Notoginsenoside R1 protects against myocardial ischemia/reperfusion injury in mice via suppressing TAK1-JNK/p38 signaling.

Previous studies show that notoginsenoside R1 (NG-R1), a novel saponin isolated from Panax notoginseng, protects kidney, intestine, lung, brain and heart from ischemia-reperfusion injury. In this study we investigated the cardioprotective mechanisms of NG-R1 in myocardial ischemia/reperfusion (MI/R) injury in vivo and in vitro. MI/R injury was induced in mice by occluding the left anterior descending coronary artery for 30 min followed by 4 h reperfusion. The mice were treated with NG-R1 (25 mg/kg, i.p.) every 2 h for 3 times starting 30 min prior to ischemic surgery. We showed that NG-R1 administration significantly decreased the myocardial infarction area, alleviated myocardial cell damage and improved cardiac function in MI/R mice. In murine neonatal cardiomyocytes (CMs) subjected to hypoxia/reoxygenation (H/R) in vitro, pretreatment with NG-R1 (25 µM) significantly inhibited apoptosis. We revealed that NG-R1 suppressed the phosphorylation of transforming growth factor ß-activated protein kinase 1 (TAK1), JNK and p38 in vivo and in vitro. Pretreatment with JNK agonist anisomycin or p38 agonist P79350 partially abolished the protective effects of NG-R1 in vivo and in vitro. Knockdown of TAK1 greatly ameliorated H/R-induced apoptosis of CMs, and NG-R1 pretreatment did not provide further protection in TAK1-silenced CMs under H/R injury. Overexpression of TAK1 abolished the anti-apoptotic effect of NG-R1 and diminished the inhibition of NG-R1 on JNK/p38 signaling in MI/R mice as well as in H/R-treated CMs. Collectively, NG-R1 alleviates MI/R injury by suppressing the activity of TAK1, subsequently inhibiting JNK/p38 signaling and attenuating cardiomyocyte apoptosis.

Aerobic Exercise Attenuates Pressure Overload-Induced Myocardial Remodeling and Myocardial Inflammation via Upregulating miR-574-3p in Mice.

BACKGROUND: Exercise training can promote cardiac rehabilitation, thereby reducing cardiovascular disease mortality and hospitalization rates. MicroRNAs (miRs) are closely related to heart disease, among which miR-574-3p plays an important role in myocardial remodeling, but its role in exercise-mediated cardioprotection is still unclear. METHODS: A mouse myocardial hypertrophy model was established by transverse aortic coarctation, and a 4-week swimming exercise training was performed 1 week after the operation. After swimming training, echocardiography was used to evaluate cardiac function in mice, and histopathologic staining was used to detect cardiac hypertrophy, myocardial fibrosis, and cardiac inflammation. Quantitative real-time polymerase chain reaction was used to detect the expression levels of miR-574-3p and cardiac hypertrophy markers. Western blotting detected the IL-6 (interleukin-6)/JAK/STAT inflammatory signaling pathway. RESULTS: Echocardiography and histochemical staining found that aerobic exercise significantly improved pressure overload-induced myocardial hypertrophy (n=6), myocardial interstitial fibrosis (n=6), and cardiac inflammation (n=6). Quantitative real-time polymerase chain reaction detection showed that aerobic exercise upregulated the expression level of miR-574-3p (n=6). After specific knockdown of miR-574-3p in mouse hearts with adeno-associated virus 9 using cardiac troponin T promoter, we found that the protective effect of exercise training on the heart was significantly reversed. Echocardiography and histopathologic staining showed that inhibiting the expression of miR-574-3p could partially block the effects of aerobic exercise on cardiac function (n=6), cardiomyocyte cross-sectional area (n=6), and myocardial fibrosis (n=6). Western blotting and immunohistochemical staining showed that the inhibitory effects of aerobic exercise on the IL-6/JAK/STAT pathway and cardiac inflammation were partially abolished after miR-574-3p knockdown. Furthermore, we also found that miR-574-3p exerts cardioprotective effects in cardiomyocytes by targeting IL-6 (n=3). CONCLUSIONS: Aerobic exercise protects cardiac hypertrophy and inflammation induced by pressure overload by upregulating miR-574-3p and inhibiting the IL-6/JAK/STAT pathway.

Efficacy of radiofrequency catheter ablation for premature ventricular contractions in children.

PURPOSE: This study evaluated the efficacy and safety of transcatheter radiofrequency ablation (RFCA) in treating ventricular premature contractions (PVCs) in children, summarized the countermeasures during intraoperative ventricular fibrillation (VF), and improved the safety of ventricular premature treatment. METHODS: A retrospective analysis was conducted on 75 children with PVCs who received RFCA in the Second Affiliated Hospital of Wenzhou Medical University from January 2010 to April 2019. Data including age, sex, body weight, ejection fraction, left ventricular end diastolic diameter, burden and number of PVCs/24 h, origin of PVCs, and its complications were collected. Paired t test was used to compare changes in cardiac function before and after surgery. RESULTS: Among the 75 cases treated with RFCA, 68 were successfully ablated, giving a success rate of 90.67%. After ablation, the left ventricular ejection fraction (LVEF) of the children was 69.13 ± 3.81%, which was significantly higher than that before surgery (69.13 ± 3.81% vs. 66.21 ± 3.22%, P = 0.012). One of the patients experienced VF during the operation, with no other complications. The initial locus of origin was the anterior septum of the right ventricular outflow tract, but VF occurred during the ablation process. Mean follow-up time was 39 ± 33 months, with two recurrent cases (2.94%). CONCLUSIONS: Performing RFCA in children is safe and effective, with a low recurrence rate and few complications. VF is not an indication to cease surgery; the key to eliminating complications is repositioning the catheter and finding a more accurate origin point.

Vagus nerve plays a pivotal role in CD4+ T cell differentiation during CVB3-induced murine acute myocarditis.

Abnormalities in CD4+ T cell (Th cell) differentiation play an important role in the pathogenesis of viral myocarditis (VMC). Our previous studies demonstrated that activation of the cholinergic anti-inflammatory pathway (CAP) alleviated the inflammatory response. In addition, we observed that right cervical vagotomy aggravates VMC by inhibiting CAP. However, the vagus nerve's effect on differentiation of CD4+ T cells has not been studied in VMC mice to date. In this study, we investigated the effects of cervical vagotomy and the α7nAChR agonist pnu282987 on CD4+ T cell differentiation in a murine myocarditis model (BALB/c) infected with coxsackievirus B3 (CVB3). Splenic CD4+ T cells from CVB3-induced mice obtained and cultured to investigate the potential mechanism of CD4+ T cell differentiation. Each Th cell subset was analyzed by flow cytometry. Our results showed that right cervical vagotomy increased proportions of Th1 and Th17 cells and decreased proportions of Th2 and Treg cells in the spleen. Vagotomy-induced upregulation of T-bet, Ror-γ, IFN-γ, and IL-17 expression while downregulating the expression of Gata3, Foxp3, and IL-4 in the heart. In addition, we observed upregulated levels of proinflammatory cytokines, aggravated myocardial lesions and cellular infiltration, and worsened cardiac function in VMC mice. Pnu282987 administration reversed these outcomes. Furthermore, vagotomy inhibited JAK2-STAT3 activation and enhanced NF-κB activation in splenic CD4+ T cells. The CD4+ T cell differentiation was related to JAK2-STAT3 and NF-κB signal pathways. In conclusion, vagus nerve modulates the inflammatory response by regulating CD4+ T cell differentiation in response to VMC.

Oxidised Low-Density Lipoprotein and Its Receptor-Mediated Endothelial Dysfunction Are Associated with Coronary Artery Lesions in Kawasaki Disease.

The study aimed to investigate the role of oxidised low-density lipoprotein (oxLDL)/lectin-like-oxLDL receptor-1 (LOX-1) in coronary artery lesions (CALs) in Kawasaki disease (KD) and of plasma oxLDL concentration in the early prediction of CALs in KD. This prospective study included 80 KD patients, 20 febrile and 20 healthy children. oxLDL, LOX-1 and other parameters were analysed in the acute phase. Plasma oxLDL concentration and LOX-1 mRNA expression in peripheral blood mononuclear cells (PBMCs) were significantly increased in KD patients compared with febrile and healthy children (P < 0.001 and P = 0.022, respectively), particularly in the group with CALs (P < 0.001 and P = 0.027, respectively). Coronary Z-score was significantly correlated with plasma oxLDL concentration and LOX-1 mRNA expression (r = 0.739 and 0.637, respectively; P < 0.01). The sensitivity and specificity of predicting CALs were 71.4% and 77.2%, respectively, at plasma oxLDL concentration ≥ 12.38 mU/L. oxLDL/LOX-1 may be involved in CAL development. The plasma oxLDL concentration in the acute phase is a potentially useful biological indicator for predicting CAL in KD patients.

[Experimental analysis of microRNAs related to cardiac development differential expression].

OBJECTIVE: To investigate the differential expression profiles of microRNAs in the knockout Pax-8 mice by miRNA microarray analysis and study the function of microRNA during cardiac development. METHODS: The knockout Pax-8 mice model was established and the total RNA derived from Pax-8 KO-/- and Pax-8 KO+/- mice heart. MicroRNA microarray containing 567 mammalian microRNA probes was used to investigate the microRNAs differential expression between Pax-8 KO-/- and Pax-8 KO+/- mice. The candidates of microRNAs were confirmed by real time RT-PCR assay. RESULTS: The heart of pax-8 KO-/- mice became spheroidal. Left ventricle enlargement, left ventricular wall and interventricular septum thickening and papillary muscles in left ventricle enlargement were found. Furthermore, many apoptotic cells were discovered in left ventricular wall and interventricular septum in pax-8 KO-/- mice. The MicroRNA microarray result displayed 10 microRNAs differential expressions, in which 2 microRNAs became down-regulated and 8 microRNAs up-regulated by more than two folds in pax-8 KO-/- mice. This was in accordance with the result of real-time RT-PCR. CONCLUSION: Some microRNAs may play important roles in cardiac development and ventricular septal defect pathogenesis.

Alteration of Cholinergic Anti-Inflammatory Pathway in Rat With Ischemic Cardiomyopathy-Modified Electrophysiological Function of Heart.

BACKGROUND: With chronic ischemia after myocardial infarction, the resulting scar tissue result in electrical and structural remodeling vulnerable to an arrhythmogenic substrate. The cholinergic anti-inflammatory pathway elicited by vagal nerve via α7 nicotinic acetylcholine receptors (α7-nAChR) can modulate local and systemic inflammatory responses. Here, we aimed to clarify a novel mechanism for the antiarrhythmogenic properties of vagal nerve during the ischemic cardiomyopathy (ICM). METHODS AND RESULTS: Left anterior descending artery of adult male Sprague-Dawley rats was ligated for 4 weeks to develop ICM. Western blot revealed that eliciting the cholinergic anti-inflammatory pathway by nicotine treatment showed a significant reduction in the amounts of collagens, cytokines, and other inflammatory mediators in the left ventricular infarcted border zone via inhibited NF-κB activation, whereas it increased the phosphorylated connexin 43. Vagotomy inhibited the anti-inflammatory, anti-fibrosis, and anti-arrhythmogenic effect of nicotine administration. And immunohistochemistry confirmed that the nicotine administration-induced increase of connexin 43 was located in intercellular junctions. Furthermore nicotine treatment suppressed NF-κB activation in lipopolysaccharide-stimulated RAW264.7 cells, and α-bungarotoxin (an α7-nAChR selective antagonist) partly inhibited the nicotine-treatment effect. In addition, 4-week nicotine administration slightly improved the cardiac function, increased cardiac parasympathetic tone, decreased the prolonged QTc, and decreased the arrhythmia score of programmed electric stimulation-induced ventricular arrhythmia. CONCLUSIONS: Eliciting the cholinergic anti-inflammatory pathway exerts anti-arrhythmogenic effects against ICM-induced ventricular arrhythmia accompanied by downregulation of cytokines, downgenerating of collagens, decrease in sympathetic/parasympathetic ratio, and prevention of the loss of phosphorylated connexin 43 during ICM. Our findings may suggest a promising therapy for the generation of ICM-induced ventricular arrhythmia by eliciting the cholinergic anti-inflammatory pathway.

Salvianolic Acid Exerts Cardioprotection through Promoting Angiogenesis in Animal Models of Acute Myocardial Infarction: Preclinical Evidence.

Radix Salviae miltiorrhizae, danshen root (danshen), is one of the widely used Chinese herbal medicines in clinics, containing rich phenolic compounds. Salvianolic acid is the main active compound responsible for the pharmacologic effects of danshen. Here, we aimed to evaluate the effects of salvianolic acid on cardioprotection through promoting angiogenesis in experimental myocardial infarction. Studies of salvianolic acid in animal models of myocardial infarction were obtained from 6 databases until April 2016. The outcome measures were vascular endothelium growth factor (VEGF), blood vessel density (BVD), and myocardial infarct size. All the data were analyzed using Rev-Man 5.3 software. Ultimately, 14 studies were identified involving 226 animals. The quality score of studies ranged from 3 to 6. The meta-analysis of six studies showed significant effects of salvianolic acid on increasing VEGF expression compared with the control group (P < 0.01). The meta-analysis of the two salvianolic acid A studies and three salvianolic acid B studies showed significantly improving BVD compared with the control group (P < 0.01). The meta-analysis of five studies showed significant effects of salvianolic acid for decreasing myocardial infarct size compared with the control group (P < 0.01). In conclusion, these findings demonstrated that salvianolic acid can exert cardioprotection through promoting angiogenesis in animal models of myocardial infarction.

Nerve growth factor-induced Akt/mTOR activation protects the ischemic heart via restoring autophagic flux and attenuating ubiquitinated protein accumulation.

The dysregulation of autophagy is related to a variety of cardiovascular diseases, such as myocardial ischemia/reperfusion (I/R). Nerve growth factor (NGF) has been shown to have therapeutic potential in ischaemic heart injury. In this study, we demonstrate that NGF administration can accelerate autophagic flux and attenuate protein ubiquitination in myocardial I/R heart. Our results showed that NGF could restored heart function and decreased the apoptosis of cardiomyocytes which induced by myocardial I/R injury. The protective effect of NGF is associated with the inhibition of autophagy related proteins. On another hand, NGF enhances the clearance of ubiquitinated protein and increases the survival of myocardial cell in vivo and in vitro. Additionally, NGF could activate the PI3K/AKT and mTOR signaling pathways. These results suggested that the cardioprotective effect of NGF is related to the restoration of autophagic flux and attenuation of protein ubiquitination via the activation of PI3K/AKT and mTOR pathway.

bFGF regulates autophagy and ubiquitinated protein accumulation induced by myocardial ischemia/reperfusion via the activation of the PI3K/Akt/mTOR pathway.

Autophagy is involved in the development and/or progression of many diseases, including myocardial ischemia/reperfusion (I/R). In this study, we hypothesized a protective role of basic fibroblast growth factor (bFGF) both in vivo and in vitro and demonstrated that excessive autophagy and ubiquitinated protein accumulation is involved in the myocardial I/R model. Our results showed that bFGF improved heart function recovery and increased the survival of cardiomyocytes in myocardial I/R model. The protective effect of bFGF is related to the inhibition of LC3II levels. Additionally, bFGF enhances the clearance of Ub by p62 and increases the survival of H9C2 cells. Moreover, silencing of p62 partially blocks the clearance of Ub and abolishes the anti-apoptosis effect of bFGF. An shRNA against the autophagic machinery Atg7 increased the survival of H9C2 cells co-treated with bFGF and rapamycin. bFGF activates the downstream signaling of the PI3K/Akt/mTOR pathway. These results indicate that the role of bFGF in myocardial I/R recovery is related to the inhibition of excessive autophagy and increased ubiquitinated protein clearance via the activation of PI3K/Akt/mTOR signaling. Overall, our study suggests a new direction for bFGF drug development for heart disease and identifies protein signaling pathways involved in bFGF action.

Pachyman treatment improves CD4+CD25+ Treg counts and serum interleukin 4 and interferon γ levels in a mouse model of Kawasaki disease.

The aim of this study was to explore the effect of pachyman, a mushroom extract, on CD4+CD25+ regulatory T cells (Tregs), serum interleukin 4 (IL-4) and interferon γ (IFN-γ) levels in a mouse model of Kawasaki disease. Lactobacillus casei cell wall extract was diluted to 1 mg/ml in PBS and administered to mice by intraperitoneal injection to establish a model of Kawasaki disease. Sixty female mice were used in this study, 40 of which were randomly assigned to a model (normal saline by gavage, n=20) or experimental group (200 mg/kg/day pachyman by gavage, n=20). The remaining 20 mice were disease and treatment-free, and were used as the control group. Compared to the control mice, mice in the model group exhibited a significantly lower percentage of CD4+CD25+ Tregs and significantly higher serum IL-4 and IFN-γ levels (P<0.05). However, CD4+CD25+ Tregs significantly increased and IL-4 and IFN-γ levels significantly decreased in experimental mice following pachyman treatment (P<0.05). Further analysis showed a negative correlation between CD4+CD25+ Tregs and IL-4/IFN-γ levels (P<0.05). In conclusion, pachyman improves immune function in a mouse model of Kawasaki disease by upregulating CD4+CD25+ Tregs, which may inhibit the cytokine secretion of Th1 and Th2 cells.

[Reducing plasma homocysteic acid lowers serum C-reactive protein level in children with Kawasaki disease].

OBJECTIVE: To investigate the effect of plasma homocysteic acid (HCA) reduction on serum C-reactive protein (CRP) level in children with Kawasaki disease (KD). METHODS: Seventy-six children with KD were divided into 2 equal groups for treatment with aspirin and IVIG, or with vitamin B6 and folic acid besides in addition to aspirin and IVIG. Serum CRP level was tested before and after the treatments, and plasma HCA level was also measured after the treatments. RESULTS: Serum CRP level was comparable between the two groups before the treatment, but significantly reduced after vitamin B6 and folic acid treatment (7.56-/+2.94 mg/L vs 12.23-/+4.16 mg/L, P<0.05). Additional vitamin B6 and folic acid treatment significantly lowered plasma HCA level (4.56-/+1.14 micromol/L vs 7.79-/+1.79 micromol/L, P<0.05), and correlation analysis demonstrated a positive correlation between plasma HCA and serum CRP levels (r=0.697, P<0.01). CONCLUSION: Lowering plasma HCA can decrease serum CRP in children with KD to enhance the therapeutic effect.

[Effects of hemoglobin oxygenase-1 inhibitor zinc protoporphyrin IX on acute viral myocarditis in mice].

OBJECTIVE: To investigate the role of heme oxygenase-1 (HO-1) and its catalyst carbon monoxide (CO) in the development of myocardial damage and the effects of zinc protoporphyrin IX (ZnPPIX), an inhibitor of HO-1 on myocardium of mice with acute viral myocarditis. METHODS: A total of 112 inbred male Balb/C mice 4 - 6 weeks of age were divided randomly into 3 groups: the control group (C group, n = 32), the viral myocarditis group (V group, n = 40) and ZnPPIX group (Z group, n = 40). The Z and V groups were inoculated intraperitoneally (i.p.) with 0.1 ml of 10(-4.36) tissue culture infectious dose 50% (TCID(50))/ml Coxsackie virus B3 (CVB(3)) to produce viral myocarditis model on day 0, C group was injected i.p. with virus-free 1640 culture culture medium 0.1 ml at the same time, then operation was done as follows: the mice of group C and group V were injected i.p. with 0.1 ml NS each day. The mice of group Z were injected i.p. with 40 micromol per kilogram of body weight ZnPPIX (HO-1 inhibitor) qod. Eight mice of each group were sacrificed on days 4, 8, 15 and 21, respectively. The blood specimens were collected by taking out the eyeballs to test for the content of carboxyhemoglobin (COHb) using spectrophotometry and cardiac troponin I (cTnI) using chemiluminescent immunoassay. The hearts tissue slides were also stained by immunohistochemistry (IHC) for HO-1 and in situ hybridization (ISH) for HO-1 mRNA. The histological and ultrastructural changes were observed under light and electron microscopes. RESULTS: (1) The histopathological changes of myocardial cells: in the V and Z groups myocardial inflammatory cells infiltration reached the peak on day 8, the Z group histopathological scores were significantly lower than those in V group on day 8 (2.40 +/- 0.31 vs. 1.73 +/- 0.24, P < 0.01) and on day 15 (1.78 +/- 0.29 vs. 1.43 +/- 0.23, P < 0.05). No inflammation was present in group C. (2) The changes of serum cTnI level in both V and Z groups were significantly higher than those in C group on day 4, 8 and 15 (P < 0.01). The level in Z group was significantly lower than that in V group on day 4 [(6.074 +/- 1.475) ng/ml vs (7.911 +/- 1.225) ng/ml, P < 0.05] and day 8 [(0.821 +/- 0.294) ng/ml vs (1.480 +/- 0.454) ng/ml, P < 0.05]. (3) The changes of blood COHb level: compared with V group, in Z group the COHb level was lower on day 4 (P < 0.05) and day 15 (P < 0.01) after CVB(3) inoculation. Surprisingly, in Z group COHb level elevated suddenly on day 8 and showed conspicuously higher than that of V group (P < 0.01). (4) The result of HO-1 IHC staining: in both V and Z group myocardial cells had positive expression, while C group did not. (5) The results of HO-1 ISH were similar to those of HO-1 IHC, the A values of group Z was significantly lower than that of group V on day 4, 15 and 21(P < 0.01), but on day 8 it was higher than that of group C (P < 0.05). CONCLUSION: HO-1 inhibitor, ZnPP not only could inhibit HO-1 overexpression but also could induce HO-1 expression temporarily and protect against myocardial injury at the early stage of acute viral myocarditis.