镉处理细胞12 h,利用Western-blot方法检测线粒体自噬相关蛋白p62、LC3、PINK1、Parkin的表达水平;利用荧光显微镜观察GFP-RFP-LC3和Bcl-XL在细胞中分布的变化。结果,5μmol/L镉处理IEC6细胞不同时间后,红色LC3聚点数量增多,与6 h组相比,镉处理细胞12 h后,LC3红色聚点数量下降;Western-blot结果显示,与镉单独处理组相比,Ad与镉共处理组Parkin蛋白表达水平升高,p62、LC3-II、PINK1表达水平降低;荧光观察结果显示,与镉单独处理组相比,Ad与镉共处理组LC3、Bcl-XL红色信号增强,绿色信号减弱。上述结果表明,镉能够引起IEC6细胞自噬流阻滞,Ad可以通过缓解镉诱导的自噬流阻滞,促进镉致IEC6细胞线粒体自噬的发生。

Plating densities, alpha-difluoromethylornithine effects and time dependence on the proliferation of IEC-6 cells

OBJECTIVE: To characterize the role of plating densities and alpha-difluoromethylornithine (DFMO) on the proliferation of IEC-6 cells in vitro. METHODS: IEC-6 cells were seeded in 96-well microplates at various densities in the presence or absence of DFMO. Cells were counted and their proliferative capability was monitored Days 1 to 7 with MTT assay at an optical density of 570 nm. RESULTS: There was a positive relationship between cell number and OD value (r = 0.954, P 0.5 x 10(4) cells/well) inhibited the growth of cells on Day 2. When the density reaches 4 x 10(4) cells/well, the OD value increased gradually and reached a peak on Day 5. After that, the OD value began to fall. The growth of IEC-6 cells was limited at a low density (0.2 x 10(4) cells/well) on Day 4. DFMO caused a complete inhibition of proliferation of IEC-6 cells on Days 1 to 3. CONCLUSION: Proliferation of IEC-6 cells is related to plating density and incubation time. It is inhibited by DFMO, but is reversible when the incubation time is prolonged.

Magnolol pretreatment attenuates heat stress-induced IEC-6 cell injury

Objective： Heat stress （HS） is an important environmental stressor that adversely influences livestock during the summer. The aim of this study was to investigate whether magnolol protects against HS-induced intestinal epithelial cell injury. Materials and methods： An intestinal epithelial cell line （IEC-6） was subjected to HS at 42℃, with and without magnolol pretreatment. Cell injury was detected by monitoring lactate dehydrogenase （LDH） release. MTS （3-（4,5-dimethyithiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium） assay was used to as- sess cell proliferation and viability, including identifying effective concentrations of magnolol. Flow cytometry confirmed Gl-phase cell-cycle arrest and its alleviation by magnolol. Active DNA synthesis was measured by incorporation of nucleic acid 5-ethynyl-2＇-deoxyuridine （EdU）. Gl-phase cell-cycle-related gene expression was assessed by real-time reverse transcription polymerase chain reaction （RT-PCR） and levels of Gl-phase-related proteins by Western blotting Results： HS induced IEC-6 cell injury and decreased cell viability, as demonstrated by data from LDH and MTS assays respectively. Based on a number of criteria, IEC-6 cells subjected to HS were arrested in the G1 phase Of the cell cycle. Magnolol pretreatment decreased HS-induced cell injury through relief of this cell-cycle arrest. Conclusions： Magnolol pretreatment attenuates HS-induced injury in IEC-6 cells. Magnolol is potentially promising as a protective strategy for HS in livestock.

益生菌用于疾病治疗的研究进展

益生菌是一类可对机体产生有益作用的微生物,可通过改善宿主肠道微生物平衡从而对机体产生有利影响。益生菌逐渐成为研究热点,其积极作用也日益体现。本文简述益生菌与肠道上皮细胞(IEC)相互作用的机制,并进一步综述益生菌用于胃肠道疾病、癌症、获得性免疫缺陷综合征(AIDS)及中枢神经系统(CNS)疾病治疗的研究进展,有助于更深入地探索肠道菌群对宿主的影响,旨在为益生菌用于疾病治疗提供理论基础。