Characterization of gut microbial and metabolite alterations in faeces of Goto Kakizaki rats using metagenomic and untargeted metabolomic approach.

BACKGROUND: In recent years, the incidence of type 2 diabetes (T2DM) has shown a rapid growth trend. Goto Kakizaki (GK) rats are a valuable model for the study of T2DM and share common glucose metabolism features with human T2DM patients. A series of studies have indicated that T2DM is associated with the gut microbiota composition and gut metabolites. We aimed to systematically characterize the faecal gut microbes and metabolites of GK rats and analyse the relationship between glucose and insulin resistance. AIM: To evaluate the gut microbial and metabolite alterations in GK rat faeces based on metagenomics and untargeted metabolomics. METHODS: Ten GK rats (model group) and Wistar rats (control group) were observed for 10 wk, and various glucose-related indexes, mainly including weight, fasting blood glucose (FBG) and insulin levels, homeostasis model assessment of insulin resistance (HOMA-IR) and homeostasis model assessment of ß cell (HOMA-ß) were assessed. The faecal gut microbiota was sequenced by metagenomics, and faecal metabolites were analysed by untargeted metabolomics. Multiple metabolic pathways were evaluated based on the differential metabolites identified, and the correlations between blood glucose and the gut microbiota and metabolites were analysed. RESULTS: The model group displayed significant differences in weight, FBG and insulin levels, HOMA-IR and HOMA-ß indexes (P < 0.05, P < 0.01) and a shift in the gut microbiota structure compared with the control group. The results demonstrated significantly decreased abundances of Prevotella sp. CAG:604 and Lactobacillus murinus (P < 0.05) and a significantly increased abundance of Allobaculum stercoricanis (P < 0.01) in the model group. A correlation analysis indicated that FBG and HOMA-IR were positively correlated with Allobaculum stercoricanis and negatively correlated with Lactobacillus murinus. An orthogonal partial least squares discriminant analysis suggested that the faecal metabolic profiles differed between the model and control groups. Fourteen potential metabolic biomarkers, including glycochenodeoxycholic acid, uric acid, 13(S)-hydroxyoctadecadienoic acid (HODE), N-acetylaspartate, ß-sitostenone, sphinganine, 4-pyridoxic acid, and linoleic acid, were identified. Moreover, FBG and HOMA-IR were found to be positively correlated with glutathione, 13(S)-HODE, uric acid, 4-pyridoxic acid and allantoic acid and ne-gatively correlated with 3-α, 7-α, chenodeoxycholic acid glycine conjugate and 26-trihydroxy-5-ß-cholestane (P < 0.05, P < 0.01). Allobaculum stercoricanis was positively correlated with linoleic acid and sphinganine (P < 0.01), and 2-methyl-3-hydroxy-5-formylpyridine-4-carboxylate was negatively associated with Prevotella sp. CAG:604 (P < 0.01). The metabolic pathways showing the largest differences were arginine biosynthesis; primary bile acid biosynthesis; purine metabolism; linoleic acid metabolism; alanine, aspartate and glutamate metabolism; and nitrogen metabolism. CONCLUSION: Metagenomics and untargeted metabolomics indicated that disordered compositions of gut microbes and metabolites may be common defects in GK rats.

Effect of berberine on hyperglycaemia and gut microbiota composition in type 2 diabetic Goto-Kakizaki rats.

BACKGROUND: A recent investigation showed that the prevalence of type 2 diabetes mellitus (T2DM) is 12.8% among individuals of Han ethnicity. Gut microbiota has been reported to play a central role in T2DM. Goto-Kakizaki (GK) rats show differences in gut microbiota compared to non-diabetic rats. Previous studies have indicated that berberine could be successfully used to manage T2DM. We sought to understand its hypoglycaemic effect and role in the regulation of the gut microbiota. AIM: To determine whether berberine can regulate glucose metabolism in GK rats via the gut microbiota. METHODS: GK rats were acclimatized for 1 wk. The GK rats were randomly divided into three groups and administered saline (Mo), metformin (Me), or berberine (Be). The observation time was 8 wk, and weight, fasting blood glucose (FBG), insulin, and glucagon-like peptide-1 (GLP-1) were measured. Pancreatic tissue was observed for pathological changes. Additionally, we sequenced the 16S rRNA V3-V4 region of the gut microbiota and analysed the structure. RESULTS: Compared with the Mo group, the Me and Be groups displayed significant differences in FBG (P < 0.01) and GLP-1 (P < 0.05). A significant decrease in weight and homeostatic model assessment-insulin resistance was noted in the Be group compared with those in the Me group (P < 0.01). The pancreatic islets of the Me- and Be-treated rats showed improvement in number, shape, and necrosis compared with those of Mo-treated rats. A total of 580 operational taxonomic units were obtained in the three groups. Compared to the Mo group, the Me and Be groups showed a shift in the structure of the gut microbiota. Correlation analysis indicated that FBG was strongly positively correlated with Clostridia_UCG-014 (P < 0.01) and negatively correlated with Allobaculum (P < 0.01). Body weight showed a positive correlation with Desulfovibrionaceae (P < 0.01) and a negative correlation with Akkermansia (P < 0.01). Importantly, our results demonstrated that Me and Be could significantly decrease Bacteroidetes (P < 0.01) and the Bacteroidetes/Firmicutes ratio (P < 0.01). Furthermore, Muribaculaceae (P < 0.01; P < 0.05) was significantly decreased in the Me and Be groups, and Allobaculum (P < 0.01) was significantly increased. CONCLUSION: Berberine has a substantial effect in improving metabolic parameters and modulating the gut microbiota composition in T2DM rats.

[Methods to establishment of serum induced syndrome of liver-depression and spleen-deficiency cell model].

To explore the method of establishing a cell model of traditional Chinese medicine (TCM) syndromes, HepG2 cells were induced by human serum of liver-depression and spleen-deficiency syndrome(LDSDS) to establish a cell model of LDSDS in this research. The concentration of cells, the content of human serum in culture medium and the growth characteristics of model-cell (cell growth curve, the survival rate and apparent morphology were investigated by MTT assay and microscopy. Evaluation of syndrome cell model: metabolomics was used to analyze the human serum of normal individuals and patients with LDSDS, and cell models induced by these serums, respectively. We obtained the difference metabolites from serums and cell models of LDSDS, respectively; then compared the biomarkers from two metabolomics and their metabolic pathways, to verify that the reliability and applicability of the model. Metabolomics data were collected by UPLC-Q-TOF-MS, and then all data were analyzed by multivariate statistical (PCA，OPLS-DA). The results showed that, model cells have the characteristics of normal growth, slow proliferation and stable morphological structure inducted by 10% serum of LDSD in 24-72 h. There were the same 19 difference metabolites which from the human serum of normal individuals and patients with LDSDS, and cell models induced by these serums; including 9 metabolic pathways that play an important role in maintaining normal physiological activities of the human body, such as lipids, amino acids, nucleotides, and energy metabolism etc. It was shown that the established syndrome cell model can reflect the biological basis of LDSDS to some extent. This research provides a reference method for the establishment of TCM syndrome cell model.

Correlation between CO2 storage at the last minute of gas insufflation and area of retroperitoneal lacuna during retroperitoneal laparoscopic radical nephrectomy.

BACKGROUND: Adequate operation interspace is the premise of laparoscopy, and carbon dioxide (CO2) was an ideal gas for forming lacuna. A retroperitoneal space is used to form operation interspace in retroperitoneal laparoscopic radical nephrectomy by making ballooning, and the retroperitoneal space has no relative complete and airtight serous membrane, therefore CO2 absorption may be greater in retroperitoneal than transperitoneal laparoscopic radical nephrectomy. Excess CO2 absorption may induce hypercapnemia and further cause physiopathological change of respiratory and circulatory system. Therefore, exact evaluation of amount of CO2 which is eliminated from body via minute ventilation is important during retroperitoneal laparoscopic radical nephrectomy. The aim of the paper is to study the correlation between CO2 storage at the last minute of gas insufflation and area of retroperitoneal lacuna during retroperitoneal laparoscopic radical nephrectomy. METHODS: Forty ASA I/II patients undergoing retroperitoneal laparoscopic radical nephrectomy were enrolled. CO2 storage at the last minute of gas insufflation and area of a retroperitoneal lacuna were observed. Linear correlation and regression were performed to determine the correlation between them. RESULTS: There was positive correlation between CO2 storage at the last minute of gas insufflation and area of retroperitoneal lacuna (r = 0.880, P = 0.000), and the equation of linear regression was y = -83.097 + 0.925x (R(2) = 0.780, t = 11.610, P = 0.000). CONCLUSIONS: Amount of CO2 which is eliminated from body via mechanical ventilation could be calculated by measuring the area of retroperitoneal lacuna during retroperitoneal laparoscopic radical nephrectomy, and an anesthetist should be aware of the size of lacuna to predict high CO2 storage at the last minute of gas insufflation.

Elevated survivin mediated multidrug resistance and reduced apoptosis in breast cancer stem cells.

PURPOSE: Overexpression of survivin in breast cancer cells is associated with aberrant inhibition of apoptosis which leads to massive proliferation of cancer cells. Downregulation of survivin by the anticancer agent prodigiosin can efficiently induce apoptosis in cancer cells. METHODS: The levels of survivin expression in breast cancer stem like side population (SP) cells were assessed. Analyzed were also the rate of apoptosis, drug resistance and the efficiency of clone formation of breast cancer SP cells after treatment with progiosin. RESULTS: Breast cancer samples contained about 2.7% of cancer stem like SP cells which possessed elevated mRNA expression of stem cell proteins Oct-4, EpCAM and ABC transporter ABCG2, essential for the maintenance of SP cells. Furthermore, the SP cells displayed overexpression of survivin in conjunction with reduced apoptosis and increased multidrug resistance. After treatment with prodigiosin, the SP cells became more sensitive to apoptosis and to several chemotherapeutic agents. CONCLUSION: These data suggest that increased expression of survivin in SP cells is one of the major factors involved in apoptosis and resistance to chemotherapy.

S100A8 contributes to postoperative cognitive dysfunction in mice undergoing tibial fracture surgery by activating the TLR4/MyD88 pathway.

Neuro-inflammation plays a key role in the occurrence and development of postoperative cognitive dysfunction (POCD). Although S100A8 and Toll-like receptor 4 (TLR4) have been increasingly recognized to contribute to neuro-inflammation, little is known about the interaction between S100A8 and TLR4/MyD88 signaling in the process of systemic inflammation that leads to neuro-inflammation. Firstly, we demonstrated that C57BL/6 wide-type mice exhibit cognitive deficit 24h after the tibial fracture surgery. Subsequently, increased S100A8 and S100A9 expression was found in the peripheral blood mononuclear cells (PBMCs), spleen, and hippocampus of C57BL/6 wide-type mice within 48h after the surgery. Pre-operative administration of S100A8 antibody significantly inhibited hippocampal microgliosis and improved cognitive function 24h after the surgery. Secondly, we also observed TLR4/MyD88 activation in the PBMCs, spleen, and hippocampus after the surgery. Compared with those in their corresponding wide-type mice, TLR4(-/-) and MyD88(-/-) mice showed lower immunoreactive area of microglia in the hippocampal CA3 region after operation. TLR4 deficiency also led to reduction of CD45(hi)CD11b(+) cells in the brain and better performance in both Y maze and open field test after surgery, suggesting a new regulatory mechanism of TLR4-dependent POCD. At last, the co-location of S100A8 and TLR4 expression in spleen after operation suggested a close relationship between them. On the one hand, S100A8 could induce TLR4 activation of CD11b(+) cells in the blood and hippocampus via intraperitoneal or intracerebroventricular injection. On the other hand, TLR4 deficiency conversely alleviated S100A8 protein-induced hippocampal microgliosis. Furthermore, the increased expression of S100A8 protein in the hippocampus induced by surgery sharply decreased in both TLR4 and MyD88 genetically deficient mice. Taken together, these data suggest that S100A8 exerts pro-inflammatory effect on the occurrence and development of neuro-inflammation and POCD by activating TLR4/MyD88 signaling in the early pathological process of the postoperative stage.