LONG NONCODING RNA; COMPETING ENDOGENOUS RNA; KAPPA-B PATHWAY; MICRORNA LET-7E; IN-VITRO; OX-LDL; EXPRESSION; DYSFUNCTION; BETA; DIFFERENTIATION

The inflammatory responses of vascular endothelial cells (VECs) are critical in the development of many cardio-cerebrovascular diseases. Let-7e is an important regulator of endothelial function and inflammation. However, the effects and mechanisms of let-7e on VECs inflammation have not been studied until recently. Thus, we investigated these issues and found that in addition to proliferation, apoptosis and cell adhesion, let-7e was also implicated in the regulation of inflammatory responses through a complex network, including I kappa B beta and lncRNA lnc-MKI67IP-3. Let-7e promoted NF-kappa B activation and translocation to the nucleus by inhibiting its target gene (I kappa B beta) expression and subsequently increased the expression of inflammatory and adhesion molecules. Meanwhile, lnc-MKI67IP- 3 acted as a sponge or competing endogenous RNA (ceRNA) for let-7e, suppressing its pro-inflammatory effects, and let-7e decreased lnc-MKI67IP-3 expression, thereby forming a positive feedback loop to aggravate inflammation. Moreover, let-7e, lnc-MKI67IP-3 and I kappa B beta were also abnormal in oxLDL-treated VECs and atherosclerotic plaques. The present study revealed let-7e as a pro-inflammatory mediator and a novel regulatory mechanism for the NF-kappa B pathway through ceRNA crosstalk, comprising let-7e and its target I kappa B beta and the ceRNA lnc-MKI67IP-3. Thus, this molecule might play important roles in the inflammatory responses of VECs and development of atherosclerosis.

QUANTIFICATION

In this study, we developed and validated a High-Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) method to determine bile acid in mouse serum. The serum samples were analysed after solid-phase extraction. The analytes were separated on a Diamonsil C18 column with a mobile phase of methanol and water containing 10 mmol/L ammonium acetate and 0.005% formic acid (70: 30) at a flow rate of 0.5 mL/min. Analytes were detected by tandem mass spectrometry in negative ion mode. The results demonstrated that the calibration curve was linear for all bile acids over a range of 10-10000 ng/L. The specificity, matrix effect, recovery, linearity, accuracy, and precision were validated for bile acid in mouse serum. The HPLC/MS/MS method was selective, sensitive, and simple, and was applied successfully to determine the bile acid in more than 200 mouse serum samples. In conclusion, this method is suitable for the quantitative detection of bile acid.

CLASS-I MOLECULES; AUTOREACTIVE T-CELLS; ACTIVATING KIR GENES; LEUKOCYTE ANTIGEN-C; MHC CLASS-I; INHIBITORY RECEPTORS; GRAVES-DISEASE; MATERNAL KIR; LYMPHOCYTES; ARTHRITIS

Objective: Natural killer (NK) cells serve as primary immune surveillance and are partially regulated by combinations of killer immunoglobulin-like receptors (KIR) and their human leukocyte antigen-C (HLA-C) ligands. Alterations in NK cell activity have been associated with Hashimoto thyroiditis (HT). The aim of this study was to determine whether certain KIR/HLA-C genotype combinations play a role in HT pathogenesis.;-;Methods: The present study enrolled 107 unrelated HT patients and 108 random healthy individuals in a case-control study. Blood was collected for DNA extraction; typing of KIR genes and HLA-C alleles was performed by polymerase chain reaction with sequence specific primers (PCR-SSP), followed by electrophoresis on agarose gels.;-;Results: Among a panel of KIR2D/HLA-C genotype combinations, the frequency of KIR2DS2/HLA-C1 was significantly increased in HT patients compared to controls (33.64% vs. 12.96%, P<.001). To further analyze the precise genotype, we investigated inhibitory or activating KIR/HLA-C gene pairs when their corresponding activating or inhibitory KIR genes were absent in the 2 groups. Only the frequency of KIR2DS2(-)2DL2/3(+)HLA-C1(+) was significantly decreased in HT patients compared to controls (48.60% vs. 70.37%, P = .001).;-;Conclusion: Our data suggest that KIR2DS2/HLA-C1 may correlate with HT pathogenesis. On the contrary, the predominance of KIR2DL2/3/HLA-C1 in the absence of KIR2DS2 suggests a potential inhibitory role in HT pathogenesis. In conclusion, our findings may further elucidate the mechanisms underlying the pathogenesis of HT and other autoimmune diseases.

INSULIN-RESISTANCE; BETA-CELL; CARDIOVASCULAR-DISEASE; DIABETES-MELLITUS; FATTY PANCREAS; ECTOPIC FAT; OBESITY; GLUCOSE; ACCUMULATION; LIPOTOXICITY

Background: Type 2 diabetes mellitus, obesity and hepatic steatosis showed a strong correlation with metabolic syndrome. However, data on the influence of pancreatic steatosis on metabolic syndrome are lacking.;-;Objective: Our aim is to perform the prevalence of pancreatic steatosis in adults and its association with metabolic syndrome in a Chinese population.;-;Methods: This was a cross-sectional study, randomly selected. A total of 1190 health examination subjects were recruited. Pancreatic steatosis or hepatic steatosis was diagnosed via trans-abdominal so-nography. The clinical and metabolic parameters were compared between the two groups, and their associations with pancreatic steatosis were examined.;-;Results: The prevalence of pancreatic steatosis was 30.7%. The presence of pancreatic steatosis was significantly increased by age, gender, central obesity, hepatic steatosis, hypertriglyceridemia and hyperglycemia. In the logistic regression analysis, age (P < 0.05), central obesity (P < 0.01), diabetes (P < 0.05), hypertriglyceridemia (P < 0.05) and hepatic steatosis (P < 0.01) were independently associated with pancreatic steatosis. The number of the parameters of the metabolic syndrome in pancreatic steatosis group was more than that in non-pancreatic steatosis group [(2.5 +/- 1.1) vs (1.4 +/- 1.2)] (P < 0.01).;-;Conclusion: The pancreatic steatosis is,strongly associated with the parameters of metabolic syndrome, such as central obesity, diabetes, and hepatic steatosis. (C) 2016 IAP and EPC. Published by Elsevier B.V. All rights reserved.

CORONARY-ARTERY-DISEASE; SMOOTH-MUSCLE-CELLS; INSULIN-RESISTANCE; GLUCOSE-TOLERANCE; PLASMA YKL-40; OBESE WOMEN; PREGNANCY; INFLAMMATION; ALBUMINURIA; CHITINASE

Objective: Serum YKL-40 levels are elevated in patients with type 1 and 2 diabetes. However, the correlation between YKL-40 and gestational diabetes mellitus (GDM) remains unknown. The present study compared serum YKL-40 levels in pregnant women with GDM and those with normal glucose tolerance and evaluated the relationship between YKL-40 and insulin-resistant syndrome.;-;Methods: Thirty-five patients with GDM and 43 age-matched healthy pregnant women at 24-28 weeks of gestation were studied. In addition to anthropometric assessments, serum glucose, insulin, YKL-40, total cholesterol, triglycerides, high-density lipoprotein, low-density lipoprotein and glycated hemoglobin were measured in all subjects. All subjects underwent a 2-h 75-g oral glucose tolerance test (OGTT). Body mass index (BMI) and the homeostasis model assessment of insulin resistance (HOMA-IR) were calculated.;-;Results: Fasting and 2 h serum YKL-40 levels were significantly higher in pregnant women with GDM compared with controls (77.3 +/- 29.3 versus 50.9 +/- 16.7 ng/mL, p < 0.001, fasting concentrations; 63.5 +/- 20.1 versus 40.6 +/- 10.7 ng/mL, p = 0.009, 2 h concentrations). OGTT had no effect on YKL-40 levels in either group (p > 0.05). There were significant correlations between YKL-40 and glycated hemoglobin (beta = 0.37, p = 0.006), fasting insulin (beta = 0.49, p = 0.001) and HOMA-IR (beta = 0.18, p = 0.015) in the GDM group.;-;Conclusions: Serum YKL-40 levels are elevated in patients with GDM but are unaffected by OGTT. YKL-40 levels are related to glycated hemoglobin, fasting insulin and HOMA-IR. These results suggest that YKL-40 may be a major contributor to GDM.

THROMBOXANE A(2); THYROID-DYSFUNCTION; DEFICIENT MICE; ALL-CAUSE; IN-VIVO; RISK; ATHEROSCLEROSIS; REPLACEMENT; CELLS; OVERT

Aim: The eicosanoids derived from phospholipids play key roles in inflammation. However, the profiles of serum eicosanoids in subclinical hypothyroidism (SH) patients and the effects of thyroxine replacement therapy (TRT) on these eicosanoids remain unclear. Many studies show that TSH regulates lipid metabolism. As eicosanoids derived from phospholipids play key roles in oxidative stress and immune function and inflammatory process, it was necessary to explore the profiles of serum eicosanoids in SH patients and the effects of thyroxine replacement therapy (TRT) on the eicosanoids.;-;Methods: A total of 50 Chinese SH patients and 22 healthy volunteers were recruited. SH patients received TRT (L-T4, 25 and 50 mcg/d for patients with TSH <= 10.0 mIU/L and TSH>10.0 mIU/L, respectively) for 3 months. Serum levels of major eicosanoids and cPLA2 were analyzed using LC-MS and clinical biochemical assays.;-;Results: The serum levels of cPLA2, eicosanoids (8-isoPGF2a, 11-dehydroTXB2 and 12-HETE) and 11-dehydroTXB2/6-Keto-PGF1a were significantly elevated in SH patients. The serum TSH levels were significantly correlated with the levels of cPLA2 (r=+0.65), 11-dehydroTXB2 (r=+0.32) and 11-dehydroTXB2/6-Keto-PGF1a (r=+0.37). After 3-month TRT, the serum levels of TSH, cPLA2 and the above-mentioned eicosanoids in SH patients were significantly decreased.;-;Conclusion: The metabolism of eicosanoids is significantly altered in Chinese SH patients, and TRT can ameliorate the abnormalities of serum eicosanoid levels.

MAMMARY-GLAND; GROWTH-HORMONE; PRL RECEPTOR; PERFORMANCE; ACTIVATION; LACTATION

In this work, we prepared a panel of monoclonal anti-idiotypic antibodies to ovine prolactin (oPRL) by the hybridoma technique. Among these antibodies, one anti-idotypic antibody (designated B7) was chosen for further characterization by a series of experiments. We first demonstrated that B7 behaved as a typical Ab2 beta based on a series of enzyme-linked immunosorbent assays. Subsequently, the results of a competitive receptor-binding assay confirmed that B7 could specifically bind to the prolactin receptor (PRLR) expressed on target cells. Finally, we examined its biological activities in CHO-PRLR and Nb2 cells and observed that B7 could activate Janus kinase 2-signal transducer and activator of transcription signalling in CHO-PRLR and Nb2 cells and induce BaF3 proliferation. The present study suggests that i) B7 can serve as a PRLR agonist or PRL mimic and has potential applications in regulating mammary gland development, milk production and maintenance of lactation in domestic animals and ii) B7 may be a biological reagent that can be used to explore the mechanism of PRLR-mediated intracellular signalling.

HEPARAN-SULFATE PROTEOGLYCAN; RNA TRANSFECTION; MESSENGER-RNA; T-CELLS; CANCER; SUPPRESSION; MICRORNAS; EXPRESSION; DELIVERY; SERUM

Background;-;MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs.;-;Methods and Results;-;Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye) could firmly bind to the surface of adherent cells (Hela) and suspended cells (K562) even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein) to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it.;-;Conclusions These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.

APOPTOSIS-INDUCING-LIGAND; VASCULAR INFLAMMATION; HEALTHY-ADULTS; OSTEOPROTEGERIN; ATHEROSCLEROSIS; ASSOCIATIONS; INVOLVEMENT; ARTHRITIS; MARKER; CELLS

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a multi-functional cytokine, which is involved in the pathophysiological processes of cardiovascular and metabolic diseases. Previously, we demonstrated that TRAIL stimulated lipid uptake and foam cell formation in macrophages in vitro. Several clinical studies have suggested that the serum concentration of TRAIL may be increased in humans with elevated blood cholesterol; however, the current data appear to be inconclusive in this regard. In the present study, we examined the relationships between the serum TRAIL concentration and cholesterol levels in 352 generally healthy subjects undergoing the routine annual health check. We showed that there were significant correlations between TRAIL concentration and levels of total and low-density lipoprotein cholesterols. The level of TRAIL was significantly elevated in subjects with hypercholesterolemia, although this relationship might be also associated with changes of other metabolic factors. Moreover, we showed that the level of blood cholesterol was significantly higher in subjects in the upper quartile of serum TRAIL. In conclusion, our data demonstrate that the serum TRAIL concentration is elevated in people with hypercholesterolemia.

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This study aims to identify the predictive value of cystatin C for diabetic retinopathy (DR) in Chinese patients with type 2 diabetes. Data from a cross-sectional hospitalbased survey of 450 type 2 diabetes patients were analyzed in the study. DR was assessed by fundus fluorescein angiography. Duration of diabetes and other related information were obtained by questionnaire. Body mass index, blood pressure, HbA1c, cystatin C, glomerular filtration rate, urinary albumin excretion, blood lipids, and uric acid were measured. Binary logistic regression was performed to evaluate potential risk factors for DR. The predictive value of cystatin C for DR was evaluated using ROC curve. Cystatin C (P=0.039) was a risk factor for DR after GFR, and other possibly related variables were adjusted. Cystatin C had a significant predictive value for any DR (AUC, 0.763, P<0.001; optimal cutoff value, 1.11 mg/L; sensitivity, 56.00 %; specificity, 83.90 %) or severe DR (AUC, 0.821, P<0.001; optimal cutoff value, 1.23 mg/L; sensitivity, 73.60 %; specificity, 88.70 %). Cystatin C is a novel risk factor for DR and should be used to screen and forecast the presence of DR (especially severe DR) in Chinese patients with type 2 diabetes. The association between cystiatin C and DR should not depend on the excellent ability of cystatin C for the estimation of GFR.