Predictive value of left ventricular ejection fraction reserve assessed by SPECT G-MPI for major adverse cardiovascular event in patients with coronary artery disease / 中华心血管病杂志

Objective: To evaluate the prognostic value of left ventricular ejection fraction (LVEF) reserve assessed by gated SPECT myocardial perfusion imaging (SPECT G-MPI) for major adverse cardiovascular event (MACE) in patients with coronary artery disease. Methods: This is a retrospective cohort study. From January 2017 to December 2019, patients with coronary artery disease and confirmed myocardial ischemia by stress and rest SPECT G-MPI, and underwent coronary angiography within 3 months were enrolled. The sum stress score (SSS) and sum resting score (SRS) were analyzed by the standard 17-segment model, and the sum difference score (SDS, SDS=SSS-SRS) was calculated. The LVEF at stress and rest were analyzed by 4DM software. The LVEF reserve (ΔLVEF) was calculated (ΔLVEF=stress LVEF-rest LVEF). The primary endpoint was MACE, which was obtained by reviewing the medical record system or by telephone follow-up once every twelve months. Patients were divided into MACE-free and MACE groups. Spearman correlation analysis was used to analyze the correlation between ΔLVEF and all MPI parameters. Cox regression analysis was used to analyze the independent factors of MACE, and the optimal SDS cutoff value for predicting MACE was determined by receiver operating characteristic curve (ROC). Kaplan-Meier survival curves were plotted to compare the difference in the incidence of MACE between different SDS groups and different ΔLVEF groups. Results: A total of 164 patients with coronary artery disease [120 male; age (58.6±10.7) years] were included. The average follow-up time was (26.5±10.4) months, and a total of 30 MACE were recorded during follow-up. Multivariate Cox regression analysis showed that SDS (HR=1.069, 95%CI: 1.005-1.137, P=0.035) and ΔLVEF (HR=0.935, 95%CI: 0.878-0.995, P=0.034) were independent predictors of MACE. According to ROC curve analysis, the optimal cut-off to predict MACE was a SDS of 5.5 with an area under the curve of 0.63 (P=0.022). Survival analysis showed that the incidence of MACE was significantly higher in the SDS≥5.5 group than in the SDS<5.5 group (27.6% vs. 13.2%, P=0.019), but the incidence of MACE was significantly lower in the ΔLVEF≥0 group than in theΔLVEF<0 group (11.0% vs. 25.6%, P=0.022). Conclusions: LVEF reserve (ΔLVEF) assessed by SPECT G-MPI serves as an independent protective factor for MACE, while SDS is an independent risk predictor in patients with coronary artery disease. SPECT G-MPI is valuable for risk stratification by assessing myocardial ischemia and LVEF.

Mechanism of Tingli Dazao Xiefeitang on Cell Pyrolysis and Apoptosis Induced by Caspase-1 in A549 Cells / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of coking death and apoptosis of A549 cells induced by Tingli Dazao Xiefeitang. MethodA549 cells were randomized into blank group， traditional Chinese medicine（TCM） low， medium， and high concentration groups， which were treated with 20， 40， 60 mg·L-1 Tingli Dazao Xiefeitang， and TCM low， medium， and high concentration groups， respectively， and blank group was treated with equal volume culture medium. After 48 h of treatment， cell migration was detected by scratch assay and cell apoptosis was detected by flow cytometry. The relative expression levels of cysteine aspartate protease-1（Caspase-1）， NOD-like receptor protein 3 （NLRP3）， dermoderin D （GSDMD）， Survivin protein and nuclear transcription factor -κB （NF-κB） pathway proteins were detected by Western blot. The levels of intracellular reactive oxygen species （ROS） were determined by DCFH-DA fluorescence probe， and the contents of tumor necrosis factor -β （TNF-β） and interleukin-1β （IL-1β） in supernatant were determined by enzyme-linked immunosorbent assay （ELISA）. ResultCompared with blank group， the scratch healing rate， apoptosis rate， relative expression of Survivin protein， Caspase-1， GSDMD， NLRP3， ROS and NF-κB phosphorylation levels were significantly increased in low， medium and high concentration groups. The contents of TNF-β and IL-1β in supernatant were significantly increased （P<0.05）. Compared with the low concentration group， the scratch healing rate， apoptosis rate， Survivin protein relative expression， Caspase-1， GSDMD， NLRP3 relative expression， ROS and NF-κB phosphorylation levels were significantly increased in the medium and high concentration groups. The contents of TNF-β and IL-1β in supernatant were significantly increased （P<0.05）. Compared with the TCM group， the scratch healing rate， apoptosis rate， Survivin protein relative expression， Caspase-1， GSDMD， NLRP3 relative expression， ROS and NF-κB phosphorylation levels were significantly increased in the high concentration group. The contents of TNF-β and IL-1β in supernatant were significantly increased （P<0.05）. ConclusionTingli Dazao Xiefeitang can improve NLRP3 protein expression， inhibit Survivin protein expression and promote apoptosis of A549 cells. At the same time， it can activate NF-κB pathway and ROS system， up-regulate the expression of Caspase-1 and GSDMD， mediate scortosis of A549 cells.

Effect of light rhythm on the expression of cryptochrom 2 in retina / 中华实验眼科杂志

Background The cryptochrom 2 (Cry2)in mammalian retina is a main influential factor of circadian clock.Objective Purpose of this study was to investigate the effect of light exposure rhythm on expression of Cry2 in retina.Methods Thirty clean healthy Sprague Dawley(SD)rats were collected and divided into two groups randomly.The rats of the control group exposed to natural light with the normal rhythm for 30 days,but rats of the experimental group exposed to the artificial light (light: dark =18 hours:6 hours) for 3 months with the light intensity of(533± 16)lx.The histopathological change and ultrastructural alteration of rat retina in both groups were examined under the light microscope and transmission electron microscope at the end of the experiment.Expressions of Cry2 protein and its mRNA were assayed by immunohistochemistry and quantitative PCR(Q-PCR).Results The rat retinal morphology and ultrastructure were clear and order-arranged under the light microscope and transmission electron microscope in the control group.However,atrophy and disorganization of retina were found under the light microscope,and liquefaction and vacuole of outer segments of photoreceptors were observed.The vacuolar degeneration of mitochondria in the inner segments of photoreceptors,cellular nuclear shrinkage,chromatin margination,nuclear notch and destruction were seen in the outer nuclear layer under the transmission electron microscope.Immunohistochemistry showed that the expression of Cry2 protein located in cytoplasm and nuclei membrane of the retinal ganglion cell layer and inner nuclear layer in both normal rats and experimental rats.The scores of Cry2 protein expression were 0.833±0.197 in the experimental group,and 1.700±0.245 in the control group,with a significant difference between them (P=0.009).The quantities of Cry2 mRNA were 0.962 ± 0.125 in the control group and 0.615±0.100 in the experimental group,showing a significant difference between the two groups (P =0.006).Conclusions Long-term light exposure under the 533 lx leads to retinal structural and functional damage probably by down-regulating Cry2 expression in retina.Whether the regulation of Cry2 expression is helpful for stabilizing the biorhythm or not is a worthy question to explore.

Identification of up-regulated genes induced by angiotensin II in cardiac fibroblasts / 生理学报

To identify up-regulated genes in adult rat cardiac fibroblasts (CF) induced by angiotensin II (Ang II), suppression subtractive hybridization (SSH) was performed between the CF stimulated by Ang II (tester) and unstimulated CF (driver) to generate subtractive cDNA library. The library was screened with dot blots hybridization to further verify the differentially expressed cDNA clones. Partial positive clones (19 up-regulated genes) were sequenced and BLAST analyzed. Twelve up-regulated genes related to extracellular matrix, cell cycle, intracellular signal transduction, cell cytoskeleton, cell metabolism and 7 new expressed sequence tags (EST) were acquired (GenBank accession number: CN382808, CN382809, CN382810, CN382811, CN382812, CN382813, CN382814). Our data reveal that SSH is a powerful technique of high sensitivity for the detection and cloning of up-regulated genes expressed in CF induced by Ang II, which may be helpful to clarify the mechanism of cardiac remodeling.

A rapid and high throughput method for apoE genotyping / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop a new method that can determine the apolipoprotein E(apoE) genotypes rapidly in high throughput.</p><p><b>METHODS</b>Genome DNA samples were extracted from the anticoagulated peripheral blood samples of 79 patients with Alzheimer's disease(AD) and 63 healthy individuals, and the 492 bp apoE gene fragments including 112 and 158 codons were amplified by polymerase chain reaction (PCR). With one PCR product, three recombined alleles (epsilon 2, epsilon 3 and epsilon 4) of apoE gene as controls were obtained by cloning and site-directed mutagenesis. The excess primers and dNTPs in all PCR products were removed by treatment with clean up reagents, then template-directed dye-terminator incorporation reaction (TDI) was performed and R110 or TAMRA labeled Acyclo-terminators were added into the mutation sites specifically. Fluorescence polarization value (FP) was measured using victor 2 multilabel counter and the polymorphisms in 112 and 158 condons of apoE gene were investigated.</p><p><b>RESULTS</b>The apoE genotypes in recombined plasmid controls and all serum samples were analyzed using the authors' TDI-FP method, and the reliability and specificity were confirmed by DNA sequencing. The frequency of epsilon 4 allele in patients was significantly higher than that in controls, suggesting that apoE epsilon 4 allele gene is a risk factor for late-onset AD.</p><p><b>CONCLUSION</b>TDI-FP is an easy, reliable and high throughput technology in analyzing polymorphism of apoE gene; it can be used in the prediction of susceptibility to AD in elderly individuals. Furthermore, it is an ideal method for large-scale screening and for studying the relationship between the allelic and genotypic frequencies of apoE and other diseases.</p>

Quantification of mRNA expression for angiotensinogen in rat arterial tissues by real-time PCR / 中国应用生理学杂志

<p><b>AIM</b>To develop a real-time PCR method for quantifying low abundance mRNA expression in rat arterial tissues and examine differential changes of angiotensinogen (AGT) gene expression in basilar and femoral arterial tissues after 8 weeks simulated weightlessness.</p><p><b>METHODS</b>After 8-wk of simulation, basilar and femoral arteries were harvested from both simulated weightless (SUS) and control (CON) rats. Then total RNA was extracted and reverse transcribed. The real-time PCR using TaqMan-MGB probe was performed to quantify AGT mRNA expression. The amplification efficiency (E) of PCR was calculated from the slope of standard curve. The threshold cycle (Ct) was detected by changes in fluorescence during a real-time PCR. Finally, the relative expression ratio of the target gene (AGT) to the reference gene (GAPDH) was calculated using E and Ct according to the mathematical model derived from the equation calculating the starting fluorescence (Ro).</p><p><b>RESULTS</b>After a 8-weeks simulated weightlessness, the AGT mRNA expression increased by 240 percent in basilar arterial, and decreased by 66 percent in femoral arterial tissues.</p><p><b>CONCLUSION</b>The specificity, sensitivity, precision, reproducibility, and simplicity of real-time PCR method using TaqMan-MGB probe make it particularly suitable for quantification of low abundance mRNA in arterial tissue from rats.</p>