HE4 Serum Levels are Associated with Poor Prognosis in Patients with Acute Heart Failure Combined with Chronic Kidney Disease.

Purpose: The levels of human epididymis protein 4 (HE4) is associated not only with the prognosis of patients with acute heart failure (AHF), but also with chronic kidney disease (CKD). Our study aims to understand the prediction value of HE4 on prognosis in patients with AHF combined with CKD. Patients and Methods: This study prospectively enrolled patients diagnosed with AHF combined with CKD at the Department of Cardiology of Hunan Provincial People's Hospital from March 2019 to December 2022. Serum levels of HE4 were measured using a chemiluminescence microparticle immunoassay. The endpoint events included heart failure readmission and cardiovascular death. Results: A total of 130 patients with AHF combined with CKD were included in the stud. The median age is 73 years (interquartile range: 65-79 years). Among the patients, 94 experienced the endpoint events. The multivariable Cox analysis reveals that LnHE4 (HR=2.280, 95% CI 1.300-3.998, P = 0.004) and age (HR=1.024, 95% CI 1.003-1.045, P = 0.025) are independent predictors of the endpoint events. The Kaplan-Meier survival curve demonstrates that patients with HE4 levels>276.15 pmol/L has a significantly higher incidence of endpoint events compared to those with HE4 levels≤276.15 pmol/L (Log rank test: χ2=19.689, P < 0.001). After adjusting for age and gender, the HR is 2.520 (95% CI: 1.626-3.906, P < 0.001). Conclusion: HE4 is an independent predictor of heart failure readmission and cardiovascular death in patients with AHF combined with CKD.

Identification of m6A regulator-mediated RNA methylation modification patterns and key immune-related genes involved in atrial fibrillation.

The role of m6A in the regulation of the immune microenvironment in atrial fibrillation (AF) remains unclear. This study systematically evaluated the RNA modification patterns mediated by differential m6A regulators in 62 AF samples, identified the pattern of immune cell infiltration in AF and identified several immune-related genes associated with AF. A total of six key differential m6A regulators between healthy subjects and AF patients were identified by the random forest classifier. Three distinct RNA modification patterns (m6A cluster-A, -B and -C) among AF samples were identified based on the expression of 6 key m6A regulators. Differential infiltrating immune cells and HALLMARKS signaling pathways between normal and AF samples as well as among samples with three distinct m6A modification patterns were identified. A total of 16 overlapping key genes were identified by weighted gene coexpression network analysis (WGCNA) combined with two machine learning methods. The expression levels of the NCF2 and HCST genes were different between controls and AF patient samples as well as among samples with the distinct m6A modification patterns. RT-qPCR also proved that the expression of NCF2 and HCST was significantly increased in AF patients compared with control participants. These results suggested that m6A modification plays a key role in the complexity and diversity of the immune microenvironment of AF. Immunotyping of patients with AF will help to develop more accurate immunotherapy strategies for those with a significant immune response. The NCF2 and HCST genes may be novel biomarkers for the accurate diagnosis and immunotherapy of AF.

Statistical Analysis of IABP-Surgery Data with the Co-use of Anticoagulants, Pulse of Dorsalis Pedis Artery, D-Dimer Data, and Coagulation Function.

Data analysis was performed on IABP (intra-aortic balloon pump) patients for the use of anticoagulants, pulse of dorsalis pedis artery, D-dimer data, and coagulation function. According to the differential diagnosis of 52 patients admitted to hospital, data on the use of anticoagulants, dorsalis pedis artery pulsation, D-dimer data, and coagulation function were collected. These data were analyzed by using a nonparametric test, linear regression analysis, adjustment effect analysis, and chi-square test. Some findings of the analysis included: (1) There were differences in the dorsalis pedis artery pulsation of samples from different sexes, all of which were significant. (2) Coagulation function has a significant positive relationship with D-dimer. (3) When the D-dimer affects the prognosis, the regulatory variable (dorsalis pedis artery pulse) is at different levels, and the influence amplitude has significant differences. (4) Samples taken with different anticoagulants all showed significant differences in the dorsalis pedis artery pulsation.

Association Between Circulating Cell-Free DNA Level at Admission and the Risk of Heart Failure Incidence in Acute Myocardial Infarction Patients.

Plasma cell-free DNA (cfDNA) was elevated in patients with acute myocardial infarction (AMI) or heart failure (HF). However, whether cfDNA could serve as a predictor for risk of HF after AMI remains unknown. In this study, we conducted a pilot prospective cohort study in which 98 AMI patients were enrolled from a single center to assess the association between cfDNA levels at admission and risk of HF in an AMI population. Patients with cfDNA above the median level (14.39 ng/mL) showed higher low-density lipoprotein cholesterol, cardiac troponin I (cTnI), and soluble suppression of tumorigenicity 2 (sST2) levels compared with patients below the median. cfDNA was positively correlated with cTnI (r = 0.377, p < 0.001) and sST2 (r = 0.443, p < 0.001). Within a median follow-up of about 345 days, 46 patients (52.6%) developed HF. Multivariate Cox analysis showed that a higher cfDNA (above the cutoff value: 9.227 ng/mL) was an effective risk predictor (C-index = 0.74, 95% confidence interval [CI]: 0.733-0.748) for HF incidence after AMI (adjusted hazard ratio [HR]: 2.805; 95% CI: 1.087-7.242; p = 0.033). Moreover, a linear association was observed between cfDNA and risk of HF incidence adjusted for by age, gender, and history of chronic kidney disease (p for linear trend = 0.044). Taken together, the cfDNA levels at admission are associated with the incidence of HF in AMI patients. A positive correlation between cfDNA and the fibrotic factor sST2 was proved, but the underlying mechanisms require further study.

Dapagliflozin, sildenafil and their combination in monocrotaline-induced pulmonary arterial hypertension.

BACKGROUND: Dapagliflozin, a selective inhibitor of sodium-glucose cotransporter 2 (SGLT2), can reduce cardiovascular events and mortality in patients with heart failure. A number of mechanisms have been proposed to explain the beneficial effects of SGLT2 inhibitors. The purpose of this study was to determine whether dapagliflozin can improve pulmonary vascular remodelling and the efficacy of dapagliflozin as an add-on therapy to sildenafil in rats with pulmonary arterial hypertension (PAH). METHODS: A monocrotaline (MCT)-induced PAH rat model was used in our study. MCT-injected rats were randomly divided into four groups and treated for 3 weeks with daily per os treatment with vehicle, dapagliflozin (1 mg/kg/day), sildenafil (25 mg/kg/day), or a combination of dapagliflozin (1 mg/kg/day) and sildenafil (25 mg/kg/day). Haemodynamic measurements, histological analysis, enzyme-linked immunosorbent assay and western blotting analysis were employed to detect the changes in PAH rats after treatments. RESULTS: Dapagliflozin significantly attenuated MCT-induced increases in right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH) in PAH rats. Dapagliflozin effectively decreased the thickening of pulmonary artery media and decreased the muscularization of pulmonary arterioles in PAH rats. Moreover, dapagliflozin attenuated nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome activation in lung tissues and the levels of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) in plasma. However, dapagliflozin as an add-on therapy to sildenafil in rats with PAH did not show a more pronounced beneficial effect on right ventricular systolic pressure and pulmonary vascular remodelling in MCT rats than sildenafil alone. CONCLUSIONS: Dapagliflozin reduces right ventricular systolic pressure and pulmonary vascular remodelling in a rat model of PAH. However, combination therapy with dapagliflozin and sildenafil was not more effective than monotherapy with sildenafil in PAH rats.

Ultrasound-targeted microbubble destruction (UTMD)-mediated miR-150-5p attenuates oxygen and glucose deprivation-induced cardiomyocyte injury by inhibiting TTC5 expression.

BACKGROUND: Cardiomyocyte injury is a typical feature in cardiovascular diseases. Changes in cardiomyocytes strongly affect the progression of cardiovascular diseases. This work aimed to investigate the biological function and potential mechanism of action of miR-150-5p in cardiomyocytes. METHODS AND RESULTS: A myocardial ischemia (MI) injury rat model was constructed to detect miR-150-5p and tetratricopeptide repeat domain 5 (TTC5) expression during heart ischemia injury. Primary cardiomyocytes were isolated for in vitro study. CCK-8 assays were used to detect cardiomyocyte viability. Western blots were used to detect TTC5 and P53 expression. qPCR was utilized to measure RNA expression of miR-150-5p and TTC5. The TUNEL assay was used to determine cell apoptosis. ELISA was used to determine cytokine (TNF-α, IL-1ß, IL-6, and IL-8) levels in heart tissues and cell culture supernatants. A dual-luciferase reporter assay was carried out to verify the binding ability between miR-150-5p and TTC5. Oxygen-glucose deprivation (OGD) treatment significantly inhibited cell viability. Ultrasound-targeted microbubble destruction (UTMD)-mediated uptake of miR-150-5p inverted these results. Additionally, UTMD-mediated uptake of miR-150-5p retarded the effects of OGD treatment on cell apoptosis. Besides, UTMD-mediated uptake of miR-150-5p counteracted the effects of OGD treatment on the inflammatory response by regulating cytokine (TNF-α, IL-1ß, IL-6, and IL-8) levels. For the mechanism of the protective effect on the heart, we predicted and confirmed that miR-150-5p bound to TTC5 and inhibited TTC5 expression. CONCLUSIONS: UTMD-mediated uptake of miR-150-5p attenuated OGD-induced primary cardiomyocyte injury by inhibiting TTC5 expression. This discovery contributes toward further understanding the progression of primary cardiomyocyte injury.

MicroRNA-33-5p inhibits cholesterol efflux in vascular endothelial cells by regulating citrate synthase and ATP-binding cassette transporter A1.

BACKGROUND: A high level of total cholesterol is associated with several lipid metabolism disorders, including atherosclerosis and cardiovascular diseases. ATP-binding cassette (ABC) transporter A1 (ABCA1) and miR-33-5p play crucial roles in atherosclerosis by controlling cholesterol efflux. While citrate is a precursor metabolite for lipid and cholesterol synthesis, little is known about the association between citrate synthase (CS) and cholesterol efflux. This study investigated the role of the miR-33-5p/ABCA1/CS axis in regulating cholesterol efflux in vascular endothelial cells (VECs). MATERIALS AND METHODS: VECs were treated with oxidized low-density lipoprotein cholesterol (ox-LDL), or pretreated with plasmids overexpressing CS, ABCA1, siRNAs against CS and ABCA1, and an miR-33-5p inhibitor. Cell apoptosis, cellular senescence-associated ß-galactosidase activity, inflammation, and cholesterol efflux were detected. RESULTS: Treatment with ox-LDL decreased ABCA1 and CS levels and increased miR-33-5p expression and apoptosis in dose-dependent manners. In contrast, treatment with the miR-33-5p inhibitor and ABCA1 and CS overexpression plasmids inhibited the above-mentioned ox-LDL-induced changes. In addition, treatment with ox-LDL decreased cholesterol efflux, induced aging, and promoted the production of inflammatory cytokines (i.e., IL-6 and tumor necrosis factor TNF-α), as well as the expression of Bax and Caspase 3 proteins in VECs. All these changes were rescued by miR-33-5p inhibition and ABCA1 and CS overexpression. The inhibition of ABCA1 and CS by siRNAs eliminated the effects mediated by the miR-33-5p inhibitor, and knockdown of CS eliminated the effects of ABCA1 on VECs. CONCLUSIONS: This study demonstrated the crucial roles played by the miR-33-5p/ABCA1/CS axis in regulating cholesterol efflux, inflammation, apoptosis, and aging in VECs, and also suggested the axis as a target for managing lipid metabolism disorders.

Evidence-Based Analysis on Observation for Nursing Care of Patients with Intra-Aortic Balloon Pumping.

In this study, data analysis was performed on 52 patients. According to the different outcomes and discharge diagnosis of patients, data on sedative use, emotions, behavioral abnormalities, hearing loss, pain, total time on board the IABP (intra-aortic balloon pumping), and days of hospitalization of patients were collected. The data were subjected to frequency analysis, paired chi-square analysis, chi-square test, Poisson regression analysis, and stepwise regression analysis. Some findings of the analysis included the following: Between outcome and admission diagnosis, the analysis showed that significant differences existed between paired data. Patients with heart failure and acute myocardial infarction are in an unhealed state, and most patients with coronary atherosclerotic heart disease, myocarditis, and heart disease showed improvement. The samples taken by different sedatives showed no significant differences in the emotional and behavioral abnormalities, hearing loss, and pain. A total of 1 item of hospital stay had a significant negative impact on the total operation time of IABP. However, discharge diagnosis and admission diagnosis did not affect the total time on board the IABP. The dorsalis pedis artery pulse condition has a significant negative effect on the total time on board the IABP.

CXCR4 protects bone marrow-derived endothelial progenitor cells against hypoxia through the PI3K/Akt signaling pathway.

The present study aimed to investigate the regulatory mechanism of chemokine (C-X-C motif) receptor 4 (CXCR4) on endothelial progenitor cells (EPCs) through the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway under hypoxic conditions. Mononuclear cells were isolated from the bone marrow (BM) of young Sprague-Dawley (SD) rats. Bone marrow-derived endothelial progenitor cells (BM-EPCs) were characterized by using Dil-labeled acetylated low-density lipoprotein (Dil-ac-LDL) and fluorescein isothiocyanate-labeled UEA (FITC-UEA-1). Phenotype identification of BM-EPCs was based on red cytoplasm and green cytomembrane. Flow cytometry was employed to examine the markers CD14, CD34, and KDR. Expression level of the EPC-specific surface marker CD14 was found to be negative, while the expression level of CD34 and KDR was positive. In addition, CXCR4 was stably overexpressed in BM-EPCs after transfection with adenovirus-CXCR4. Cell proliferation, migration and apoptosis abilities were measured through the application of CCK-8, followed by Transwell and flow cytometry assays. The expression level of CXCR4, PI3K and Akt was determined by reverse transcription-quantitative PCR and western blotting assays. Functional experiments demonstrated that hypoxia inhibited BM-EPC proliferation and migration, while accelerating BM-EPC apoptosis. Additionally, CXCR4 was found to promote proliferation and migration, and suppress apoptosis in BM-EPCs with or without hypoxia treatment. Evidence also demonstrated that CXCR4 markedly upregulated the expression levels of PI3K and Akt. Furthermore, PI3K inhibitor (LY294002) and CXCR4 inhibitor (AMD3100) effectively inhibited the proliferation, migration and resistance to apoptosis of CXCR4-mediated BM-EPCs under hypoxic conditions.

Resveratrol inhibits necroptosis by mediating the TNF-α/RIP1/RIP3/MLKL pathway in myocardial hypoxia/reoxygenation injury.

Resveratrol (RES) protects myocardial cells from hypoxia/reoxygenation (H/R)-caused injury. However, the mechanism of this effect has not been clarified. Thus, in this study, we aimed to determine whether RES attenuates H/R-induced cell necroptosis by inhibiting the tumor necrosis factor-alpha (TNF-α)/receptor-interacting protein kinase 1 (RIP1)/RIP3/mixed-lineage kinase domain-like (MLKL) signaling pathway. Rat myocardial ischemia/reperfusion (I/R) models and H/R-injured cell models were constructed. Our study showed that myocardial H/R injury significantly increased the levels of TNF-α, RIP1, RIP3, and p-MLKL/MLKL by western blot analysis. Cell viability assay and 4,6-dianmidino-2-phenylindole (DAPI)-propidium iodide staining showed that the cell viability was decreased, and necroptosis was increased after myocardial H/R injury. The expressions of TNF-α, RIP1, RIP3, and p-MLKL/MLKL in H/R myocardial cells treated with different concentrations of RES were significantly downregulated. In addition, we also found that the cell viability was increased and necroptosis was decreased in dose-dependent manners when H/R-injured cells were treated with RES. In addition, the enhanced effect of TNF-α on necroptosis in myocardial H/R-injured cells was improved by RES, and the effect of RES was confirmed in vivo in I/R rats. This study also showed that RES suppresses necroptosis in H9c2 cells, which may occur through the inhibition of the TNF-α/RIP1/RIP3/MLKL signaling pathway. Our data suggest that necroptosis is a promising therapeutic target and may be a promising therapeutic target for the treatment of myocardial I/R injury.

miR-520d suppresses rapid pacing-induced apoptosis of atrial myocytes through mediation of ADAM10.

MicroRNAs (miRNAs) play a key role in various pathological processes like atrial fibrillation (AF). However, the mechanisms remain unclear. Herein, this study was undertaken to probe the roles of ADAM10 and its targeting miR-520d in rapid pacing-induced apoptosis in atrial myocytes. In this study, the atrial myocytes grew adherently with irregular morphology. Immunofluorescence showed that more than 90% of atrial myocytes were α-sarcomeric actin (α-SCA) positive, indicating that the primary cells were positive for α-SCA staining and atrial myocytes were successfully isolated. The pacing atrial myocyte model was established after rapid pacing stimulation and we found the rapid pacing stimulation caused elevated ADAM10 and suppressed miR-520d. CCK-8 assay was applied for evaluation of cell viability, TUNEL staining for assessment of cell apoptosis and dual-luciferase reporter gene assay for verification of the targeting relationship between miR-520d and ADAM10. Overexpression of miR-520d or silencing of ADAM10 could enhance cell viability and reduce cell apoptosis in the rapid pacing-induced atrial myocytes. ADAM10 was a target gene of miR-520d. MiR-520d negatively targeted ADAM10, thereby promoting cell viability and inhibiting apoptosis in rapid pacing atrial myocyte model. In summary, miR-520d enhances atrial myocyte viability and inhibits cell apoptosis in rapid pacing-induced AF mouse model through negative mediation of ADAM10.

Genotype-Guided Dosing of Warfarin in Chinese Adults: A Multicenter Randomized Clinical Trial.

BACKGROUND: Warfarin is an effective treatment for thromboembolic disease but has a narrow therapeutic index; optimal anticoagulation dosage can differ tremendously among individuals. We aimed to evaluate whether genotype-guided warfarin dosing is superior to routine clinical dosing for the outcomes of interest in Chinese patients. METHODS: We conducted a multicenter, randomized, single-blind, parallel-controlled trial from September 2014 to April 2017 in 15 hospitals in China. Eligible patients were ≥18 years of age, with atrial fibrillation or deep vein thrombosis without previous treatment of warfarin or a bleeding disorder. Nine follow-up visits were performed during the 12-week study period. The primary outcome measure was the percentage of time in the therapeutic range of the international normalized ratio during the first 12 weeks after starting warfarin therapy. RESULTS: A total of 660 participants were enrolled and randomly assigned to a genotype-guided dosing group or a control group under standard dosing. The genotype-guided dosing group had a significantly higher percentage of time in the therapeutic range than the control group (58.8% versus 53.2% [95% CI of group difference, 1.1-10.2]; P=0.01). The genotype-guided dosing group also achieved the target international normalized ratio sooner than the control group. In subgroup analyses, warfarin normal sensitivity group had an even higher percentage of time in the therapeutic range during the first 12 weeks compared with the control group (60.8% versus 48.9% [95% CI, 1.1-24.4]). The incidence of adverse events was low in both groups. CONCLUSIONS: The outcomes of genotype-guided warfarin dosing were superior to those of clinical standard dosing. These findings raise the prospect of precision warfarin treatment in China. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02211326.

Long non-coding RNA KCNQ1OT1/microRNA-204-5p/LGALS3 axis regulates myocardial ischemia/reperfusion injury in mice.

Myocardial ischemia/reperfusion (IR) injury is one of the most prevalent cardiovascular diseases, known for its high mortality and morbidity worldwide. Based on pre-existing evidence, LGALS3 has been found to be closely associated with cardiac diseases. Hence, the objective of our study is to explore the potential function of KCNQ1OT1/microRNA-204-5p (miR-204-5p)/ LGALS3 axis on myocardial IR injury and the underlying mechanism. A myocardial IR injury mouse model was established in vivo and an in vitro cardiomyocyte model was induced by hypoxia/Reoxygenation exposure. Next, gain- and loss-of-function experiments were employed in order to measure the viability and apoptosis of cardiomyocytes and the area of ischemic infarct by CCK-8, TUNEL staining and Evans blue/TTC double staining. LGALS3 was found to be highly expressed in myocardial IR injury. The downregulation of LGALS3 resulted in the alleviation of myocardial IR injury in mouse models. In addition, KCNQ1OT1 could promote the LGALS3 expression by binding to miR-204-5p, which led to aggravated myocardial IR injury. In conclusion, KCNQ1OT1 binds to miR-204-5p to exacerbate myocardial IR injury in mice through the up-regulation of LGALS3, providing a novel insight for myocardial IR injury treatment.

Prostaglandin E1 protects cardiomyocytes against hypoxia-reperfusion induced injury via the miR-21-5p/FASLG axis.

BACKGROUND: Prostaglandin-E1 (PGE1) is a potent vasodilator with anti-inflammatory and antiplatelet effects. However, the mechanism by which PGE1 contributes to the amelioration of cardiac injury remains unclear. METHODS: The present study was designed to investigate how PGE1 protects against hypoxia/reoxygenation (H/R)-induced injuries by regulating microRNA-21-5p (miR-21-5p) and fas ligand (FASLG). Rat H9C2 cells and isolated primary cardiomyocytes were cultured under hypoxic conditions for 6 h (6H, hypoxia for 6 h), and reoxygenated for periods of 6 (6R, reoxygenation for 6 h), 12, and 24 h, respectively. Cells from the 6H/6R group were treated with various doses of PGE1; after which, their levels of viability and apoptosis were detected. RESULTS: The 6H/6R treatment regimen induced the maximum level of H9C2 cell apoptosis, which was accompanied by the highest levels of Bcl-2-associated X protein (Bax) and cleaved-caspase-3 expression and the lowest level of B-cell lymphoma 2 (Bcl-2) expression. Treatment with PGE1 significantly diminished the cell cytotoxicity and apoptosis induced by the 6H/6R regimen, and also decreased expression of IL-2, IL-6, P-p65, TNF-α, and cleaved-caspase-3. In addition, we proved that PGE1 up-regulated miR-21-5p expression in rat cardiomyocytes exposed to conditions that produce H/R injury. FASLG was a direct target of miR-21-5p, and PGE1 reduced the ability of H/R-injured rat cardiomyocytes to undergo apoptosis by affecting the miR-21-5p/FASLG axis. In addition, we proved that PGE1 could protect primary cardiomyocytes against H/R-induced injuries. CONCLUSIONS: These results indicate that PGE1 exerts cardioprotective effects in H9C2 cells during H/R by regulating the miR-21-5p/FASLG axis.

Downregulated microRNA-330 suppresses left ventricular remodeling via the TGF-ß1/Smad3 signaling pathway by targeting SRY in mice with myocardial ischemia-reperfusion injury.

microRNAs (miRs) are essential in the development of heart failure. The aim of this study is to investigate the effect of microRNA-330 (miR-330) on left ventricular remodeling via the TGF-ß1/Smad3 signaling pathway by targeting the sex-determining region Y (SRY) in mice with myocardial ischemia-reperfusion injury (MIRI). Differentially expressed gene (DEG) in myocardial ischemia-reperfusion (IR) was screened out and the miR that targeted the DEG was also predicted and verified. A model of MIRI was established to detect the expression of miR-330, SRY, transforming growth factor-ß (TGF-ß1), and Sekelsky mothers against dpp3 (Smad3). To further investigate the role of miR-330 in MIRI with the involvement of SRY and TGF-ß1/Smad3 signaling pathway, the modeled mice were treated with different mimic, inhibitor, or small interfering RNA (siRNA) to observe the changes of the related gene expression, as well as the myocardial infarction size and volume of myocardial collagen. SRY was screened out and verified as a target gene of miR-330. The MIRI mice showed enlarged myocardial infarction size, increased volume of myocardial collagen, increased expression of miR-330, TGF-ß1 and Smad3, while decreased the expression of SRY. The MIRI mice treated with miR-330 inhibitor showed decreased myocardial infarction size, the volume of myocardial collagen, and expression of TGF-ß1 and Smad3 but promoted expression of SRY. Our findings demonstrated that downregulated miR-330 could suppress left ventricular remodeling to inhibit the activation of the TGF-ß1/Smad3 signaling pathway via negatively targeting of SRY in mice with MIRI. This can be a potential target in the strategy to attenuate patient suffering.

[Effect of miR-21 down-regulated by H 2O 2 on osteogenic differentiation of MC3T3-E1 cells].

Objective: To explore the effect and mechanism of miR-21 down-regulated which was induced by H 2O 2 on osteogenic differentiation of MC3T3-E1 cells. Methods: MC3T3-E1 cells were cultured and passaged, and the 7th generation cells were harvested to use in experiment. The MC3T3-E1 cells were treated with different concentrations (0, 40, 80, 160, and 320 µmol/L) of H 2O 2. The expression of miR-21 was detected by real-time quantitative PCR (RT-PCR) and the cell viability was determined by MTS. Then the appropriate concentration of H 2O 2 was obtained. To analyze the effect of H 2O 2 on osteogenic differentiation of MC3T3-E1 cells, the MC3T3-E1 cells were divided into blank control group (group A), H 2O 2 group (group B), osteogenic induction group (group C), and H 2O 2+osteogenic induction group (group D). The expression of miR-21 and the osteogenesis related genes expressions of Runx2, osteopontin (OPN), and collagen type Ⅰ alpha 1 (Col1a1) were detected by RT-PCR. The expression of phosphatase and tensin homolog (PTEN) was detected by Western blot. The extracellular calcium deposition was detected by alizarin red staining. To analyze the effect on osteogenic differentiation of MC3T3-E1 cells after the transfection of miR-21 inhibitor and siRNA-PTEN, the MC3T3-E1 cells were divided into H 2O 2 group (group A1), H 2O 2+osteogenic induction group (group B1), H 2O 2+osteogenic induction+miR-21 inhibitor group (group C1), and H 2O 2+osteogenic induction+miR-21 inhibitor negative control group (group D1); and H 2O 2 group (group A2), H 2O 2+osteogenic induction group (group B2), H 2O 2+osteogenic induction+siRNA-PTEN negative control group (group C2), and H 2O 2+osteogenic induction+siRNA-PTEN group (group D2). The osteogenesis related genes were detected by RT-PCR and the extracellular calcium deposition was detected by alizarin red staining. Results: The results of MTS and RT-PCR showed that the appropriate concentration of H 2O 2 was 160 µmol/L. The expression of miR-21 was significantly lower in group B than in group A at 1 and 2 weeks ( P<0.05). The expression of miR-21 was significantly lower in group D than in group C at 1 and 2 weeks ( P<0.05). The expression of PTEN protein was significantly lower in group C than in groups A and D ( P<0.05). The mRNA expressions of Runx2, OPN, and Col1a1 were significantly lower in group D than in group C at 1 and 2 weeks ( P<0.05). The extracellular calcium deposition in group D was obviously less than that in group C. The expression of PTEN protein was significantly higher in group C1 than in group D1 ( P<0.05). The mRNA expressions of Runx2 and OPN were significantly lower in group C1 than in groups B1 and D1 at 1 and 2 weeks ( P<0.05). The mRNA expression of Col1a1 was significantly lower in group C1 than in groups B1 and D1 at 2 weeks ( P<0.05). The extracellular calcium deposition in group C1 was obviously less than those in groups B1 and D1. The mRNA expressions of OPN and Col1a1 were significantly higher in group D2 than in groups B2 and C2 at 1 week ( P<0.05). The extracellular calcium deposition in group D2 was obviously more than those in groups B2 and C2. Conclusion: H 2O 2 inhibits the osteogenic differentiation of MC3T3-E1 cells, which may be induced by down-regulating the expression of miR-21.

Notch1 signaling activation protected myocardium against hypoxia injury via reducing programmed cell death.

Programmed cell death plays an important role in cardio protection, and Notch1 was an important factor related to programmed cell death. The role of Notch1 on ischemia myocardium remains unclear.H9C2 myocardial cells were cultured with routine medium, transfected with Notch1 over expression plasmid, Notch1-siRNA-overexpression plasmid and vehicle plasmid for further hypoxic experiment. Condition of hypoxic experiment was 1% oxygen centration and culturing for 12hours, then the cell proliferation activity and apoptosis rate was assessed by MTS kit and flow cytometry, respectively. The expressions of Caspase-3, Caspase-9 and Bcl-2 were determined by RT-qPCR and Western Blot, respectively. Compared with normoxia treatment, hypoxia could decrease H9C2 cell proliferation activity as well as Bcl-2 mRNA expression, and increase cell apoptosis rate as well as Caspase-3 and Caspase-9 mRNA expression. Notch1 activation could increase proliferation activity as well as Bcl-2 mRNA expression, while decrease apoptosis rate as well as Caspase-3 and Caspase-9 mRNA expression. Compared with Notch1 activation H9C2 cells, the opposite effect on programmed cell death was observed in cells with Notch1-siRNA-overexpression plasmid. Targeted activation of Notch1 gene to reduce hypoxia-induced programmed cell death in myocardial cells via up-regulating the expression of Caspase-3 and Caspase-9 and inhibiting the expression of Bcl-2.

Effects of lercanidipine hydrochloride versus felodipine sustained-release on day-to-day home blood pressure variability.

OBJECTIVE: The objective of this study was to compare the effectiveness of lercanidipine with felodipine in patients with mild-to-moderate hypertension on day-to-day home blood pressure variability. METHODS: This is a sub-study of a multicenter, randomized, open-label, parallel group and active controlled clinical trial. Hypertensive patients aged 18-75 (i.e. diastolic blood pressure ≥90 mmHg and <110 mmHg; systolic blood pressure ≥140 mmHg and <180 mmHg) and 24 h mean BP >130/80 mmHg) were eligible for this study. During the study, blood pressure (BP) and heart rate (HR) were recorded. The day-to-day BP variability (BPV) and HR variability (HRV) were obtained by the standard deviation (SD) of daily BP/HR average (of six readings) in 7 days. RESULTS: There were 186 patients (89 and 97 patients in the lercanidipine and felodipine groups, respectively) included in this study. Lercanidipine hydrochloride 10 mg/d and felodipine sustained-release tablets 5 mg/d were given to their respective groups. After 6 weeks of treatment, SD of home BP significantly reduced compared with baseline in both groups (P < .05) while SD of home HR also changed significantly after treatment (P < .05). There was no significant difference in SD of home BPV between the lercanidipine and felodipine groups after treatment. CONCLUSION: Treatment with lercanidipine and felodipine both resulted in reduction of BPV and HRV. There was no significant inter-group difference in reduction of BPV between the groups. Lercanidipine is an effective antihypertensive drug in improving BPV. National clinical trial: NCT01520285.

[EFFECT OF ACTIVED RAW264.7 INDUCED BY H2O2 ON MIGRATION, PROLIFERATION AND OSTEOGENESIS GENE EXPRESSION OF MC3T3-E1].

OBJECTIVE: To explore the effect of H2O2-actived RAW264.7 macrophages on the migration, proliferation, and osteogenesis gene expression of MC3T3-E1 in mice. METHODS: MC3T3-E1 cells and RAW264.7 cells were cultured to the 7th generation. RAW264.7 macrophages were stimulated with 0, 25, 50 and 100 µmol/L H2O2, the cell proliferation rate was detected by MTS at 1, 3, and 6 hours after stimulated, and superoxide dismutase (SOD) content by SOD assay kit at 1 hour after stimulated. The appropriate concentration and action time of H2O2-actived RAW264.7 were obtained. The supernatant of RAW264.7 macrophages stimulated by H2O2 or not was collected at 24 hours. Then, the supernatant was used to culture MC3T3-E1 cells in groups B (not stimulated by H2O2) and C (stimulated by H2O2), and DMEM was used as a control in group A. The migration of MC3T3-E1 cells was detected at 12 and 24 hours by cell scratch test, the proliferation of MC3T3-E1 cells at 24, 48, and 72 hours by MTS assay. MC3T3-E1 cells were cultured with only complete medium in blank control group, with complete medium containing 50 µg/mL vitamin C + 10 nmol/L ß sodium glycerophosphate in positive group, normal control group (adding the supernatant not stimulated by H2O2), and experimental group (adding the supernatant stimulated by H2O2). At 3, 7, and 14 days, RT-PCR was used to determine the osteogenesis related mRNA expressions of alkaline phosphatase (ALP), Runx2, osteopontin (OPN), osteocalcin (OC), bone sialoprotein (BSP), and collagen type I (COL-I). RESULTS: The results of MTS and SOD assay showed that the appropriate concentration and action time of H2O2-actived RAW264.7 macrophages were 25 µmol/L and 1 hour, respectively. MTS assay showed that the proliferation rate of MC3T3-E1 cells was significant higher in groups B and C than group A (P<0.05), in group B than group C, and significant difference was shown between groups at 2 and 3 days (P<0.05). The cell scratch test indicated that the migration of MC3T3-E1 cells was significantly faster in groups B and C than group A, and in group C than group B at 12 hours (P<0.05); many migrated cells were observed in all scratch sites of groups B and C at 24 hours. When compared with positive control group, the mRNA expressions of ALP, Runx2, OC and BSP in experimental group were significantly down-regulated at 7 and 14 days (P<0.05). When compared blank control group, the mRNA expressions of OPN and COL-I in experimental group were significantly down-regulated at 7 and 14 days (P<0.05). CONCLUSIONS: The appropriate concentration and action time of H2O2-actived RAW264.7 macrophages are 25 µmol/L and 1 hour. The H2O2-actived RAW264.7 cells can promote MC3T3-E1 cells migration, and suppress MC3T3-E1 cells proliferation and expressions of osteogenesis related genes.