基于ERIC-PCR指纹图谱技术的小鼠肠道菌群失调分析方法的建立

目的采用常规菌群分析方法和ERIC-PCR技术对正常小鼠和抗生素相关性腹泻小鼠模型分别进行肠道菌群检测,结合细菌培养和DNA指纹图谱检测结果分析小鼠肠道内主导菌群数量和种类的改变情况,建立利用ERIC-PCR技术分析小鼠肠道菌群失调的检测方法。方法先利用常规菌群分析方法鉴定正常小鼠和抗生素相关性腹泻小鼠的菌群状况,再提取其基因组DNA,最后以肠杆菌科基因间重复序列(ERIC)为模板,利用ERIC-PCR方法获得正常小鼠和模型小鼠的肠道菌群指纹图谱,与常规菌群分析结果作比较,验证ERIC-PCR技术的准确性。结果常规菌群分析结果表明四种优势菌群在数量上出现明显的变化,证实造模成功。经ERIC-PCR技术成功获得两组小鼠粪便基因组DNA图谱,两组间呈现具有一定对比性的特异性指纹图谱。结论从小鼠粪便基因组经ERIC-PCR后的图谱中特异性条带的分布、数目和亮度来看,能说明肠道菌群的分布状况存在明显差异,结合常规菌群分析方法作对比,说明ERIC-PCR技术是一种分析小鼠肠道菌群失调高效快捷的检测方法。     

小鼠肠道菌群代谢产物与糖尿病的相关性研究

目的研究糖尿病小鼠粪便中肠道菌群代谢产物与血糖之间的相关性,探讨肠道菌群与糖尿病之间的关系。方法采用高脂饮料喂养加腹腔注射链脲佐菌素(STZ)的方法建立糖尿病小鼠模型;将实验动物随机分为正常组、高脂组、糖尿病组及模型给药组,连续给药5周后,采血测血糖血脂,同步收集动物粪便,测粪便中短链脂肪酸(Short-chain fatty acids,SCFA)及D-乳酸。SCFA的检测使用气相色谱法,D-乳酸的检测使用紫外酶促法。结果糖尿病组小鼠粪便中乙酸、丙酸和正丁酸含量明显低于正常组及高脂组(P<0.01),D-乳酸含量明显高于正常组及高脂组(P<0.01);给药组乙酸、丙酸和正丁酸含量明显高于糖尿病组(P<0.01),D-乳酸含量明显低于糖尿病组(P<0.01)。给药组丙酸、正丁酸的含量与正常组间差异无统计学意义(P>0.05),但乙酸的含量仍低于正常组(P<0.01),D-乳酸的含量仍高于正常组(P<0.01)。结论糖尿病小鼠粪便中的肠道菌群代谢产物与血糖之间存在着密切的关系,代谢产物的差异性,提示肠道菌群的差异性,反映出糖尿病小鼠存在肠道菌群紊乱。     

大蒜素对急性酒精性肝损伤小鼠 肠道菌群失调的预防作用研究

目的应用肠杆菌基因间重复共有序列基因扩增(ERIC-PCR)和聚合酶链式反应—变性梯度凝胶电泳(PCR-DGGE)研究大蒜素对急性酒精性肝损伤小鼠肠道菌群失调的预防作用。方法 SPF级昆明小鼠40只,分为正常对照组、急性酒精性肝损伤组、护肝片组、大蒜素高和低浓度组,每组8只。灌服相应药物30d,除正常对照组,其他组灌胃14 mL/kg红星二锅头,12h后收集鼠便,提取粪便细菌DNA,利用ERIC-PCR和PCR-DGGE电泳获得肠道菌群指纹图谱,主成分分析(PCA)和聚类分析(UPGMA)研究肠道菌群整体差异并鉴定优势条带序列。结果 ERIC-PCR表明急性酒精性肝损伤小鼠肠道中细菌条带较少,以300bp和500bp左右的条带为特征条带,PCR-DGGE显示肠球菌为优势菌型;大蒜素灌胃小鼠肠道菌群结构组成发生改变,优杆菌属为优势菌型。结论大蒜素预防给药可以扶持肠道中优杆菌属等益生菌生长,抑制肠球菌等病原菌的增殖,调节急性酒精性肝损伤伴有的肠道菌群失调。     

大蒜素对急性酒精性肝损伤小鼠肠道菌群失调的预防作用

目的应用肠杆菌基因间重复共有序列基因扩增(ERIC-PCR)和聚合酶链式反应—变性梯度凝胶电泳(PCR-DGGE)研究大蒜素对急性酒精性肝损伤小鼠肠道菌群失调的预防作用。方法 SPF级昆明小鼠40只,分为正常对照组、急性酒精性肝损伤组、护肝片组、大蒜素高和低浓度组,每组8只。灌服相应药物30d,除正常对照组,其他组灌胃14 mL/kg红星二锅头,12h后收集鼠便,提取粪便细菌DNA,利用ERIC-PCR和PCR-DGGE电泳获得肠道菌群指纹图谱,主成分分析(PCA)和聚类分析(UPGMA)研究肠道菌群整体差异并鉴定优势条带序列。结果 ERIC-PCR表明急性酒精性肝损伤小鼠肠道中细菌条带较少,以300bp和500bp左右的条带为特征条带,PCR-DGGE显示肠球菌为优势菌型;大蒜素灌胃小鼠肠道菌群结构组成发生改变,优杆菌属为优势菌型。结论大蒜素预防给药可以扶持肠道中优杆菌属等益生菌生长,抑制肠球菌等病原菌的增殖,调节急性酒精性肝损伤伴有的肠道菌群失调。     

中药复方对醋酸胃溃疡脾虚大鼠的治疗作用比较

ERIC-PCR(enterobacterial repetitive intergenic consensus-PCR)指纹图谱方法研究四君子汤、理中汤、补中益气汤、益胃汤四种中药复方对醋酸胃溃疡脾虚大鼠的治疗作用.方法:利用食醋灌胃塑造大鼠醋酸胃溃疡脾虚模型,分别提取大鼠健康、模型和复方中药治疗后粪便中的肠道菌群总DNA,并进行ERIC-PCR,建立醋酸模型脾虚大鼠肠道菌群ERIC-PCR指纹图谱,确定其相关特征条带,以各阶段特征条带的净面积和相似性系数为指标,比较四种复方对醋酸胃溃疡脾虚大鼠的治疗作用.结果;益胃汤低中高剂量和四君子汤中剂量治疗后,特征条带500bp净面积显著性降低,相似性系数与模型组相比显著性增加,疗效较好.结论:醋酸模型脾虚主要造成胃溃疡,单纯补气健脾中药治疗效果不明显,益胃汤通过滋养脾胃,对胃溃疡型脾虚有较好治疗效果.     

ERIC-PCR和PCR-DGGE技术分析壳聚糖对抗生素引起肠道菌群失调小鼠的影响

目的应用肠杆菌基因间重复共有序列基因扩增(ERIC-PCR)和聚合酶链式反应——变性梯度凝胶电泳(PCR-DGGE)分析壳聚糖对抗生素致肠道菌群失调小鼠的影响。方法 SPF级昆明小鼠24只,每组8只,依次分为模型组(N)、壳聚糖高(CS-H)和低浓度组(CS-L),盐酸左氧氟沙星灌胃6d后使用相应药物灌胃24d,收集鼠便,提取粪便细菌DNA,利用ERIC-PCR和PCR-DGGE电泳获得肠道菌群指纹图谱,主成分分析(PCA)和聚类分析(UPGMA)研究肠道菌群整体差异,并鉴定优势条带序列。结果 ERIC-PCR表明菌群失调组小鼠肠道中细菌条带减少明显,以300bp左右的条带为特征条带,PCR-DGGE显示屎肠球菌为优势菌型;壳聚糖灌胃小鼠肠道菌群结构组成发生改变,乳酸菌成为优势菌型。结论壳聚糖扶持乳酸菌等益生菌、抑制肠球菌等病原菌的增殖,起到益生元的作用进而调节肠道微生态均衡。     

ERIC-PCR指纹图谱技术分析糖尿病小鼠肠道细菌群落变化

目的通过比较1型糖尿病模型组和空白对照组雄性小鼠肠道菌群结构的变化,探索糖尿病造模与肠道菌群的关系。方法收集造模2周后空白对照组(n=5)、STZ造模成功组(n=5)和造模不成功组(n=3)ICR小鼠的新鲜粪便样品,提取粪便样品的总DNA,ERIC-PCR扩增形成DNA指纹图谱,借助多变量统计分析方法研究各组样品肠道菌群结构上的异同。结果ERIC-PCR指纹图谱结合偏最小二乘法(PLS-DA)分析表明造模成功组和造模不成功组小鼠的肠道菌群结构显著区别于空白对照组,而造模不成功组小鼠的肠道菌群结构与造模成功组仍有一定的区别。结论STZ诱导的1型糖尿病会造成小鼠的肠道菌群结构的变化,而部分小鼠造模失败可能与这些小鼠的肠道菌群结构有关。     

基于16S rRNA基因和细菌基因组间重复序列的DNA指纹图谱技术对肝硬化大鼠肝移植后肠道菌群多样性的研究

目的应用PCR-DGGE和rep-PCR技术对肝硬化大鼠肝移植后肠道菌群多样性进行研究,探讨肠道菌群多样性的变化,并比较这两种方法在菌群分析中的作用。方法首先建立CCL4诱导肝硬化大鼠的肝移植模型,收取对照组、肝硬化成模时、肝移植后7 d和肝移植后30 d的大鼠粪便,提取细菌基因组DNA,采用PCR-DGGE和rep-PCR[BOX-PCR,ERIC-PCR,ERIC2-PCR,(GTG)5-PCR,REP-PCR]进行DNA指纹图谱分析。结果PCR-DGGE可明确将正常大鼠、肝硬化大鼠、肝移植大鼠分为3个簇,并显示出肝硬化、肝移植大鼠肠道菌群多样性明显增多。rep-PCR技术也可将各组分开,其中ERIC-PCR、ERIC2-PCR及REP-PCR三者扩增条带各组差异有显著性,鉴别效果更好。结论应用基于16S rRNA基因和细菌基因组间重复序列的指纹图谱技术对肝硬化大鼠肝移植后肠道微生态的研究具有重要价值,可对临床肝硬化、肝移植患者肠道微生态制剂的使用起到指导作用。     

组方食品对2型糖尿病小鼠血糖及 肠道菌群的影响

目的研究含有浒苔等植物的功能性食品对糖尿病小鼠血糖浓度的影响,并应用PCR-DGGE技术评价其对昆明小鼠肠道菌群稳态的影响。方法采用高脂饲料喂养昆明小鼠加腹腔注射链脲佐菌素(STZ)的方法建立糖尿病小鼠模型;将实验动物随机分为正常对照组、自然恢复组和功能性食品喂养组,连续给药4周,尾静脉采血测量血糖水平。收集小鼠新鲜粪便,提取粪便细菌基因组DNA,通过PCR-DGGE获得细菌群落指纹图谱,并进行相关软件分析,同时切离差异显著条带进行序列分析。结果本实验采用的功能性食品对糖尿病小鼠有降糖作用,并使血糖值稳定地维持在较低水平。4周后,功能性食品喂养的糖尿病小鼠血糖水平相对普通饲料喂养的糖尿病小鼠血糖发生显著性下降(t=4.19,P0.01);给2型糖尿病小鼠提供普通饲料时,其肠道菌群种类和数目相对较少;而提供功能性食品时,肠道菌群种类和数目相对增多,特别是双歧杆菌、Prevotella oryzae、Barnesiella intestinihominis、Culturomica和Parabacteroides distasonis明显增多,但是Muribaculum较少。结论高脂饲料结合STZ诱导的2型糖尿病小鼠肠道菌群发生显著改变,组方食品通过扶持肠道菌群,降低血糖水平。     

健康儿童与发育不佳儿童肠道菌群结构的比较研究

目的对健康儿童与发育不佳(FTT)儿童肠道中微生物区系的ERIC-PCR指纹图谱异同进行研究。方法根据美国疾病预防控制中心(CDC)对儿童生长发育的评价指标对某幼儿园200例4～6岁儿童进行评价,筛选出16例健康儿童和13例FTT儿童,每周1次连续3周跟踪取样,提取粪便样品中细菌总DNA,获得其ERIC-PCR指纹图谱,再将其中一个样品的ERIC-PCR产物作为混合探针通过杂交对指纹图谱上DNA条带序列的异同进一步比较。结果同一个体的肠道菌群结构在取样期间稳定性较好;虽然健康儿童间的肠道菌群结构也有一定差异,但它们却有着共同的结构特征;而健康儿童与FTT儿童的肠道菌群结构差异较大。结论儿童发育状况与肠道菌群结构有一定的关系。     

基于ERIC-PCR技术研究酪蛋白糖巨肽对小鼠肠道菌群结构的影响

目的了解酪蛋白糖巨肽(CGMP)对小鼠肠道中微生物群落结构及其动态变化的影响。方法采用ER IC-PCR技术分析鉴定在灌胃小鼠CGMP期间其肠道菌群结构的变化情况。在实验的第0、3、5、7、10、15和21天(灌胃停止后1周)分别提取对照组和CGMP组小鼠粪便总DNA,以此为模板进行PCR反应,获得肠道微生物群落的ER IC-PCR指纹图谱。结果小鼠个体在一段时间内微生物群落演替不明显,群落相似性较高。对照组小鼠肠道菌群多样性指数范围为1.75±0.06,CGMP组多样性指数范围为1.89±0.04,二者之间差异有统计学意义。结论小鼠肠道内的微生物群落非常丰富,普遍存在共有的优势菌群,且优势菌群的群落结构较为稳定;CGMP能够显著增加小鼠肠道菌群的多样性;聚类分析结果显示:对照组小鼠个体在不同时间的肠道菌群结构相似性较高,且ER IC-PCR指纹图谱没有明显的规律;CGMP组小鼠个体的ER IC-PCR指纹图谱被明显地分成2个亚族,说明小鼠灌胃CGMP 3~5 d后,其肠道菌群的群落结构开始发生明显变化。     

高脂饮食中添加短链菊粉对小鼠肠道菌群的影响

目的:探究高脂饮食中添加短链菊粉对小鼠肠道菌群的影响。方法:选择8周龄雄性小鼠,5只喂食高脂饲料,5只喂食10%菊粉复合型高脂饲料,喂食8周后收集小鼠粪便,检测小鼠粪便中三种主要的短链脂肪酸。同时,提取小鼠粪便中的细菌基因组,对菌群基因组16S rRNA基因V4高变区进行测序,对数据进行PCoA分析、Alpha多样性分析、LEfSe分析和16S功能预测。结果:菊粉添加后,小鼠粪便中含有的细菌DNA量增多,短链脂肪酸增加。菊粉组和对照组PCoA图可以看到明显聚类。菊粉组物种多样性低于对照组。菊粉组小鼠粪便中S24_7菌科丰度上升;Lachnospiraceae(毛螺菌科),Ruminococcaceae(瘤胃菌科)和Deferribacteraceae(脱铁杆菌科)丰度下降。16S基因功能预测发现22个第二层级的KEGG通路发生变化。结论:高脂饮食情况下短链菊粉的添加会改变小鼠肠道菌群,继而影响肠道菌群的功能。     

Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces

Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota.     

Cyclocarya paliurus polysaccharides alleviate type 2 diabetic symptoms by modulating gut microbiota and short-chain fatty acids

BackgroundCyclocarya paliurus polysaccharide (CCPP), a primary active component in the leaves of Cyclocarya paliurus (Batal.) Iljinsk (C. paliurus), has the ability to treat type 2 diabetes mellitus (T2DM), but cannot be digested by our digestive system. Therefore, mechanisms of regulating the gut microbiota and intestinal metabolites might exist.PurposeTo reveal the potential mechanism of CCPP treatment, this study aimed to investigate the alterations of the gut microbiota and intestinal metabolites especially short chain fatty acids (SCFAs) in type 2 diabetic rats.Study design and methodsType 2 diabetic rat models were developed, and the therapeutic effects of CCPP were evaluated. Metagenomics analysis was utilized to analyze the alterations to the gut microbiota, and UHPLC-QTOF/MS-based untargeted metabolomics analysis of colon contents was used to identify the differential intestinal metabolites. GC/MS was used to measure the SCFAs in rat's colon contents and human fecal inoculums. Furthermore, the expression of SCFA receptors including GPR41, GPR43 and GPR109a was verified by qRT-PCR and the concentration of glucagon-like peptide-1(GLP-1) and peptide tyrosinetyrosine (PYY) was measured by Elisa.ResultsInhibition of the blood glucose levels and improvements in glucose tolerance and serum lipid parameters were observed after CCPP treatment. Eleven SCFA-producing species including Ruminococcus_bromii, Anaerotruncus_colihominis, Clostridium_methylpentosum, Roseburia_intestinalis, Roseburia_hominis, Clostridium_asparagiforme, Pseudoflavonifractor_capillosus, Intestinimonas_butyriciproducens, Intestinimonas_sp._GD2, Oscillibacter_valericigenes and Oscillibacter_ruminantium were clearly increased in the CCPP group. Furthermore, our study indicated that CCPP increases the production of SCFAs both in vivo and in vitro, and the gut microbiota are the key factor of this process. The SCFA receptors including GPR41, GPR43 and GPR109a, were significantly stimulated in the CCPP treated rats, which was accompanied by the upregulated expression of GLP-1 and PYY.ConclusionThese results demonstrated that CCPP could alleviate type 2 diabetic symptoms by increasing the SCFA-producing bacteria, promoting the production of SCFAs and upregulating SCFA-GLP1/PYY associated sensory mediators.     

Utilisation of enterobacterial repetitive intergenic consensus (ERIC) sequence-based PCR to fingerprint the genomes of Bifidobacterium isolates and other probiotic bacteria

Enterobacterial repetitive intergenic consensus based on PCR (ERIC-PCR) was used to generate DNA fingerprints for bifidobacteria and other probiotic bacteria. Two primers (ERIC 1R and ERIC 2) used in ERIC-PCR revealed that all of the probiotic bacteria tested possess enterobacterial repetitive intergenic consensus sequences with the PCR products ranging from 250 bp to 5000 bp. The bacterial strains can be differentiated by comparing fingerprint patterns. The dendrogram of the fingerprints revealed that most of the bifidobacterial wild type strains fell into one cluster at similarity level of approximately 79%.     

Alterations in gut microbiota and metabolites associated with altitude-induced cardiac hypertrophy in rats during hypobaric hypoxia challenge

The gut microbiota is involved in host responses to high altitude. However, the dynamics of intestinal microecology and their association with altitude-related illness are poorly understood. Here, we used a rat model of hypobaric hypoxia challenge to mimic plateau exposure and monitored the gut microbiome, short-chain fatty acids (SCFAs), and bile acids (BAs) over 28 d. We identified weight loss, polycythemia, and pathological cardiac hypertrophy in hypoxic rats, accompanied by a large compositional shift in the gut microbiota, which is mainly driven by the bacterial families of Prevotellaceae, Porphyromonadaceae, and Streptococcaceae. The aberrant gut microbiota was characterized by increased abundance of the Parabacteroides, Alistipes, and Lactococcus genera and a larger Bacteroides to Prevotella ratio. Trans-omics analyses showed that the gut microbiome was significantly correlated with the metabolic abnormalities of SCFAs and BAs in feces, suggesting an interaction network remodeling of the microbiome-metabolome after the hypobaric hypoxia challenge. Interestingly, the transplantation of fecal microbiota significantly increased the diversity of the gut microbiota, partially inhibited the increased abundance of the Bacteroides and Alistipes genera, restored the decrease of plasma propionate, and moderately ameliorated cardiac hypertrophy in hypoxic rats. Our results provide an insight into the longitudinal changes in intestinal microecology during the hypobaric hypoxia challenge. Abnormalities in the gut microbiota and microbial metabolites contribute to the development of high-altitude heart disease in rats.

     

ERIC-PCR fingerprinting-based community DNA hybridization to pinpoint genome-specific fragments as molecular markers to identify and track populations common to healthy human guts

Bacterial populations common to healthy human guts may play important roles in human health. A new strategy for discovering genomic sequences as markers for these bacteria was developed using Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR fingerprinting. Structural features within microbial communities are compared with ERIC-PCR followed by DNA hybridization to identify genomic fragments shared by samples from healthy human individuals. ERIC-PCR profiles of fecal samples from 12 diseased or healthy human and piglet subjects demonstrated stable, unique banding patterns for each individual tested. Sequence homology of DNA fragments in bands of identical size was examined between samples by hybridization under high stringency conditions with DIG-labeled ERIC-PCR products derived from the fecal sample of one healthy child. Comparative analysis of the hybridization profiles with the original agarose fingerprints identified three predominant bands as signatures for populations associated with healthy human guts with sizes of 500, 800 and 1000 bp. Clone library profiling of the three bands produced 17 genome fragments, three of which showed high similarity only with regions of the Bacteroides thetaiotaomicron genome, while the remainder were orphan sequences. Association of these sequences with healthy guts was validated by sequence-selective PCR experiments, which showed that a single fragment was present in all 32 healthy humans and 13 healthy piglets tested. Two fragments were present in the healthy human group and in 18 children with non-infectious diarrhea but not in eight children with infectious diarrhea. Genome fragments identified with this novel strategy may be used as genome-specific markers for dynamic monitoring and sequence-guided isolation of functionally important bacterial populations in complex communities such as human gut microflora.     

Chronic coffee consumption in the diet-induced obese rat: impact on gut microbiota and serum metabolomics

Epidemiological data confirms a strong negative association between regular coffee consumption and the prevalence of type 2 diabetes. Coffee is initially absorbed in the stomach and small intestine but is further fermented in the colon by gut microbiota. The bioavailability, production and biological activity of coffee polyphenols is modulated, in part, by gut microbiota. The purpose of this study was to determine if chronic coffee consumption could mitigate negative gut microbiota and metabolomic profile changes induced by a high-fat diet. Male Sprague–Dawley rats were randomized to chow (12% kcal fat) or high-fat (60% kcal fat) diet. Each group was further divided into water or caffeinated coffee for 10 weeks. Coffee consumption in high-fat-fed rats was associated with decreased body weight, adiposity, liver triglycerides and energy intake. Despite a more favorable body composition, rats displayed profound systemic insulin resistance, likely due to caffeine. Coffee consumption attenuated the increase in Firmicutes (F)-to-Bacteroidetes (B) ratio and Clostridium Cluster XI normally associated with high-fat feeding but also resulted in augmented levels of Enterobacteria. In the serum metabolome, coffee had a distinct impact, increasing levels of aromatic and circulating short-chain fatty acids while lowering levels of branched-chain amino acids. In summary, coffee consumption is able to alter gut microbiota in high-fat-fed rats although the role of these changes in reducing diabetes risk is unclear given the increased insulin resistance observed with coffee in this study.