Arrhythmias in patients with coronavirus disease 2019 (COVID-19) in Wuhan, China: Incidences and implications.

BACKGROUND: Coronavirus disease 2019 (COVID-19) continues to impact populations around the globe. Information regarding the incidences and implications of arrhythmias in COVID-19 is limited. METHODS: A total of 463 patients with COVID-19 and who had at least one electrocardiogram recording from February 1 to March 19, 2020, in Wuhan Union Hospital were enrolled in the study. RESULTS: Arrhythmias occurred in 85 of 463 (18.4%) patients: atrial arrhythmias in 10.2%, junctional arrhythmias in 0.2%, ventricular arrhythmias in 3.5%, and conduction block in 7.3%. Compared with patients without arrhythmias, those with arrhythmias had higher mortality, both during the time from symptom onset (p < 0.001) and from admission to follow-up (p < 0.001). The frequencies of severe COVID-19 (44.7% vs. 21.2%; p < 0.001) and death (25.9% vs. 10.1%; p < 0.001) were higher in patients with arrhythmias than in those without arrhythmias. Atrial arrhythmias and ventricular arrhythmias could predict severity and mortality, their odds ratios (OR) were 4.45 (95% confidence interval [CI] 2.35 to 8.40), 5.80 (95% CI 1.89 to 17.76) respectively for severity, and were 3.51 (95% CI 1.74 to 7.08), 3.41 (95% CI 1.13 to 10.24) respectively for mortality. High levels of interleukin-6 (IL-6) and IL-10 were associated with the occurrence of arrhythmias (all p < 0.05). CONCLUSION: Arrhythmias were significantly associated with COVID-19 severity and mortality. Atrial arrhythmia was the most frequent arrhythmia type. IL-6 and IL-10 levels can predict the risk of arrhythmias in COVID-19 patients.

Circulating MiR-19b-3p, MiR-134-5p and MiR-186-5p are Promising Novel Biomarkers for Early Diagnosis of Acute Myocardial Infarction.

BACKGROUND/AIMS: Recent studies have shown that circulating microRNAs (miRNAs) are emerging as promising biomarkers for cardiovascular diseases. This study aimed to determine whether miR-19b-3p, miR-134-5p and miR-186-5p can be used as novel indicators for acute myocardial infarction (AMI). METHODS: To investigate the kinetic expression of the three selected miRNAs, we enrolled 18 patients with AMI and 20 matched controls. Plasma samples were collected from each participant, and total RNA was extracted. Quantitative real-time PCR and ELISA assays were used to investigate the expression of circulating miRNAs and cardiac troponin I (cTnI), respectively. Plasma samples from another age- and gender-matched cohort were collected to investigate the impact of medications for AMI on the expression of the selected miRNAs. RESULTS: Levels of plasma miR-19b-3p, miR-134-5p and miR-186-5p were significantly increased in early stage of AMI. Plasma miR-19b-3p and miR-134-5p levels reached peak expression immediately after admission (T0), whereas miR-186-5p achieved peak expression at 4 h after T0. All of these times were earlier than the peak for cTnI (8 h after T0). In addition, all three miRNAs were positively correlated with cTnI. Receiver Operating Characteristic (ROC) analysis indicated that each single miRNA showed considerable diagnostic efficiency for predicting AMI. Furthermore, combining all three miRNAs in a panel increased the efficiency of distinguishing between patients with AMI and controls. Moreover, we found that heparin and medications for AMI did not impact the expression of these circulating miRNAs. CONCLUSION: Circulating miR-19b-3p, miR-134-5p and miR-186-5p could be considered promising novel diagnostic biomarkers for the early phase of AMI.

MicroRNA-101a inhibits cardiac fibrosis induced by hypoxia via targeting TGFßRI on cardiac fibroblasts.

BACKGROUND/AIMS: Hypoxia is a basic pathological challenge that is associated with numerous cardiovascular disorders including aberrant cardiac remodeling. Transforming growth factor beta (TGF-ß) signaling pathway plays a pivotal role in mediating cardiac fibroblast (CF) function and cardiac fibrosis. Recent data suggested that microRNA-101a (miR-101a) exerted anti-fibrotic effects in post-infarct cardiac remodeling and improved cardiac function. This study aimed to investigate the potential relationship between hypoxia, miR-101a and TGF-ß signaling pathway in CFs. METHODS AND RESULTS: Two weeks following coronary artery occlusion in rats, the expression levels of both TGFß1 and TGFßRI were increased, but the expression of miR-101a was decreased at the site of the infarct and along its border. Cultured rat neonatal CFs treated with hypoxia were characterized by the up-regulation of TGFß1 and TGFßRI and the down-regulation of miR-101a. Delivery of miR-101a mimics significantly suppressed the expression of TGFßRI and p-Smad 3, CF differentiation and collagen content of CFs. These anti-fibrotic effects were abrogated by co-transfection with AMO-miR-101a, an antisense inhibitor of miR-101a. The repression of TGFßRI, a target of miR-101a, was validated by luciferase reporter assays targeting the 3'UTR of TGFßRI. Additionally, we found that overexpression of miR-101a reversed the improved migration ability of CFs and further reduced CF proliferation caused by hypoxia. CONCLUSION: Our study illustrates that miR-101a exerts anti-fibrotic effects by targeting TGFßRI, suggesting that miR-101a plays a multi-faceted role in modulating TGF-ß signaling pathway and cardiac fibrosis.

Plasma exosomes from patients with acute myocardial infarction alleviate myocardial injury by inhibiting ferroptosis through miR-26b-5p/SLC7A11 axis.

AIMS: Ferroptosis promotes myocardial injury in acute myocardial infarction (AMI). Increasing evidence suggests the crucial role of exosomes in post-AMI pathophysiological regulation. We aimed to investigate the effects and underlying mechanisms of plasma exosomes derived from patients with AMI in inhibiting ferroptosis after AMI. METHODS: Plasma exosomes were isolated from controls (Con-Exo) and patients with AMI (MI-Exo). These exosomes were incubated with hypoxic cardiomyocytes or intramyocardially injected into the AMI mice. Histopathological changes, cell viability, and cell death were measured to evaluate the myocardial injury. For the ferroptosis evaluation, iron particle deposition, Fe2+, ROS, MDA, GSH, and GPX4 levels were detected. Exosomal miR-26b-5p expression was detected by qRT-PCR, and the targeting relationship between miR-26b-5p and SLC7A11 was confirmed by dual luciferase reporter gene assay. The role of the miR-26b-5p/SLC7A11 axis in the regulation of ferroptosis was validated by rescue experiments in cardiomyocytes. FINDINGS: Hypoxia-treatment induced ferroptosis and injury in H9C2 cells and primary cardiomyocytes. MI-Exo performed better than Con-Exo in inhibiting hypoxia-induced ferroptosis. miR-26b-5p expression was downregulated in MI-Exo, and miR-26b-5p overexpression significantly eliminated the inhibitory effect of MI-Exo on ferroptosis. Mechanistically, knockdown of miR-26b-5p upregulated SLC7A11/GSH/GPX4 expressions by directly targeting SLC7A11. Moreover, SLC7A11 silencing also reversed the inhibitory effect of MI-Exo on hypoxia-induced ferroptosis. In vivo, MI-Exo significantly inhibited ferroptosis, reduced myocardial injury, and improved the cardiac function of AMI mice. SIGNIFICANCE: Our findings revealed a novel mechanism of myocardial protection that downregulation of miR-26b-5p in MI-Exo notably upregulated SLC7A11 expression, thereby inhibiting post-AMI ferroptosis and alleviating myocardial injury.

MicroRNA-130a attenuates cardiac fibrosis after myocardial infarction through TGF-ß/Smad signaling by directly targeting TGF-ß receptor 1.

Cardiac fibrosis is a common pathophysiological change associated with myocardial infarction (MI), and while there is evidence that miR-130a plays an important role in a variety of fibrotic diseases, its role in the cardiac fibrosis during MI is unclear. Our study aimed to assess miR-130a's ability to modulate cardiac fibrosis post-MI and uncover its potential molecular mechanisms. miR-130a was significantly downregulated in infarcted myocardium and hypoxic cardiac fibroblasts (CFs), whereas TGF-ß, α-SMA, collagen 1 (Col-1), and TGF-ß receptor 1 (TGFBR1) were upregulated. We transfected mice with AAV-9 carrying miR-130a and found that miR-130a overexpression statistically improved cardiac function and reduced the area of cardiac fibrosis in mice post-MI. Eukaryotic transcriptome sequencing and dual-luciferase reporter assay results verified that Tgfbr1 was a target gene of miR-130a. miR-130a inhibition heightened Col-1, α-SMA, and TGFBR1 expressions and Smad3 phosphorylation levels in CFs; however, these increments were suppressed by the overexpression of miR-130a. Meanwhile, co-transfection with TGFBR1 weakened miR-130a's ability to inhibit α-SMA and Col-1 expression. These findings suggest that miR-130a exerts antifibrotic properties by directly targeting TGFBR1 to regulate TGF-ß/Smad signaling and inhibit the conversion of CFs to myofibroblasts. Thus, miR-130a is a promising therapeutic target for alleviating cardiac fibrosis.

Human umbilical vein endothelial cells-derived exosomes enhance cardiac function after acute myocardial infarction by activating the PI3K/AKT signaling pathway.

Currently, acute myocardial infarction (AMI) is one of the leading causes of human health issues worldwide. The sudden and continuous occlusion of the coronary artery results in myocardial hypoxic-ischemic necrosis, which is accompanied by inflammatory infiltration and fibrosis, leading to pathological cardiac remodeling. Exosome-based therapy is a promising cell-free approach for repairing the ischemic myocardium. This study aimed to explore the effects and mechanism of human umbilical vein endothelial cells (HUVECs)-derived exosomes on AMI. The results indicated that the localized injection of HUVECs-derived exosomes in the infarcted area could significantly improve cardiac function in AMI mouse models. It could also ameliorate myocardial fibrosis and decrease infarct size after AMI. Additionally, HUVECs-derived exosomes had cardioprotective effects on the H9C2 cells in hypoxic culture conditions, including increased cell viability and decreased lactate dehydrogenase (LDH) release. In both the in-vivo and in-vitro experiments, HUVECs-derived exosomes could effectively inhibit cardiomyocyte apoptosis. The low expression levels of Bcl-2-associated X protein (Bax) and cleaved caspase-3, high expression levels of B-cell lymphoma 2 (Bcl-2), phosphorylated phosphatidylinositol 3-kinase (p-PI3K), and phosphorylated protein kinase B (p-AKT) were detected in AMI mouse models treated with HUVECs-derived exosomes in-vivo. In conclusion, HUVECs-derived exosomes effectively enhanced cardiac function after AMI and inhibited cardiomyocyte apoptosis, which might be regulated through the phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT) signaling pathway.

Identification of Immune Markers in Dilated Cardiomyopathies with Heart Failure by Integrated Weighted Gene Coexpression Network Analysis.

Dilated cardiomyopathy (DCM), a heterogeneous cardiomyopathy, is a major cause of heart failure and heart transplant. Currently, immunotherapy is believed to be an effective treatment method for DCM. However, individual differences are so obvious that the clinical effect is not satisfactory. In order to find immune-related biomarkers of DCM to guide treatment and improve clinical efficacy, we downloaded a GSE120895 dataset from the Gene Expression Omnibus (GEO) database using CIBERSORT and WGCNA algorithms in RStudio and visualizing the protein-protein interaction (PPI) network for key modules by Cytoscape, and finally obtained six hub genes. A GSE17800 dataset was downloaded from the GEO dataset to verify the diagnostic values of hub genes, MYG1, FLOT1, and ATG13, which were excellent. Our study revealed unpublished potential immune mechanisms, biomarkers, and therapeutic targets of DCM.

miR-186-5p Dysregulation in Serum Exosomes from Patients with AMI Aggravates Atherosclerosis via Targeting LOX-1.

Purpose: The formation of macrophage-derived foam cells via the uptake of modified lipoproteins is a pivotal development event in atherosclerosis. It has been reported that clinical and experimental myocardial infarction could accelerate atherosclerosis. Several studies have suggested the critical role of exosomes in cardiovascular diseases. However, the role of exosomes from patients with acute myocardial infarction (AMI) patients in atherogenesis remains unclear. Patients and Methods: Serum exosomes from AMI patients (AMI-Exo) and control individuals (Con-Exo) were isolated and characterized. These exosomes were studied in vitro and in vivo to determine their impact on macrophage foaming and atherogenesis. Results: Our results showed that AMI-Exo promoted foam cell formation in oxidized low-density lipoprotein (ox-LDL)-treated macrophages and progression of atherosclerosis in high-fat/cholesterol diet-fed ApoE-/- mice together with a significantly upregulated levels of lectin-like ox-LDL receptor-1 (LOX-1). The miR-186-5p was found to be downregulated in AMI-Exo and macrophages administered with AMI-Exo. Moreover, serum exosomal miR-186-5p achieved high diagnostic performance for AMI. Luciferase reporter assay indicated that miR-186-5p directly inhibited LOX-1. The endogenous or exogenous miR-186-5p deficiency enhanced lipid accumulation by upregulating LOX-1, whereas miR-186-5p mimics had a reverse effect. Conclusion: In conclusion, the current findings suggest that dysregulated miR-186-5p in AMI-Exo may explain the contribution of acute ischemia events to the advancement of atherosclerosis by enhancing macrophage foaming via its target, LOX-1.

Total flavonoids of sea buckthorn (Hippophae rhamnoides L.) improve MC903-induced atopic dermatitis-like lesions.

ETHNOPHARMACOLOGICAL RELEVANCE: Sea buckthorn (Hippophae rhamnoides L.) is popularly used as a herbal medicine and food additive in the world. Total flavonoids of Hippophae rhamnoides (TFH) are reported to have anti-inflammatory and immunomodulatory activities. AIM: The effects of TFH on atopic dermatitis (AD)-like lesions induced by MC903 in mice was elucidated in the study. METHODS: To induce AD-like lesions, MC903 was adopted to apply repeatedly on the left ear in C57BL/6 mice. After induction of AD-like lesions, 0.5% and 1% TFH cream was applied topically on ears of mice once a day for 8 days. The degree of skin lesions was evaluated by macroscopical and histological methods. Expressions of filaggrin (FLG) was evaluated by Western blotting. Real-time polymerase chain reaction (qPCR) was adopted to detect the mRNA expression of thymic stromal lymphopoietin (TSLP), interferon (IFN)-γ, interleukin (IL-4), tumor necrosis factor (TNF)-α in skin lesions. In vitro, Cytokine Antibody Arrays were performed to measure production of cytokines in IFN-γ/TNF-α-treated HaCaT cells, Western blotting was employed to detect the expressions of p-NF-κB, p-ERK and p-P38. RESULTS: Topical application of TFH significantly improved the severity of dermatitis by inhibiting the infiltration of mast cell, increasing expression of FLG, decreasing the expressions of TNF-α, IL-4, IFN-γ and TSLP in skin lesions. TFH decreased the levels of IL-1α, IL-1ß, IL-6, monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage-derived chemokine (MDC), platelet-derived growth factor (PDGF)-BB, thymus and activation regulated chemokine (TARC) in the supernatants of the HaCaT cells treated by IFN-γ/TNF-α. Furthermore, expressions of p-NF-κB, p-ERK and p-P38 were also decreased by TFH administration with dose dependent manner in HaCaT cells treated by IFN-γ/TNF-α. CONCLUSIONS: Topical application of TFH improved AD-like lesions in mice induced by MC903. Which exerted the effects of anti-inflammation and repairing skin barrier by regulating Th1/Th2 balance. This finding indicates that TFH is a novel potential agent for the external treatment of AD.

Corrected QT interval in hospitalized patients with coronavirus disease 2019: Focus on drugs therapy.

ABSTRACT: Corrected QT (QTc) interval prolongation has been associated with poor patient prognosis. In this study, we assessed the effects of different drugs and cardiac injury on QTc interval prolongation in patients with coronavirus disease 2019 (COVID-19).The study cohort consisted of 395 confirmed COVID-19 cases from the Wuhan Union Hospital West Campus. All hospitalized patients were treated with chloroquine/hydroxychloroquine (CQ/HCQ), lopinavir/ritonavir (LPV/r), quinolones, interferon, Arbidol, or Qingfei Paidu decoction (QPD) and received at least 1 electrocardiogram after drug administration.Fifty one (12.9%) patients exhibited QTc prolongation (QTc ≥ 470 ms). QTc interval prolongation was associated with COVID-19 severity and mortality (both P < .001). Administration of CQ/HCQ (odds ratio [OR], 2.759; 95% confidence interval [CI], 1.318-5.775; P = .007), LPV/r (OR, 2.342; 95% CI, 1.152-4.760; P = .019), and quinolones (OR, 2.268; 95% CI, 1.171-4.392; P = .015) increased the risk of QTc prolongation. In contrast, the administration of Arbidol, interferon, or QPD did not increase the risk of QTc prolongation. Notably, patients treated with QPD had a shorter QTc duration than those without QPD treatment (412.10 [384.39-433.77] vs 420.86 [388.19-459.58]; P = .042). The QTc interval was positively correlated with the levels of cardiac biomarkers (creatine kinase-MB fraction [rho = 0.14, P = .016], high-sensitivity troponin I [rho = .22, P < .001], and B-type natriuretic peptide [rho = 0.27, P < .001]).In conclusion, QTc prolongation was associated with COVID-19 severity and mortality. The risk of QTc prolongation was higher in patients receiving CQ/HCQ, LPV/r, and quinolones. QPD had less significant effects on QTc prolongation than other antiviral agents.

Ameliorative effects of sea buckthorn oil on DNCB induced atopic dermatitis model mice via regulation the balance of Th1/Th2.

BACKGROUND: Atopic dermatitis (AD) is a worldwide chronic skin disease which burden public health. Sea buckthorn (SBT) (Hippophae rhamnoides L., Elaeagnaceae) oil, as a traditional herbal medicine, has been used for disease treatment for many years. The effects of SBT oil on AD mouse model induced by repeated administration of 2,4-dinitrochlorobenzene (DNCB) in BALB/c mice was evaluated in this study. METHODS: Mice were divided into four groups including the normal control group, AD model group, AD model group treated with SBT oil (5 ml/kg) and AD model group treated with SBT oil (10 ml/kg). Same volume at different concentrations of SBT oil was applied daily on the latter two groups by gavage for 15 days following AD model induction. The function of skin barrier and the production of IL-4, IFN-γ, TNF-α and TSLP were examined after animal sacrifice. The migration and mature of langerhans cell (LCs) in lymph node was further assessed by flow cytometry. RESULTS: SBT oil alleviated dermatitis scores, decreased ear thickness, prevented infiltration of mast cell, reduced lymph node weight and depressed activity of Th2 cells. SBT oil also reduced the expression of IL-4, IFN-γ, TNF-α and TSLP in ear tissue, IgE level in serum and mRNA relative expression of IL-4, IFN-γ, TNF-α in lymph node. Moreover, SBT oil inhibited the migration of LCs cells from local lesions to lymph node and it's mature in lymph node. CONCLUSIONS: These results suggest SBT oil had a beneficial effect either systemic or regional on DNCB-induced AD mice via maintain the balance of Th1/Th2 and may be a potential complementary candidate for AD treatment.

Clinical Characteristics and Prognosis of 244 Cardiovascular Patients Suffering From Coronavirus Disease in Wuhan, China.

Background The coronavirus disease 2019 (COVID-19) has developed into a global outbreak. Patients with cardiovascular disease (CVD) with COVID-19 have different clinical characteristics and prognostic outcomes. This study aimed to summarize the clinical characteristics and laboratory indicators of patients with COVID-19 with CVD, especially the critically ill patients. Methods and Results This study included 244 patients diagnosed with COVID-19 and CVD (hypertension, coronary heart disease, or heart failure). The patients were categorized into critical (n=36) and noncritical (n=208) groups according to the interim guidance of China's National Health Commission. Clinical, laboratory, and outcome data were collected from the patients' medical records and compared between the 2 groups. The average body mass index of patients was significantly higher in the critical group than in the noncritical group. Neutrophil/lymphocyte ratio, and C-reactive protein, procalcitonin, and fibrinogen, and d-dimer levels at admission were significantly increased in the critical group. The all-cause mortality rate among cases of COVID-19 combined with CVD was 19.26%; the proportion of coronary heart disease and heart failure was significantly higher in deceased patients than in recovered patients. High body mass index, previous history of coronary heart disease, lactic acid accumulation, and a decrease in the partial pressure of oxygen were associated with death. Conclusions All-cause mortality in patients with COVID-19 with CVD in hospitals is high. The high neutrophil/lymphocyte ratio may be a predictor of critical patients. Overweight/obesity combined with coronary heart disease, severe hypoxia, and lactic acid accumulation resulting from respiratory failure are related to poor outcomes. Registration URL: https://www.chictr.org.cn; Unique identifier: ChiCTR2000029865.

Magnolol-induced H460 cells death via autophagy but not apoptosis.

We have reported that the protective effect of Magnolol on TBHP-induced injury in human nonsmall lung cancer H460 cells is partially via a p53 dependent mechanism. In this study, we found that Magnolol displayed a stimulatory effect at low concentrations (< or = 20 microM) whilst inhibitory effect at high concentrations (> or = 40 microM) in H460 cells. To investigate the mechanism of inducing the biphasic effect in H460 cells with Magnolol, we showed that Magnolol stimulated DNA synthesis at low concentrations and displayed an inhibition effect at high concentrations in H460 cells. More importantly, the inhibition of DNA synthesis was accompanied by the S phase cell cycle arrest and the appearance of intense intracytoplasmic vacuoles. These vacuoles can be labeled by autophagic marker monodansylcadaverin (MDC), 3-methyladenine (3-MA), an inhibitor of autophagy, was able to inhibit the occurrence of autophagy. The results of the LDH activity assay and TUNEL assay also showed that Magnolol at high concentrations inhibiting H460 cell growth was not via apoptotic pathway. Furthermore, accompanied by the occurrence of autophagy, the expression of phospho-Akt was down-regulated but PTEN significantly was up-regulated. In conclusion, Magnolol induces H460 cells death by autophagy but not apoptotic pathway. Blockade of PI3K/PTEN/Akt pathway is maybe related to Magnolol-induced autophagy. Autophagic cells death induction by Magnolol underlines the potential utility of its induction as a new cancer treatment modality.

Effects of total flavonoids of sea buckthorn ( Hippophae rhamnoides L.) on cytotoxicity of NK92-MI cells.

Sea buckthorn ( Hippophae rhamnoides L.) has multifarious medicinal properties including immunoregulatory effect. The total flavonoids of Hippophae rhamnoides L. (TFH) are the main active components isolated from berries of sea buckthorn. The aim of this study was to evaluate the effects of TFH on the cytotoxicity of NK92-MI cells and its possible mechanisms. NK92-MI cells were treated with TFH (2.5 or 5.0 mg/L) or phosphate-buffered saline (PBS) for 24 h, the cytotoxicity against K562 was detected by measuring the release of lactate dehydrogenase (LDH), expression levels of NCRs (NKp30, NKp44, NKp46) and NKG2D were detected by flow cytometry, and expression levels of perforin and granzyme B were detected by western blot. Cytokine Antibody Arrays with 80 cytokine proteins were used to profile the effect of TFH on cytokines. Western blot was adopted to detect the effects of TFH on STAT1, STAT4, and STAT5 signal pathway. Compared with the normal control group, TFH could significantly enhance NK92-MI cell cytotoxicity against K562 cells, upregulate expressions of NKp44, NKp46, perforin, and granzyme B. TFH could upregulate expressions of IL-1α, IL-2, IL-7, IL-15, CSF-2, CSF-3, MCP-1, MIG, IFN-γ, TNF-α, and TNF-ß and downregulate expressions of IL-16, MIP-1ß, CX3CL-1, and MIF. TFH could increase expressions of phospho-STAT1 and phospho-STAT5. The results suggest that TFH stimulated NK92-MI cells to activate and enhance cytotoxicity of NK92-MI cells.

Effect of ginseng polysaccharides on NK cell cytotoxicity in immunosuppressed mice.

The aim of the present study was to investigate the effects of Ginseng polysaccharides (GPS) on natural killer (NK) cell cytotoxicity in immunosuppressed mice. Cyclophosphamide (Cy) was used to construct an immunosuppressed mouse model. The mice in each group were submitted to gavages with 200 or 400 mg/kg GPS every day for 10 days. Magnetic-activated cell sorting was used to isolate spleen NK cells, and the NK cell cytotoxicity, blood distribution, expression levels of perforin and granzyme, and the mRNA expression levels of interferon (IFN)-γ were detected. Compared with the normal control group, the cytotoxicity and proportion of NK cells in the blood, and the expression levels of perforin, granzyme and IFN-γ mRNA in the Cy model group were significantly reduced (P<0.05). In addition, compared with the Cy model group, the cytotoxicity and proportion of NK cells in the whole blood, and the expression levels of perforin and granzyme in the NK cells in the Cy + low-dose GPS and Cy + high-dose GPS groups were significantly increased (P<0.05). However, the mRNA expression levels of IFN-γ in the NK cells did not significantly change (P>0.05). Compared with the normal control group, the cytotoxicity and proportion of NK cells in the whole blood, and the expression levels of perforin in the Cy + low-dose GPS and the Cy + high-dose GPS groups were significantly lower (P<0.05). However, the expression levels of granzyme in the NK cells was not significantly different, as compared with the normal control group (P>0.05). These results suggested that GPS promotes NK cell cytotoxicity in immunosuppressed mice by increasing the number of NK cells in the whole blood and upregulating the expression of perforin and granzyme. Thus, the present study investigated the molecular mechanism underlying NK cell activation by GPS, the research showed that GPS have a wide application prospects in the treatment of cancer and immunodeficiency diseases.

MicroRNA-101 protects cardiac fibroblasts from hypoxia-induced apoptosis via inhibition of the TGF-ß signaling pathway.

Cardiac fibroblasts (CFs) are the most numerous cells in the heart and are recognized primarily for their ability to maintain both the structural integrity and the physiological functions of the heart. The transforming growth factor beta (TGF-ß) signaling pathway is reportedly involved in the modulation of CF functions, including apoptosis. Recent studies have indicated that microRNA-101 (miR-101) attenuates the TGF-ß signaling pathway, either by inhibiting the expression of TGFß1 or by targeting transforming growth factor-ß receptor type I (TGFßRI). The present study aimed to determine whether miR-101 protects CFs from hypoxia-induced apoptosis and to investigate the mechanisms underlying its protective effects. The CCK-8 test, electron microscopy and TUNEL assay results demonstrated that miR-101a/b significantly inhibited hypoxia-induced CF apoptosis. The results of Western blotting, quantitative RT-PCR and immunofluorescence assays indicated that miR-101a dramatically inhibited the hypoxia-induced up-regulation of both TGFßRI and p-Smad 3 but not TGFß1 in CFs. Additionally, miR-101a significantly reversed the hypoxia-induced up-regulation of Bax and Caspase-3, the down-regulation of Bcl-2 and the activation of Caspase-3 in CFs. Moreover, miR-101a markedly inhibited the intracellular Ca(2+) ([Ca(2+)]i) overload caused by hypoxia. Taken together, our results suggest that miR-101a protects CFs against hypoxia-induced apoptosis by inhibiting the TGF-ß signaling pathway, which may be a potential therapeutic target for heart injury.

[Effects of acupoint area and non-acupoint area of eye-acupuncture on expressions of VIP and AQP 8 in colonic tissues in rats with D-IBS].

OBJECTIVE: To explore the point specificity of eye-acupuncture and the mechanism of eye-acupuncture on diarrhea-predominant irritable bowel syndrome (D-IBS). METHODS: Forty male Wistar rats of SPF grade were randomly divided into a normal group, a model group, a eye-acupuncture point (AA) group and a non-point (NA) group. The D-IBS rat model was established with the combination methods of the chronic stress and binding limbs. The AA group was treated by acupuncture at "low energizer area", "large intestine area", "liver area" and "spleen area", and the NA group by acupuncture at 3 mm apart from the same points area mentioned above, and the normal group and the model group with no intervention. The rate of feces moisture content was detected. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA of aquaporin 8 (AQP 8) in colon. Protein expressions of the vasoactive intestinal peptide (VIP) and AQP 8 in colon were detected by SABC immunohistochemistry method. RESULTS: Compared with normal group, the rate of feces moisture content at the 18th and 25th days, VIP protein in colon mucosa, myenteric nerve plexus and hypo-mucosa nerve plexus increased significantly (all P < 0.01), and AQP 8 mRNA in colon mucosa decreased significantly in model, AA and NA group (P < 0.05, P < 0.01); AQP 8 protein in colon mucosa decreased significantly in model group and NA group (both P < 0.01). Compared with model group, the rate of feces moisture content at the 25th day and VIP protein in colon mucosa decreased significantly (P < 0.01, P < 0.05), and AQP 8 mRNA and protein increased significantly (P < 0.05, P < 0.01) in AA group. Compared with AA group, the rate of feces moisture content at the 25th day and VIP protein in colon mucosa increased significantly (both P < 0.01), and AQP 8 mRNA and protein decreased significantly (both P < 0.01) in NA group. CONCLUSION: Eye-acupuncture has a good therapeutic effect on D-IBS. It is suggested that one of the mechanism is relate to increase AQP 8 in colon tissue and restrain the expression of VIP. Non-point area of eye-acupuncture has no obviously therapeutic effect and so to illustrate the point specificity of eye-acupuncture.

[Effects of eye-acupuncture therapy on the expression of AQP4 in brain tissue of rats with acute cerebral ischemia-reperfusion injury].

OBJECTIVE: To explore the mechanism of the eye-acupuncture for treatment of acute cerebral ischemia-reperfusion injury. METHODS: Thirty-two healthy SD rats were randomly divided into a normal group, a sham operation group, a model group and an eye-acupuncture group, 8 rats in each group. The rat model of cerebral ischemia-reperfusion was established with thread occlusion method in the model group and the eye-acupuncture group. The eye-acupuncture group was treated by eye-acupuncture at "liver region", "upper energizer area", "lower energizer area" and "kidney region" for 20 min immediately after reperfusion and at 30 min before sampling. No treatment was done in the normal group and the sham operation group, and no thread occlusion was performed in the sham operation group. The Neurologic impairment was scored and the methods of immunohistochemistry staining, western-blotting and real-time fluorescent quantitation polymerase chain reaction (RQ-PCR) were taken to detect the expression of the aquaporin protein 4 (AQP4) and its mRNA in cerebral cortex after reperfusion for 3 hours. RESULTS: The neurologic impairment score of 1.50 +/- 0.54 in the eye-acupuncture group was significant lower than 2.63 +/- 0.92 in the model group (P < 0.01). The expression of the AQP4 protein by immunohistochemistry and western-blot respectively were 116.33 +/- 10.24 and 0.53 +/- 0.04 in the normal group, 118.97 +/- 12.72 and 0.55 +/- 0.07 in the sham operation group, and 129.30 +/- 18.36 and 0.67 +/- 0.08 in the eye-acupuncture group, with statistical significance compared to 150.88 +/- 15.82 and 0.94 +/- 0.04 in the model group (all P < 0.01), and there were significant differences between the eye-acupuncture group and the normal group (both P < 0.01). The tendency in the expression of AQP4 protein and its mRNA in all the group were almost the same. CONCLUSION: The eye-acupuncture therapy can relieve the cerebral ischemia-reperfusion injury and the protective mechanism is related to the downregulation of the cerebral AQP4 expression.

[Effect of eye-acupuncture on cerebral intracellular adhesion molecule-1 expression in rats with acute cerebral ischemia-reperfusion injury].

OBJECTIVE: To investigate the effect of the eye-acupuncture therapy on cerebral intercellular adhesion molecule-1 (ICAM-1) expression in rats with acute cerebral ischemia-reperfusion injury (CI/RI) so as to reveal its underlying mechanism. METHODS: Forty male SD rats were randomly and equally divided into normal, sham operation (sham), model and eye-acupuncture groups. Acute CI/RI model was established by occlusion of the middle cerebral artery with a modified suture-blocking method. Filiform needles were inserted into the "Qan" (Liver Area), "Shangjiao" (Upper Energizer Area), "Xiajiao" (Lower Energizer Area), etc. of the eye-acupuncture acupoints, and retained for 20 min. The treatment was conducted twice daily, 7 times altogether. Seventy-two hours after Acute CI/RI, the neurological behavior was assessed by ZeaLonga neurophysical impairment scale. Cerebral ICAM-1 protein and ICAM-1 mRNA expression levels were detected by Western blot (Penumbra area of the right cerebral infarction region) and real time-polymerase chain reaction (PCR) methods, respectively. RESULTS: After modeling, the neurophysical impairment score of the model group was increased from 0 to (2.63 +/- 0.92) points on the first day and (2.63 +/- 0.74) points 72 h following Acute CI/RI. Correspondingly, cerebral ICAM-1 protein and mRNA expression was up-regulated considerably in the model group than in the normal control group (P < 0. 01). In comparison with the model group, the neurologic impairment score, and cerebral ICAM-1 protein and mRNA expression levels of the eye-acupuncture group were down-regulated obviously (P < 0.01, P < 0.05). No significant differences were found between the normal control and sham groups in neurologic impairment score and the expression levels of the ICAM-1 protein and ICAM-1 mRNA(P > 0.05). CONCLUSION: Eye-acupuncture therapy can improve the cerebral ischemia-reperfusion injury induced behavior changes, which may be related to its effects in down-regulating the expression of cerebral ICAM-1 protein and gene.

[Effects of eye-acupuncture therapy on serum and colonic SP and VIP contents in rats with irritable bowel syndrome].

OBJECTIVE: To observe the effect of the eye-acupuncture therapy on serum and colonic substance P (SP) and vasoactive intestinal peptide (VIP) contents in rats with irritable bowel syndrome (IBS) so as to explore its underlying mechanism. METHODS: Forty male Wistar rats were equally randomized into control group, IBS model group, eye-acupuncture group and medication (Pinaverium bromide, 7.5 mg/kg, twice daily, intragastric administration) group. IBS model was established by giving the rat with chronic stress stimulation (cold-water swimming, tail clamping, electrical shock, etc.) for 18 days. Eye-acupuncture of Xiajiao (Low Energizer) Area, Pi (Spleen) Area, Gan (Liver) Area and Dachang (Large Intestine) Area was given to the rat 20 min, twice daily for 7 d. Histopathological changes of the colon tissue were displayed by HE staining; and serum and colonic SP and VIP contents were detected by enzyme linked immunosorbent assay (ELISA). RESULTS: No significant difference was found among 4 groups in the histopathological changes of the colon. In comparison with normal control group, both serum and colonic SP and VIP contents in model group increased significantly (P < 0.01), while compared with model group, those in eye-acupuncture and medication groups lowered considerably (P < 0.01). CONCLUSION: Eye-acupuncture can reduce serum and coIonic SP and VIP contents in IBS rats, which may play a role in relieving IBS in eye-acupuncture clinic.