Project description:Our previous studies have shown that the combination of Astragalus membranaceus and Salvia miltiorrhiza (HD) had a good antihypertensive effect, but its potential mechanism remained unclear. This study aimed to investigate the role of intestinal flora and serum metabolism induced by HD against hypertension. 16 spontaneous hypertensive rats (SHRs) were divided into HD group (5.9?g/kg) and model group (M) (normal saline), with eight Wistar-Kyoto (WKY) rats as control group (W) (normal saline). Rats were fed by gavage once a day for 28 days. The changes of intestinal flora and serum metabolism were analyzed by 16S rDNA sequencing and LC-MS/MS assay. HD decreased blood pressure steadily, improved the structure and composition of imbalance flora in SHRs, increased the abundance and diversity of flora, and decreased flora Firmicutes to Bacteroidetes (F/B) ratio. Rumen bacterium NK4A214, Clostridium sp. MC 40 increased remarkably in M group. Akkermansia, Akkermansia muciniphila, and Lactobacillus intestinalis increased significantly in HD group, which were functionally related to the significant increase of Lachnoclostridium, Faecalibaculum, and Lactobacillus reuteri in W group, which were all probiotics producing butyric acid, lactic acid, and regulating inflammation and other antihypertensive related factors. HD also changed the serum metabolic pattern of SHRs. 16 potential biomarkers related to inflammation, vasodilation, steroid hormones, oxidative stress, and etc. changed significantly, mainly enriched in arachidonic acid metabolism, tryptophan metabolism, steroid hormone biosynthesis, and glutathione metabolism. The correlation analysis demonstrated that the dominant genius and species in three groups were highly correlated with steroid hormone biosynthesis, arachidonic acid metabolism, tryptophan metabolism, and vitamin B6 metabolism. Our research indicated that HD had a good antihypertensive effect, which may be driven by the protective intestinal flora and beneficial metabolites induced by it, and the metabolites were closely related to the changes of intestinal flora. It provided new insights for the antihypertensive mechanism of HD.
Project description:Caerulomycin A (CRM A) is the first example of natural caerulomycins with a 2,2'-bipyridyl ring core and 6-aldoxime functional group from Streptomyces caeruleus and recently from marine-derived Actinoalloteichus cyanogriseus WH1-2216-6. Our previous study revealed that CRM A showed anti-tumor activity against human colorectal cancer (CRC) both in vitro and in vivo. Because some intestinal flora can affect the occurrence and development of CRC, the influence of CRM A on the intestinal flora is worthy of study in Sprague-Dawley (SD) rats. The high throughput sequencing of the V3-V4 hypervariable region in bacterial 16S rDNA gene results showed that the CRM A affected the diversity of intestinal flora of the SD rats treated with CRM A for 2, 3 and 4 weeks. Further analysis indicated that the abundance of genera Prevotella_1, Prevotellaceae_UCG-001, and Lactobacillus were increased while the that of genera Alloprevotella and Ruminiclostridium_1 were decreased. For the CRC related intestinal flora, the abundance of genera Bacteroides, Fusobacterium, Enterococcus, Escherichia-Shigella, Klebsiella, Streptococcus, Ruminococcus_2, and Peptococcus of SD rats treated with CRM A were decreased, while that of abundance of genera Bifidobacterium, Lactobacillus, Faecalibacterium, Blautia, Oscillibacter, and Clostridium were increased. The results indicated that CRM A could influence the intestinal flora by inhibiting some species of harmful flora and improving the beneficial bacteria in intestinal flora in the SD rats. The results may provide a new idea for revealing the mechanism of the anti-CRC activity of CRM A.
Project description:The study of the composition of the intestinal flora is important to the health of the host, playing a key role in maintaining intestinal homeostasis and the evolution of the immune system. For these studies, various universal primers of the 16S rDNA gene are used in microbial taxonomy. Here, we report an evaluation of 5 universal primers to explore the presence of microbial DNA in colon biopsies preserved in RNAlater solution. The DNA extracted was used for the amplification of PCR products containing the variable (V) regions of the microbial 16S rDNA gene. The PCR products were studied by restriction fragment length polymorphism (RFLP) analysis and DNA sequence, whose percent of homology with microbial sequences reported in GenBank was verified using bioinformatics tools. The presence of microbes in the colon of rats was quantified by the quantitative PCR (qPCR) technique. We obtained microbial DNA from rat, useful for PCR analysis with the universal primers for the bacteria 16S rDNA. The sequences of PCR products obtained from a colon biopsy of the animal showed homology with the classes bacilli (Lactobacillus spp) and proteobacteria, normally represented in the colon of rats. The proposed methodology allowed the attainment of DNA of bacteria with the quality and integrity for use in qPCR, sequencing, and PCR-RFLP analysis. The selected universal primers provided knowledge of the abundance of microorganisms and the formation of a preliminary test of bacterial diversity in rat colon biopsies.
Project description:Sodium carboxymethyl starch (CMS-Na), a kind of food additive with high degree of substitution, is also known as a prebiotic. The aim of this study was to determine the effect of CMS-Na on defecation. Constipated mouse model was prepared by loperamide. Normal rats were also used in the study. Short-chain fatty acids in rat feces were detected by gas chromatography. The bacterial communities in rat feces were identified by 16S rDNA gene sequencing. 5-hydroxytryptamine (5-HT) and tryptophan hydroxylase 1 (Tph1) were measured by ELISA. The results showed that CMS-Na increased the fecal granule counts and intestinal propulsion rate in constipated mice. The contents of water, acetic acid, propionic acid and n-butyrate in feces, Tph1 in colon and 5-HT in serum of rats were increased. In addition, CMS-Na shortened the colonic transport time in rats. The 16S rDNA gene sequencing results indicated that CMS-Na increased the relative abundance of Alloprevotella and decreased the proportion of Lactobacillus. However, the biodiversity of the normal intestinal flora was not altered. In conclusion, CMS-Na can promote defecation in constipated mice. The mechanism may be related to the regulation of Alloprevotella and Lactobacillus in colon, the increase of short-chain fatty acids, and the promotion of the synthesis of Tph1 and 5-HT.
Project description:Mice deficient in interleukin-2 are well suited for use as an animal model for inflammatory bowel disease. Raised under specific-pathogen-free conditions, interleukin-2-deficient mice develop an inflammatory bowel disease resembling ulcerative colitis in humans. The finding that colitis was attenuated when the mice were kept under germfree conditions implies that the resident intestinal flora is involved in the pathogenesis of colitis. The present study addresses the composition of the mucosa-associated bacterial flora in colon samples from interleukin-2-deficient mice that developed colitis. This was investigated by comparative 16S ribosomal DNA (rDNA) sequence analysis and fluorescence in situ hybridization using rRNA-targeted fluorescent probes to quantify the bacterial populations of the mucosa-associated flora. The investigations revealed distinct differences in the bacterial composition of the mucosa-associated flora between interleukin-2-deficient mice and healthy controls. Fluorescence in situ hybridization identified up to 10% of the mucosa-associated flora in interleukin-2-deficient mice as Escherichia coli, whereas no E. coli was detected in the mucosa from healthy wild-type mice. This finding was consistent with the results from comparative 16S rDNA analysis. About one-third of the clones analyzed from 16S rDNA libraries of interleukin-2-deficient mice represented Enterobacteriaceae, whereas none of the clones analyzed from the healthy controls harbored 16S rDNA from Enterobacteriaceae. The abundance of E. coli in the colonic mucosa of interleukin-2-deficient mice strongly suggests a participation in the pathogenesis of colitis in the interleukin-2-deficient mouse model for inflammatory bowel disease.
Project description:With the improvement of living standards and dietary changes, childhood obesity has increased worldwide. This study aimed to understand the differences of intestinal flora structure between obese and normal children at school-age. Using the next generation sequencing platform, Illumina Miseq, 16S rDNA high-throughput sequencing technology, we analyzed the diversity and relative abundance of intestinal flora in 39 obese and 38 normal control school-age children. First, we categorized gut bacteria on the basis of their Operational taxonomic units (OTUs) using the RDP 16s rRNA database in RDP classifier. The alpha (?) diversity was used to measure the diversity within a sample and is calculated as a value for each sample. The beta (?) diversity was used to compare different samples and to measure the dissimilarity between each other sample. Our results indicated that intestinal flora in obese children showed lower diversity than normal controls. Significant differences of relative abundance of intestinal flora were detected at multiple levels of classifications. Identification of intestinal flora with significant difference between obese and normal children may provide important information to uncover the roles of these specific bacteria in the development of obesity and find new strategy to prevent and treat obesity through intervening the intestinal flora.
Project description:Neuroinflammation plays a key role in the progression and pathogenesis of postoperative cognitive dysfunction, but it does not always occur in the local response to primary injury. In this study, we revealed that probiotics alleviate cognitive dysfunction associated with neuroinflammation in cardiac surgery. Rats were administered a probiotic or placebo once a day by gavage for 2 weeks until the day of surgery. Cardiac surgery was induced by ischemia/reperfusion of the left coronary artery. Key factors, such as the gut microbiome, the gut barrier and the blood-brain barrier (BBB), were systematically investigated to determine whether changes in the gut microbiome lead to neuroinflammation. We used 16S rDNA sequencing to confirm that cardiac surgery induced intestinal flora dysbiosis by altering the number of organisms rather than the structure in the cecum microbiome, which occurs at the same time as damage to the gut barrier. Cardiac surgery also increased BBB permeability, suggesting that disruption of the microbiome may increase the likelihood of neuroinflammation. Probiotics-induced alterations in the intestinal flora significantly reduced the level of inflammatory cytokines (IL-6 and IL-1?). Importantly, we found that the administration of probiotics significantly improved spatial memory impairment in rats after cardiac surgery, as measured by the Morris water maze. Overall, dysbiosis of the gut flora may aggravate cognitive impairment associated with neuroinflammation after cardiac surgery, and probiotics may attenuate this effect.
Project description:There are 10 mice in the experiment, named REC. The mice were fed with high salt diets (5% NaCl) for 4 weeks and then fed with normal salt diets for 4 weeks. Then extracted DNA from mice gastric flora to detect changes in the gastric flora of mice. Overall design: 16s rDNA-sequencing for 10 samples.
Project description:The seed of <i>Ziziphus jujuba</i> Mill. var. <i>spinosa</i> (Bunge) Hu ex H. F. Chou (ZSS) is often used as a traditional Chinese medicine for insomnia due to its sedative and hypnotic effects, but the mechanism underlying this effect has not been thoroughly elucidated. In this study, an insomnia model induced by intraperitoneal injection of DL-4-chlorophenylalanine suspension in Sprague-Dawley rats was adopted to investigate the therapeutic effect of ZSS extract. Metabolomics analyses of plasma and urine as well as 16S rRNA gene sequencing of the intestinal flora were performed. The relationships between the plasma and urine metabolites and the intestinal flora in insomnia rats were also analyzed. The results showed that changes in plasma and urine metabolites caused by insomnia were reversed after administration of ZSS, and these changes were mainly related to amino acid metabolism, especially phenylalanine metabolism. The results of 16S rRNA gene sequencing and short-chain fatty acid determination showed that the ZSS extract could reverse the imbalance of intestinal flora caused by insomnia and increase the contents of SCFAs in feces. All of these improvements are mainly related to the regulation of inflammation. Therefore, it is concluded that insomnia, which alters metabolic profiles and the intestinal flora, could be alleviated effectively by ZSS extract.
Project description:<h4>Background</h4>Qiweibaizhu decoction (QBD), a classic Chinese herbal formula, has been widely used for treating diarrhea in infants and children with spleen deficiency syndrome for centuries, but its mechanism of action remains unclear. The gut microbiota, short-chain fatty acids (SCFAs), and intestinal mucus are closely associated with diarrhea.<h4>Methods</h4>In this study, the composition of the gut microbiota in diarrheal rats was analyzed by 16S rDNA amplicon sequencing. The concentrations of colon SCFAs were determined using gas chromatography-mass spectrometry (GC-MS). The expression of mucin 2 (MUC2) in the colon was detected by immunofluorescence.<h4>Results</h4>Diarrhea significantly changed the diversity and structure of the gut microbiota and disrupted the mucus barrier in juvenile rats. QBD did not significantly change the diversity and structure of the intestinal flora, but it enhanced the increasing tendencies of Verrucomicrobia and <i>Akkermansia</i> and decreased the abundance of <i>Turicibacter</i> (<i>P</i>=0.037) and <i>Flavonifractor</i> (<i>P</i>=0.043). QBD tends to repair the mucus layer and promote MUC2 expression in juvenile rats with diarrhea. Moreover, <i>S. boulardii</i> significantly increased the abundance of <i>Parasutterella</i> (<i>P</i>=0.043). In addition, QBD treatment tends to increase the propionic acid concentration during diarrhea, but its levels of acetic acid, propionic acid, butyric acid, and total SCFAs were lower than those in the <i>S. boulardii</i> group.<h4>Conclusion</h4><i>S. boulardii</i> significantly increased the abundance of <i>Parasutterella</i>, leading to increased production of acetic acid, propionic acid, and butyric acid, consequently leading to alleviation of diarrhea. In comparison, QBD affected diarrhea via regulation of the intestinal flora, especially by increasing the abundance of Verrucomicrobia and <i>Akkermansia,</i> resulting in mucus barrier repair, protection of the intestines, and treatment of diarrhea.