Epoxyeicosatrienoic acid: the signal transduction

Cytochrome P450 monoxygenase converts arachionic acid to four epoxyeicosatrienoic acid regiosomes: 5, 6-EET (epoxyeicosatrienoic acid); 8, 9-EET; 11, 12-EET and 14, 15-EET. Recent studies show that EETs are involved in signal transduction. EETs open Ca²⁺-sensit ive K⁺ channel and inhibit Na⁺ channel, Ca²⁺-sensitive Cl^- channel and so on. What is more, EETs have been demonstrated to activate PP60^c-src and initiate a tyrosine kinase cascade that mediates mitogenic effects.     

Expression of GST fusion proteins of human cytochrome P2B6 and preparation of anti-cytochrome P2B6 polyclonal antibody

AIM: To get large amounts of pure antigens to raise specific antibodies and to perform quantifications.METHODS: CYP2B6 (cytochrome P) cDNA fragments was ligated into BamHI restricted PGEX-3b to generate recombinants PGEX/2B6. We identified recombinants PGEX/2B6 by EcoRI digestion. The expression of fusion proteins were induced by adding isopropyl-thiogalactoside(IPTG). Several clones showed high-level expression of fusion proteins. Insoluble proteins was isolated from the bacteria and the fusion proteins was recovered and purified from a preparative (2mm) SDS-PAGE. The polyarcrylamide gel containing the fusion proteins glutathione S-transferase(GST-2B6) were used to immunize BALB/C mice from which polyclonal ascites fluid was prepared. The purified fusion proteins GST-1A1(GST fusion protein of CYP1A1 cDNA246~386aa expressed in this library, purified by preparative SDS-PAGE), GST-2B6 were used to test the specificity of 2B6pAb. RESULTS:Fusion proteins constructed between GST and CYP2B6 was expressed in Escherichia coli DH5α. Mouse antibodies are raised against the fusion proteins GST-2B6. 2B6pAb was fond to be specific antibody.CONCLUSION:Recombinant PGEX/2B6 were constructed and purified fusion proteins GST-2B6, and specific 2B6pAb were obtained.     

Effects of atorvastatin on expression of cytochrome P450 hydroaylase in spontaneously hypertensive rats

AIM: To investigate the effects of atorvastatin on blood pressure and expression of cytochrome P450 hydroaylase (CYP) 4A1 in spontaneously hypertensive rats (SHR). METHODS: SHRs (n=18) were randomly divided into three groups: SHR control group, 50 mg atorvastatin (HATV) group and 10 mg (LATV) group. Six male Wistar-Kyoto rats were selected as normal control group (WKY group). All rats were given vehicle once a day by gavage for 10 weeks. Systolic blood pressure (SBP) was measured before and after treatment every 2 weeks. The expressions of CYP 4A1 mRNA and protein in heart, liver, kidney, and aorta were detected by RT-PCR and Western blotting analysis, respectively. Plasma lipids were also measured.RESULTS: SBP in all SHR groups was much higher than that in WKY group before experiment (P<0.01). Compared with SHR control group, SBP significantly decreased in HATV group at 6, 8, 10 weeks and in LATV group merely at 10 weeks (P<0.01 or P<0.05). The expressions of CYP 4A1 mRNA and protein in heart, liver, kidney and aorta tissues of SHR control group were significantly higher than those in WKY group (P<0.01 or P<0.05). After 10 weeks, the levels of CYP 4A1 mRNA, protein and plasma lipids in HATV and LATV groups were markedly lower than those in SHR control group (P<0.01 or P<0.05).CONCLUSION: Atorvastatin down-regulates the expressions of CYP 4A1 mRNA and protein in SHR, which may be the mechanism of the favorable effects of statins on regulation of hypertension.     

Epoxyeicosatrienoic acids: A kind of endothelium derived hyperpolarizing factor

The endothelium derived hyperpolarizing factor (EDHF) has been paied attention to since the late 1980s. But it is not clear which are EDHF. The article reviewed the EDHF works and epoxyeicosatrienoic acids (EETs), an important kind of EDHF.     

CYP450 epoxygenase/EET pathway attenuates adipose inflammation of obese mice through HIF-1α

AIM To investigate the effects of cytochrome P450 （CYP450） epoxygenase/epoxyeicosatrienoic acid （EET） pathway on insulin resistance in obese mice, and to explore the possible mechanisms. METHODS High-fat diet-induced obesity model was established in C57BL/6Cnc mice, and the obese mice were randomly divided into 3 groups, including obesity group （treated with saline; n=10）, EET group （treated with 11,12-EET; n=10） and EET inhibitor 14,15-epoxyeicosa-5（Z）-enoic acid （EEZE） group （n=10）. Normal C57BL/6Cnc mice （n=10） treated with saline served as control. Protein expression of CYP2J2 （one of CYP450 epoxygenases） and hypoxia-inducible factor-1α （HIF-1α） was measured by Western blot. Vessel-like structure was detected by immunofluorescence staining. The serum levels of insulin, tumor necrosis factor-α （TNF-α）, interleukin （IL）-1β, IL-6 and monocyte chemoattractant protein-1 （MCP-1） were measured by ELISA. RESULTS In obese mice, homeostasis model assessment of insulin resistance （HOMA-IR） values were increased, the protein level of CYP2J2 was reduced, and the protein level of HIF-1α was increased in adipose tissues as compared with the controls （P<0.05）. The serum levels of MCP-1, IL-1β, IL-6 and TNF-α were also significantly increased in obese mice （P<0.05）. After treatment with 11, 12-EET, the HOMA-IR values were decreased compared with vehicle-treated obese mice, HIF-1α expression levels were decreased in the adipose tissue, and the serum levels of MCP-1, IL-1β, IL-6 and TNF-α were reduced （P<0.05）. Immunohistochemical results of adipose tissue from vehicle-treated obese mice showed a marked decrease in vessel-like structures （CD31-positive） compared with normal control mice （P<0.05）. EET treatment significantly increased the newly formed vessel-like structures in the visceral adipose tissues of obese mice as compared with vehicle-treated obese mice （P<0.05）. CONCLUSION High-fat diet-induced obesity and insulin resistance are closely related to the CYP450 pathway. Exogenous EETs effectively decrease obesity-induced insulin resistance possibly through pro-angiogenesis and attenuation of hypoxia and inflammation.     

Induction of apoptosis in mouse fibroblast cell line L929 by arachidonic acid

AIM: To observe whether arachidonic acid (AA) could induce apoptosis in mouse fibroblast cell line L929 and the potential mechanism involved. METHODS: The viability and damaged degree of L929 was monitored by MTT and the release of lactate dehydrogenase (LDH). Lipid peroxidation in L929 was measured as malondialdehyde (MDA) content by colorimetric assay. Hoechst 33258 staining was used to observe AA-induced morphological changes. Agarose gel electrophoresis was used to detect DNA fragmentation. RESULTS: Treatment of L929 cell with AA for 24 h, in the range of 40-160 μmol/L, caused a great decrease in cell survival and increased MDA contents and the release of LDH simultaneously (P＜0.01). Cells treated by 160 μmol/L AA showed typical morphological changes of apoptosis. A "ladder" pattern representing fragmentation of DNA into oligonucleosome length fragments was observed 24 h after AA treatment. CONCLUSION: Higher concentration of arachidonic acid (80-160 μmol/L) induced apoptosis in mouse fibroblast cell line L929. The mechanism of its action might be related to lipid peroxidation.     

Mitophagy and heart failure

Heart failure (HF) is the end stage of various cardiovascular diseases. The prognosis of patients with HF is poor. The precise pathophysiological mechanism of HF is still not fully elucidated. Recent studies have shown that mitochondrial dysfunction is closely associated with HF. Mitophagy plays a crucial role in mitochondrial quality control. In this article, we review the present evidence implicating the role of mitophagy in HF, and provide the potential ther?apeutic strategies targeting mitophagy to improve outcome in the patients with HF.     

Effects of CYP2J3/EETs on myocardial apoptosis under the condition of hypoxic postconditioning

AIM: To investigate the effects and underlying mechanism of endogenous cytochrome P450 2J3/epoxyeicosatrienoic acids (CYP2J3/EETs) system on myocardial apoptosis under the condition of hypoxic postconditioning (HPost).METHODS: Primary myocardial cells from neonatal Wistar rats (12 h-24 h) were cultured and divided into 7 groups as follows: control group, hypoxia/reoxygenation (H/R) group, HPost group, CYP2J3 transfection group, empty vector group, 6-(2-propargyloxyphenyl)hexanoic acid (PPOH, an inhibitor of CYP2J3) group and dimethyl sulfoxide (DMSO, as solvent) group. The H/R treatment was conducted in all the groups except control group. The cell viability was tested by MTT method. The concentration of 11,12-EET in the cell culture medium was measured by high-perfor-mance liquid chromatography (HPLC). The expression of caspase-3 was detected by Western blotting and its activity was determined by caspase-3 activity assay kit.RESULTS: Compared with H/R group, the cell viability and the 11,12-EET concentration were significantly elevated in HPost group (P<0.01). The two indexes in CYP2J3 transfection group were higher than those in HPost group (P<0.01), but they were lowered in PPOH group than those in HPost group (P<0.01). The comparisons of the expression and activity of caspase-3 among groups were as follows: H/R group > HPost group (P<0.01), PPOH group > HPost group (P<0.01) and empty vector group > CYP2J3 transfection group (P<0.05). Additionally, the caspase-3 expression in CYP2J3 transfection group was lower than that in HPost group (P<0.01).CONCLUSION: The CYP2J3/EETs system protects myocardial cells through inhibiting caspase-3-induced apoptosis.     

Pivotal role of microRNAs in cardiac development and heart diseases

MicroRNAs (miRNAs) are non-coding small RNAs, which bind to the 3'-UTR of target mRNAs and negatively regulate the gene expression. Accumulating evidence demonstrates that miRNAs are involved in many biological processes such as embryo development, cell proliferation, differentiation, apoptosis and tumorigenesis. Heart development and heart diseases are complex processes controlled by various signaling pathways. Recent researches indicate the importance of miRNAs in the process of cardiac development and heart diseases. In this review, the role of miRNAs in cardiac development and the pathogenesis of heart diseases are overviewed. The insight into the regulating miRNAs will significantly expand the cardiovascular therapeutic strategies beyond classical pharmacology.     

竹荪深层发酵产物的氨基酸及其多糖分析

本文通过对深层发酵竹荪菌丝体和发酵液的多糖组份及其氨基酸分析，表明竹荪菌丝体氨基酸含量为２５．４％，较竹荪子实体含量１３．８％高；竹荪菌丝体多糖由葡萄糖、甘露糖及半乳糖组成；发酵液中游离氨基酸含量为１．８６ｍｇ／ｍｌ。多糖由一种单糖即葡萄糖构成。     

Role of cytokines in the pathogenesis of heart failure

Previous reports have shown that cytokines and cytokine receptors are independent predictors of mortality in patients with advanced heart failure. Recent studies demonstrate that cytokines, such as tumor necrosis factor, interleukin 1 and interleukin 6, play an important role in the pathogenesis of heart dysfunction in patients with heart failure. In this review, we summarized the importance, expression and mechanism of cytokines in the development and progression of heart failure.     

Relationship between hypothermia and heart rate variability,blood pressure variability

AIM: To study the function of autonomic nervous system by the method of heart rate variability (HRV) and arterial blood pressure variability (BPV).METHODS: The rectal temperature of rats decreased gradually with their body surfaces contacted with ice,the range of the rectal temperature varied from 19 ℃ to 37 ℃.Both the dynamic electrocardiogram (ECG) signals and dynamic blood pressure signals were recorded,respectively.RESULTS: Using the method of HRV,there was an increase in RR interval (RRI),which implied a corresponding decrease in heart rate with the rectal temperature varying from 19 ℃ to 29 ℃ (P<0.01).There was a decrease in the value of normalized low frequency power (LFn) (P<0.05) and an increase in the value of normalized high frequency power (HFn) (P<0.01) with the rectal temperatures varying from 19 ℃ to 21 ℃.Therefore,the balance control of cardiac autonomic nervous was transferred to increase of vagal nerve drive (P<0.05).Using the method of BPV,there was an increase (P<0.01) in the value of HFn related to respiratory with the body temperatures falling to 31 ℃.While the body temperature varied from 19 ℃ to 29 ℃ (except 27 ℃),there was an increase in the value of HFn related to respiratory rates (P<0.05 or P<0.01) and the balance control of autonomic nervous was transferred to increase of vagal nerve drive (P<0.05).CONCLUSION: As the body temperature reduces,the vagal nerve activity in cardiovascular system increases,and the effect of hypothermia on HRV is sensitive to BPV.     

Effects of Qili Qiangxin capsule on serum concentration of adiponectin and heart function in patients with coronary heart disease combined with congestive heart failure

AIM：To investigate the effects of Qili Qiangxin capsule on serum adiponectin (APN), serum N-terminal pro-brain natriuretic peptide (NT-proBNP) and heart function in the patients of coronary heart disease combined with congestive heart failure. METHODS：One hundred and twenty patients were randomly divided into treatment group and control group, and both groups were given anti-failure routine therapy. The patients in treatment group were treated with Qili Qiangxin capsule and the patients in control group were treated with placebo. The patients in the 2 groups were given a certain dose of the drugs for 6 months. The New York Heart Association (NYHA) heart function classification, left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD),left ventricular ejection fraction (LVEF), and 6-min walking test (6-MWT) were observed before and after treatment. The levels of APN, NT-proBNP were measured by ELISA before and after treatment. RESULTS：With the increase in the class of NYHA heart function, the serum concentrations of APN and NT-proBNP in the heart failure cases increased significantly. After 6-month treatment, the effective rate in experimental group was 91.7% and that in control group was 75.0%. A significant difference was found between the 2 groups (P<0.01). After treatment, LVEDD and LVESD in both groups were decreased significantly, and LVEF in both groups was increased significantly. The serum concentrations of APN and NT-proBNP decreased significantly (P<0.05). 6-MWT result was improved significantly. Compared with control group, more obvious effect was observed in experimental group (P<0.05). CONCLUSION：Treatment with Qili Qiangxin capsule reduces the levels of APN and NT-proBNP in the patients with coronary heart disease combined with congestive heart failure.     

Effect of atorvastatin on progression of heart failure and association with sodium-calcium exchanger expression

AIM: To investigate the effect of atorvastatin on the development of heart failure after myocardial infarction and the association with sodium-calcium exchanger (NCX) expression.METHODS: The model of heart failure was established by ligation of the anterior descending coronary artery for 8 weeks. The rats were randomly divided into control group, heart failure group, and atorvastatin group. Either atorvastatin (10 mg·kg^-1·d^-1) or vehicle was orally administered to the rats on the next day after the surgery. The left ventricular function and NCX expression were analyzed 8 weeks after ligation. Neonatal rat cardiomyocytes were cultured to investigate the effect of atorvastatin on the changes of calcium concentration induced by hypoxemia.RESULTS: A decrease in left ventricular diastolic dimension, an increase in left ventricular fractional shortening, and reductions of BNP level and NCX expression were observed in atorvastatin group. The hypoxemia-induced calcium overload in cultured cardiomyocytes was inhibited by atorvastatin, and it was inhibited by the inhibitor of NCX.CONCLUSION: Atorvastatin treatment improves cardiac function, which may be related to the expression and function of sodium calcium exchanger in heart failure.     

Bio-effects of salusins on isolated rat heart and neonatal cardiomyocytes

AIM: To investigate the bio-effects of salusins on rat heart and cardiomyocytes. METHODS: The cardiac function was determined by multipurpose polygraph in isolated rat heart treated with various concentrations of salusin-α or salusin-β.[⁴⁵Ca²⁺] and[³H]-Leu incorporation were determined in cultured neonatal rat cardiomyocytes with β-liquid scintillation counter. RESULTS: 10^-12-10^-7mol/L salusin-α and salusin-β had no effects on isolated rat cardiac function. However, salusin-α and salusin-β stimulated uptake and[³H]-Leu incorporation. The [⁴⁵Ca²⁺] uptake induced by salusins were inhibited by nicardipine, and were synergistically increased by endothelin-1. The[³H]-Leu incorporation induced by salusin-α and salusin-β was inhibited by nicardipine, FK506 (a special inhibitor of carcineulin), PD₉₈₀₅₉ (inhibitor of MAPK) and chelerthine (inhibitor of PKC). The effects of salusin-β[⁴⁵Ca²⁺] on uptake was stronger than those of salusin-α. But there were no statistical difference in[³H]-Leu incorporation between salusin-α and salusin-β. CONCLUSIONS: Salusin-α and salusin-β did not affect directly cardiac function in rat hearts. But salusins improved calcium uptake and protein synthesize in neonatal rat cardiomyocytes. Those effects of salusins were related with calcium channel, carcinuelin, MAPK and PKC signal pathways. Salusins may be the regulatory factors for myocardium growth and hypertrophy.     

Dexmedetomidine attenuates acute alcoholic hepatic injury in mice

AIM: To investigate the effects of dexmedetomidine (DEX) on acute alcoholic hepatic injury in mice and to explore the possible mechanisms. METHODS: Kunming mice (n=50) were randomly divided into 5 groups (n=10): normal saline control (NS) group, acute alcoholic hepatic injury model (E) group, low-dose (10 μg/kg) DEX (E+L) group, medium-dose (50 μg/kg) DEX (E+M) group and high-dose (100 μg/kg) DEX (E+H) group. The animals were sacrificed at 6 h after gavage of alcohol or normal saline. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD) were measured. The livers were removed for evaluation of histological characteristics and determining the content of tumor necrosis factor-α (TNF-α) amd interleukin-1β (IL-1β) in the liver tissues by ELISA. The expression levels of cytochrome P450 2E1 (CYP2E1) and nuclear factor-κB (NF-κB) in the liver tissues were evaluated by Western blot. RESULTS: Compared with NS group, the levels of ALT, AST and TG were obviously increased in E group, which were obviously decreased in E+M and E+H groups. Compared with NS group, the levels of TNF-α, IL-1β and MDA were obviously increase in E group, which were obviously decreased in E+M and E+H groups. Compared with NS group, the activity of SOD and the content of GSH were obviously decreased in E group, which were obviously increased in E+M and E+H groups. Compared with NS group, the expression of CYP2E1 and NF-κB was obviously increase in E group, which was obviously decreased in E+M and E+H groups. Compared with NS group, ethanol induced marked liver histological injury, which was less pronounced in E+M and E+H groups. CONCLUSION: DEX has a protective effect on mouse liver with acute alcoholic injury by the involvement in the processes of antioxidation and antiinflammation, and its mechanism may be associated with the inhibition of CYP2E1 and NF-κB expression.     

Cardiac L-type calcium channel and its alterations in cardiac hypertrophy and heart failure

There are two type of cardiac calcium channel current, both are inward current: (1) L-type Ca²⁺channel current (I_ca-L), plays a major role in determining myocyte action potential plateau characteristics as well as initiating the myocyte excitation-contraction coupling; (2) T-type Ca²⁺ channel current (I_ca-T), probably plays an important role in pacemaker activity. The alterations of L-type calcium channel abundance and function in cardiac hypertrophy and heart failure are determined by the the species difference, especially by the stage of the disease process and the degree of the disease. Both abundance and function of L-type calcium channel decrease in severe hypertrophy and heart failure.