Klotho prevents epithelial-mesenchymal transition through Egr-1 downregulation in diabetic kidney disease.

INTRODUCTION: As a key event leading to tubulointerstitial fibrosis in diabetic kidney disease (DKD), epithelial-mesenchymal transition (EMT) has drawn increasing attention from researchers. The antiaging protein Klotho attenuates renal fibrosis in part by inhibiting ERK1/2 signaling in DKD. Early growth response factor 1 (Egr-1), which is activated mainly by ERK1/2, has been shown to play an important role in EMT. However, whether Klotho prevents EMT by inhibiting ERK1/2-dependent Egr-1 expression in DKD is unclear.The aim of this study was to investigate whether Klotho prevents EMT through Egr-1 downregulation by inhibiting the ERK1/2 signaling pathway in DKD. RESEARCH DESIGN AND METHODS: Male C57BL/6J mice fed an high-fat diet for 4 weeks received 120 mg/kg streptozotocin (STZ), which was injected intraperitoneally. Klotho and Egr-1 expression was detected in the renal cortices of these mice on their sacrifice at 6 and 12 weeks after STZ treatment. In In vitro studies, we incubated HK2 cells under high-glucose (HG) or transforming growth factor-ß1 (TGF-ß1) conditions to mimic DKD. We then transfected the cells with an Klotho-containing plasmid, Klotho small interfering RNA. RESULTS: Klotho expression was significantly decreased in the renal cortices of mice with diabetes mellitus (DM) compared with the renal cortices of control mice at 6 weeks after treatment and even more significantly decreased at 12 weeks. In contrast, Egr-1 expression was significantly increased in mice with DM compared with control mice only at 12 weeks. We also found that Klotho overexpression downregulated Egr-1 expression and the (p-ERK1/2):(ERK1/2) ratio in HG-treated or TGF-ß1-treated HK2 cells. Conversely, Klotho silencing upregulated Egr-1 expression and the (p-ERK1/2):(ERK1/2) ratio in HG-treated or TGF-ß1-treated HK2 cells. Moreover, the effects of si-Klotho were abolished by the ERK1/2 inhibitor PD98059. CONCLUSIONS: Klotho prevents EMT during DKD progression, an effect that has been partially attributed to Egr-1 downregulation mediated by ERK1/2 signaling pathway inhibition.

Intestinal Flora is a Key Factor in Insulin Resistance and Contributes to the Development of Polycystic Ovary Syndrome.

CONTEXT: The key gut microbial biomarkers for polycystic ovarian syndrome (PCOS) and how dysbiosis causes insulin resistance and PCOS remain unclear. OBJECTIVE: To assess the characteristics of intestinal flora in PCOS and explore whether abnormal intestinal flora can affect insulin resistance and promote PCOS and whether chenodeoxycholic acid (CDCA) can activate intestinal farnesoid X receptor (FXR), improving glucose metabolism in PCOS. SETTING AND DESIGN: The intestinal flora of treatment-naïve PCOS patients and hormonally healthy controls was analyzed. Phenotype analysis, intestinal flora analysis, and global metabolomic profiling of caecal contents were performed on a letrozole-induced PCOS mouse model; similar analyses were conducted after 35 days of antibiotic treatment on the PCOS mouse model, and glucose tolerance testing was performed on the PCOS mouse model after a 35-day CDCA treatment. Mice receiving fecal microbiota transplants from PCOS patients or healthy controls were evaluated after 10 weeks. RESULTS: Bacteroides was significantly enriched in treatment-naïve PCOS patients. The enrichment in Bacteroides was reproduced in the PCOS mouse model. Gut microbiota removal ameliorated the PCOS phenotype and insulin resistance and increased relative FXR mRNA levels in the ileum and serum fibroblast growth factor 15 levels. PCOS stool-transplanted mice exhibited insulin resistance at 10 weeks but not PCOS. Treating the PCOS mouse model with CDCA improved glucose metabolism. CONCLUSIONS: Bacteroides is a key microbial biomarker in PCOS and shows diagnostic value. Gut dysbiosis can cause insulin resistance. FXR activation might play a beneficial rather than detrimental role in glucose metabolism in PCOS.

Safety and immunogenicity of an alum-adjuvanted whole-virion H7N9 influenza vaccine: a randomized, blinded, clinical trial.

OBJECTIVES: A case of H7N9 influenza virus infection was first identified in China in 2013. This virus is considered to have high pandemic potential. Here we developed an H7N9 influenza vaccine containing an aluminium adjuvant and evaluated the safety and immunogenicity of the vaccine. METHODS: From October 2017 through August 2018 we conducted a randomized, double-blinded, single-centre phase I clinical trial in China among 360 participants aged ≥12 years. All participants received two doses of the vaccine (7.5, 15 or 30 µg haemagglutinin antigen) or placebo at an interval of 21 days. Adverse event data were collected for 30 days after vaccination. Serum samples were collected on days 0, 21 and 42 for the haemagglutinin inhibition (HI) antibody assay. RESULTS: A total of 347 participants (347/360, 96.4%) completed the study. The proportions of vaccine-related adverse events after one injection were 56.7% (34/60) in the 7.5-µg group, 86.7% (52/60) in the 15-µg group and 86.7% (52/60) in the 30-µg group. The proportions of adverse events after two injections were less than those reported after the first dose. None of the serious adverse events were related to the vaccine. After receiving two doses of the 7.5-µg vaccine, the proportion of participants achieving an HI titre of ≥40 was 98.2% (55/56, 95%CI 72.3~100.0%), with a geometric mean titre (GMT) of 192.6 (95%CI 162.9~227.8). CONCLUSIONS: The alum-adjuvanted H7N9 whole-virion inactivated vaccine was safe and strongly immunogenic in a population aged ≥12 years.

The relationship between pancreatic cancer and type 2 diabetes: cause and consequence.

Pancreatic cancer (PC) is a devastating and lethal malignant disease and it is well known that there is a complex bidirectional relationship between PC and type 2 diabetes mellitus (T2DM). In order to more deeply summarize the relationship between them, this article summarizes the epidemiological data on the relationship between PC and T2DM in the past 5 years, and further explains the mechanism of interaction between them. Meanwhile, it also summed up the effects of drug therapy for T2DM on PC and the impact of T2DM on surgical resection of PC. Epidemiological studies clearly indicate that the risk of PC is increased in patients with T2DM. But increasing epidemiological data points out that PC also acts as a cause of T2DM and new-onset T2DM is sign and consequence of PC. Insulin resistance, hyperinsulinemia, hyperglycemia, and chronic inflammation are the mechanisms of T2DM-Associated PC. Metformin decreases the risk of PC, while insulin therapy increases the risk of PC. Besides, studies have shown that T2DM decreases the survival in patients with PC resection.

[A machine learning model based on initial gut microbiome data for predicting changes of Bifidobacterium after prebiotics consumption].

OBJECTIVE: To investigate the effects of prebiotics supplementation for 9 days on gut microbiota structure and function and establish a machine learning model based on the initial gut microbiota data for predicting the variation of Bifidobacterium after prebiotic intake. METHODS: With a randomized double-blind self-controlled design, 35 healthy volunteers were asked to consume fructo-oligosaccharides (FOS) or galacto-oligosaccharides (GOS) for 9 days (16 g per day). 16S rRNA gene high-throughput sequencing was performed to investigate the changes of gut microbiota after prebiotics intake. PICRUSt was used to infer the differences between the functional modules of the bacterial communities. Random forest model based on the initial gut microbiota data was used to identify the changes in Bifidobacterium after 5 days of prebiotic intake and then to build a continuous index to predict the changes of Bifidobacterium. The data of fecal samples collected after 9 days of GOS intervention were used to validate the model. RESULTS: Fecal samples analysis with QIIME revealed that FOS intervention for 5 days reduced the intestinal flora alpha diversity, which rebounded on day 9; in GOS group, gut microbiota alpha diversity decreased progressively during the intervention. Neither FOS nor GOS supplement caused significant changes in ß diversity of gut microbiota. The area under the curve (AUC) of the prediction model was 89.6%. The continuous index could successfully predict the changes in Bifidobacterium (R=0.45, P=0.01), and the prediction accuracy was verified by the validation model (R=0.62, P=0.01). CONCLUSION: Short-term prebiotics intervention can significantly decrease α-diversity of the intestinal flora. The machine learning model based on initial gut microbiota data can accurately predict the changes in Bifidobacterium, which sheds light on personalized nutrition intervention and precise modulation of the intestinal flora.

Evaluation and implementation of a membrane filter method for Cronobacter detection in drinking water.

A membrane filter (MF) method was evaluated for its suitability for qualitative and quantitative analyses of Cronobacter spp. in drinking water by pure strains of Cronobacter and non-Cronobacter, and samples spiked with chlorinated Cronobacter sakazakii ATCC 29544. The applicability was verified by the tests: for pure strains, the sensitivity and the specificity were both 100%; for spiked samples, the MF method recovered 82.8 ± 10.4% chlorinated ATCC 29544 cells. The MF method was also applied to screen Cronobacter spp. in drinking water samples from municipal water supplies on premises (MWSP) and small community water supplies on premises (SCWSP). The isolation rate of Cronobacter spp. from SCWSP samples was 31/114, which was significantly higher than that from MWSP samples which was 1/131. Besides, the study confirmed the possibility of using total coliform as an indicator of contamination level of Cronobacter spp. in drinking water, and the acquired correct positive rate was 96%.

Detection of Mycoplasma pneumoniae by colorimetric loop-mediated isothermal amplification.

Mycoplasma pneumoniae (M. pneumoniae) is one of the most important pathogens that cause respiratory tract infection in children and adults. In this study, we describe a rapid and sensitive colorimetric loop mediated isothermal amplification (LAMP) method to detect M. pneumoniae. The specificity and sensitivity of this assay were detected with 21 common respiratory pathogens and 39 M. pneumoniae DNA. The sensitivity of LAMP was 100% among 39 M. pneumoniae isolates and the specificity was 100% among 9 members of other Mycoplasma and 12 common respiratory pathogens. The lowest detectable limit (LDL) of this assay was 102 copies, which detected by a series of standard M. pneumoniae DNA. To evaluate the clinical applicability of the LAMP assay, a total of 80 clinical samples were examined by conventional PCR, real-time PCR and the LAMP assays, respectively. The positive rates were 15.0%, 32.5% and 26.3%, respectively. This colorimetric LAMP assay demonstrated a high level of sensitivity comparable with that of conventional PCR for the detection of M. pneumoniae. It is a valuable method for simple, cost-effective and rapid detection of M. pneumoniae in the rural areas and basic clinical of China.

[Plasma membrane-related Ca(2+)-ATPase-1 gene silencing promotes insulin secretion in islet beta cells NIT].

OBJECTIVE: To assess the effect of RNA interference-mediated gene silencing of plasma membrane-related Ca(2+)-ATPase-1 (PMR1) gene on the insulin secretion in islet beta cells NIT-1 in vitro. METHODS: A small interfering RNA duplex (siPMR1) corresponding to the nucleotides 337-357 of mouse PMR1 cDNA was introduced into NIT-1 cells via liposomes. The gene silencing effect was assessed by RT-PCR, and the total insulin level in the transfected cells was measured by radioimmunoassay. RESULTS: Transfection with siPMR1 resulted in obviously reduced PMR1 expression and increased insulin secretion in NIT-1 cells. CONCLUSION: The synthesized siPMR1 can significantly silence the expression of PMR1 and promote the secretion of insulin in the islet cells in vitro, which shed light on further studies of RNAi-based therapy of diabetes.

Effects of high concentration glucose on the expression of NF-kappaB, Bax and cytochrome C and apoptosis of islet cells in mice.

The roles of NF-kappaB (NF-kappaB) expression, Bax activity and cytochrome C (Cyt C) release, apoptosis of islet cells induced by high concentration glucose were explored in vitro. Pancreatic islet cells, which were isolated from Kunming mice, were cultured with different concentrations of glucose in DMEM, and divided into the following groups: G1, G2, G3, G4, G5, and G6 groups, corresponding to the glucose concentrations of 5.6, 7.8, 11.1, 16.7, 22.5, and 27.6 mmol/L, respectively. After culture for 120 h, insulin secretion was evaluated by radioimmunoassay, and the NF-kappaB expression was detected by immunocytochemistry. Bax activity and Cyt C release were measured by immunofluorescence, and apoptosis was examined by Hoechst33342 assay. The results showed that in G1, G2 and G3 groups, insulin secretion was enhanced with the increase of glucose concentration, and the NF-kappaB expression was also increased (P<0.05), but Bax activity, Cyt C release and apoptosis rate showed no significant difference among them. However, in G4, G5, and G6 groups, apoptosis rate of islet cells, NF-kappaB expression, Bax activity, and Cyt C release were all significantly increased, and insulin secretion was impaired as compared with G1, G2, and G3 groups (P<0.05). It was concluded that the exposure of islet cells to high glucose could induce islet cells apoptosis as well as impaired insulin secretion. The NF-kappaB signaling pathway and mitochondria pathway in islet cells might play some roles in the progressive loss of islet cells in diabetes. The inhibition of the NF-kappaB expression could be an effective strategy for protecting pancreatic islet cells.

[High glucose lowers insulin receptor substrate 2 expression and induces apoptosis in mouse islet cells in vitro].

OBJECTIVE: To investigate the role of insulin receptor substrate 2 (IRS2) and Bax on mouse islet cell apoptosis in the presence of high glucose in vitro. METHODS: The pancreatic islet cells were isolated from Kunming mice and divided into 6 groups (G1-G6 groups) for a 72-h culture in the media containing different concentrations of glucose (5.6, 7.8, 11.1, 16.7, 22.2, and 27.6 mmol/L, respectively). Insulin secretion by the cells was evaluated by radioimmunoassay, and the expressions of IRS2 and Bax were detected using immunocytochemistry and immunofluorescence assay, respectively. Hoechst33342 staining was employed to observe the cell apoptosis. RESULTS: Exposure to 5.6-11.1 mmol/L glucose resulted in increased insulin secretion and progressive elevation of IRS2 and Bax expression, whereas the cell apoptosis underwent no obvious changes. In the presence of glucose above 16.7 mmol/L, the percentages of apoptotic islet cells increased with glucose concentration, but insulin secretion and IRS2 expression decreased; Bax expression significantly increased in the presence of high-concentration glucose. CONCLUSION: Prolonged exposure of mouse islet cells to high glucose induces apoptosis and impairs insulin secretion of the cells. Decreased IRS2 expression and increased Bax expression may play an important role in the glucotoxicity in mouse islet cells.

[Effects of sustained-release alpha-lipoic acid tablet on blood lipid, blood sugar and insulin in hyperlipidemic New Zealand rabbits].

OBJECTIVE: To evaluate the effect of sustained-release alpha-lipoic acid tablets (SRLA) on blood lipid, glucose and insulin levels in hyperlipidemic New Zealand rabbits. METHODS: Twenty-four New Zealand rabbits were randomized into normal diet group, high-fat diet group, and high-fat diet + SRLA (300 mg/tablet) group with corresponding feed. At the beginning and 4 weeks after the feeding, the serum levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), blood glucose, and serum insulin were measured, and insulin sensitivity index (ISI) was calculated. RESULTS: Four weeks after feeding with high-fat diet, the insulin levels was elevated and the ISI lowered in the New Zealand rabbits, indicating successful establishment of the animal model of hyperlipidemia. Compared with the high-fat diet group, the serum levels of TG, TC, LDL-C and insulin were significantly reduced (P<0.05), and the ISI was significantly increased (P<0.05) in high fat diet + SRLA group. But no statistically significant difference was found in the blood glucose among the 3 groups. CONCLUSION: SRLA can significantly correct blood lipid and insulin disorders in hyperlipidemic New Zealand rabbits and prevent the occurrence of insulin resistance and hyperlipidemia.

Effects of High Concentration Glucose on the Expression of NF-κB, Bax and Cytochrome C and Apoptosis of Islet Cells in Mice / 华中科技大学学报（医学）（英德文版）

abetes. The inhibition of the NF-κB expres-sion could be an effective strategy for protecting pancreatic islet cells.

[High-performance liquid chromatography for determining plasma alpha-lipoic acid in New Zealand rabbits].

OBJECTIVE: To establish a method for determining the content of alpha-lipoic acid in New Zealand rabbit plasma. METHODS: Alpha-lipoic acid in the plasma samples was purified by solid-phase extractor and analyzed on an HYPERSIL C18 column with isocratic mobile phase consisting of potassium dihydrogen phosphate-acetonitrile (50:50, v/v) at a flow rate of 1.0 ml/min and detection wavelength of 230 nm. RESULTS: The standard curve was linear in the range of 5-100 microg/L (r=1) and the average recovery was 77.4%-82.1%. The relative standard deviations of intra-day and inter-day assay were within 1.5%-8.9%. CONCLUSION: The method is sensitive, accurate and simple for determining plasma alpha-lipoic acid levels in New Zealand rabbits.