Regulation of A 9-cis-epoxycarotenoid Dioxygenase(NCED)Gene from Raspberry on High Salinity and Cold Tolerance

Abscisic acid(ABA)is a plant hormone that plays an important role in plant development and abiotic stress.The 9-cis-epoxycarotenoid dioxygenase(NCED)is a key rate-limiting enzyme in the ABA biosynthetic pathway.The physiological and molecular mechanisms of NCED regulating plant development and abiotic stress tolerance have been reported in many plant species,but gene function of RiNCEDs in Rubus idaeus L.is rarely reported.In this study,the open reading frame(ORF)sequence of RiNCED2 in red raspberry fruit was isolated and the function of this gene under abiotic stress was investigated.While RiNCED2 was induced by cold,high salinity,drought and ABA,it was highly expressed in new leaves as measured by real-time qPCR.Overexpression of RiNCED2 in Arabidopsis under both high-salt and cold stress increased ABA content,demonstrating that RiNCED2 was involved in ABA biosynthesis.Meanwhile,the leaf wilting degree of transgenic Arabidopsis was less,while the content of malondialdehyde(MDA)was significantly reduced,and the chlorophyll content,proline content,peroxidase(POD),catalase(CAT)and superoxide dismutase(SOD)activities were significantly increased.These results indicated that overexpression of RiNCED2 enhanced the resistance of transgenic Arabidopsis to high salt and cold.

拟南芥Alpha-dioxygenase1的原核表达、纯化及活性的检测

从经水杨酸处理的拟南芥开花期植株中获得cDNA,扩增得到Alpha-dioxygenase 1(DOX1)基因,进行原核表达、纯化和生物活性检测。利用原核表达载体pMAL-c4x在T7 Express CompetentE.coli和BL21(DE3)-RIPL codon+菌株中表达DOX1,经Amylose Resin亲和层析柱纯化。SDS-PAGE结果表明,重组融合蛋白在BL21(DE3)-RIPL codon+中的表达通过灰度值比较分析以可溶性为主,表达率约3.7%,纯化的DOX1纯度可达45%。愈创木酚法表明,可溶性重组蛋白不具有过氧化物酶活性;2,4-DNP法测试表明可溶性重组蛋白具有脂肪酸双加氧酶活性。

三角梅甜菜色素代谢酶4,5-DOPA-dioxygenase基因的分离和表达

甜菜色素在自然界中与花青素排斥分布,主要在石竹目(Caryophyllales)的9个科的植物中合成。虽然甜菜色素的生物合成已经有很多猜测,酶催化的生物合成关键步骤仍然缺少足够的实验数据支持。本研究首先从三角梅属的3个种(B.spectabilis‘Splendens’,B.buttiana‘Mahara’,B.pruviana‘Thimma’)的苞片中分离了甜菜色素合成途径末端的关键酶4,5-DOPA-dioxygenase完整的cDNA,大小分别为902、899、899 bp,分别编码298、297、297个氨基酸,并对这3个同源基因与已经报道的来自B.glabra的该基因进行比较。推测的分子量分别为33 758、33 076、33 233 u,等电点(pI)分别是5.62、5.54和5.94。对分属于4个种(B.spectabilis‘Splendens’,B.buttiana‘Mahara’,B.glabra‘Alba’,B.pruviana‘Thimma’)花的4,5-DOPAdioxygenase的Real-time分析结果表明,该基因在4个种的花中,种间变化较大,总体趋势是开花早期该基因的表达较弱,盛花期表达迅速增强,后期又呈现下降。其中,在红色的B.buttiana‘Mahara’中,该基因的表达最强,在白色的B.glabra‘Alba’中表达最弱。这些结果表明,该基因的表达与花的颜色鲜艳程度直接相关。

Cloning and Functional Characterisation of Carotenoid Cleavage Dioxygenase 4 from Wolfberry

Carotenoid cleavage dioxygenases (CCDs) are a class of enzymes in plants involved in the biosynthesis of apocarotenoids, such as phytohormones, flavour compounds, and other compounds with yet unknown functions. To date, several CCDs have been functionally characterised in plants, but little is known about the CCD4 members. A carotenoid cleavage dioxygenase 4 gene (LcCCD4) was isolated from the leaves of wolfberry (Lycium chinense) to gain insight into its biological function. Multiple sequence alignment and phylogenetic analyses showed that the deduced amino acid sequence of LcCCD4 shares high homology with that of CCD4 proteins from other plants. Expression analysis using semi-quantitative polymerase chain reaction revealed that LcCCD4 was strongly expressed in leaves and flowers and that the expression level was in accordance with β-carotene concentration. LcCCD4 transcripts in fruits tended to decrease as carotenoids accumulated. Recombinant expression of LcCCD4 cleaved β-carotene to produce β-ionone in in vivo assays. These results show that LcCCD4 is a CCD gene that may be involved in producing aromatic apocarotenoids in leaves and flowers, whereas it may be involved in controlling carotenoid accumulation in fruits. © 2016, Tianjin University and Springer-Verlag Berlin Heidelberg.

Indoleamine-2,3-dioxygenase 1/cyclooxygenase 2 expression prediction for adverse prognosis in colorectal cancer

AIM To evaluate indoleamine-2,3-dioxygenase 1/cyclooxygenase 2(IDO1/COX2) expression as an independent prognostic biomarker for colorectal cancer(CRC) patients.METHODS We retrospectively studied the medical records of 95 patients who received surgical resection from August 2008 to January 2010. All patients were randomly assigned to adjuvant treatment with or without celecoxib groups after surgery. We performed standard immunohistochemistry to assess the expression levels of IDO1/COX2 and evaluated the correlation of IDO1/COX2 with clinicopathological factors and overall survival(OS) outcomes.RESULTS The expression of nuclear IDO1 was significantly correlated with body mass index(P < 0.001), and IDO1 expression displayed no association with sex, age, tumor differentiation, T stage, N stage, carcinoembryonic antigen, cancer antigen 19-9, CD3+ and CD8+ tumor infiltrating lymphocytes, and COX2. In univariate analysis, we found that nuclear IDO1(P = 0.039), nuclear/cytoplasmic IDO1 [hazard ratio(HR) = 2.044, 95% confidence interval(CI): 0.871-4.798, P = 0.039], nuclear IDO1/COX2(HR = 3.048, 95%CI: 0.868-10.7, P = 0.0049) and cytoplasmic IDO1/COX2(HR = 2.109, 95%CI: 0.976-4.558, P = 0.022) all yielded significantly poor OS outcomes. Nuclear IDO1(P = 0.041), nuclear/cytoplasmic IDO1(HR = 3.023, 95%CI: 0.585-15.61, P = 0.041) and cytoplasmic IDO1/COX2(HR = 2.740, 95%CI: 0.764-9.831, P = 0.038) have significantly poor OS outcomes for the CRC celecoxib subgroup. In our multivariate Cox model, high coexpression of cytoplasmic IDO1/COX2 was found to be an independent predictor of poor outcome in CRC(HR = 2.218, 95%CI: 1.011-4.48, P = 0.047) and celecoxib subgroup patients(HR = 3.210, 95%CI: 1.074-9.590, P = 0.037).CONCLUSION Our results showed that cytoplasmic IDO1/COX2 coexpression could be used as an independent poor predictor for OS in CRC.

Inhibition of allogeneic T-cell response by Kupffer cells expressing indoleamine 2,3-dioxygenase

AIM:To explore the possibility and mechanism of inhibiting allogeneic T-cell responses by Kupffer cells (KC)pretreated with interferon-γ(IFN-γ)in vitro. METHODS:The expressions of indoleamine 2,3-dioxygenase(IDO)mRNA and FasL mRNA in KC pretreated with IFN-γwere studied with real-time polymerase chain reaction(PCR).The catabolism of tryptophan by IDO from KC was analyzed by high performance liquid chromatography.Allogeneic T-cell response was used to confirm the inhibition of KC in vitro.The proliferation of lymphocytes was detected using[ 3 H]thymidine incorporation.Cell cycle and lymphocyte apoptosis were evaluated by flow cytometric assay. RESULTS:Real-time PCR revealed IDO mRNA and FasL mRNA expressions in KC pretreated with IFN-γ,and IDO catabolic effect was confirmed by a decrease in tryptophan and increase in kynurenine concentration. KC expressing IDO and FasL in BABL/c mice acquired the ability to suppress the proliferation of T-cells from C57BL/6,which could be blocked by addition of 1-methyl-tryptophan and anti-FasL antibody.KC expressing IDO could induce allogeneic T-cell apoptosisCONCLUSION:In addition to Fas/FasL pathway,IDO may be another mechanism for KC to induce immune tolerance.

Indoleamine 2,3-dioxygenase: As a potential prognostic marker and immunotherapeutic target for hepatocellular carcinoma

Tumor cells induce an immunosuppressive microen-vironment which leads towards tumor immune escape. Understanding the intricacy of immunomodulation by tumor cells is essential for immunotherapy. Indoleamine 2,3-dioxygenase(IDO) is an immunosuppressive enzyme which mediates tumor immune escape in various cancers including hepatocellular carcinoma(HCC). IDO up-regulation in HCC may lead to recruitment of regulatory T-cells into tumor microenvironment and therefore inhibit local immune responses and promote metastasis. HCC associated fibroblasts stimulate natural killer cells dysfunction through prostaglandin E2 and subsequently IDO promotes favorable condition for tumor metastasis. IDO up-regulation induces immuno-suppression and may enhance the risk of hepatitis C virus and hepatitis B virus induced HCC. Therefore, IDO inhibitors as adjuvant therapeutic agents may have clinical implications in HCC. This review proposes future prospects of IDO not only as a therapeutic target but also as a prognostic marker for HCC.

Expression of indoleamine 2,3-dioxygenase in a murine model of Aspergillus fumigatus keratitis

AIM： To observe the presence and expression of indoleamine 2,3-dioxygenase（IDO） during the corneal immunity to Aspergillus fumigatus（A. fumigatus） in the murine models.·METHODS： The murine model of fungal keratitis was established by smearing with colonies of A. fumigatus after scraping central epithelium of cornea and covering with contact lenses in C57BL/6 mice. The mice were randomly divided into control group, sham group and A.fumigatus keratitis group. The cornea was monitored daily using a slit lamp and recorded disease score after infection. Corneal lesion was detected by immunofluorescence staining. IDO m RNA and protein were also detected by quantitative reverse transcription-polymerase chain reaction（q RT-PCR） and Western blot.· RESULTS： The disease score and slit lamp photography indicated that disease severity was consistent with corneal inflammation in the murine models, and the disease scores in A. fumigatus keratitis group were obviously higher than those in the sham group. By immunofluorescence staining, IDO was mainly localized in corneal epithelium and stroma in the murine corneal tissues with A. fumigatus keratitis. Compared with the sham group, IDO m RNA expression was significantly enhanced in corneal epithelium infected by A. fumigatus. Furthermore, IDO protein expression detected by Western blot was in accord with transcript levels of IDO m RNA measured by q RT-PCR. IDO protein expression was enhanced after A. fumigatus infection compared with the sham group.·CONCLUSION： IDO is detected in corneal epithelium and stroma locally, which indicates IDO takes part in the pathogenesis of A. fumigatus keratitis and plays a key role in immune regulation at the early stage.

Molecular Cloning and Characterization of a New Cold-active Extradiol Dioxygenase from a Metagenomic Library Derived from Polychlorinated Biphenyl-contaminated Soil

To find new extradiol dioxygenases（EDOs,EC 1.13.11.2）,a metagenomics library was constructed from polychlorinated biphenyl-contaminated soil and was screened for some dioxygenase with aromatic ring cleavage activity.A novel EDO,designated as BphC_A,was identified and heterologously expressed in Escherichia coli.The deduced amino acid sequence of BphC_A exhibited a homology of less than 60% with other known EDOs.Phylogenetic analysis of BphC_A suggests that the protein is a novel member of the EDO family.The enzyme exhibits higher substrate affinity and catalytic efficiency toward 3-methylcatechol than toward 2,3-dihydroxybiphenyl or catechol,the preferred substrate of other known EDOs.The optimum activity of purified BphC_A occurred at pH=8.5 and 35 °C,and BphC_A showed more than 40% of its initial activity at 5 °C.The activity of purified BphC_A was significantly induced by Mn^2＋ and slightly reduced by Al^3＋,Cu^2＋ and Zn^2＋.

Subcloning and high level expression of xylE gene coding for the catechol 2, 3 dioxygenase in E. coli

To construct a high-level expression of xylE gene in E. coli for quick detection of environmental pollution of aromatic compounds. Methods and Results: XylE gene coding for the catechol 2, 3 dioxygenase (CatO2ase ) was amplified from the recombinant plasmid pTG402 by using PCR technique and was subcloned into pUC118N and pUC119N. The single stranded recombinant phage DNA from the transformed E. coli MV1184 cells was used for sequencing. The sequence of xylE gene was proved to be the same as reported. The gene was then subcloned into the high expression plasmid pJLA503, its expression amount being about 34. 2% of the total bacterial proteins. Conclusion: xylE gene is highly expressed in host E. coli TG1.

2-Oxoglutarate-dependent dioxygenases in plants:biosynthesis,mechanism and evolution

2-Oxoglutarate(2OG)-dependent dioxygenases(2-ODDs)are omnipresent iron-containing non-heme enzymes that catalyze various oxidation-reduction reactions in plant growth and development,nucleic acid modification and secondary metabolism.We systematically summarized recent research on the oxidative modifications of plant 2-ODDs and related enzymes,their vital importance in the biosynthesis of plant special metabolites,and their catalytic specificity/flexibility,and discussed the potential of 2-ODD as a new approach for the identification of pivotal genes and the elucidation of biosynthetic pathway.

Expression of the C-Terminal Domain of Mammalian TET3 DNA Dioxygenase in Arabidopsis thaliana Induces Heritable Methylation Changes at rDNA Loci

In plants, demethylation of 5-methylcytosine (5 mC) residues is controlled by DNA glycosylases, while in mammals it requires oxidation of 5 mC by TET proteins, a group of Fe(II)/2-oxoglutaratedependent dioxygenases. We analysed the effects of expressing the C-terminal catalytic domain of the human TET3 gene (TET3c) in Arabidopsis thaliana, using an rDNA region as a methylation reporter. In TET3c transformants, epialleles with hypomethylation or hypermethylation patterns can be induced, which is each stably retained in progeny lines even after removal of the TET3c transgene. In TET3c transformants, 5-hydroxymethylcytosine (5 hmC) marks are detected, indicative of the oxidative activity of the transgenic enzyme. 5-formylcytosine (5 fC) is only detectable in TET3c transformants with a DNA glycosylase mutant background suggesting further oxidation of 5 hmC residues to 5 fC by TET3c, and efficient recognition and removal of 5 fC by plant glycosylases. The results suggest that TET3c can be employed to induce heritable locus-specific changes in DNA methylation, and that accumulation of 5 hmC can be used as a marker for TET3c target regions.

Relationship of Abortion and the Expression of Indoleamine 2,3- dioxygenase (IDO) in Villus and Syncytiotrophoblasts

Objective To study the relationship of abortion and the expression of indoleamine 2, 3- dioxygenase （IDO） in villus and syncytiotrophoblast in vitro. Methods RT-PCR was applied to analyze the mRNA transcription of lDO in villus of normal pregnancy and inevitable abortion and JAR cells as well. Immunohistochemistry was applied to analyze the expression of IDO protein in villus. Western blot was applied to determinate the expression of IDO protein on cultured syncytiotrophoblast. Highperformance liquid chromatography was applied to determinate whether there was kynurenine in cell culture medium of syncytiotrophoblast. Results The expression of IDO mRNA and protein in villus of inevitable abortion was lower than that of normal pregnancy; IDO mRNA did not express in JAR cells. IDO protein expressed on cultured syncytiotrophoblast, and there was kynurenine in cell culture medium of syncytiotrophoblast. Conclusion Appropriate expression of IDO in villus is necessary.for maintenance of normal pregnancy and an active IDO protein expresses in syncytiotrophoblast.

Molecular Cloning and Characterization of Porcine Indoleamine 2,3-Dioxygenase and Its Expression in Various Tissues

In order to confirm the existence of indoleamine 2,3-dioxygenase(IDO) gene in swine,and to clone the novel gene followed by the molecule structure properties and expression pattern analysis,the porcine mRNA sequences homologous to human IDO were obtained from GenBank database by bioinformatics method.By using RT-PCR,the IDO gene was cloned from porcine endothelial cell line and the accuracy of the nucleic acid sequence was confirmed,and the expression pattern of the gene was detected.The three-dimensional structure model of porcine IDO was built referring to the tertiary structure of human IDO using biological sequence analysis software and database.The results showed that the porcine IDO was identified by sequencing.The nucleotide sequences were confirmed as a novel gene after submitted to Genbank.Porcine IDO was expressed in the lung,thymus,epididymis and anterior chamber with a basic level,however in peripheral blood mononuclear cells(PBMCs) the IDO gene was highly expressed.The three-dimensional structure model of porcine IDO was similar to that of human IDO.It was suggested that identification of the structure information of porcine IDO is essential to further investigate the immunologic function of the gene.Study of IDO on NK cells-mediated xenograft rejection will be a novel therapeutic target for the development of xenotransplantation.

Transcriptional Analysis of 9-Cis-Epoxycarotenoid Dioxygenase, Glucosyltransferase, 8＇-Hydroxylase and B-Glucosidase Genes that Regulate Abscisic Acid Homeostasis around the Onset of Grape Berry Ripening

The mechanisms for fine-tuning ABA level related to grape berry ripening remain elusive. Here, transcription analysis showed that the mRNA expression level of 9-cis-epoxycarotenoid dioxygenase gene （VvNCED1） increases first, rapidly in mesocarp before the onset of grape berry ripening. After VvNCED1 peaks its expression level, ABA content increases rapidly in mesocarp coupled with an increase in both soluble sugar content and pH value. On the onset of berry ripening, VvNCED1 transcripts decline rapidly to its lowest point, then increases slightly. Whereas, the mRNA expression level of B-glucosidase gene VvBGI, on the whole, increases constantly during grape berry ripening. During berry de-greening, ABA glucosyltransferase （VvGT） and ABA 8＇-hydroxylase （VvCYPI） equilibrate ABA level; during berry coloring-up, VvGT predominantly equilibrates ABA level, namely, the up-regulation of ABA level mainly leads from VvNCED1 and VvBG1 gene high expression; the down-regulation of ABA level leads mainly from VvCYP! transcript level both in ABA content- and developmental phase-dependence manner. In conclusion, our main results show that VvNCED1 and VvBG1 genes are closely related to grape berry ripening.

26S proteasome inhibitors inhibit dexamethasone-dependent increase of tyrosine aminotransferase and tryptophan 2,3-dioxygenase mRNA levels in primary cultured rat hepatocytes

Dexamethasone (Dex), a ligand for transcriptional enhancement of tyrosine aminotransferase (TAT) and tryptophan 2,3-dioxygenase (TO) genes, (100 nM) maximally increased these mRNA levels at 12 h and 7 h in primary cultured rat hepatocytes and the nuclear fraction, respectively. Lactacystin (5 μM) and epoxomicin (0.5 μM), 26S proteasome inhibitors, significantly suppressed the Dex-dependent maximum increase of TAT and TO mRNA levels in the cells and the nuclear fraction. Electrophoretic mobility shift assay demonstrated that lactacystin did not affect binding of glucocorticoid receptor to glucocorticoid responsive element. Furthermore, lactacystin did not affect the activation of GRE luciferase reporter by Dex transfected to the cells. The results demonstrate that 26S proteasome is positively involved in the Dex-dependent increase of TAT and TO mRNA levels in the cells and suggest that the mechanism of action of 26S proteasome may be degradation of some RNase(s), which breaks down TAT and TO mRNAs.

干酪乳杆菌对母婴分离诱导子代抑郁大鼠外周及中枢神经炎症的影响

目的观察干酪乳杆菌对产后进行母婴分离(maternal separation,MS)诱导子代大鼠抑郁样行为的影响,探索益生菌改善抑郁样行为潜在的中枢神经炎症的作用机制。方法采用母婴分离应激方法建立子代抑郁动物模型,并将SD子鼠采用随机数字表法分为对照组(CON组)、母婴分离组(MS组)、母婴分离+干酪乳杆菌组(MS+干酪乳杆菌组),每组8只。其中对照组雌鼠在产后与子鼠不进行母婴分离干预;母婴分离组雌鼠在正常分娩后与子鼠每天分离3 h,连续分离14 d;MS+干酪乳杆菌组的子鼠在MS组基础上给予为期4周的干酪乳杆菌干预,每天按体质量灌胃8×10^(8) CFU/(kg·d)。采用糖水偏好实验(SPT)和强迫游泳实验(FST)评估各组子代大鼠的抑郁样行为,采用ELISA法测定各组子代大鼠血清和海马组织中TNF-α、IL-6、IL-1β、IL-10的水平,采用荧光实时定量PCR法检测各组子代大鼠海马组织中IDO1及5-HT mRNA的表达水平。结果与对照组相比,母婴分离组子代大鼠的糖水偏好百分比明显降低(P<0.05),虽然自发活动实验中穿格次数的减少差异无统计学意义,但强迫游泳实验中的不动时间显著延长(P<0.05),说明母婴分离组子代大鼠产生了明显的抑郁样行为。而干酪乳杆菌显著改善了母婴分离应激引起的子代大鼠的行为学变化,明显提高了糖水偏好百分比(P<0.05),缩短了强迫游泳的不动时间(P<0.05)。与MS组相比,MS+干酪乳杆菌组子代大鼠海马和外周血中IL-6、IL-1β、TNF-α的含量显著降低(P<0.01),IL-10的含量显著增加(P<0.05);子代大鼠海马中IDO1 mRNA的表达显著降低(P<0.01),5-HT mRNA的表达显著升高(P<0.01)。结论母婴分离诱导子代大鼠产生了抑郁样行为,干酪乳杆菌通过调节外周血和海马炎性因子的表达,抑制海马中IDO1的表达、增加海马中5-HT的表达,可改善子代大鼠抑郁样行为。

比较蛋白质组学揭示茉莉酸甲酯诱导的雷公藤甲素生物合成

目的:探索雷公藤甲素生物合成途径,以提高其产量或用于合成生物学异源生产。方法:使用无标记(lable-free)比较蛋白质组学对茉莉酸甲酯(MeJA)诱导的雷公藤悬浮细胞进行测序,通过京都基因与基因组百科全书(KEGG)、基因本体(GO)分析等方法,结合基因转录表达分析和基因体内功能研究,逐步筛选参与雷公藤甲素生物合成的功能基因。结果:MeJA诱导不同时间的样品组鉴定得到376个差异蛋白质,包括8个细胞色素P450酶(CYP450)、3个转录因子(TF)和2-氧戊二酸依赖的双加氧酶(2ODD)。其中CYP71BE89、CYP82AQ2和Tw2ODD基因在MeJA诱导及植物不同组织部位中,均与已知参与雷公藤甲素母核环化的二萜合酶TwTPS7(v2)和TwTPS27(v2)基因具有相似的表达模式,表明其可能与雷公藤甲素生物合成相关。进一步利用植物细胞体内功能研究,验证了CYP71BE89、CYP82AQ2和Tw2ODD基因干扰使雷公藤甲素积累分别降低了24.1%、41.4%和71.4%,表明了3个基因与雷公藤甲素生物合成直接相关。结论:利用比较蛋白质组学技术揭示了几种与雷公藤甲素生物合成相关的蛋白,进一步表征了雷公藤甲素生物合成与外源激素刺激之间的关系,并为发掘中药活性成分生物合成途径提供了一种有效方法。

TET2重塑CXCR4 DNA甲基化对急性心肌梗死小鼠心肌组织自噬、炎症反应及凋亡的影响

目的:探究急性心肌梗死(AMI)过程中内皮细胞tet甲基胞嘧啶双加氧酶2(TET2)对趋化因子受体4(CXCR4)DNA甲基化的影响以及对AMI小鼠心肌组织自噬、炎症反应及组织细胞凋亡的影响机制,为临床探究AMI发展的分子机制提供理论依据。方法:8周龄雄性C57/BL6小鼠50只,制备AMI模型,尾部注射TET2、CXCR4过表达质粒;蛋白免疫印迹(Western Blot)法检测心肌组织TET2、CXCR4、微管相关蛋白3(LC3)、P62、B细胞淋巴瘤/白血病-2基因(Bcl-2)关联X蛋白(Bax)、半胱氨酸蛋白酶3(Caspase-3)、Bcl-2表达;甲基化检测CXCR4 DNA甲基化水平;酶联免疫吸附法(ELISA)检测心肌组织内炎性因子白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)水平;原位末端转移酶标记技术(TUNEL)检测各组心肌组织细胞凋亡指数。结果:与假手术组比较,模型组心肌组织内TET2、CXCR4均表达上调,TET2、CXCR4均在心肌组织内过表达,TET2过表达促进CXCR4表达,差异有统计学意义(P<0.05);与模型组比较,TET2 mimic组CXCR4启动子区域DNA甲基化程度降低,CXCR4蛋白表达升高,差异有统计学意义(P<0.05);与假手术组比较,模型组小鼠心肌组织自噬蛋白LC3、抑制细胞凋亡蛋白Bcl-2表达下调,炎性因子IL-6、TNF-α、IL-1β水平、自噬蛋白P62、促细胞凋亡蛋白Bax、cleaved Caspase-3表达上调,差异有统计学意义(P<0.05);TET2、CXCR4过表达进一步下调LC3、Bcl-2蛋白表达,上调炎性因子IL-6、TNF-α、IL-1β水平,P62、Bax、cleaved Caspase-3蛋白表达;TET2、CXCR4二者联合体现出最低LC3、Bcl-2蛋白表达,最高炎性因子IL-6、TNF-α、IL-1β水平以及P62、Bax、cleaved Caspase-3蛋白表达,差异有统计学意义(P<0.05)。结论:AMI发展中,TET2通过降低CXCR4 DNA甲基化,促进CXCR4基因表达,进而抑制AMI小鼠心肌组织自噬,上调炎症反应及细胞凋亡程度,促进疾病发展。

肠道菌群通过Treg/IDO信号通路参与动脉粥样硬化进展的实验研究

目的:探讨肠道菌群通过调节性T细胞(Treg)/吲哚胺2,3-双加氧酶-1(IDO1)轴信号通路参与动脉粥样硬化的进展。方法:将C57BL无特定病原体(SPF)级的5周雌性载脂蛋白E基因敲除(ApoE^(-/-))小鼠随机分为普通饲料处理组(NC组)和高脂饮食(含0.2%胆固醇,42%脂肪)处理组(HF组),每组10只。通过主动脉表面油红O染色和苏木精-伊红染色分析主动脉斑块和硬化损伤面积。采用流式细胞术分析脾细胞中CD_(4)^(+)CD_(25)^(+)叉头框蛋白p3(Foxp3)+Treg在CD_(4)^(+)T细胞中的百分比;蛋白免疫印迹法定量动脉吲哚胺2,3-双加氧酶(IDO),聚合酶链式反应(PCR)进行粪便微生物群分析。结果:与NC组比较,HF组小鼠主动脉斑块和硬化损伤面积增加(P<0.001),CD_(4)^(+)CD_(25)^(+)Foxp^(3+)/CD_(4)^(+)T比例和Foxp3 mRNA水平均降低(P<0.001)。与NC组比较,HF组小鼠IDO1含量降低(P<0.001)。小鼠斑块与Treg、IDO1呈负相关(r值分别为-0.87,-0.66,P<0.01)。厚壁菌门与动脉粥样硬化斑块呈正相关(r=0.64,P<0.01);放线菌与动脉粥样硬化斑块呈负相关(r=-0.74,P<0.01)。厚壁菌门与IDO1呈负相关(r=-0.59,P<0.05);拟杆菌门与IDO1呈正相关(r=0.67,P<0.01)。厚壁菌门与Treg呈负相关(r=-0.63,P<0.01);变形菌门与Treg呈正相关(r=0.61,P<0.01)。结论:肠道菌群改变可能通过Treg/IDO1信号通路,参与高脂饮食诱导的动脉粥样硬化进展。