Effects of drynaria total flavonoids on serum levels of leptin,interleukin 6, prostaglandin E2 and expression of bone β2-adrenergic receptor in ovariectomized osteoporosis rats

AIM: To investigate the effects of drynaria total flavonoids on serum levels of leptin (LEP), interleukin 6(IL-6), prostaglandin E₂(PGE₂) and the expression of bone β₂-adrenergic receptor (ADRB2) in a rat model of ovariectomized osteoporosis(OP). METHODS: The osteoporosis model was established by ovariectomy. Twelve weeks after modeling,bone mineral density (BMD) was determined by dual-energy X-ray absorptiometry to verify successful modeling.Enzyme-linked immunosorbent assay was applied to detect the concentrations of LEP, IL-6 and PGE₂ in serum. The expression of ADRB2 was determined by immunohistochemical technique. RESULTS: Compared with sham group,BMD of the rats in model group significantly decreased in multiple regions 12 weeks after modeling(P<0.01). The serum levels of LEP, IL-6 and PGE₂ in model group were significantly higher than those in sham group(P<0.05). The levels of LEP, IL-6 and PGE₂ in drynaria total flavonoids group were significantly lower than those in model group(P<0.01). No significant difference of PGE₂ between these 2 groups was observed. The ADRB2 expression in sham group and treatment group was significantly different from that in model group, and no significant difference between sham group and treatment group was found. CONCLUSION: The serum levels of LEP, IL-6 and PGE₂ and the expression of bone ADRB2 increased in OP rats.Drynaria total flavonoids reduce the production of LEP, IL-6 and the expression of ADRB2, and suppress the bone absorption, which may be one of the mechanisms in treating OP.     

Lipoxin A4 reduces lipopolysaccharide-induced expression of cyclooxygenase 2 and prostaglandin E2 in human bronchial epithelial cells

AIM: To explore the effects of lipoxin A₄ on the expression of cyclooxygenase 2 (COX-2) in human bronchial epithelial cells (HBECs). METHODS: HBECs were incubated with various concentrations (0.1, 1 and 10 mg/L) of lipopolysaccharide(LPS) for 9 h, or 1 mg/L LPS for different time (3 h, 6 h and 9 h). The levels of COX-2 mRNA in HBECs and prostaglandin E₂ (PGE₂) in the culture supernatant were measured. In addition, the HBECs were exposed to lipoxin A₄ at concentration of 0, 100 and 400 μmol/L after stimulated with LPS at concentration of 1 mg/L for 9 h, and the supernatant of the culture cells was collected for determining the content of PGE₂ by ELISA. The cells were also harvested, and the mRNA and protein levels of COX-2 were analyzed by RT-PCR and Western blotting, respectively. RESULTS: LPS increased the mRNA expression of COX-2 and production of PGE₂ in a dose and time dependent manners in HBECs. Induction of COX-2 mRNA and protein by LPS were inhibited by lipoxin A₄ in a dose-dependent manner. Lipoxin A₄ also significantly decreased LPS-induced production of PGE₂. CONCLUSION: Lipoxin A₄ down-regulates LPS-induced expression of COX-2 and consequently inhibits the production of PGE₂ in HBECs.     

Effect of IL-10 on IL-1β-induced cyclooxygenase-2 expression and prostaglandin E2 release in human mesangial cells

AIM: To investigate the effect of IL-10 on IL-1β-induced prostaglandin E₂(PGE₂) release and cyclooxygenase-2(COX-2) expression in human mesangial cells and to examine whether IL-10 has effect on the biological function of IL-1β.METHODS: The PGE₂ concentration in supernatants of HMC was measured by radioimmunoassay. The COX-2 mRNA and protein were measured by RT-PCR and Western blot, respectively. RESULTS: PGE₂ and COX-2 were significantly increased after treatment with IL-1β(P＜0.01 for both) in cultured human mesangial cells. IL-10 had no effects on basical production of COX-2 and PGE₂(P＞0.05, respectively), while it inhibited IL-1β-elicited PGE₂ production, as well as COX-2 mRNA and protein expression in a concentration-dependent fashion. CONCLUSIONS: These results indicated that IL-10 depressed the IL-1β-induced release of PGE₂ and expression of COX-2. These data suggested that IL-10 could exert anti-inflammatory actions at several levels, not only by inhibiting the production of pro-inflammatory cytokines but also by suppressing their biological function.     

Changes of PGE2, PGI2 and TXA2 concentration in rat traumatic pulpitis

AIM: To study the relationship between prostaglandins and acute pulpitis. METHODS: Rat traumatic pulpitis model was established by pulp exposure. The kinetic pathological changes in dental pulpal tissues and changes of PGE₂,6-Keto-PGF_1α and TXB₂ concentration in dental pulp were observed. RESULTS: After pulpal trauma, the dental pulp showed inflammatory changes and the concentrations of PGE₂,6-Keto-PGF_1α and TXB₂ were increased, which peaked at 6 hour post-trauma. CONCLUSION: Prostaglandins play a significant role in the pathogenesis of pulpitis.     

Effects of sodium ferulate on function of macrophages in the colonic tissue of colitis rats

AIM: To explore the effects of sodium ferulate (SF) on function of macrophages in colonic tissue of the colitis rats in vivo. METHODS: The immunological colitis model of rats was produced. SF was used intracolonically for 21 days. The contents of malondialdehyde (MDA), nitric oxide (NO), prostaglandin E₂ (PGE₂) and the activity of superoxide dismutase (SOD), interleukin-1 (IL-1), TNF-α, myelopexoxidase (MPO), and the expression level of NF-κB p65 in colonic tissue of the rats were detected. RESULTS: SF (200,400,800 mg/kg) decreased the elevated contents of MDA, NO, PGE₂, the activity of IL-1, TNF-α, MPO, and the expression level of NF-κB p65, while increased the reduced activity of SOD in colonic tissue of the colitis rats in a dose-depended manner. CONCLUSION: SF restrained the activity of activated colonic macrophages and relieved the colonic inflammation reaction in vivo in colitis rats, which may be related to the suppression of NF-κB activation.     

Role of central PGE2 on sympathetic excitation in chronic heart failure

AIM: To observe the effect of central prostaglandin E₂ (PGE₂) on sympathetic activation in chronic heart failure (CHF) and to explore the underlying mechanism. METHODS: Male SD rats were subjected to coronary artery ligation to induce heart failure (HF), and the intracerebroventricular infusion was performed by osmotic pump continuously. The rats in sham group and HF group were given artificial cerebrospinal fluid (0.25 μL/h). The rats in HF plus treatment group was given celecoxib (CLB; 20 mg/h). After 4 weeks, the levels of PGE₂ in cerebrospinal fluid (CSF), the sympathetic nerve excitability and cardiac function were measured, and the changes of corticotropin-hormone releasing hormone (CRH)-containing neurons activation and neurotransmitter contents in the hypothalamic paraventricular nucleus (PVN) were also determined. RESULTS: Compared with the sham-operated rats, the HF rats had raised level of PGE₂ in CSF, up-regulated renal sympathetic nerve activity and plasma norepinephrine, increased left ventricular end diastolic pressure, lung-to-body weight and right ventricular-to-body weight ratios, and decreased maximal increase and decreased rate of left ventricular pressure (P<0.05). In addition, the number of CRH positive neurons in PVN and the level of plasma adrenocorticotropic hormone were higher in HF rats than those in sham-operated rats (P<0.05). After administration of CLB into the lateral ventricle of HF rats, the contents of PGE₂ in CSF were significantly reduced, the number of activation CRH neurons in PVN was decreased, the excitability of sympathetic nerves was down-regulated and cardiac function was improved (P<0.05). Compared with the sham-operated rats, the content of glutamic acid in PVN of HF rats was increased, the content of γ-aminobutyric acid and the number of glutamate decarboxylase 67-positive neurons were decreased (P<0.05). After the CLB was given, the above indexes were reversed (P<0.05). CONCLUSION: These findings indicate that in CHF, the increased central PGE₂ may activate CRH-containing PVN neurons and contribute to the augmented sympathetic drive possibly by modulating the neurotransmitters within the PVN.     

Cross talk of CCK8 and EGF in mouse neurons

AIM: To explore the mechanism of signaling transduction and cross talk between cholecystokinin octapeptide (CCK₈) and epidermal growth factor (EGF) in mouse neurons and to observe the effect of CCK₈ in coordination with EGF on neuron growth and cell viability. METHODS: For determining which kind of CCK receptor mediated the phosphorylation of EGF receptor, the cultured neurons were randomly divided into control group, CCK₈ stimulation group, CCK_A receptor antagonist group, CCK_B receptor antagonist group, and CCK_A+CCK_B receptor antagonist group. Control and stimulation groups were stimulated with DMEM and CCK₈ (10^-7 mol/L) for 5 min, respectively, while antagonist groups were pre-incubated with different types of receptor antagonists (10^-8 mol/L) for 10 min and followed by stimulating the neurons with CCK₈. For observing the effect of CCK₈ and EGF on the phosphorylation of EGFR in neurons and on neuron growth and cell viability, the cultured neurons were randomly divided into control group, CCK₈ stimulation group, EGF stimulation group and CCK₈+EGF stimulation group, which were stimulated with DMEM, CCK₈ (10^-7 mol/L), EGF (40 μg/L) and CCK₈+EGF for 5 min, respectively. Reactions were terminated by freezing the neurons in liquid nitrogen and the phosphorylated EGFR was detected by Western blotting. Meanwhile, the viability of the neurons was observed by MTT method after stimulated for 24 h, 48 h, 72 h and 96 h. RESULTS: The phosphorylation levels of EGFR were decreased in the neurons treated with either of the two CCK receptor antagonists, and more obvious decrease was observed when the two CCK receptor antagonists were used in combination. Compared with control group, the phosphorylation levels of EGFR in the neurons were significantly increased(P<0.05) after stimulated with CCK₈ or EGF, and the increase was more remarkable in CCK₈+EGF stimulation group. CCK₈ or EGF improved the viability and prolonged the life span of the neuron, and synergism of these two reagents was observed. CONCLUSION: Both CCK_A and CCK_B receptors are involved in the phosphorylation of EGFR in the neurons stimulated by CCK₈, and the type A receptor may play a more important role. There is cross-talk between CCK₈ and EGF signaling pathways in neurons. The signaling cross-talk between CCK₈ and EGF may be the underlying molecular mechanism responsible for the synergistic effect on the neuron growth and viability in vitro.     

Expression of cyclooxygenase-2 in IL-1β-stimulated mesangial cells is medicated by NF-κB/IκB signal pathway

AIM: To investigate the role of NF-κB/IκB signal pathway in the regulation of cyclooxygenase-2 (COX-2) expression in human mesangial cells (HMC). METHODS: The PGE₂ concentration in supernatants of HMC was measured by radioimmunoassay. COX-2 mRNA and protein expression were determined by RT-PCR and Western blot. Electrophoretic mobility shift assay (EMSA) and Western blot were used to detect the activity of NF-κB and degradation of IκB. RESULTS: IL-1β significantly upregulated COX-2 expression and PGE₂ production in HMC. Significant up-regulation of NF-κB activation, nuclear translocation of p65 subunit, and degradation of IκB α and IκB β were observed in IL-1β-induced HMC. CONCLUSION: Expression of COX-2 in IL-1β-induced HMC is mediated by NF-κB/IκB signal pathway.     

Effects of folic acid on antioxidant enzyme,nitric oxide synthase and nitric oxide in ovariectomized rats

AIM: To observe the effects of folic acid (FA) on antioxidant enzyme, nitric oxide synthase (NOS) and nitric oxide (NO) in ovariectomized (OVX) rats.METHODS: Forty three-month-old female SD rats were randomly divided into 5 groups: sham group, OVX group, diethylstilbestrol group (0.03 mg·kg^-1·d^-1), low-dose FA group (5 mg·kg^-1·d^-1) and high-dose FA group (20 mg·kg^-1·d^-1). Gastric gavage started 1 week after operation and lasted for 10 weeks. The rats in sham group and OVX group were given distilled water instead of FA as controls. At the end of the 10th week, the L₅ vertebra and right femur were removed for determination of bone mineral density (BMD). The bone homogenates were made using the L₃ and L₄ vertebrae. The levels of the total antioxidant capacity (TAC), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), NOS and NO were detected in plasma and bone homogenates.RESULTS: Compared with sham group, the BMD levels in L₅ vertebra and right femur and the levels of GSH-Px and NO in the plasma were all decreased. The levels of TAC, GSH-Px, NOS and NO in the bone homogenates were also decreased, while the MDA concentration was increased in OVX group (all P < 0.01). Compared with OVX group, the levels of TAC, GSH-Px, NOS, NO and BMD of the L₅ vertebra and right femur were all increased, while the MDA concentration was decreased in high-dose FA group (all P < 0.01). CONCLUSION: In female SD rats, ovariectomy leads to a significant reduction of antioxidant enzyme, NOS and NO levels. Oxidative stress is possibly involved in the development of osteoporosis. Protection against osteoporosis by high-dose FA may be linked to improvement of antioxidant enzyme activity, the levels of NOS and NO as well as a reduction of oxidative stress in ovariectomized rats.     

Inhibitory effect of zacopride on ouabain-induced arrhythmias in adult rats

AIM: To investigate the effect of zacopride, an inward rectifier potassium channel agonist, on ouabain-induced arrhythmias in adult rats, and to explore the underlying electrophysiological mechanism.METHODS: Using ouabain to establish in vitro and in vivo arrhythmic rat models, the effects of zacopride on ouabain-induced arrhythmias were observed. The technique of whole-cell patch clamp was used to observe the effects of zacopride on inward rectifier potassium current (I_K1), resting membrane potential (RMP) and delayed afterdepolarizations (DADs) in single rat ventricular myocyte. RESULTS: Zacopride at 1 μmol/L significantly reduced total number of premature ventricular beats, and the duration and incidence of ventricular tachycardia and ventricular fibrillation induced by ouabain in rat hearts in vitro (P<0.05). In anesthetized rats, zacopride at 15 μg/kg significantly reduced total number of premature ventricular beats, and the duration and incidence of ventricular tachycardia and ventricular fibrillation induced by ouabain (P<0.05). I_K1 was significantly inhibited by ouabain (P<0.05), which was partially and even completely reversed by zacopride at 0.1~10 μmol/L. RMP value was significantly reduced by ouabain (P<0.05), and then increased to different levels after treatment with zacopride (0.1~10 μmol/L). Zacopride at 1 μmol/L showed its maximal effect and RMP was restored to normal level. Moreover, zacopride at 1 μmol/L markedly suppressed ouabain-induced DADs in single rat ventricular myocyte. The incidence of DADs decreased from 91.67% to 12.50% after zacopride was applied (P<0.05), and this effect was abolished by 1 μmol/L BaCl₂. CONCLUSION: Inward rectifier potassium channel agonist zacopride significantly inhibits ouabain-induced ventricular arrhythmias in adult rats. The mechanism is related to increased RMP level and inhibition of DADs by activation of I_K1 channel.     

Influence of lanthanum nitrate on the expression of aquaporin 7 in the testis of rats

AIM: To find out whether different dosage of rare earth element-lanthanum can influence the expression of aquaporin 7(AQP 7) in the testis of rats. METHODS:Rats were fed with lanthanum nitrate[La(NO₃)₃]and killed 6 months later.Testes were then removed immediately to extract total RNA.Northern blot analysis is performed finally. RESULTS:0.1 mg/kg La(NO₃)₃ depressed the expression of AQP 7 in rat testis, while 20 mg/kg La(NO₃)₃ had no significant effect on it. CONCLUSION: AQP 7 expession is found in the rat testis; La(NO₃)₃ can depress the expression of AQP 7 in the rat testis.     

Total flavonoids of onion attenuate hydrogen peroxide-induced oxidative damage in retinal pigment epithelial cells

AIM: To investigate the effects of total flavonoids of onion (FO) on hydrogen peroxide (H₂O₂)-induced oxidative damage in retinal pigment epithelial cells. METHODS: The retinal pigment epithelium ARPE-19 cells were divided into 5 groups:control group, H₂O₂ group (treated with H₂O₂), FO-L+H₂O₂ group (treated with H₂O₂ and low concentration of FO), FO-M+H₂O₂ group (treated with H₂O₂ and medium concentration of FO) and FO-H+H₂O₂ group (treated with H₂O₂ and high concentration of FO). The cell viability was measured by MTT assay. Apoptosis was analyzed by flow cytometry. DCFH-DA staining was used to detect reactive oxygen species (ROS) level in the cells. WST assay was used to detect superoxide dismutase (SOD) activity. The content of malonaldehyde (MDA) was measured by TBA method. Mitochondrial membrane potential was analyzed by JC-1 staining. The protein levels of cytochrome C (Cyt C) in the cytoplasm, and cleaved caspase-3 and cleaved caspase-9 in the cells were determined by Western blot. RESULTS: Treatment with H₂O₂ decreased ARPE-19 cell viability, increased the apoptotic rate and the level of ROS in the cells, decreased SOD activity, increased the content of MDA, decreased mitochondrial membrane potential, and increased the protein levels of Cyt C in the cytoplasm and cleaved caspase-3 and cleaved caspase-9 in the cells (P<0.05). Compared with H₂O₂ group, the cell viability in FO-L+H₂O₂ group, FO-M+H₂O₂ group and FO-H+H₂O₂ group was increased, the apoptotic rates were decreased, the levels of ROS were decreased, SOD activity was increased, the content of MDA was decreased, mitochondrial membrane potential was increased, the protein level of Cyt C was decreased in the cytoplasm, and the protein levels of cleaved caspase-3 and cleaved caspase-9 protein in the cells were decreased gradually (P<0.05). CONCLUSION: Total flavonoids of onion reduce H₂O₂-induced oxidative damage in retinal pigment epithelial cells.     

Effects of cimetidine on platelet function and thrombosis

AIM:To observe the effects of cimetidine(Cim) on platelet function and thrombosis. METHODS:After incubated with Cimin vitro, rat platelets were activated with ADP or thrombin. The platelet aggregation, platelet malondialdehyde(MDA) formation, platelet intracellular free calcium( [Ca²⁺]_i), and thromboxane B₂ (TXB₂) were measured. The effects of Cim on electric-induced thrombosis in rat carotid artery were examined. RESULTS:Cim potentiated ADP induced platelet aggregation, increased the thrombin induced [Ca²⁺]_i and MDA formation, decreased TXB₂. Also, Cim shortened the duration of electric-stimulated occlution time in rat carotid artery. CONCLUSION:Cim increased platelet function and accelerated thrombosis.     

Expression of VEGF and bFGF in rat model of pressure ulcer

AIM:To study the effects of vascular endothelial growth factor(VEGF) and basic fibroblast growth factor(bFGF) on the molecular pathogenesis of pressure ulcer.METHODS:SD rats were randomly divided into control group and experiment group. The pressure ulcer model was established by magnetic disk circulating compression method. HE staining was used to observe the pathological changes of the skin in the rats. The expression of VEGF and bFGF in the tissues was detected by immunohistochemical method. RESULTS:The expression of VEGF and bFGF in the tissues of rat Ⅲ-degree pressure ulcer was lower than that in the surrounding tissues and normal skin(P<0.01). The changes of VEGF and bFGF were consistent(κ=0.58). CONCLUSION:The expression levels of VEGF and bFGF are decreased in the tissues of rat pressure ulcer, suggesting that they may be the potential key factors in the difficult healing of pressure ulcer.     

Phasic change of water soluble major crystallins with aging in rats

AIM: To study the correlation between phasic change of the relative quantity of major crystallins with aging in rats. METHODS: Six groups of SD rats (age 1 d， 8 d，2 weeks，8 weeks，8 months and 1.5 years) were raised routinely. Water soluble crystallins were extracted and separated by two－dimensional polyacrylamide gelelectrophoresis. After comassize blue staining，the crystallins patterns were scanned and analyzed. RESULTS: (1) Out of the eighteen water soluble major rat crystallins tested in each group, seven showed gradual phasic changes in relative quantity of crystallins, but there were no significant changes in total quantity of water soluble crystallins. (2) Phasic changes in these crystallins presented four different patterns: increasing (βB₄、αB₂、αA₂、βA₁), decreasing (β₇、β₈、γ_2,3、γ_5,6),relatively stable(βA₃、βB₅), and irregular. (3) The ratio of βB₄ /αA₂ increased gradually with the rat aging process. CONCLUSION: The gradual phasic changes in relative quantity of crystallins reflect the aging status of rat crystalline.     

氮、磷、钾肥对红枣产量和品质的影响

研究了氮磷钾肥对6a树龄红枣产量和品质的影响。结果表明:新疆南部红枣最高产量施肥量为N691.5 kg/hm²,P₂O₅ 577.5 kg/hm²,K₂O 72 kg/hm²,施用比例1:0.84:0.11。采用平衡施肥能增加红枣产量,降低红枣果实中总酸度,并能增加红枣果实中水解还原糖和VC含量,从而提高红枣产品品质,增加效益。红枣果实中总酸度随氮肥施用量增加而增加;磷钾肥能降低总酸度。当氮、磷、钾肥分别控制在600、180～450、75kg/hm²时能显著提高红枣果实中水解还原糖和VC含量。     

Cytological characteristics and gene micro-array analysis of a mouse brain microvascular endothelial cell strain: bEnd.3

AIM: To study the cytological characteristics and gene expression of normal cultured bEnd.3, a mouse brain microvascular endothelial cell strain. METHODS: The morphology of bEnd.3 was studied by light and electronic microscopy, its molecular markers were observed by immunocytochemistry. Cell proliferation kinetics and apoptosis were analyzed by flow cytometry and MTT assay, PGE₂ level was measured by ELISA, and expression of the genes that closely related with vascular endothelial functions was studied by gene micro-array. RESULTS: bEnd.3 had morphological characteristics of microvascular endothelial cells (MVEC) growing in a cobblestone pattern, forming tube-like structure or capillary network and having microvilli. Furthermore, bEnd.3 showed positive staining for vW factor and CD34 and secreted high level of PGE₂ (644.55±30.24 ng/L). Gene micro-array analysis showed CD31, CD36, CD105 expression, and other genes closely related to microvascular endothelial functions also expressed at relatively high level. In addition, bEnd.3 responsed sensitively to mitogen such as basic fibroblast growth factor. CONCLUSION: bEnd.3 is a kind of MVEC, and it can be utilized to study the mechanisms of some diseases such as cancers and cardio- cerebral vascular diseases.     

The effect of arachidonic acid and its derivatives on superoxide release of neutrophils primed by TNFα

AIM: TO study the control mechanism of the developing of inflammation diseases, two inflammatory mediators, TNFa and arachidonic acid(AA) and its derivatives have been used as stimuli to induce human peripheral blood neutrophils to produce superoxide radicals. METHODS: The method of lucigeni - dependent light release has been used to exam the ability of releasing superoxide of neutrophils induced by the cooperation of TNFa and high class fatty acids as well as their derivatives. RESULTS: TNFa pretreatment markedly augmented the amount of superoxide produced in response to AA. Similarly, polyunsaturated fatty acids(PUFA) such as eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, linlenic acid as well as monounsaturated fatty acid oleic acid were effected by TNFa. However, hydroxide (C_20:4, n - 6 hydroxy) and hydroperoxide(C_20:4, n - 6 hydroperoxy) of AA were not effected by TNFa. Leukotriene B₄(LTB₄) and PGE₂ which are products of AA metabolism via the lipoxygenase and cyclooxygenase pathways respectively. The two major major of mediators are generated during an inflammatory response. The former acted on neutrophils in the generation of reactive oxygen species while the latter played an inhibition function. CONCLUSION: The complexity of the mediator network operating in various immune reactions and interaction between unsaturated lipids and cytokines may play an important role in disease processes and regulation of immune responses.