Protective effect of quercetin on rat cardiomyocytes against anoxia/reoxygenation injury is mediated by PKCε signaling pathway

AIM：To investigate whether quercetin (Que) protects cardiomyocytes from anoxia/reoxygenation (A/R) injury through protein kinase C epsilon (PKCε) pathway. METHODS：Primary cardiomyocytes were isolated from neonatal SD rats and exposed to A/R (3 h of anoxia followed by 2 h of reoxygenation) as well as Que and/or εV1-2 (a selective PKCε inhibitor) preconditioning. The expression of PKCε in the cells was detected by Western blotting. The levels of lactate dehydrogenase (LDH) and creatine kinase (CK) in cell culture supernatants, the reactive oxygen species (ROS) and mitochondrial membrane potential in the cells, the opening of mitochondrial permeability transition pore (mPTP) and the cell viability and apoptosis were also measured. RESULTS：The expression of PKCε protein was significantly increased in the cardiomyocytes pretreated with 40 μmol/L Que 72 h before A/R (P<0.01 vs A/R group). Meanwhile, Que preconditioning could increase cell survival rate, decrease ROS production and cell apoptosis, alleviate the loss of mitochondrial membrane potential and inhibit the opening of mPTP induced by A/R injury (P<0.01 vs A/R group). However, pretreatment with Que and εV1-2 attenuated these protective effects of Que (P<0.01 vs Que+A/R group). CONCLUSION：One of the mechanisms underlying the cardioprotective effect of Que might be the increase in PKCε protein expression and the activation of its downstream pathway.     

The basic mechanism of increased microvascular permeability

The increased microvascular permeability appears mainly in venule during inflammation, shock, and burns. Endothelial cells play an important role in venule permeability enhancement. There are two kinds of pathway for macromolecule extravasation. One is paracellular pathway and another is transcellular pathway, which are related to the formation of endothelial gap or transcellular openings seperately. The alteration of intercellular related protein, such as occludin, claudin, zona occludens (ZO), junctional adhesion molecule (JAM), VE cadherin, catenin, integrin, etc, and the alteration of endothelial cytoskeleton, such as rearrangement of actin filament, formation of stress fiber and focal adhesion, etc, involve in the pathogenesis of increased microvascular permeability.     

Role of cytokeratin 8 on changes of intestinal epithelial permeability induced by corticotropin-releasing factor

AIM: To investigate the role of cytokeratin 8 (CK8) on the change of intercellular permeability of intestinal epithelial cells induced by corticotropin-releasing factor (CRF). METHODS: The expression levels of CRF receptor 1 (CRFR1) and CRFR2 on human colon adenocarcinoma HT29 cell surface were determined by immunofluorescence staining. After treatment with 100 nmol/L CRF for 72 h, the translocation of FITC-labelled dextran was measured in a Transwell chamber. The structural changes of tight junctions were observed under transmission electron microscope. The expression levels of CK8, and tight junction proteins ZO-1 and occludin were determined by Western blot. The activity of protein kinase C (PKC) was detected by ELISA. Furthermore, the effects of CRF on intestinal epithelial permeability were examined in CK8-silencing HT29 cells, which were constructed by infection with sh-CK8 lentivirus. RESULTS: CRF treatment increased the permeability of FITC-labelled dextran (P<0.05), caused the opening of tight junctions, and induced increased fluorescence intensity of CK8. The expression levels of occludin and ZO-1 were down-regulated (P<0.05). PKC activity was decreased at 1 h after CRF treatment (P<0.05). CRF-induced increase in the permeability and down-regulation of occludin were not blocked by CK8 silencing. Nevertheless,CK8 silencing blocked the effects of CRF regarding the decrease in the expression levels of ZO-1 and the increase in PKC activity (P<0.05). CONCLUSION: CK8 may be involved in CRF-induced increase in intestinal epithelial permeability by inhibiting the activity of PKC, and there may be other signaling pathways involved.     

Effect of protein kinase C signal pathway on resistin expression in 3T3-L1 adipocytes

AIM：To investigate the effect of protein kinase C on resistin expression in 3T3-L1 adipocytes.METHODS：The differentiated 3T3-L1 adipocytes were incubated with 50 nmol/L phorbol 12-myristate 13-acetate (PMA) or 5 μmol/L Ro-31-8220 for 24 h.Expression of resistin mRNA was detected by RT-PCR and expression of resistin protein was detected by Western blotting.RESULTS：Compared with control,PMA increased the expression of resistin mRNA and protein in 3T3-L1 adipocytes significantly (P<0.01),while Ro-31-8220 decreased the expression of resistin mRNA and protein in 3T3-L1 adipocytes obviously (P<0.01).CONCLUSION：Protein kinase C signal pathway may regulate resistin expression in 3T3-L1 adipocytes.     

Signaling transduction of noxious visceral stimuli in postsynaptic dorsal column neurons in spinal cord

The spinal postsynaptic dorsal column pathway plays a critical role in the visceral pain transmission in spinal cord. The noxious visceral stimuli might induce complex receptor expression and intracellular signaling transductions in postsynaptic dorsal column neurons. It has been demonstrated that neurokinin-1(NK-1) receptor, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), and intracellular protein kinase C (PKC), protein kinase A (PKA), mitogen-activated protein kinases (MAPKs) and cAMP response element-binding (CREB) protein are involved in the signaling transduction of visceral stimuli. All these processes contribute to the sensitization of postsynaptic dorsal column neurons and enhance the spinal transmission of visceral pain, indicating a potential and promising way of visceral pain therapy to inhibit the sensitization of postsynaptic dorsal column neurons.     

植物丝裂原活化蛋白激酶激酶的生物信息学分析

丝裂原活化蛋白激酶(MAPK)信号传导通路是植物细胞内重要的信号传导系统,在多种生物和非生物胁迫,激素、细胞分化和发育进程中发挥关键作用。丝裂原活化蛋白激酶激酶(MAPKK)是信号传导中的重要成员,执行整合上游信号到丝裂原活化蛋白激酶的重要功能。基于BLAST搜索,得到植物丝裂原活化蛋白激酶激酶基因序列。采用CLUTALW、SMART、SwissModel和MEGA3,1软件分析这些序列。多序列比对揭示了它们具有典型的蛋白激酶ATP结合域签名和丝氨酸、苏氨酸蛋白激酶激活位点签名。空间结构预测表明它们与人MAPKK的晶体结构相似。系统进化树中烟草和番茄的丝裂原活化蛋白激酶激酶、野生稻和印度栽培稻丝裂原活化蛋白激酶激酶形成独立的分支,说明它们分别具有较近的亲缘关系。     

RISK signaling pathway in myocardial preconditioning and postconditioning

The anti-apoptotic pro-survival kinase signaling cascades, phosphatidylinositol-3-OH kinase (PI3K)-Akt and p42/p44 extra-cellular signal-regulated protein kinases (ERK 1/2), which have been termed the reperfusion injury salvage kinase (RISK) pathway, are involved in cellular survival. In myocardial ischemic preconditioning, pharmacological preconditioning, ischemic postconditioning and pharmacological postconditioning, the activation of these kinase cascades at the time of reperfusion has been demonstrated to confer cardioprotection against reperfusion-induced injury. Targeting the RISK signaling pathway may provide a novel strategy to salvaging viable myocardium and limiting infarct size during myocardial ischemia-reperfusion.     

Effects of Dryk1A,ASF and alternative splicing of CaMKⅡδ on myocardial hypertrophy in renovascular hypertensive rats

AIM: To investigate the role of dual-specificity tyrosine phosporylation-regulated kinase 1A (Dyrk1A)-alternative splicing factor (ASF)-calcium/calmodulin-dependent protein kinase Ⅱδ (CaMK Ⅱδ) pathway in the progression of myocardial hypertrophy in renovascular hypertensive rats. METHODS: The renovascular hypertension was induced by two-kidney one-clip (2K1C) method. The changes of blood pressure and myocardial hypertrophy were measured. The techniques of RT-PCR and Western blotting were used to detect CaMKⅡδ alternative splicing and the protein expression of Dyrk1A and ASF, respectively. RESULTS: Eight weeks after operation, systolic blood pressure (SBP) and diastolic blood pressure (DBP) in 2K1C rats increased (P<0.05). The increases in left ventricular weight (LVW), the ratio of LVW to body weight (BW) and the area of myocardial cells indicated that the hypertensive rats developed significant cardiac hypertrophy. The protein expression of Dyrk1A and mRNA expression of CaMKⅡδA and δB were significantly increased, while the protein expression of ASF and mRNA expression of CaMKⅡδC were decreased compared with sham-operated control rats (P<0.05). Treatment with Dryk1A inhibitor epigallocatechin gallate (EGCG) or harmine effectively attenuated cardiac hypertrophy and reversed the changes in the protein expression of Dyrk1A, ASF and alternative splicing of CaMKⅡδ (all P<0.05). CONCLUSION: Dyrk1A-ASF-CaMKⅡδ pathway plays a role in the development of myocardial hypertrophy in renovascular hypertensive rats.     

Activation of JNK/SAPK pathway in vero cells induced by N-methyl-N'-nitro-N-nitrosoguanidine

AIM and METHODES: To evaluate the possible signal transduction mechanism of nontargeted mutagenesis in vero cells induced by DNA damaging agent N-methyl-N'-nitro-N-nitrosoguanidine(MNNG),the activation of c-Jun NH₂-terminal kinase/stress activated protein kinase(SAPK/JNK) pathway in vero cells induced by MNNG was studied. Western Blot analysis and Solid-phase kinase assay were used to measure the phosphorylation of JNK1 and kinase activity of JNKs, respectively. RESULTS: After 0.2 μmol/L, 2.5 h MNNG or 1 mg/L, 1 h cycloheximide (CHM) treatment, the proportion of phosphorylated JNK1 in cell extract increased significantly, simultaneously the kinase activity of JNKs increased dramatically(6.7 and 3.0 folds respectively), as measured by the phosphorylation of c-Jun, a substrate of JNKs. CONCLUSION: Both 0.2 μmol/L 2.5 h MNNG and 1 mg/L 1 h CHM treatment can induce the activation of JNK/SAPK pathway, one of the stress signal transduction pathways, in vero cells.     

Effects of insulin on mitogen-activited protein kinase of vascular smooth muscle cells in spontaneously hypertensive rat

AIM:To explose the possible existing pathway of intracellular signaling transduction in hypertensive induced by insulin in rat vascular smooth muscle cells proliferation which involved mitogen-activated protein kinase. METHODS:Male spontaneously hypertensive rat (SHR) aorta and WKY(6 weeks old) were isolated and then cultured to make the purified vascular smooth muscle cells.6-8th generation of VSMC were interfered with insulin in vitro. MAPK activity was determined by myelin basic protein method and its volume was measured with Western Blot. And [³H]-TdR was used to measure DNA synthesis in VSMC proliferation. RESULTS: After the interfered with insulin the DNA synthesis was increased obviously in SHR group. MAPK activity and its contains in SHR were increased more than the control group. Protein kinase C inhibitor decreased MAPK activity induced by insulin. CONCLUSION:Proliferation of SHR VSMC in vitro was correlated with increased activity of MAPK. Insulin can affect MAPK induced activity. So an insulin-PKC-MAPK axis may exist in hypertensive VSMC.     

Effect of Akt1 gene transfection on mitochondrial permeability transition after myocardium ischemia-reperfusion in rat

AIM: To investigate the effects of Akt1 gene transfection into myocardium after ischemia-reperfusion (I/R) on mitochondrial permeability transition. METHODS: Forty adult male SD rats were divided randomly into five groups with 8 rats each: control group, I/R group, Ad-gene group, Ad-blank group and Ad-inhibitor group. The rats in Ad-gene group were injected with 30 μL Lipofectamine 2000 solution including Akt1 gene to the myocardium 48 h before ischemia while those in control group and I/R group were injected with PBS of the same volume. Rats in Ad-blank group were injected with Lipofectamine 2000 of the same volume into myocardium. In Ad-inhibitor group 30 μL Lipofectamine 2000 and gene complexes with LY294002 were injected. Hemodynamics, apoptotic index, the concentrations of lactate dehydrogenase, creatine kinase, the expression of Akt1, cytosolic, mitochondrial cytochrome C and MPT were also measured. RESULTS: The lowest level of Akt1 protein expression was observed in control group. The protein expression of Akt1 in Ad-gene group was higher than that in I/R group, Ad-blank group and Ad-inhibitor group. The AI, LDH and CK in Ad-gene group were significantly lower than those in other groups except control group. Transfection of Akt1 markedly reduced the loss of mitochondrial cytochome C after I/R injury. Ad-gene transfection led to a significant increase in absorbance at 540 nm compared to I/R group, Ad - black group and Ad-inhibitor group (P<0.05). CONCLUSION: Akt1 gene prevents myocardial apoptosis after I/R injury. Akt1 gene also inhibits the opening of mitochondria permeability transition and protects mitochondrial functions of myocardium in I/R injury.     

EGFR-p38 MAPK signaling pathway is involved in expression of HMGB1 in pulmonary tissues of rats with ventilator-induced lung injury

AIM: To investigate the role of epidermal growth factor receptor (EGFR)-p38 mitogen-activated protein kinase (MAPK) pathway in the expression of high mobility group box 1 protein (HMGB1) in the lung tissues of rats with ventilator-induced lung injury (VILI).METHODS: Thirty-two healthy Sprague-Dawley (SD) rats were randomly divided into 4 groups (n=8 each): group A, spontaneous breathing; group B, small tidal volume ventilation (VT=8 mL/kg); group C, high tidal volume ventilation (VT=40 mL/kg); group D, high tidal volume ventilation plus EGFR antagonist AG-1478. The rats in group B, group C and group D were mechanically ventilated for 4 h and then all animals were sacrificed.Total protein content and white blood cell (WBC) count in bronchoalveolar lavage fluid (BALF), the lung wet/dry weight ratio (W/D) and myeloperoxidase (MPO) activity were determined. The histological changes of lung tissues were observed by HE staining. The EGFR protein and mRNA expression, p38 MAPK activity and HMGB1 protein expression in the lung tissues were also detected.RESULTS: The inflammatory responses as evidenced by lung HE staining, total protein and WBC in BALF, the lung W/D and MPO activity were significantly higher in group C than those in group A (P<0.05). The mRNA expression of EGFR, EGFR activity, p38 activity and HMGB1 protein level also significantly increased in group C (P<0.05) as compared with group A. Significant decreases in the above indexes in group D were observed as compared with group C.CONCLUSION: High tidal volume ventilation induces acute lung injury, which may be related to up-regulation of HMGB1 expression through EGFR-p38 MAPK signal pathway.     

Mechanism of coronary artery constriction induced by 5-HT

AIM:To investigate the possible mechanism of coronary artery contraction induced by 5-hydroxytryptamine (5-HT). METHODS:Isolated coronary artery rings were obtained from male Wistar rats, and the vascular tension meter was used to determine the tension of the coronary artery rings. The effects of inhibitors of different signaling pathway on vascular contraction tension induced by 5-HT were observed. RESULTS:Firstly, we found that 5-HT_2A receptor antagonist sarpogrelate (1 μmol/L) completely eliminated the coronary artery contraction induced by 5-HT. Phospholipase C_β (PLC_β) inhibitor U73122 (10 μmol/L and 50 μmol/L), Rho-related protein kinase inhibitor Y-27632 (3 μmol/L and 10 μmol/L) and protein kinase C δ subunit (PKCδ) inhibitor rottlerin (3 μmol/L and 10 μmol/L) significantly inhibited the contraction of coronary artery ring caused by 5-HT (P<0.05). In addition, compared with the untreated group, vascular contraction tension induced by 5-HT was also decreased significantly by L-type calcium channel (Cav1.2) blocker nifedipine (1 μmol/L), store-operated Ca²⁺ entry (SOCE) inhibitor SKF96365 (10 μmol/L and 30 μmol/L) and 2-aminoethoxydiphenyl borate (2-APB, 50 μmol/L and 100 μmol/L) (P<0.05). At the same time, 5-HT also induced vasoconstriction after treated with nifedipine (1 μmol/L) Kerbs-Henseleit (K-H) liquid without calcium (P<0.05). CONCLUSION:5-HT activates 5-HT_2A receptor induced coronary artery contraction, possibly related to the PKC/Rho kinase signaling pathway and calcium regulation.     

Effects of lipopolysaccharide,IL-6 and TNFα on the tissue factor expression of astrocytes

AIM:To study the effects of lipopolysaccharide(LPS), interleukin-6(IL-6)and tumor necrosis factor α (TNFα) on tissue factor(TF) expression of astrocytes. METHODS:Astrocytes were identified with anti-glial fibrillary acidic protein antibody. The TF activity of cell lysate was measured with one stage clotting assay. RESULTS:TF activity of astrocytes of LPS,IL-6,TNFα groups were obviously higher than that of the control group(P <0.05); While LPS,IL-6 and TNFα were combined with trifluoperazine or H₇, their inductive effects were inhibited. CONCLUSION:LPS,IL-6 and TNFα promoted the TF expression of astrocytes and its mechanisms may connected with Calcium/Camodulin and protein kinase C pathway.     

间歇浸没式生物反应器在辰星草培养苗扩繁中的应用

以茎尖培养得到的辰星草(Limonium hybrid‘MistyBIue’)培养苗(带2叶片)为试材。培养基为MS BA 1．0mg／L 蔗糖30g／L，pH调节为5．8。固体培养和振荡培养使用250mL三角瓶，每瓶100mL培养基。生物反应器培养使用5L气球型反应器，2L培养基。完全浸没式生物反应器培养时，无“载桥”，培养苗在培养基中始终处于浸泡状态；间歇浸没式生物反应器培养时，离反应器底部15cm处架有“载桥”，培养基定时与‘‘载桥”上的苗接触，每     

Salinomycin promotes the apoptosis-inducing effect of gefitinib on human lung adenocarcinoma cell line A549

AIM: To investigate the effect of salinomycin alone or in combination with gefitinib (an inhibitor of epidermal growth factor receptor tyrosine kinase) on the growth and apoptosis of human non-small-cell lung cancer cell line A549. METHODS: The inhibitory effect of salinomycin on the growth of A549 cells was tested by MTT assay. The cell apoptosis and the level of mitochondrial membrane potential were determined by flow cytometry. The activity of caspase-3, -8, and -9 was measured by the method of colorimetry. The protein levels of cytochrome C, Bcl- 2, p-EGFR, p-Akt and p-ERK were detected by Western blotting. RESULTS: Salinomycin or gefitinib alone inhibited the growth of A549 cells in a dose-dependent manner. Salinomycin or gefitinib also induced apoptosis of the cells. Salinomycin combined with gefitinib produced stronger inhibitory effect on the cell proliferation, and a significant increase in cell apoptosis was also observed. Compared with control group, salinomycin alone significantly reduced mitochondrial membrane potential, transitorily increased the levels of intracellular reactive oxygen species (ROS), cytoplasmic cytochrome C and Ca2+, and increased the activity of caspase-3, -8 and -9 in A549 cells. Gefitinib alone inhibited the protein expression of p-EGFR, p-Akt and p-ERK, but no obvious effect on the release of cytochrome C and the activity of caspase-3, -8 and -9 was found. The combination of salinomycin and gefitinib significantly reduced the protein levels of Bcl-2, p-EGFR, p-Akt and p-ERK, but the protein levels of EGFR, Akt and ERK were not obviously changed. CONCLUSION: The synergy of salinomycin and gefitinib is observed. Salinomycin inhibits the growth and induces apoptosis of human lung carcinoma A549 cells through Bcl-2 pathway and mitochondrial apoptosis pathway. Salinomycin also increases the sensitivity of A549 cells to gefitinib.     

Effects of temperature and seed coat treatments on germination of sweet pea

Tests on seeds of sweet pea (Lathyrus odoratus L.) were set up to examine responses to temperature during germination and to investigate the effects of mechanical and chemical treatments on the permeability of the seed coat to water.Optimum temperatures for germination occurred at about 21 °C, and higher temperatures resulted in the production of fewer seedlings. Although temperatures below c. 15 °C also reduced the numbers of seeds which germinated, even at 2 °C more than 50 % germinated within 60 days.Treatments with concentrated sulphuric acid or the mechanical removal of part of the seed coat by filing greatly increased permeability to water and under some conditions improved germination responses. Filing, followed by exposure to excess free water, reduced germination rates, possibly as a result of loss of solutes from the seed during imbibition.     

The effect of homocysteine on airway smooth muscle cells and fibroblasts

AIM: To study the effect of homocysteine (HCY) on proliferation of airway smooth muscle cells and fibroblasts and the effect of HCY on collagen prodution of airway fibroblasts. METHODS: [³H]-TdR incorpora- tion was measured in cultured airway smooth muscle cells. The [³H]-TdR and [³H]-proline incorporation were mea- sured in cultured airway fibroblasts. RESULTS: HCY induced proliferation of airway smooth muscle cells and fibroblasts in a concentration - dependent manner. HCY also induced collagen production of airway fibroblasts in a concentration - dependent manner. The inhibitors of protein kinase C, H7 and polymyxin B, inhibited HCY - induced proliferation of airway smooth muscle cells. CONCLUSIONS: HCY induced proliferation of airway smooth muscle cells and fibroblasts, HCY also induced collagen production of airway fibroblasts. The HCY - induced proliferation of airway smooth muscle cells may be related to the pathway of PKC signal transduction.