Platelet derived TGF-ß promotes cervical carcinoma cell growth by suppressing KLF6 expression.

Platelets in the primary tumor microenvironment play crucial roles in regulating tumor growth, metastasis, and angiogenesis, but the underlying mechanisms are unclear. Here, we show that platelet releasates exhibited a proliferative effect on HeLa cells, and this effect correlated with a reduction of KLF6 expression. After incubation with either washed human platelets or collagen-related peptide (CRP) activated platelet releasates, expression of KLF6 in the HeLa cervical tumor cell line was markedly reduced. However, no significant difference was observed between control HeLa cells and HeLa cells incubated with resuspended activated platelet pellet. Moreover, the platelets' promoting effect on HeLa cell growth was significantly abolished in KLF6 silenced HeLa cells. In addition, blocking TGF-ß signaling with SB431542, a TGF-ß receptor inhibitor, also counteracted the effect of platelets on proliferation and KLF6 expression in HeLa cells. From these findings, we conclude that platelet derived TGF-ß promotes proliferation of HeLa cells by decreasing the expression of KLF6. The discovery that KLF6 is a key target of platelet-derived TGF-ß signaling in HeLa cells identifies a potential new therapeutic target for the prevention and treatment of cervical carcinoma.

Platelet releasates promote the proliferation of hepatocellular carcinoma cells by suppressing the expression of KLF6.

Platelets in the primary tumor microenvironment play crucial roles in the regulation of tumor progression, but the mechanisms underlying are poorly understood. Here, we report that platelet releasates exerted a proliferative effect on hepatocellular carcinoma (HCC) cells both in vitro and in vivo. This effect depended on a reduction of KLF6 expression in HCC cells. After incubation with either platelets or platelet granule contents, SMMC.7721 and HepG2 cells exhibited significant increases in proliferation and decreases in apoptosis. However, no effect was observed when incubating cancer cells with resuspended activated platelet pellet which exhausted of releasates. Platelet releasates also increased the population of HCC cells in the S and G2/M phases of the cell cycle and reduced the cell population in the G0/G1 phase. Moreover, knocking down KLF6 expression significantly diminished the platelet-mediated enhancement of HCC growth. In addition, blocking TGF-ß signaling with the TGF-ß receptor inhibitor SB431542 counteracted the effect of platelets on KLF6 expression and proliferation of HCC cells. Based on these findings, we conclude that platelet releasates, especially TGF-ß, promote the proliferation of SMMC.7721 and HepG2 cells by decreasing expression of KLF6. This discovery identifies a potential new therapeutic target for the prevention and treatment of hepatocellular carcinoma.

Inhibitory effects of hesperetin on Nav1.5 channels stably expressed in HEK 293 cells and on the voltage-gated cardiac sodium current in human atrial myocytes.

AIM: Voltage-gated sodium channels composed of a pore-forming α subunit and auxiliary ß subunits are responsible for the upstroke of the action potential in cardiac myocytes. The pore-forming subunit of the cardiac sodium channel Nav1.5, which is encoded by SCN5A, is the main ion channel that conducts the voltage-gated cardiac sodium current (INa) in cardiac cells. The current study sought to investigate the inhibitory effects of hesperetin on human cardiac Nav1.5 channels stably expressed in human embryonic kidney 293 (HEK 293) cells and on the voltage-gated cardiac sodium current (INa) in human atrial myocytes. METHODS: The effects of hesperetin on human cardiac Nav1.5 channels expressed in HEK 293 cells and on cardiac Na+ currents in human atrial myocytes were examined through whole-cell patch-clamp techniques. RESULTS: Nav1.5 currents were potently and reversibly suppressed in a concentration- and voltage-dependent manner by hesperetin, which exhibited an IC50 of 62.99 µmol/L. Hesperetin significantly and negatively shifted the voltage-dependent activation and inactivation curves. Hesperetin also markedly decelerated Nav1.5 current inactivation and slowed the recovery from Nav1.5 channel inactivation. The hesperetin-dependent blockage of Nav1.5 currents was frequency-dependent. Hesperetin also potently and reversibly inhibited Na+ current (INa) in human atrial myocytes, consistently with its effects on Nav1.5 currents in HEK 293 cells. CONCLUSION: Hesperetin is a potent inhibitor of INa in human atrial myocytes and Nav1.5 channels expressed in human embryonic kidney 293 cells. Hesperetin probably functions by blocking the open state and the inactivated state of these channels.

Inhibitory effects of neferine on Nav1.5 channels expressed in HEK293 cells.

Neferine, a bisbenzylisoquinoline alkaloid in Lotus Plumule, was proved to have a wide range of biological activities. In the present study, using whole-cell patch-clamp technique, we investigated the effects of neferine on Nav1.5 channels that are stably expressed in HEK 293 cells. We found that neferine potently and reversibly inhibited Nav1.5 currents in a concentration dependent manner with a half-maximal inhibition (IC50) being 26.15 µmol/L. The inhibitory effects of neferine on Nav1.5 currents were weaker than those of quinidine at the same concentration. The steady-state inactivation curve was significantly shifted towards hyperpolarizing direction in the presence of 30 µmol/L neferine, while the voltage-dependent activation was unaltered. Neferine prolonged the time to peak of activation, increased the inactivation time constants of Nav1.5 currents and markedly slowed the recovery from inactivation. The inhibitory effect of neferine could be potentiated in a frequency-dependent manner. These results suggested that neferine can block Nav1.5 channels under the open state and inactivating state and it is an open channel blocker of Nav1.5 channels.

Inhibitory effects of hesperetin on Kv1.5 potassium channels stably expressed in HEK 293 cells and ultra-rapid delayed rectifier K(+) current in human atrial myocytes.

In the present study, the inhibitory effects of hesperetin (HSP) on human cardiac Kv1.5 channels expressed in HEK 293 cells and the ultra-rapid delayed rectifier K(+) current (Ikur) in human atrial myocytes were examined by using the whole-cell configuration of the patch-clamp techniques. We found that hesperetin rapidly and reversibly suppressed human Kv1.5 current in a concentration dependent manner with a half-maximal inhibition (IC50) of 23.15 µΜ with a Hill coefficient of 0.89. The current was maximally diminished about 71.36% at a concentration of 300µM hesperetin. Hesperetin significantly positive shifted the steady-state activation curve of Kv1.5, while negative shifted the steady-state inactivation curve. Hesperetin also accelerated the inactivation and markedly slowed the recovery from the inactivation of Kv1.5 currents. Block of Kv1.5 currents by hesperetin was in a frequency dependent manner. However, inclusion of 30µM hesperetin in pipette solution produced no effect on Kv1.5 channel current, while the current were remarkable and reversibly inhibited by extracellular application of 30µM hesperetin. We also found that hesperetin potently and reversibly inhibited the ultra-repaid delayed K(+) current (Ikur) in human atrial myocytes, which is in consistent with the effects of hesperetin on Kv1.5 currents in HEK 293 cells. In conclusion, hesperetin is a potent inhibitor of Ikur (which is encoded by Kv1.5), with blockade probably due to blocking of both open state and inactivated state channels from outside of the cell.

Antiplatelet activity of chrysin via inhibiting platelet αIIbß3-mediated signaling pathway.

SCOPE: Propolis is thought to help prevent thrombotic and related cardiovascular diseases in humans. Chrysin, a bioflavonoids compound found in high levels in propolis and in honey, has been reported to possess antiplatelet activity. However, the mechanism by which it inhibits platelet function is unclear. METHODS AND RESULTS: The effects of chrysin on agonist-activated platelet-aggregation, granule-secretion, and integrin αIIbß3 activation were examined. Its effects on the phosphorylation of Akt, GSK3ß, MAPKs, and several proteins of the glycoprotein VI (GPVI) signaling pathway were also studied on collaged-activated platelets. In addition, human platelet spreading on immobilized fibrinogen was also tested. We found that chrysin dose dependently inhibited platelet aggregation and granule secretion induced by collagen, as well as platelet aggregation induced by ADP, thrombin, and U46619. Chrysin also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen. Biochemical analysis revealed that chrysin inhibited collagen-induced activation of Syk, PLCγ2, PKC, as well as the phosphorylation of Akt and ERK1/2. Additionally, chrysin attenuated phosphorylation of molecules such as FcγRIIa, FAK, Akt, and GSK3ß in platelet spreading on immobilized fibrinogen. CONCLUSIONS: Our findings indicate that chrysin suppresses not only integrin αIIbß3-mediated "inside-out" signaling, but also the "outside-in" signal transmission. This implies that chrysin may represent a potential candidate for an antiplatelet agent.

Tacrolimus inhibits vasoconstriction by increasing Ca(2+) sparks in rat aorta.

The present study attempted to test a novel hypothesis that Ca(2+) sparks play an important role in arterial relaxation induced by tacrolimus. Recorded with confocal laser scanning microscopy, tacrolimus (10 µmol/L) increased the frequency of Ca(2+) sparks, which could be reversed by ryanodine (10 µmol/L). Electrophysiological experiments revealed that tacrolimus (10 µmol/L) increased the large-conductance Ca(2+)-activated K(+) currents (BKCa) in rat aortic vascular smooth muscle cells (AVSMCs), which could be blocked by ryanodine (10 µmol/L). Furthermore, tacrolimus (10 and 50 µmol/L) reduced the contractile force induced by norepinephrine (NE) or KCl in aortic vascular smooth muscle in a concentration-dependent manner, which could be also significantly attenuated by iberiotoxin (100 nmol/L) and ryanodine (10 µmol/L) respectively. In conclusion, tacrolimus could indirectly activate BKCa currents by increasing Ca(2+) sparks released from ryanodine receptors, which inhibited the NE- or KCl-induced contraction in rat aorta.

Effects of neferine on Kv4.3 channels expressed in HEK293 cells and ex vivo electrophysiology of rabbit hearts.

AIM: Neferine is an isoquinoline alkaloid isolated from seed embryos of Nelumbo nucifera (Gaertn), which has a variety of biological activities. In this study we examined the effects of neferine on Kv4.3 channels, a major contributor to the transient outward current (I(to)) in rabbit heart, and on ex vivo electrophysiology of rabbit hearts. METHODS: Whole-cell Kv4.3 currents were recorded in HEK293 cells expressing human cardiac Kv4.3 channels using patch-clamp technique. Arterially perfused wedges of rabbit left ventricles (LV) were prepared, and transmembrane action potentials were simultaneously recorded from epicardial (Epi) and endocardial (Endo) sites with floating microelectrodes together with transmural electrocardiography (ECG). RESULTS: Neferine (0.1-100 µmol/L) dose-dependently and reversibly inhibited Kv4.3 currents (the IC50 value was 8.437 µmol/L, and the maximal inhibition at 100 µmol/L was 44.12%). Neferine (10 µmol/L) caused a positive shift of the steady-state activation curve of Kv4.3 currents, and a negative shift of the steady-state inactivation curve. Furthermore, neferine (10 µmol/L) accelerated the inactivation but not the activation of Kv4.3 currents, and markedly slowed the recovery of Kv4.3 currents from inactivation. Neferine-induced blocking of Kv4.3 currents was frequency-dependent. In arterially perfused wedges of rabbit LV, neferine (1, 3, and 10 µmol/L) dose-dependently prolonged the QT intervals and action potential durations (APD) at both Epi and Endo sites, and caused dramatic increase of APD10 at Epi sites. CONCLUSION: Neferine inhibits Kv4.3 channels likely by blocking the open state and inactivating state channels, which contributes to neferine-induced dramatic increase of APD10 at Epi sites of rabbit heart.

Pentamethylquercetin (PMQ) reduces thrombus formation by inhibiting platelet function.

Flavonoids exert both anti-oxidant and anti-platelet activities in vitro and in vivo. Pentamethylquercetin (PMQ), a polymethoxylated flavone derivative, has been screened for anti-carcinogenic and cardioprotective effects. However, it is unclear whether PMQ has anti-thrombotic effects. In the present study, PMQ (20 mg/kg) significantly inhibited thrombus formation in the collagen- epinephrine- induced acute pulmonary thrombosis mouse model and the ferric chloride-induced carotid injury model. To explore the mechanism, we evaluated the effects of PMQ on platelet function. We found that PMQ inhibited platelet aggregation and granule secretion induced by low dose agonists, including ADP, collagen, thrombin and U46619. Biochemical analysis revealed that PMQ inhibited collagen-, thrombin- and U46619-induced activation of Syk, PLCγ2, Akt, GSK3ß and Erk1/2. Therefore, we provide the first report to show that PMQ possesses anti-thrombotic activity in vivo and inhibited platelet function in vitro, suggesting that PMQ may represent a potential therapeutic candidate for the prevention or treatment of thrombotic disorders.

Antiplatelet activity of loureirin A by attenuating Akt phosphorylation: In vitro studies.

Loureirin A is a flavonoid extracted from Dragon׳s Blood that has been used to promote blood circulation and remove stasis in Chinese traditional medicine. However, the mechanisms of these effects are not fully understood. We explored the anti-platelet activity and underlying mechanism of loureirin A in vitro. Our results indicated that loureirin A negatively affected agonist-induced platelet aggregation such as collagen, collagen-related peptide (CRP), ADP and thrombin. Loureirin A inhibited collagen-induced platelet ATP secretion and thrombin-stimulated P-selectin expression in a dose-dependent manner. Platelet spreading on immobilized fibrinogen was significantly impaired in the presence of loureirin A. Immunoblotting analysis indicated that 100µM of loureirin A almost completely eliminated collagen-induced Akt phosphorylation at Ser473. Interestingly, a submaximal dose (50µM) of loureirin A had an additive inhibitory effect with the phosphoinositide 3-kinase (PI3K) inhibitor Ly294002 on collage-induced Akt phosphorylation in platelets. Taken together, loureirin A had an inhibitory effect on platelet activation, perhaps through an impairment of PI3K/Akt signaling.

JAK2/STAT5/Bcl-xL signalling is essential for erythropoietin-mediated protection against apoptosis induced in PC12 cells by the amyloid ß-peptide Aß25-35.

BACKGROUND AND PURPOSE: Erythropoietin (EPO) exerts neuroprotective actions in the CNS, including protection against apoptosis induced by the amyloid ß-peptide Aß25-35 . However, it remains unclear which signalling pathway activated by EPO is involved in this neuroprotection. Here, we have investigated whether JAK2/STAT5/Bcl-xL and ERK1/2 signalling pathways are essential for EPO-mediated protection against apoptosis induced by Aß25-35 . EXPERIMENTAL APPROACH: EPO was added to cultures of PC12 cells, 1 h before Aß25-35 . For kinase inhibitor studies, AG490 and PD98059 were added to PC12 cells, 0.5 h before the addition of EPO. Transfection with siRNA was used to knockdown STAT5. Activation of JAK2/STAT5/Bcl-xL and ERK1/2 signalling pathways were investigated by Western blotting. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide assay and apoptosis was detected by TUNEL and acridine orange-ethidium bromide double staining. KEY RESULTS: EPO increased phosphorylation of JAK2 and STAT5 in PC12 cells treated with Aß25-35 . Furthermore, EPO modulated the nuclear translocation of phospho-STAT5, which increased expression of Bcl-xL and decreased levels of caspase-3. These beneficial effects were blocked by the JAK2 inhibitor, AG490 or STAT5 knockdown. However, the ERK1/2 pathway did not play a crucial role in our model. CONCLUSIONS AND IMPLICATIONS: EPO protected PC12 cells against Aß25-35 -induced neurotoxicity. Activation of JAK2/STAT5/Bcl-xL pathway was important in EPO-mediated neuroprotection. EPO may serve as a novel protective agent against Aß25-35 -induced cytotoxicity in, for instance, Alzheimer's disease.

2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (THSG) attenuates human platelet aggregation, secretion and spreading in vitro.

INTRODUCTION: 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside(THSG) is a water-soluble component of the rhizome extract from the traditional Chinese herb Polygonum multiflorum. Recent studies have demonstrated that THSG has potent anti-oxidative and anti-inflammatory effects. In this study, we investigated the anti-platelet aggregation, secretion and spreading of THSG with different methods. The purpose was to explore the anti-platelet effect of THSG and the underlying mechanism. MATERIALS AND METHODS: We investigated the anti-platelet activity of THSG on platelet aggregation induced by collagen (2 µg/mL), thrombin(0.04U/mL), U46619 (3 µM) and ADP (2 µM). ATP secretion induced by collagen (2 µg/mL) was also investigated. P-selectin expression and PAC-1 binding were measured by flow cytometry. In addition, human platelet spreading on immobilized fibrinogen and immunoblotting were also tested. RESULTS: THSG dose-dependently inhibited platelet aggregation and ATP secretion induced by collagen. It inhibited platelet P-selectin expression and PAC-1 binding induced by thrombin(0.1U/mL). THSG also inhibited human platelet spreading on immobilized fibrinogen, a process mediated by platelet outside-in signaling. Western blot analysis showed that THSG could inhibit platelet Fc γ RIIa, Akt(Ser473)and GSK3ß(Ser9) phosphorylation. CONCLUSIONS: Our study indicates that THSG has potent anti-platelet activity to collagen induced aggregation. THSG is likely to exert protective effects in platelet-associated thromboembolic disorders by modulating human platelet.

Neferine exerts its antithrombotic effect by inhibiting platelet aggregation and promoting dissociation of platelet aggregates.

INTRODUCTION: Neferine, a kind of isoquinoline alkaloid, extracted from the seed embryo of Nelumbo nucifera Gaertn, has long been recognized in traditional medicine as a medicinal plant with various usages. Neferine has many biological activities, including anti-hypertensive, anti-arrhythmic, negative inotropic effect and relaxation on vascular smooth muscle. Although previous studies have reported its antithrombotic effect, the mechanisms by which it exerts antithrombotic effect have not been thoroughly studied. METHOD: Washed mice platelets and mice platelet-rich-plasma (PRP) were used to investigate the effects of neferine on platelet aggregation, secretion induced by various agonists and dissociation of agonist-formed platelet aggregates. Bioflux plates coated with collagen were used to investigate the effect of neferine on platelet adhesion and thrombosis in vitro. With collagen-epinephrine-induced acute pulmonary thrombus formation mouse model, the effect of neferine on thrombosis in vivo was also examined. RESULTS: Neferine, significantly and dose-dependently, inhibited collagen-, thrombin-, U46619-induced platelet aggregation in mice washed platelets, or ADP-induced platelet aggregation in PRP. Neferine treatment decreased platelet dense granule secretion initiated by collagen, thrombin and U46619. Also, Neferine dramatically and dose-dependently promoted the dissociation of platelet aggregates pre-formed by various agonists including collagen, thrombin, U46619 or ADP. Neferine can significantly reduce the area of mice platelets adhesion to the collagen and inhibit thrombosis in vitro. In collagen-epinephrine-induced acute pulmonary thrombus mouse model, neferine, at 6 mg/kg, significantly attenuated thrombus formation. CONCLUSIONS: Neferine remarkably prevents thrombus formation by inhibiting platelet activation, adhesion and aggregation, as well as promoting disassembly of pre-formed platelet aggregates. The inhibitory effects of neferine on platelet activation might be relevant in cases involving aberrant platelet activation where neferine could be used as an anti-platelet and antithrombotic agent.

Induction of Dickkopf-1 contributes to the neurotoxicity of MPP+ in PC12 cells via inhibition of the canonical Wnt signaling pathway.

The secreted glycoprotein Dickkopf-1 (Dkk1), an antagonist of the Wnt/ß-catenin pathway, has been implicated in many neurodegenerative diseases. However, it is unknown whether Dkk1 is involved in the pathogenesis of Parkinson's disease (PD). In this study, we discovered that Dkk1 was induced in MPP(+)-treated PC12 cells and the increase of Dkk1 preceded PC12 cell loss. RhDkk1 aggravated the neurotoxicity of MPP(+) in PC12 cells. Furthermore, the level of Dkk1 was correlated with the number of apoptotic PC12 cells. The apoptosis could be decreased by Dkk1-siRNA in MPP(+)-induced PC12 cells and Dkk1-siRNA regulated the expression of ß-catenin and p-Ser9-GSK-3ß in MPP(+)-induced PC12 cells. LiCl (an inhibitor of GSK-3ß) also rescued the loss of PC12 cell viability and the apoptosis induced by MPP(+). These data suggest that the induction of Dkk1 contributes to the MPP(+)-induced neurotoxicity in PC12 cells via inhibition of the canonical Wnt pathway and Dkk1 antagonists which could rescue the Wnt pathway might be neuroprotective in PD.

Curcumin alleviates diabetic cardiomyopathy in experimental diabetic rats.

OBJECTIVES: Diabetic cardiomyopathy (DCM), characterized by myocardial structural and functional changes, is an independent cardiomyopathy that develops in diabetic individuals. The present study was sought to investigate the effect of curcumin on modulating DCM and the mechanisms involved. METHODS: An experimental diabetic rat model was induced by low dose of streptozoticin(STZ) combined with high energy intake on rats. Curcumin was orally administrated at a dose of 100 or 200 mg · kg(-1) · d(-1), respectively. Cardiac function was evaluated by serial echocardiography. Myocardial ultrastructure, fibrosis area and apoptosis were assessed by histopathologic analyses. Metabolic profiles, myocardial enzymes and oxidative stress were examined by biochemical tests. Inflammatory factors were detected by ELISA, and interrelated proteins were measured by western blot. RESULTS: Rats with DCM showed declined systolic myocardial performance associated with myocardial hypertrophy and fibrosis, which were accompanied with metabolism abnormalities, aberrant myocardial enzymes, increased AGEs (advanced glycation end products) accumulation and RAGE (receptor for AGEs) expression, elevated markers of oxidative stress (MDA, SOD, the ratio of NADP(+)/NADPH, Rac1 activity, NADPH oxidase subunits expression of gp91(phox) and p47(phox) ), raised inflammatory factor (TNF-α and IL-1ß), enhanced apoptotic cell death (ratio of bax/bcl-2, caspase-3 activity and TUNEL), diminished Akt and GSK-3ß phosphorylation. Remarkably, curcumin attenuated myocardial dysfunction, cardiac fibrosis, AGEs accumulation, oxidative stress, inflammation and apoptosis in the heart of diabetic rats. The inhibited phosphorylation of Akt and GSK-3ß was also restored by curcumin treatment. CONCLUSIONS: Taken together, these results suggest that curcumin may have great therapeutic potential in the treatment of DCM, and perhaps other cardiovascular disorders, by attenuating fibrosis, oxidative stress, inflammation and cell death. Furthermore, Akt/GSK-3ß signaling pathway may be involved in mediating these effects.

Inhibition of the canonical Wnt pathway by Dickkopf-1 contributes to the neurodegeneration in 6-OHDA-lesioned rats.

Dickkopf-1 (Dkk1), an antagonist of the Wnt/ß-catenin pathway, has been implicated in many neurodegenerative diseases. However, it's unknown whether Dkk1 is involved in the pathogenesis of Parkinson's disease. In this study, we discovered that Dkk1 was increased in 6-hydroxydopamin(6-OHDA)-lesioned rats. In the meanwhile, inhibition of the canonical Wnt signaling pathway, including the activation of glycogen synthase kinase-3ß (GSK-3ß) and decrease of ß-catenin, was also found in 6-OHDA-lesioned rats. Treatment with rhDkk1 aggravated the dopaminergic neuron damage of the substantia nigra and the inhibition of the canonical Wnt signaling pathway in 6-OHDA-lesioned rats, while the above effects in these rats were abolished by pretreatment with LiCl, an inhibitor of GSK-3ß, for consecutive 7 d. These data suggest that Dkk1 plays an important role in the etiology of PD models and it contributes to the neurodegeneration in 6-OHDA-lesioned rats via inhibition of the canonical Wnt pathway.

Activation of epidermal growth factor receptor mediates reperfusion arrhythmias in anaesthetized rats.

AIMS: Epidermal growth factor receptor (EGFR) plays a critical role in the development and function of the heart. Previous studies have demonstrated that EGFR is involved in regulating electrical excitability of the heart. The present study was designed to investigate whether EGFR activation would mediate cardiac arrhythmias induced by reperfusion in anaesthetized rats. METHODS AND RESULTS: Reperfusion arrhythmias were induced by 10 min ligation of the left anterior descending coronary artery, followed by a 30 min reperfusion in anaesthetized rats. The incidence and severity of cardiac arrhythmias were significantly reduced by pre-treatment with the EGFR kinase inhibitor AG556. The phosphorylation level of myocardial EGFR was increased during ischaemia and at early reperfusion. Intramyocardial transfection of EGFR siRNA reduced EGFR mRNA and protein, and decreased the incidence of ventricular fibrillation induced by reperfusion. Interestingly, tyrosine phosphorylation levels of cardiac Na(+) channels (I(Na)) and L-type Ca(2+) channels (I(Ca,L)) were significantly increased at time points corresponding to the alteration of EGFR phosphorylation levels during reperfusion. AG556 pre-treatment countered the increased tyrosine phosphorylation level of Na(+) and L-type Ca(2+) channels induced by reperfusion. Patch-clamp studies proved that AG556 could inhibit I(Na) and I(Ca,L) in rat ventricular myocytes. No significant alteration was observed in tyrosine phosphorylation levels of cardiac Kv4.2 and Kir2.1 channels during reperfusion. CONCLUSION: These results demonstrate for the first time that EGFR plays an important role in the genesis of arrhythmias induced by reperfusion, which is likely mediated at least in part by enhancing tyrosine phosphorylation of cardiac Na(+) and L-type Ca(2+) channels.

Simultaneous determination of liensinine, isoliensinine and neferine from seed embryo of Nelumbo nucifera Gaertn. in rat plasma by a rapid HPLC method and its application to a pharmacokinetic study.

A rapid and specific HPLC method has been developed and validated for the simultaneous determination of liensinine (CAS 2586-96-1), isoliensinine (CAS 6817-41-0) and neferine (CAS 2292-16-2) in rat plasma. The sample was prepared by a liquid-liquid extraction with diethyl ether and the recovery was above 80% from the plasma for the three compounds. Chromatographic separation was achieved with a Hypersil BDS C18 column (4.0 mm x 250 mm, particle size 5 microm). A mobile phase consisting of methanol: 0.2 M KH2PO4:0.2 M NaOH:triethylamine (71:17:12:0.002, v/v/v/v, pH 9.2-9.3) was slowly delivered at 0.8 ml/min in isocratic mode with a detection wavelength of 282 nm. The linearity of calibration curves were good (r > 0.999) in the concentration range of 0.031-2.00 microg/ ml. The lower limit of quantification can reach 0.03 microg/ml for the three compounds. The intra-day and inter-day variations estimated with QC samples were less than 8% for the three tested concentration levels. This developed method was applied in the plasma pharmacokinetic study of total bisbenzylisoquinoline alkaloids (TAL) of the lotus flower (Lian Zi Xin) following a single oral and intravenous administration of TAL in rats.

[Resveratrol derived from rhizoma et radix polygoni cuspidati and its liposomal form protect nigral cells of Parkinsonian rats].

OBJECTIVE: Oxidative stress is a hallmark in the pathogenesis of Parkinson disease (PD), which involves the selective loss of nigral dopaminergic neurons in PD. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is well known for its powerful antioxidant property and a wide range of other biological effects. In this study, we investigated the protective effect of resveratrol derived from Rhizoma Et Radix Polygoni Cuspidati and its liposomal form on the nigral cells of PD rats induced by unilateral microinjection of 6-hydroxy dopamine in the striatum. The results showed that after 14 days gavage of resveratrol and resveratrol liposome respectively (20 mg x kg(-1) WB per day), the abnormal rotational behavior of PD rats were deceased evidently, the numbers of total nigral cells, total nigral neurons and TH immuno-positive neurons were more than that of PD rats without given resveratrol or resveratrol liposome, simultaneously, the number of apoptotic nigral cells were decreased obviously. The results also showed that resveratrol and resveratrol liposome could decrease the total ROS activity, increase the total antioxidant capability of the nigral tissues. All the data indicated that resveratrol liposome performed stronger effects than resveratrol except for behavioral improvement. Our study confirmed that resveratrol derived from Rhizoma Et Radix Polygoni Cuspidati and its liposomal form could inhibit the loss of dopaminergic neurons of PD rats, the underlying mechanism may be attributed to their radical scavenging effect and antioxidant property. Due to presumably increased bioavailability, resveratrol liposome possesses the stronger therapeutic effect and may become a better clinical agent for the treatment of PD than free resveratrol.

Protective effect of resveratrol derived from Polygonum cuspidatum and its liposomal form on nigral cells in parkinsonian rats.

OBJECTIVE: Oxidative stress which is an important factor in the pathogenesis of Parkinson's disease (PD) leads to the selective loss of nigral dopaminergic neurons in PD. Resveratrol is a well known antioxidant that exerts extensive pharmacological effects. This study aimed to investigate the protective effect of resveratrol derived from Polygonum cuspidatum and its liposomal form on nigral cells of PD rats. METHODS: A rat PD model was established by unilateral microinjection of 6-hydroxy dopamine in the striatum. Then, P. cuspidatum and its liposomal form were intragastrically administered once daily for 2 consecutive weeks. The behaviors, tyrosine hydroxylase positive cells, apoptotic cells, ROS level and total antioxidant capacity were determined. RESULTS: Our results showed after 14 days of oral treatment with resveratrol or resveratrol liposome (20 mg/kg per day), the abnormal rotational behavior, the loss and apoptosis of nigral cells, and the levels of total reactive oxygen species were markedly decreased and the total antioxidant capability of nigral tissues significantly improved. Furthermore, resveratrol liposome showed more profound effects than free resveratrol. CONCLUSION: Resveratrol derived from P. cuspidatum and its liposomal form could protect the dopaminergic neurons in PD rats, to which their radical scavenging ability and antioxidant properties may attribute. Presumably due to the increased bioavailability, resveratrol liposome exerts more potent protection and may become a promising agent for the treatment of PD than free resveratrol.