Mingzhang Guo

MicroRNA profiling of rats with ochratoxin A nephrotoxicity

BackgroundNephrotoxicity is the most prominent one among the various toxicities of ochratoxin A (OTA). MicroRNAs (miRNAs) are small non-coding RNAs that have an impact on a wide range of biological processes by regulating gene expression at post-transcriptional level or protein systhesis level. The objective of this study is to analyze miRNA profiling in the kidneys of rats gavaged with OTA.ResultsTo profile miRNAs in the kidneys of rats with OTA nephrotoxicity, high-throughput sequencing and bioinformatics approaches were applied to analyze the miRNAs in the kidney of rats following OTA treatment. A total of 409 known miRNAs and 8 novel miRNAs were identified in the kidney and the levels of the novel miRNAs were varied in response to different doses of OTA. Expression of miR-129, miR-130a, miR-130b, miR-141, miR-218b and miR-3588 were uniquely suppressed in mid dose but then elevated in high dose, with opposite expression to their target genes. The expression pattern was closely related with the “MAPK signaling pathway”. Dicer1 and Drosha were significantly suppressed, indicating an impairment of miRNA biogenesis in response to OTA.ConclusionsThe abrogation of miRNA maturation process suggests a new target of OTA toxicity. Moreover, the identification of the differentially expressed miRNAs provides us a molecular insight into the nephrtoxicity of OTA.

Combination of metagenomics and culture-based methods to study the interaction between ochratoxin a and gut microbiota.

Gut microbiota represent an important bridge between environmental substances and host metabolism. Here we reported a comprehensive study of gut microbiota interaction with ochratoxin A (OTA), a major food-contaminating mycotoxin, using the combination of metagenomics and culture-based methods. Rats were given OTA (0, 70, or 210 μg/kg body weight) by gavage and fecal samples were collected at day 0 and day 28. Bacterial genomic DNA was extracted from the fecal samples and both 16S rRNA and shotgun sequencing (two main methods of metagenomics) were performed. The results indicated OTA treatment decreased the within-subject diversity of the gut microbiota, and the relative abundance of Lactobacillus increased considerably. Changes in functional genes of gut microbiota including signal transduction, carbohydrate transport, transposase, amino acid transport system, and mismatch repair were observed. To further understand the biological sense of increased Lactobacillus, Lactobacillus selective medium was used to isolate Lactobacillus species from fecal samples, and a strain with 99.8% 16S rRNA similarity with Lactobacillus plantarum strain PFK2 was obtained. Thin-layer chromatography showed that this strain could absorb but not degrade OTA, which was in agreement with the result in metagenomics that no genes related to OTA degradation increased. In conclusion, combination of metagenomics and culture-based methods can be a new strategy to study intestinal toxicity of toxins and find applicable bacterial strains for detoxification. When it comes to OTA, this kind of mycotoxin can cause compositional and functional changes of gut microbiota, and Lactobacillus are key genus to detoxify OTA in vivo.

Red Ginseng and Semen Coicis can improve the structure of gut microbiota and relieve the symptoms of ulcerative colitis.

ETHNOPHARMACOLOGICAL RELEVANCE Many Chinese herbs are traditionally used as medicine to improve the functions of gastrointestinal tract. Some of these herbs are also promising agents for the improvement of the gut microbiota and the treatment of ulcerative colitis. MATERIALS AND METHODS By screening seven traditional Chinese herbs, we found that Red Ginseng and Semen Coicis were the most effective in promoting the growth of probiotics including Lactobacillus and Bifidobacterium in vitro. We then evaluated the effects of Red Ginseng and Semen Coicis on the growth of the bacterial pathogens (Escherichia coli, Staphylococcus aureus, and Salmonella spp.) in vitro. In in vivo experiment, we gavage administrated trinitro-benzene-sulfonic acid induced ulcerative colitis (UC) rats with Red Ginseng and Semen Coicis extracts. After two weeks treatment, we analyzed the structure of the gut microbiota and examined the UC symptoms by employing qPCR and animal pathology detection techniques. RESULTS Both Red Ginseng and Semen Coicis promoted the growth of probiotics - Bifidobacterium and Lactobacillus in vitro. Red Ginseng also inhibited the growth of some pathogen strains. In vivo, Red Ginseng and Semen Coicis improved the structure of gut microbiota and relieved the symptoms of ulcerative colitis in vivo. Compared with Semen Coicis, Red Ginseng was more effective in relieving the symptoms of ulcerative colitis. CONCLUSIONS Red Ginseng could promote the growth of probiotic bacteria in vitro. Red Ginseng and, to a lesser extent Semen Coicis, gave positive results in an experimental in vivo model for ulcerative colitis.

Subchronic toxicity study in vivo and allergenicity study in vitro for genetically modified rice that expresses pharmaceutical protein (human serum albumin)

Genetically modified (GM) crops that express pharmaceutical proteins have become an important focus of recent genetic engineering research. Food safety assessment is necessary for the commercial development of these crops. Subchronic toxicity study in vivo and allergenicity study in vitro were designed to evaluate the food safety of the rice variety expressing human serum albumin (HSA). Animals were fed rodent diets containing 12.5%, 25.0% and 50.0% GM or non-GM rice for 90 days. The composition analysis of the GM rice demonstrated several significant differences. However, most of the differences remained within the ranges reported in the literature. In the animal study, a range of indexes including clinical observation, feed efficiency, hematology, serum chemistry, organ weights and histopathology were examined. Random changes unrelated to the GM rice exposure, within the range of historical control values and not associated with any signs of illness were observed. The results of heat stability and in vitro digestion of HSA indicated no evidence of potential allergenicity of the protein. Overall, the results of these studies suggest that the GM rice appears to be safe as a dietary ingredient when it is used at up to 50% in the diet on a subchronic basis.

Safety assessment of genetically modified rice expressing human serum albumin from urine metabonomics and fecal bacterial profile

The genetically modified (GM) rice expressing human serum albumin (HSA) is used for non-food purposes; however, its food safety assessment should be conducted due to the probability of accidental mixture with conventional food. In this research, Sprague Dawley rats were fed diets containing 50% (wt/wt) GM rice expressing HSA or non-GM rice for 90 days. Urine metabolites were detected by (1)H NMR to examine the changes of the metabolites in the dynamic process of metabolism. Fecal bacterial profiles were detected by denaturing gradient gel electrophoresis to reflect intestinal health. Additionally, short chain fatty acids and fecal enzymes were investigated. The results showed that compared with rats fed the non-GM rice, some significant differences were observed in rats fed with the GM rice; however, these changes were not significantly different from the control diet group. Additionally, the gut microbiota was associated with blood indexes and urine metabolites. In conclusion, the GM rice diet is as safe as the traditional daily diet. Furthermore, urine metabonomics and fecal bacterial profiles provide a non-invasive food safety assessment rat model for genetically modified crops that are used for non-food/feed purposes. Fecal bacterial profiles have the potential for predicting the change of blood indexes in future.

Discovery of systematic responses and potential biomarkers induced by ochratoxin A using metabolomics

Ochratoxin A (OTA) is known to be nephrotoxic and hepatotoxic in rodents when exposed orally. To understand the systematic responses to OTA exposure, GC-MS- and 1H-NMR-based metabolomic techniques together with histopathological assessments were applied to analyse the urine and plasma of OTA-exposed rats. It was found that OTA exposure caused significant elevation of amino acids (alanine, glycine, leucine etc.), pentose (ribose, glucitol, xylitol etc.) and nucleic acid metabolites (pseudouridine, adenosine, uridine). Moreover, myo-inositol, trimethylamine-oxide (TMAO), pseudouridine and leucine were identified as potential biomarkers for OTA toxicity. The primary pathways included the pentose phosphate pathway (PPP), the Krebs cycle (TCA), the creatine pathway and gluconeogenesis. The activated PPP was attributed to the high requirements for nicotinamide adenine dinucleotide phosphate (NADPH), which is involved in OTA metabolism through cytochrome P450. The elevated gluconeogenesis and TCA suggest that energy metabolism was involved. The up-regulated synthesis of creatinine reveals the elevated catabolism of proteins. These findings provide an overview of systematic responses to OTA exposure and metabolomic insight into the toxicological mechanism of OTA. Graphical Abstract

Toxicity study of ochratoxin A using HEK293 and HepG2 cell lines based on microRNA profiling

Ochratoxin A (OTA) induced DNA damage, cytotoxicity, and apoptosis in mammalian cell lines. Micro RNAs (miRNAs) are involved in physiological and developmental processes and contribute to cancer development and progression. In our study, high-throughput miRNA profiling and Kyoto Encyclopedia of Genes and Genomes analysis were applied to comparatively study the toxicity of OTA in HEK293 cells and HepG2 cells treated with 25 μM OTA for 24 h. In these two cells, the same changing miRNAs were mostly related to signal transduction pathways, whereas the different changing miRNAs were mostly related to human cancer pathways. DGCR8, Dicer1, and Drosha were significantly suppressed in HEK293 cells, indicating an impairment of miRNA biogenesis. The damage seemed more extensive in HEK293 cells. Cell models and in vivo models were also compared. Many miRNAs in vitro were markedly different from those in vivo; however, OTA toxicity was observed both in vitro and in vivo. The classification of deregulated pathways is similar. The biogenesis of miRNA was impaired in both lines. In conclusion, deregulated miRNAs in vitro are mostly related to human cancer and signal transduction pathways. The deregulated pathways in vivo are similar to those in vitro.

Insoluble Dietary Fiber from Pear Pomace Can Prevent High-Fat Diet-Induced Obesity in Rats Mainly by Improving the Structure of the Gut Microbiota

Supplement of dietary fibers (DF) is regarded as one of the most effective way to prevent and relieve chronic diseases caused by long-term intake of a high-fat diet in the current society. The health benefits of soluble dietary fibers (SDF) have been widely researched and applied, whereas the insoluble dietary fibers (IDF), which represent a higher proportion in plant food, were mistakenly thought to have effects only in fecal bulking. In this article, we proved the anti-obesity and glucose homeostasis improvement effects of IDF from pear pomace at first, and then the mechanisms responsible for these effects were analyzed. The preliminary study by real-time PCR and ELISA showed that this kind of IDF caused more changes in the gut microbiota compared with in satiety hormone or in hepatic metabolism. Further analysis of the gut microbiota by high-throughput amplicon sequencing showed IDF from pear pomace obviously improved the structure of the gut microbiota. Specifically, it promoted the growth of Bacteroidetes and inhibited the growth of Firmicutes. These results are coincident with previous hypothesis that the ratio of Bacteroidetes/Firmicutes is negatively related with obesity. In conclusion, our results demonstrated IDF from pear pomace could prevent high-fat diet-induced obesity in rats mainly by improving the structure of the gut microbiota.

Rice- or pork-based diets with similar calorie and content result in different rat gut microbiota

Abstract Rice is the most important food crop, and pork is the most widely eaten meat in the world. In this study, we compared the gut microbiota of the rats fed with rice or pork mixed diets, which have similar caloric contents. The physiological indices (body weights, hematology, serum chemistry, organ weights and histopathology) of two groups were all within the normal range. Two diets did not induce difference in the diversity of gut bacteria. However, Firmicutes were significantly higher in rice diet group, while Bacteroidetes were enriched in pork diet group. Butyrate and the bacteria enzymes β-glucuronidase, β-glucosidase and nitroreductase in the feces were all drastically higher in pork diet group. This study indicates that different diets with similar calorie and nutritional composition could change the community structure but not the diversity of rat fecal microbiota.

In Vivo Effects of Pichia Pastoris-Expressed Antimicrobial Peptide Hepcidin on the Community Composition and Metabolism Gut Microbiota of Rats.

Hepcidin, one kind of antimicrobial peptides, is one of the promising alternatives to antibiotics with broad spectrum of antimicrobial activity. Hepcidins cloned from different kinds of fishes have been produced using exogenous expression systems, and their in vitro antimicrobial effects have been verified. However their in vivo effects on gut microbiota and gut health of hosts remain unclear. Here we performed a safety study of hepcidin so that it can be used to reduce microbial contaminations in the food and feed. In this study, Pichia pastoris-expressed Pseudosciaena crocea hepcidin (PC-hepc) was first assessed by simulated digestion tests and then administered to male and female Sprague-Dawley (SD) rats in different concentrations. Subchronic toxicity testing, high throughput 16S rRNA sequencing of gut microbiota, and examinations on gut metabolism and permeability were conducted. The results showed PC-hepc could be digested in simulated intestinal fluid but not in simulated gastric fluid. PC-hepc had no adverse effects on general health, except causing increase of blood glucose (still in the normal value range of this index) in all trial groups of female rats and intestinal inflammation in HD group of female rats. Community composition of gut microbiota of female MD and HD groups shifted compared with control group, of which the decrease of genus Akkermansia might be related to the increase of blood glucose and intestinal inflammation. Significant increase of fecal nitroreductase activity was also observed in female MD and HD groups. Our results suggest the uses of exogenous PC-hepc in normal dosage are safe, however excess dosage of it may cause intestinal disorder of animals.