Alternariol induces DNA polymerase β expression via the PKA-CREB pathway in NIH3T3 cells

AIM: To investigate the signal transduction induced DNA polymerase β expression by alternariol(AOH) in NIH3T3 cells. METHODS: The NIH3T3 cells were treated with 15 μmol/L AOH for 16 h or the cells were pretreated with an inhibitor of PKA (H89) for 1 h, and then exposed to AOH. The changes of DNA polβ expression and phospho-CREB in the cells with different treatments were determined by Western blotting and immunocytochemistry analysis. RESULTS: The expression of DNA polymerase β and phospho-CREB in NIH3T3 cells induced by AOH were significantly higher than that in control group (P<0.05). However, H89 partly blocked the AOH-induced phospho-CREB and DNA polβ expression. CONCLUSION: Alternariol up-regulates DNA polymerase β expression via the PKA-CREB pathway in NIH3T3 cells.     

Asymmetric dimethylarginine protects PC12 cells against glutamate-induced excitotoxic damage

AIM: To investigate the effects of asymmetric dimethylaoyoinine (ADMA) on glutamate-induced PC12 cell damage and its mechanisms. METHODS: PC12 cells were treated with different concentrations of glutamate as an in vitro excitotoxic trauma model. The cell viability was measured by MTT assay. Glutamate cytotoxicity was evaluated by lactate dehydrogenase (LDH) release assay. Intercellular reactive oxygen species (ROS) was detected by dihydrorhodamine123 (DHR) staining and flow cytometric (FCM) analysis. Nitric oxide synthase (NOS) activity and nitric oxide (NO) production were detected by using commercial kits with a spectrophotometer. RESULTS: Glutamate at concentrations of 1 mmol/L to 6 mmol/L dose-dependently decreased PC12 cell viability. Pretreatment 30 min with ADMA prior to administration of glutamate significantly attenuated the inhibition of cell viability, LDH release and ROS accumulation induced by glutamate. Pretreatment with ADMA significantly inhibited the increases in NOS activity and NO production caused by glutamate. CONCLUSION: ADMA obviously protects PC12 cells against glutamate-induced excitotoxicity by inhibiting NOS activity, overproduction of NO and accumulation of intracellular ROS.     

Relationship between endothelial dysfunction and renin-angiotensin-aldosterone system under the condition of fatigue stress and effect of herbs to dredge collaterals

AIM: To investigate the mechanisms and relationship among endothelial dysfunction, renin-angiotensin-aldosterone system (RAAS) and family of interleukins under the condition of excessive fatigue, by using the rats with fatigue stress. METHODS: Healthy male Wistar rats were randomly divided into control group, homocysteine (HCY) group, fatigue stress group, renshen group, shuangshen group and tongxinluo group. Radioimmunoassay was carried out to detect plasma renin activity (PRA), angiotensin II (AngⅡ), aldosterone (ALD), endothelin (ET), thromboxane-2 (TXA2), prostaglandin I2 (PGI2) in plasma and interleukin-1β, 2, 6, 10 (IL -1β, IL -2, IL -6) in sera. ELISA was used to detect NE and IL -10. The content of nitric oxide (NO) in sera was also detected. The bioinformatical analysis was used to determine the relationship between RAAS and endothelial dysfunction. RESULTS: Compared with control group, the levels of ET-1 and TXA2 in fatigue stress group were significantly increased (P<0.01, P<0.05), but the content of PGI2 and NO was significantly decreased (P<0.01, P<0.01). Compared with control group, the renin activity in plasma of animals in fatigue stress group was significantly decreased (P<0.01), the AngⅡ, IL -1β, IL -6 level was significantly increased compared with the control group (P<0.01, P<0.01), and was significantly increased compared with the HCY group (P<0.05, P<0.01, P<0.01). The NE level showed the tendency of decrease in different degree. After the intervention of three kinds of herbs to dredge collaterals, the ET-1, AngⅡ, IL -1β, IL-6 level in plasma was decreased in different degree (P<0.05, P<0.01, P<0.01), and at the same time, the contents of NO and NE level were significantly increased (P<0.05). The ALD level in tongxinluo groups was apparently higher than that in control group and the fatigue stress group (P<0.05). The bioinformatics analysis showed that Ang II and ET, IL-1; PGI2 and ALD; NO and ALD composed of three subsystems and interrelated according to the principle of optimality of complex system, and gradual change regulation was also observed in fatigue stress group. However, in control group, HCY group and tongxinluo group, the same interrelation among subsystems was not existed. CONCLUSION: In a state of long-term excessive fatigue, vascular endothelial dysfunction may be induced, and is related with renin-angiotensin-aldosterone system imbalance and serious turbulence of the autonomic dysfunction. Herbs to dredge collaterals could improve it significantly. The results suggest that bearing excessive fatigue and pressure in long-term may be the potential risk factors to induce vascular endothelial dysfunction and further result in cardio-cerebro vascular diseases, Tongluo therapy may be one of the useful ways to prevent such diseases.     

Effects of salvianolic acid B on neurons during oxygen/glucose deprivation and reintroduction

AIM: To observe the damage induced in the primary cultured rat cortical neurons by oxygen/glucose deprivation and reintroduction, and to investigate the neuroprotective mechanism of salvianolic acid B (SalB). METHODS: Primary cultured rat cortical neurons were randomly divided into the control group, the model group and the SalB group. The cell model was established by oxygen/glucose deprivation for 3 h followed oxygen/glucose reintroduction for 24 h. The cortical neurons viability was determined by MTT assay. The leakage rate of lactate dehydrogenase (LDH) was measured by chromatometry. The mitochondrial membrane potentials (MMP) and the apoptosis rate were quantitatively analyzed by flow cytometry. The cytosolic free calcium was assessed using LSCM. The morphologic changes of neuronal nuclei were observed by Hoechst 33342 fluorescence staining. RESULTS: Compared to the model group, the cortical neurons viability, the survival rate and the fluorescence value of MMP in the SalB group were obviously increased (P<0.05,P<0.01). In addition, in the SalB group, the leakage rate of LDH, the fluorescence intensity of cytosolic free calcium and the apoptosis rate were significantly lower than those in the model group (P<0.01). CONCLUSION: The neuroprotective mechanism of SalB in the oxygen/glucose deprivation and reintroduction neurons would be due to the fact that SalB maintains the MMP and the calcium homeostasis.     

Role of A3 adenosine receptor on vascular reactivity of superior mesenteric artery in hemorrhagic shock in rat

AIM: To explore whether A3 adenosine receptor plays a role in the modulation of vascular reactivity after hemorrhagic shock in rat, and to find out the prospective drug target to restore the decreased vascular reactivity following hemorrhagic shock. METHODS: The hemorrhagic shock (40 mmHg) model was established in rat, and the reactivity of superior mesenteric artery (SMA) to norepinephrine (NE) was observed. A3AR expression at protein level and mRNA level were measured by Western blotting and RT-PCR respectively. RESULTS: The vascular reactivity of SMA to NE after hemorrhagic shock (40 mmHg) was decreased significantly in a biphasic response manner. The expression of A3AR mRNA in SMA after hemorrhagic shock decreased without significant difference. The expression of A3AR protein has a slight increase without statistical difference after 30 min of hemorrhagic shock and then has a significant decrease (especially at 2 h and 4 h after hemorrhagic shock). The usage of IB-MECA, a selective A3AR agonist, significantly increased the responsiveness of SMA to NE in hemorrhagic shock in rat. MRS1523, the selective A3AR antagonist, significantly abolished the restoration of the vascular reactivity to NE by IB-MECA in hemorrhagic shock in rat. CONCLUSION: A3AR plays a role in the modulation of vascular responsiveness to NE in hemorrhagic shock in rat, and the selective agonist of A3AR could restore the reactivity of SMA to NE in hemorrhagic shock in rat.     

Mechanism of RhoA on vascular reactivity following hemorrhagic shock in rats

AIM: To observe the mechanisms of RhoA on vascular reactivity following hemorrhagic shock (HS) in rats. METHODS: The superior mesenteric artery (SMA) in rats subjected to hemorrhagic shock was adopted to assay the vascular reactivity via observing the contraction initiated by norepinephrine (NE) with isolated organ perfusion system. Meanwhile, the effects of Rho kinase, myosin light chain phosphatase (MLCP), myosin light chain kinase (MLCK) on RhoA regulating vascular reactivity were observed. The effects of RhoA agonist U-46619 and inhibitor C3 enzyme on the activities of Rho kianse, MLCP, MLCK and phosphorylation of MLC20 in the vascular smooth muscle cells (VSMC) with hypoxia were also measured. RESULTS: As compared to control group, the cumulative dose-response curves of SMA to NE at 2 h after shock shifted to the right, the maximal contractions (Emax) of NE was significantly decreased. RhoA agonist U-46619 increased the vascular reactivity in the late period of shock. C3 enzyme abolished U-46619 induced the increase in the contractile response of SMA to NE. Rho kinase inhibitor Y-27632 decreased U-46619-induced the increase in the vascular reactivity, MLCP inhibitor calyculin further promoted the increase in the vascular reactivity. However, MLCK inhibitor had no effect on the U-46619-induced change of vascular reactivity. After hypoxia, the activities of Rho kinase and MLCK, and the level of MLC20 phosphorylation were decreased, MLCP activity was increased. RhoA agonist U-46619 increased the activity of Rho kinase and phosphorylation of MLC20, decreased the activity of MLCP, but had no effects on MLCK activity. CONCLUSION: RhoA plays an important role in the regulation of vascular reactivity following shock. The mechanism is closely related to regulating the activities of Rho kinase and MLCP, and increasing the phosphorylation of MLC20 in VSMC.     

Inhibition of hypothermic preservation-induced Smac/DIABLO protein expression by diazoxide in donor rat myocardium

AIM: To investigate the effect of a mitochondrial ATP-sensitive potassium channel (mitoKATP) opener diazoxide (DE) on Smac/DIABLO protein expressions in rat heart suffered from different duration of hypothermic preservation. METHODS: The Langendorff model of isolated rat heart was used. After stored in 4 ℃ Celsior solution with or without DE (30 μmol/L) for different time (0, 3, 6, 9 or 12 h). Cell apoptosis was detected by TUNEL technique. The expression of Smac/DIABLO protein in cytoplasm and total caspase-3 protein in myocardia tissue was also analyzed by Western blotting. RESULTS: (1) Compared to the hypothermic preservation groups, DE reduced the percentage of apoptotic cells and the expression of caspase-3 protein in myocardia tissue. (2) The peak of Smac/DIABLO protein expression level appeared at 6 h after hypothermic preservation, and which was postponed to 9 h by DE. (3) The above effects of DE were attenuated by a mitoKATP channel inhibitor 5-hydroxydecanoate (5-HD). CONCLUSION: The findings indicate that in the isolated rat heart, DE protects myocardium against different duration of hypothermic preservation injury via opening of mitoKATP channel and inhibition of Smac/DIABLO protein expression.     

Inhibitory effect of T-bet gene transfer on airway inflammation in a established murine allergic asthmatic model

AIM: To investigate the effect of T-bet plasmid gene transfer to airway on allergen induced airway inflammation in a murine asthmatic model. METHODS: A mouse asthma model was established by sensitization with ovalbumin (OVA). Forty C57BL/6 mice were divided into 4 groups (10 mice in each group): the normal control group (group A), the asthmatic model group (group B), the pcDNA3 plasmid group (group C), and the pcDNA3-T-bet group (group D). The animals in group B, C and D were sensitized and challenged with OVA. The animals in group A were applied with normal saline. pcDNA3 plasmid at dose of 50 μg was intranasally administered at 24 h before intranasal challenges to the mice in group C, and the 50 μg pcDNA3-T-bet plasmid for the mice in group D. Bronchial alveolar lavage fluid (BALF) was collected and lung tissues were resected at 48 h after OVA challenge for later assay. RESULTS: After administration with pcDNA3-T-bet plasmid, high level of T-bet expression at 48 h was detected in the lung tissue by Western blotting. In pcDNA3-T-bet treated asthmatic models, histological evaluation revealed the significant suppression of eosinophil peribronchial and perivascular infiltration, and reduction of epithelial damage. The numbers of eosinophils, neutrophils and lymphocytes in BALF from pcDNA3-T-bet treated mice were significantly reduced compared to those in asthmatic control group (P<0.05). The level of IL-4 in BALF was significantly decreased in pcDNA3-T-bet group compared to that in asthmatic control group (P<0.05), while the level of IFN-γ in BALF was significantly increased in pcDNA3-T-bet group. No significant change of inflammation cells and cytokines in pcDNA3 plasmid group and asthmatic control group was observed (P>0.05). CONCLUSION: Intranasal pcDNA3-T-bet plasmid transfer inhibits asthmatic airway inflammation in the murine asthmatic model, suggesting a new therapeutic strategy for allergic asthma.     

沼渣基质对温室番茄生长和产量的影响

利用沼渣和炉渣以不同体积比混合作基质,研究沼渣对温室番茄生长和产量的影响。结果表明,沼渣和炉渣复合基质的理化性状完全达标;沼渣促进了番茄植株的生长,提高了番茄果实产量和品质,其中,3份沼渣+1份炉渣处理的番茄植株根系活力最强,前3穗果实产量最高;为0.93 kg·株-1;糖酸比最高,为6.24。     

食用菊花扦插繁殖影响因素的初步研究

从基质选择、插穗长度、生根粉处理及食用菊花品种几个方面,对影响食用菊花扦插繁殖成活率的有关因素进行了研究。结果表明,珍珠岩是食用菊花扦插的首选基质;插穗长度为10 cm时生根情况较好;生根粉能够显著促进插穗生根;食用菊花插穗生根能力不同品种间差异较大。