Hepatic Knockdown of Splicing Regulator Slu7 Ameliorates Inflammation and Attenuates Liver Injury in Ethanol-Fed Mice.

Aberrant precursor mRNA splicing plays a pivotal role in liver diseases. However, roles of splicing regulators in alcoholic liver disease are unknown. Herein, we investigated a splicing regulator, Slu7, in the development of alcoholic steatohepatitis. Adenovirus-mediated alteration of hepatic Slu7 expression in mice pair fed either with or without (as control) ethanol in their diet was used. Knockdown of hepatic Slu7 by adenovirus-Slu7shRNA treatment ameliorated inflammation and attenuated liver injury in mice after ethanol administration. Mechanistically, reducing liver Slu7 expression increased the expression of sirtuin 1 (SIRT1) full-length and repressed the splicing of SIRT1 into SIRT1-ΔExon8 isoform in ethanol-fed mice. Knockdown of hepatic Slu7 in the ethanol-fed mice also ameliorated splicing of lipin-1 and serine/arginine-rich splicing factor 3 (Srsf3). In concordance with ameliorated splicing of SIRT1, lipin-1, and Srsf3, knockdown of hepatic Slu7 inhibited the activity of NF-κB, normalized iron and zinc homeostasis, reduced oxidative stress, and attenuated liver damage in ethanol-fed mice. In addition, hepatic Slu7 was significantly elevated in patients with alcoholic steatohepatitis. Our present study illustrates a novel role of Slu7 in alcoholic liver injury and suggests that dysregulated Slu7 may contribute to the pathogenesis of human alcoholic steatohepatitis.

MitoNEET Deficiency Alleviates Experimental Alcoholic Steatohepatitis in Mice by Stimulating Endocrine Adiponectin-Fgf15 Axis.

MitoNEET (mNT) (CDGSH iron-sulfur domain-containing protein 1 or CISD1) is an outer mitochondrial membrane protein that donates 2Fe-2S clusters to apo-acceptor proteins. In the present study, using a global mNT knock-out (mNTKO) mouse model, we investigated the in vivo functional role of mNT in the development of alcoholic steatohepatitis. Experimental alcoholic steatohepatitis was achieved by pair feeding wild-type (WT) and mNTKO mice with Lieber-DeCarli ethanol-containing diets for 4 weeks. Strikingly, chronically ethanol-fed mNTKO mice were completely resistant to ethanol-induced steatohepatitis as revealed by dramatically reduced hepatic triglycerides, decreased hepatic cholesterol level, diminished liver inflammatory response, and normalized serum ALT levels. Mechanistic studies demonstrated that ethanol administration to mNTKO mice induced two pivotal endocrine hormones, namely, adipose-derived adiponectin and gut-derived fibroblast growth factor 15 (Fgf15). The elevation in circulating levels of adiponectin and Fgf15 led to normalized hepatic and serum levels of bile acids, limited hepatic accumulation of toxic bile, attenuated inflammation, and amelioration of liver injury in the ethanol-fed mNTKO mice. Other potential mechanisms such as reduced oxidative stress, activated Sirt1 signaling, and diminished NF-κB activity also contribute to hepatic improvement in the ethanol-fed mNTKO mice. In conclusion, the present study identified adiponectin and Fgf15 as pivotal adipose-gut-liver metabolic coordinators in mediating the protective action of mNT deficiency against development of alcoholic steatohepatitis in mice. Our findings may help to establish mNT as a novel therapeutic target and pharmacological inhibition of mNT may be beneficial for the prevention and treatment of human alcoholic steatohepatitis.

The Detrimental Role Played by Lipocalin-2 in Alcoholic Fatty Liver in Mice.

We have previously shown that the ethanol-mediated elevation of lipocaline-2 (LCN2) is closely associated with the development of alcoholic fatty liver disease (AFLD) in mice. Herein, we aimed to understand the functional significance of LCN2 induction by ethanol and to explore its underlying mechanisms. We evaluated the effects of LCN2 in an in vitro cellular alcoholic steatosis model and in an animal study using wild-type and LCN2 knockout mice fed for 4 weeks with an ethanol-supplemented Lieber-DeCarli diet. In the cellular model of alcoholic steatosis, recombinant LCN2 or overexpression of LCN2 exacerbated ethanol-induced fat accumulation, whereas knocking down LCN2 prevented steatosis in hepatocytes exposed to ethanol. Consistently, removal of LCN2 partially but significantly alleviated alcoholic fatty liver injury in mice. Mechanistically, LCN2 mediates detrimental effects of ethanol in the liver via disrupted multiple signaling pathways, including aberrant nicotinamide phosphoribosyltransferase-sirtuin 1 axis, perturbed endocrine metabolic regulatory fibroblast growth factor 15/19 signaling, and impaired chaperone-mediated autophagy. Finally, compared with healthy human livers, liver samples from patients with AFLD had lower gene expression of several LCN2-regualted molecules. Our study demonstrated a pivotal and causal role of LCN2 in the development of AFLD and suggested that targeting the LCN2 could be of great value for the treatment of human AFLD.

Expression of the sialyltransferase, ST3Gal4, impacts cardiac voltage-gated sodium channel activity, refractory period and ventricular conduction.

The sequential glycosylation process typically ends with sialic acid residues added through trans-Golgi sialyltransferase activity. Individuals afflicted with congenital disorders of glycosylation often have reduced glycoprotein sialylation and present with multi-system symptoms including hypotonia, seizures, arrhythmia and cardiomyopathy. Cardiac voltage-gated Na(+) channel (Nav) activity can be influenced by sialic acids likely contributing to an external surface potential causing channels to gate at less depolarized voltages. Here, a possible pathophysiological role for reduced sialylation is investigated by questioning the impact of gene deletion of the uniformly expressed beta-galactoside alpha-2,3-sialyltransferase 4 (ST3Gal4) on cardiac Nav activity, cellular refractory period and ventricular conduction. Whole-cell patch-clamp experiments showed that ventricular Nav from ST3Gal4 deficient mice (ST3Gal4(-/-)) gated at more depolarized potentials, inactivated more slowly and recovered from fast inactivation more rapidly than WT controls. Current-clamp recordings indicated a 20% increase in time to action potential peak and a 30ms decrease in ST3Gal4(-/-) myocyte refractory period, concurrent with increased Nav recovery rate. Nav expression, distribution and maximal Na(+) current levels were unaffected by ST3Gal4 expression, indicating that reduced sialylation does not impact Nav surface expression and distribution. However, enzymatic desialylation suggested that ST3Gal4(-/-) ventricular Nav are less sialylated. Consistent with the shortened myocyte refractory period, epicardial conduction experiments using optical mapping techniques demonstrated a 27% reduction in minimum ventricular refractory period and increased susceptibility to arrhythmias in ST3Gal4(-/-) ventricles. Thus, deletion of a single sialyltransferase significantly impacts ventricular myocyte electrical signaling. These studies offer insight into diseases of glycosylation that are often associated with pathological changes in excitability and highlight the importance of glycosylation in cardiac physiology.

Magnetic resonance monitoring of focused ultrasound/magnetic nanoparticle targeting delivery of therapeutic agents to the brain.

The superparamagnetic properties of magnetic nanoparticles (MNPs) allow them to be guided by an externally positioned magnet and also provide contrast for MRI. However, their therapeutic use in treating CNS pathologies in vivo is limited by insufficient local accumulation and retention resulting from their inability to traverse biological barriers. The combined use of focused ultrasound and magnetic targeting synergistically delivers therapeutic MNPs across the blood-brain barrier to enter the brain both passively and actively. Therapeutic MNPs were characterized and evaluated both in vitro and in vivo, and MRI was used to monitor and quantify their distribution in vivo. The technique could be used in normal brains or in those with tumors, and significantly increased the deposition of therapeutic MNPs in brains with intact or compromised blood-brain barriers. Synergistic targeting and image monitoring are powerful techniques for the delivery of macromolecular chemotherapeutic agents into the CNS under the guidance of MRI.

Clinical pathways based on integrative medicine in chinese hospitals improve treatment outcomes for patients with acute myocardial infarction: a multicentre, nonrandomized historically controlled trial.

Objective. To determine the impact of an integrative medicine clinical pathways (CPs) on the length of in-hospital stay and on outcomes for patients with acute myocardial infarction (AMI). Methods. A multicenter nonrandomized controlled trial enrolling 197 consecutive patients with AMI at eight urban TCM hospitals was conducted between 1 January 2010 and 31 October 2010. These patients were enrolled in the interventional group after the CPs had been implemented. The control group included 405 patients with AMI from eight hospitals; these patients were treated between 1 January 2008 and 31 December 2009, before the CPs were implemented. Outcome measures were the length of hospital stay costs of medical care, and major cardiovascular events (MACEs) during hospitalization. Results. Compared with the control group, the patients in intervention group had a shorter length of hospital stay (9.2 ± 4.2 days versus 12.7 ± 8.6 days, P < 0.05), and reduced healthcare costs in hospital (46365.7 ± 18266.9 versus 52866.0 ± 35404.4, P < 0.05). There were statistically significant differences in MACE between the two groups during the hospitalization period (2.5% versus 6.9%, P = 0.03). Conclusion. These data suggest that the development and implementation of the clinical pathways based in Integrative Medicine could further improve quality of care and outcome for patients with AMI.

In vivo MR quantification of superparamagnetic iron oxide nanoparticle leakage during low-frequency-ultrasound-induced blood-brain barrier opening in swine.

PURPOSE: To verify that low-frequency planar ultrasound can be used to disrupt the BBB in large animals, and the usefulness of MRI to quantitatively monitor the delivery of superparamagnetic iron oxide (SPIO) nanoparticles into the disrupted regions. MATERIALS AND METHODS: Two groups of swine subjected to craniotomy were sonicated with burst lengths of 30 or 100 ms, and one group of experiment was also performed to confirm the ability of 28-kHz sonication to open the BBB transcranially. SPIO nanoparticles were administered to the animals after BBB disruption. Procedures were monitored by MRI; SPIO concentrations were estimated by relaxivity mapping. RESULTS: Sonication for 30 ms created shallow disruptions near the probe tip; 100-ms sonications after craniotomy can create larger and more penetrating openings, increasing SPIO leakage ∼3.6-fold than 30-ms sonications. However, this was accompanied by off-target effects possibly caused by ultrasonic wave reflection. SPIO concentrations estimated from transverse relaxation rate maps correlated well with direct measurements of SPIO concentration by optical emission spectrometry. We have also shown that transcranial low-frequency 28-kHz sonication can induce secure BBB opening from longitudinal MR image follow up to 7 days. CONCLUSION: This study provides valuable information regarding the use low-frequency ultrasound for BBB disruption and suggest that SPIO nanoparticles has the potential to serve as a thernostic agent in MRI-guided ultrasound-enhanced brain drug delivery.

Songling Xuemaikang Capsule inhibits isoproterenol-induced cardiac hypertrophy via CaMKIIδ and ERK1/2 pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Cardiac hypertrophy is a key pathologic process in heart failure. Songling Xuemaikang Capsule (SXC), is a formulae of Chinese Medicine commonly used in China to treat hypertension and heart failure. However, its mechanism of effects on cardiac hypertrophy is still unclear. AIM OF THE STUDY: The aims of the present study were to investigate the cardio-protection roles and detailed mechanisms of SXC on cardiac hypertrophy in vivo and in vitro. MATERIALS AND METHODS: A rat model of cardiac hypertrophy was constructed by isoproterenol (ISO) intraperitoneal injection (i.p), 10 mg/kg/day for 3 days, and 4 groups were compared: CON (n = 8), ISO (n = 8), MET (metoprolol, positive drug treatment, n = 7), and SXC (SXC treatment, n = 6). Cardiac structure and function were evaluated with echocardiography in vivo. Dose-dependent curve was obtained with SXC different concentrations. In addition, H9C2 rat cardiomyocytes were cultured in vitro and the phosphorylation of ERK1/2, p38, JNK, AKT, and protein expression of CaN, CaMKIIδ, GATA4 were detected with Western blot test. RESULTS: The results showed that SXC reduced diastolic thickness of left ventricular posterior wall, while did not change ejection fraction and fraction shortening significantly (P > 0.05). SXC inhibit ISO-induced cardiac hypertrophy dose-dependently with 50% inhibiting concentration (IC50) is 0.504 g/kg/day. Moreover, SXC inhibited the protein expression of CaMKIIδ, and the phosphorylation of ERK1/2, so inhibiting protein expression of GATA4 in nucleus, and brain natriuretic peptide in serum (P < 0.001). CONCLUSION: The mechanism of SXC in the treatment of heart diseases involves SXC dose-dependently inhibited the ISO-induced cardiac hypertrophy via inhibiting CaMKIIδ and ERK1/2/GATA4 signaling pathway.

Fragmented QRS as a marker of conduction abnormality and a predictor of prognosis of Brugada syndrome.

BACKGROUND: Conduction abnormalities serve as a substrate for ventricular fibrillation (VF) in patients with Brugada syndrome (BS). Signal-averaged electrograms can detect late potentials, but the significance of conduction abnormalities within the QRS complex is still unknown. The latter can present as multiple spikes within the QRS complex (fragmented QRS [f-QRS]). We hypothesized that f-QRS could indicate a substrate for VF and might predict a high risk of VF for patients with BS. METHODS AND RESULTS: In study 1, we analyzed the incidence of f-QRS in 115 patients with BS (13 resuscitated from VF, 28 with syncope, and 74 asymptomatic). f-QRS was observed in 43% of patients, more often in the VF group (incidence of f-QRS: VF 85%, syncope 50%, and asymptomatic 34%, P<0.01). SCN5A mutations occurred more often in patients with f-QRS (33%) than in patients without f-QRS (5%). In patients with syncope or VF, only 6% without f-QRS experienced VF during follow-up (43+/-25 months), but 58% of patients with f-QRS had recurrent syncope due to VF (P<0.01). In study 2, to investigate the mechanism of f-QRS, we studied in vitro models of BS in canine right ventricular tissues (n=4) and optically mapped multisite action potentials. In the experimental model of BS, ST elevation resulted from a large phase 1 notch of the action potential in the epicardium, and local epicardial activation delay reproduced f-QRS in the transmural ECG. CONCLUSIONS: f-QRS appears to be a marker for the substrate for spontaneous VF in BS and predicts patients at high risk of syncope.

The QT syndromes: long and short.

This Seminar presents the most recent information about the congenital long and short QT syndromes, emphasising the varied genotype-phenotype association in the ten different long QT syndromes and the five different short QT syndromes. Although uncommon, these syndromes serve as a Rosetta stone for the understanding of inherited ion-channel disorders leading to life-threatening cardiac arrhythmias. Ionic abnormal changes mainly affecting K(+), Na(+), or Ca(2+) currents, which either prolong or shorten ventricular repolarisation, can create a substrate of electrophysiological heterogeneity that predisposes to the development of ventricular tachyarrhythmias and sudden death. The understanding of the genetic basis of the syndromes is hoped to lead to genetic therapy that can restore repolarisation. Presently, symptomatic individuals are generally best treated with an implantable cardioverter defibrillator. Clinicians should be aware of these syndromes and realise that drugs, ischaemia, exercise, and emotions can precipitate sudden death in susceptible individuals.

Analysis of action potentials in the canine ventricular septum: no phenotypic expression of M cells.

OBJECTIVE: Transmural heterogeneity in the ventricular free wall, enhanced by the midmyocardial long action potential duration (APD) of M cells, plays an important role in the arrhythmogenesis of long QT syndrome. Although we observed dynamic expression of M cell phenotypes in the canine ventricular free wall, it is still unclear whether similar phenomena are present in the interventricular septum. This study evaluated transmural heterogeneity of APD in the septum. METHODS: We isolated and perfused 22 canine septal preparations through the septal branch of the anterior descending coronary artery, and optically mapped 256 channels of action potentials on their cut-exposed transseptal surfaces before and after treatment with sotalol (I(Kr) blocker), anemone toxin II (ATX-II, which slows the inactivation of I(Na)), or drug-free state in 6, 9, and 22 preparations, respectively. The preparations were paced from the left ventricular endocardium at cycle lengths of 500, 1000, 2000, and 4000 ms. RESULTS: We observed progressively lengthening of APD across the septum from the right ventricular to the left ventricular endocardium without a midmyocardial maximum under all conditions. All action potentials had minor phase-1 notches, resembling the endocardial action potential in the ventricular free wall. Increasing cycle lengths and concentrations of sotalol and ATX-II prolonged APD without midmyocardial preference and increased the transseptal dispersion of APDs. CONCLUSIONS: Canine interventricular septal action potentials are similar in shape to the endocardial action potentials in the ventricular free wall, with smooth transseptal transition in APD. We found no phenotypical expression of M cells in the canine interventricular septum.

Mechanism of U wave and polymorphic ventricular tachycardia in a canine tissue model of Andersen-Tawil syndrome.

OBJECTIVE: Andersen-Tawil syndrome (ATS) is a channelopathy affecting inward rectifier potassium I(K1) with QT prolongation, large U waves, and frequent ventricular tachycardia (VT). Although ATS is clinically defined and genetically identified, its electrophysiological mechanism is still unclear, and thus, was the subject of the current study. METHODS AND RESULTS: We replicated the major electrophysiological features of ATS with cesium chloride (CsCl, at I(K1) blockade concentration of 5-10 mmol/l) in 23 isolated canine left ventricular tissues perfused arterially with Tyrode's solution having normal or low potassium concentrations, [K(+)](o). We mapped action potentials (APs) on the cut-exposed transmural surface of the wedges in control, after CsCl, and CsCl with 0.15 mumol/l isoproterenol (CsCl+ISP). CsCl delayed late phase 3 repolarization and prolonged the duration of the AP, more so during low [K(+)](o) perfusion. Rapid pacing induced delayed afterdepolarizations (DADs) in all low [K(+)](o) and in 71% of normal [K(+)](o) preparations after CsCl treatment. Addition of ISP induced DADs in all preparations. DADs originated in mid-to-endocardium, and initiated VT after CsCl+ISP. Migration of DAD-VT foci resulted in multifocal VT. Alternating DADs at 2 foci resulted in bidirectional VT. There were more foci and longer durations of VT at low [K(+)](o) than at normal [K(+)](o). Delayed late phase 3 repolarization of APs and DADs generated U waves. Verapamil abolished all DADs and VT. CONCLUSIONS: CsCl blockade of I(K1) produced a ventricular wedge model of ATS. Suppressing I(K1) generated U waves by delaying late repolarization of APs and creating DADs, and promoted polymorphic VT by triggering DADs at multiple shifting sites.

Differences in arrhythmogenicity between the canine right ventricular outflow tract and anteroinferior right ventricle in a model of Brugada syndrome.

BACKGROUND: The Brugada syndrome is characterized by ST-segment elevation on the ECG, especially in the right precordial leads sensitive to the right ventricular outflow tract (RVOT). OBJECTIVES: The purpose of this study was to evaluate the hypothesis that right ventricular electrophysiologic heterogeneity caused arrhythmogenicity in the Brugada syndrome. METHODS: Action potentials (APs) were mapped on the epicardium of 14 RVOT preparations and on the transmural surfaces of 15 pairs of RVOT and right ventricular anteroinferior (RVAI) preparations isolated from canine hearts. Brugada ECG and arrhythmias were induced with pilsicainide (2.5-12.5 micromol/L), pinacidil (1.25-12.5 micromol/L), and terfenadine (2.0 micromol/L). RESULTS: Low doses of drugs elevated the J-ST segment and induced APs with both short and long action potential durations (APDs) in contiguous RVOT epicardial regions. In addition, APs in the RVOT had a larger phase 1 notch and longer APD than in RVAI. The longest APDs were in the epicardium in RVOT but in the endocardium in RVAI regions. High doses of drugs eliminated the phase 2 dome of the AP and abbreviated APDs in the epicardium but not in endocardium and reduced the epicardial heterogeneity of APs but increased the transmural gradient of APD in 14 (93%) of the RVOT preparations. In contrast, abbreviations of epicardial APDs occurred in only 4 (27%) of the RVAI preparations. Ventricular tachycardia occurred more frequently in the RVOT (47%) than in paired RVAI preparations (7%). Blocking the transient outward current reduced the heterogeneity of APs and eliminated arrhythmogenicity in all preparations. CONCLUSION: Compared with the RVAI region, the RVOT has greater electrophysiologic heterogeneity that contributes to arrhythmogenicity in this model of Brugada syndrome.

Temperature modulation of ventricular arrhythmogenicity in a canine tissue model of Brugada syndrome.

BACKGROUND: Fever promotes ventricular arrhythmias in Brugada syndrome (BrS). Hypothermia can induce BrS electrocardiogram (ECG) and arrhythmia. However, the mechanisms are unclear. OBJECTIVE: We evaluated the hypothesis that pathological temperatures promoted arrhythmogenesis by modulating the spatial heterogeneity and functional dynamics of right ventricular electrophysiological activity. METHODS: We mapped action potentials (APs) on the epicardial or cut-exposed transmural surfaces and recorded transmural ECGs in 27 arterially perfused canine right ventricular preparations before and after inducing BrS at 32 degrees C, 36.5 degrees C, and 40 degrees C. RESULTS: We observed major intraepicardial dispersion of AP duration (APD) and reversal of transmural gradient of APD in association with manifestation of BrS at 36.5 degrees C. Reducing the temperature to 32 degrees C prolonged APDs and enhanced the phase 1 notch of epicardial APs, while 40 degrees C caused opposite changes. Prominent phase 2 domes of APs frequently led to spontaneous premature ventricular activations (PVAs), which conducted to surrounding regions having shorter APDs. Longer APDs at 32 degrees C and 36.5 degrees C frequently blocked reentry, although they promoted PVA, while shortened APDs at 40 degrees C facilitated reentrant ventricular tachycardia. During bradycardia (2,000 ms), the J-ST elevation in the ECG was enhanced at 32 degrees C and attenuated at 40 degrees C. Rapid pacing (500 ms) eliminated the dome of epicardial APs and enhanced J-ST elevation at each temperature. Blocking the transient outward current, I(to), with 4-aminopyridine reduced J-ST elevation and eliminated the PVA and reentry. CONCLUSIONS: In this BrS model, prolongation and increased dispersion of APDs promoted spontaneous activation during hypothermia, while APD abbreviation facilitated reentry during hyperthermia. I(to) mediated the arrhythmogenicity.

Signal Transduction Mechanisms of Alcoholic Fatty Liver Disease: Emer ging Role of Lipin-1.

Lipin-1, a mammalian phosphatidic acid phosphatase (PAP), is a bi-functional molecule involved in various signaling pathways via its function as a PAP enzyme in the triglyceride synthesis pathway and in the nucleus as a transcriptional co-regulator. In the liver, lipin-1 is known to play a vital role in controlling the lipid metabolism and inflammation process at multiple regulatory levels. Alcoholic fatty liver disease (AFLD) is one of the earliest forms of liver injury and approximately 8-20% of patients with simple steatosis can develop into more severe forms of liver injury, including steatohepatitis, fibrosis/ cirrhosis, and eventually hepatocellular carcinoma (HCC). The signal transduction mechanisms for alcohol-induced detrimental effects in liver involves alteration of complex and multiple signaling pathways largely governed by a central and upstream signaling system, namely, sirtuin 1 (SIRT1)-AMP activated kinase (AMPK) axis. Emerging evidence suggests a pivotal role of lipin-1 as a crucial downstream regulator of SIRT1-AMPK signaling system that is likely to be ultimately responsible for development and progression of AFLD. Several lines of evidence demonstrate that ethanol exposure significantly induces lipin-1 gene and protein expression levels in cultured hepatocytes and in the livers of rodents, induces lipin-1-PAP activity, impairs the functional activity of nuclear lipin-1, disrupts lipin-1 mRNA alternative splicing and induces lipin-1 nucleocytoplasmic shuttling. Such impairment in response to ethanol leads to derangement of hepatic lipid metabolism, and excessive production of inflammatory cytokines in the livers of the rodents and human alcoholics. This review summarizes current knowledge about the role of lipin-1 in the pathogenesis of AFLD and its potential signal transduction mechanisms.

Shexiang Tongxin dropping pill protects against isoproterenol-induced myocardial ischemia in vivo and in vitro.

Shexiang Tongxin dropping pill (STDP) is a formulae of Chinese Medicine commonly used to treating angina pectoris in China. However, its mechanism of action is still yet unclear. This study investigated the roles of STDP on myocardial ischemia injury. We constructed a rat model of myocardial injury (isoproterenol subcutaneous injection, i.h, 85 mg/kg/day for 2 days), and compared among 4 groups: CON (control), ISO (ischemic injury model), MET (metoprolol), and STDP. Serum contents of Troponin I (cTnI), creatine kinase (CK), CK-MB, lactate dehydrogenase (LDH), alpha-hydroxybutyric dehydrogenase (α-HBD), and Aspartate Aminotransferase were detected and five STDP doses (1, 10, 100, 1000 and 10000 mg/kg/day) were chosen to obtain a dose-response curve. Western-blot was used to detect phosphorylations of extracellular signal-regulated kinase 1/2 (ERK1/2), protein kinase B (AKT), and camodulin kinase II (CamkII). Furthermore, an ERK1/2 inhibitor PD98059, a phosphatidylinositol-3-kinase inhibitor, LY294002, and a CamKII inhibitor, KN-93 were administered i.h. RESULTS: cTnI, CK, CK-MB, α-HBD, and LDH were significantly lower in STDP than ISO (P<0.05). STDP exhibited a dose-dependent effect with a half maximal inhibitory concentration of 42 mg/kg/day. Phosphorylation of ERK1/2 was enhanced in the STDP group (vs. ISO, P<0.05), while AKT and CamkII were not changed. Further, the protective effects of STDP were offset by PD98059 administration i.h. In conclusion, STDP protected against the ISO-induced myocardial ischemic injury via an ERK1/2 signaling pathway, which provided a mechanism to support clinical applications of STDP as treatment for ischemic heart disease.

Mechanisms of Chinese Medicine Xinmailong's protection against heart failure in pressure-overloaded mice and cultured cardiomyocytes.

Patients with heart failure (HF) have high mortality and mobility. Xinmailong (XML) injection, a Chinese Medicine, is clinically effective in treating HF. However, the mechanism of XML's effectiveness on HF was unclear, and thus, was the target of the present study. We created a mouse model of pressure-overload-induced HF with transverse aortic constriction (TAC) surgery and compared among 4 study groups: SHAM (n = 10), TAC (n = 12), MET (metoprolol, positive drug treatment, n = 7) and XML (XML treatment, n = 14). Dynamic changes in cardiac structure and function were evaluated with echocardiography in vivo. In addition, H9C2 rat cardiomyocytes were cultured in vitro and the phosphorylation of ERK1/2, AKT, GSK3ß and protein expression of GATA4 in nucleus were detected with Western blot experiment. The results showed that XML reduced diastolic thickness of left ventricular posterior wall, increased ejection fraction and fraction shortening, so as to inhibit HF at 2 weeks after TAC. Moreover, XML inhibited the phosphorylation of ERK1/2, AKT and GSK3ß, subsequently inhibiting protein expression of GATA4 in nucleus (P < 0.001). Together, our data demonstrated that XML inhibited the TAC-induced HF via inactivating the ERK1/2, AKT/GSK3ß, and GATA4 signaling pathway.

Salvianolate reduces murine myocardial ischemia and reperfusion injury via ERK1/2 signaling pathways in vivo.

OBJECTIVE: To analyze the effects of salvianolate on myocardial infarction in a murine in vivo model of ischemia and reperfusion (I/R) injury. METHODS: Myocardial I/R injury model was constructed in mice by 30 min of coronary occlusion followed by 24 h of reperfusion and pretreated with salvianolate 30 min before I/R (SAL group). The SAL group was compared with SHAM (no I/R and no salvianolate), I/R (no salvianolate), and ischemia preconditioning (IPC) groups. Furthermore, an ERK1/2 inhibitor PD98059 (1 mg/kg), and a phosphatidylinositol-3-kinase (PI3-K) inhibitor, LY294002 (7.5 mg/kg), were administered intraperitoneal injection (i.p) for 30 min prior to salvianolate, followed by I/R surgery in LY and PD groups. By using a double staining method, the ratio of the infarct size (IS) to left ventricle (LV) and of risk region (RR) to LV were compared among the groups. Correlations between IS and RR were analyzed. Western-blot was used to detect the extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (AKT) phosphorylation changes. RESULTS: There were no significant differences between RR to LV ratio among the SHAM, I/R, IPC and SAL groups (P>0.05). The SAL and IPC groups had IS of 26.1%±1.4% and 22.3%±2.9% of RR, respectively, both of which were significantly smaller than the I/R group (38.5%±2.9% of RR, P<0.05, P<0.01, respectively). Moreover, the phosphorylation of ERK1/2 was increased in SAL group (P<0.05), while AKT had no significant change. LY294002 further reduced IS, whereas the protective role of salvianolate could be attenuated by PD98059, which increased the IS. Additionally, the IS was not linearly related to the RR (r=0.23, 0.45, 0.62, 0.17, and 0.52 in the SHAM, I/R, SAL, LY and PD groups, respectively). CONCLUSION: Salvianolate could reduce myocardial I/R injury in mice in vivo, which involves an ERK1/2 pathway, but not a PI3-K signaling pathway.

Salvianolic Acid B Alleviates Heart Failure by Inactivating ERK1/2/GATA4 Signaling Pathway after Pressure Overload in Mice.

BACKGROUND: Heart failure(HF) is a dangerous disease that affects millions of patients. Radix Salvia is widely used in Chinese clinics to treat heart diseases. Salvianolic acid B(SalB) is the major active component of Radix Salvia. This study investigated the mechanisms of action and effects of SalB on HF in an experimental mouse model of HF. METHODS: We created a mouse model of HF by inducing pressure overload with transverse aortic constriction(TAC) surgery for 2 weeks and compared among 4 study groups: SHAM group (n = 10), TAC group (n = 9), TAC+MET group (metprolol, positive drug treatment, n = 9) and TAC+SalB group (SalB, 240 mg•kg-1•day-1, n = 9). Echocardiography was used to evaluate the dynamic changes in cardiac structure and function in vivo. Plasma brain natriuretic peptide (BNP) concentration was detected by Elisa method. In addition, H9C2 rat cardiomyocytes were cultured and Western blot were implemented to evaluate the phosphorylation of ERK1/2, AKT, and protein expression of GATA4. RESULTS: SalB significantly inhibited the phosphorylation of Thr202/Tyr204 sites of ERK1/2, but not Ser473 site of AKT, subsequently inhibited protein expression of GATA4 and plasma BNP(P < 0.001), and then inhibited HF at 2 weeks after TAC surgery. CONCLUSIONS: Our data provide a mechanism of inactivating the ERK1/2/GATA4 signaling pathway for SalB inhibition of the TAC-induced HF.

Myeloid Cell-Specific Lipin-1 Deficiency Stimulates Endocrine Adiponectin-FGF15 Axis and Ameliorates Ethanol-Induced Liver Injury in Mice.

Lipin-1 is a phosphatidate phosphohydrolase (PAP) required for the generation of diacylglycerol during glycerolipid synthesis, and exhibits dual functions in the regulation of lipid metabolism. Lipin-1 has been implicated in the pathogenesis of alcoholic liver disease (ALD). In the present study, we assessed lipin-1 function in myeloid cells in ALD using a myeloid cell-specific lipin-1 knockout (mLipin-1KO) mouse model. Utilizing the Gao-binge ethanol feeding protocol, matched mLipin-1KO mice and littermate loxP control (WT) mice were pair-fed with either an ethanol-containing diet or an ethanol-free diet (control). Surprisingly, deletion of lipin-1 in myeloid cells dramatically attenuated liver inflammatory responses and ameliorated liver injury that would normally occur following the ethanol feeding protocol, but slightly exacerbated the ethanol-induced steatosis in mice. Mechanistically, myeloid cell-specific lipin-1 deficiency concomitantly increased the fat-derived adiponectin and ileum-derived fibroblast growth factor (FGF) 15. In concordance with concerted elevation of circulating adiponectin and FGF15, myeloid cell-specific lipin-1 deficiency diminished hepatic nuclear factor kappa B (NF-κB) activity, limited liver inflammatory responses, normalized serum levels of bile acids, and protected mice from liver damage after ethanol challenge. Our novel data demonstrate that myeloid cell-specific deletion of lipin-1 ameliorated inflammation and alcoholic hepatitis in mice via activation of endocrine adiponectin-FGF15 signaling.