Electrophysiological effects of lidocaine on myocardial tissue in guinea-pig left ventricular outflow tract under conditions of hypoxia,acidosis and treatment with epinephrine

AIM：To study the electrophysiological effects of lidocaine on the myocardial tissue in guinea-pig left ventricular outflow tract under the conditions of hypoxia, acidosis and treatment with epinephrine. METHODS：The action potentials of pacemaker cells in guinea-pig left ventricular outflow tract were recorded by conventional technique with intracellular microelectrodes. The effects of lidocaine on the spontaneous slow response potentials were investigated under the conditions of hypoxia, acidosis and treatment with epinephrine (EPI). RESULTS：Lidocaine markedly decreased the rate of pacemaker firing (RPF), the velocity of diastolic depolarization (VDD), the maximal rate of depolarization (Vmax), the maximal diastolic potential (MDP) and the amplitude of action potential (APA). Lidocaine also shortened the 50% and 80% of duration of action potential (APD50 and APD80). At the concentrations from 0.1 μmol/L to 10 μmol/L, the effects of lidocaine were more significant. Under the condition of hypoxia and perfusion with deprived glucose content for 15 min, VDD, RPF, Vmax, MDP and APA significantly decreased, and APD50 notably shortened. Under the condition of hypoxia, lidocaine at 1 μmol/L significantly decreased VDD, RPF, Vmax and APA as compared with the cells treated with hypoxia only. Perfusion with pH 6.8 solution for 10 min, VDD, RPF, Vmax and APA significantly decreased, MDP notably increased, and APD50 and APD80 markedly shortened. Under the condition of acidosis for 10 min, lidocaine significantly decreased VDD, RPF and Vmax, and lengthened APD50 and APD80 as compared with the cells under the condition of acidosis alone. Perfusion with EPI at 10 μmol/L for 10 min resulted in significant increases in VDD, RPF, Vmax, MDP and APA, and notable shortenings of APD50 and APD80 were also observed. Compared with 10 μmol/L EPI group, 1 μmol/L lidocaine+10 μmol/L EPI significantly reduced VDD, RPF, MDP and APA, and lengthened APD50 and APD80. CONCLUSION：Lidocaine markedly decreases the autorhythmicity of the pacemaker cells in guinea-pig left ventricular outflow tract and influences the electrophysiological effects of hypoxia, acidosis and EPI.     

Effects of nitric oxide on spontaneous action potentials of guinea-pig left ventricular outflow tract under the conditions of ischemia/reperfusion

AIM: To study the electrophysiological effects of nitric oxide (NO) on the pacemaker cells in guinea-pig left ventricular outflow tract under the condition of ischemia/reperfusion (I/R). METHODS: The spontaneous slow action potentials of guinea-pig left ventricular outflow tract were recorded by conventional intracellular microelectrode technique. The effects of NO donor sodium nitroprusside (SNP) on the spontaneous slow action potentials under normal or I/R condition were investigated. RESULTS: SNP at concentrations of 1, 10 and 100 μmol/L but not 1000 μmol/L significantly increased velocity of diastolic depolarization (VDD) and rate of pacemaker firing (RPF), SNP at concentrations of 1, 10, 100 and 1 000 μmol/L notably increased maximal diastolic potential (MDP), amplitude of action potential (APA) and maximal rate of depolarization (V_max), shortened 50% and 90% of the duration (APD₅₀ and APD₉₀). In ischemia 10 min group, VDD and RPF were significantly decreased, APA and V_max were notably increased, and APD₅₀ and APD₉₀ were markedly lengthened compared with control group. In reperfusion 10 min group, VDD and RPF were significantly increased, MDP and APA were notably decreased, and APD₅₀ and APD₉₀ were markedly shortened compared with I 10 min group. In reperfusin 10 min group, the pacemaker activity was always irregular. In reperfusion 10 min group, the parameters of spontaneous slow action potentials restored to the levels of control group except for VDD and V_max. In 1, 10 and 100 μmol/L SNP+R groups, VDD and RPF were significantly increased than in ischemia 10 min group. In 1, 10, 100 and 1 000 μmol/L SNP+R groups, APA, APD₅₀ and APD₉₀ were restored to the levels of control group. CONCLUSION: SNP significantly increases the spontaneous activity of left ventricular outflow tract, and relieves the effects of I/R on the spontaneous slow action potential markedly.     

Electrophysiological changes in rat ventricular myocardium of experimental diabetes and action of CVB-D

AIM: To investigate the electrophysiological changes of diabetic myocardium and effects of cyclovirobuxine D (CVB-D) on its electrophysiology. METHODS: Diabetes was induced in male SD rats, using a single injection of alloxan into tail vein. Untreated age-matched animals were used as controls. Animal electrocardiogram (ECG) was recorded by 2 weeks. Effects of CVB-D on isolated right ventricular papillary muscle from experimental diabetic rats and control group were observed by recording the transmembrane potentials with conventional glass microelectrodes. RESULTS: QT intervals in ECG and action potential duration (APD) at all levels were significantly lengthened in myocardium from week 2 of diabetes. Within the concentration of 13.3-63.3 μmol·L^-1, CVB-D prologated APD of diabetes in dose-dependent manner and more than that of control. Within the concentration of 33.3-63.3 μmol·L^-1, CVB-D depressed RP, APA, Vmax and OS of diabetes in dose-dependent way and more than that of control. In addition, CVB-D at concentration of 20 μmol·L^-1 prologated APD in a time-dependent manner. The most prologation of APD was attained about 40 min in control, while more than 40 min in diabetes. CONCLUSION: The results show that QT intervals in ECG and APD at all levels are significantly lengthened in myocardium from week 2 of diabetes. CVB-D prolongates APD and inhibits RP, APA, OS and Vmax more in diabetes than in control.     

Effect of panax notoginseng saponins on electrophysiological characteristic of atrial cardiomyocytes in guinea pig

AIM: To study the effect of panax notoginseng saponins on the left atrial appendage (LAA) of the guinea pigs. METHODS: Standard microelectrode intracellular recording technique was used to record the LAA action potential (AP) and the effective refractory period (ERP) in the guinea pigs during the pouring with different density of PNS. RESULTS: The effect of PNS in concentration of 0.7, 7, 70, 700 mg/L was significant at 20 min, the APD50 and APD80 of the pouring at 20 min, 30 min, 40 min of the 70 mg/L was longer than the reference group (P<0.05). The rest potential (RP), action potential amplitude (APA) and Vmax kept no change during the pouring at 20 min with 4 difference densities from 0.7 to 700 mg/L. The pouring at 20 min of 7 mg/L and 70 mg/L obviously prolonged the ERP of the atrial cardiomyocytes. compared to the reference group, it prolonged from (127.00±7.26) ms to (153.00±9.19) ms and (161.00±10.21) ms (P<0.01). However, pouring at 10 min with 7 000 mg/L caused the arrhythmias. CONCLUSION: PNS prolongs the APD and ERP in guinea pig atrial cardiomyocytes. It may be the mechanism of PNS against arrhythmias in cellular level, which is similar to amiodarone.     

Electrophysiological changes in rat ventricular myocardium at different stages of experimental diabetes

AIM: To explore the probable mechanisms of diabetes-induced arrhythmias. METHODS: Diabetes was induced in male SD rats, using a single injection of alloxan into tail vein. Untreated age-matched animals were used as controls. All animals were observed by 2, 4, 6 and 8 weeks, respectively. Transmembrane potentials were recorded with conventional glass microelectrodes. RESULTS: Action potential duration(APD) at all level (APD10, APD20, APD30, APD50, APD70, APD90) was significantly lengthened in right ventricular papillary muscle from week 2 of diabetes. At week 8, APD was more lengthened at any level of repolarization than that at week 2. No differences were observed in the maximum rate of depolarization(V_max), overshoot(OS) and action potential amplitude(APA) as well as the resting membrane potential(RP) from the 2th to 8th week of diabetes. CONCLUSION: The results indicate that prolongation of APD may be prominently responsible for the increased incidence of cardiac re-entry-arrhythmias and sudden death, especially at late stages of diabetes.     

Effect of captopril on prolongation of action potential duration and reduction Ik in ischemia pig ventricular myocytes

AIM: To evaluate the effect of captopril on action potential duration and outward delayed rectification potassium current (Ik). METHODS: Action potentials were recorded using a conventional glass microelectrode filled with 3 mol/L KCl solution. Membrane patch clamp whole cell recording technique was used to investigate the Ik current maximum in the holding potential -50 mV, lasting time 100 ms, command potential +40 mV. RESULTS: The action potential duration of 30%, 50% repolarization (APD30, APD50) and ERP were significantly prolonged, but APD90 wasn't prolonged significantly when captopril group compared with ischemic group. The amplitude of Ik increased significantly in ischemic group, but significantly decreased in captopril group and in captopril+ischemic group. The shapes of current-voltage relationship were unchanged among groups, but significantly upward in ischemic group and downward in captopril and captopril+ischemic group. CONCLUSION: Captopril exerts electrophysiologic action due to decreasing delay outward rectification potassium current and prolonging action potential duration of APD30, APD50 and ERP.     

Effects of mechanical stretch on electrophysiological characteristics of calcium preconditioning myocardium

AIM: To investigate the effects of mechanical stretch under simulated anoxia and reoxygenation conditions on action potential (AP) and effective refractory period (ERP) of calcium preconditioning (CPC) in isolated papillary muscles of guinea pig hearts. METHODS: The intracellular standard glass microelectrode technique was used, and effects of stretch (intensity: 200 mg) on AP and ERP were recorded and observed in CPC papillary muscles. RESULTS: After stretching was implied during anoxia, the reductions of Vmax, RP, APA, APD50, ERP and APD90 in CPC group were less than those in anoxia-reoxygenation (AR) group, and the CT in CPC group was less elongated. Moreover, after the papillary muscles were stretched during reoxygenated reperfusion, the decreases in Vmax, RP, APA, APD50 and ERP in CPC group were less than those in AR group, and the CT and APD90 in CPC group were less extended. Streptomycin inhibited the effect of stretch on AP and ERP in CPC group. CONCLUSIONS: Under simulated anoxia and reoxygenation conditions, papillary muscles in CPC group may have better tolerance to the same stretch than those in the AR. Furthermore, streptomycin (a blockade of stretch-activated ionic channels) may inhibit the effect of mechanical stretch on action potential changes in CPC papillary muscles.     

Relationship between zinc and zinc transporter expression in breast cancer MCF-7 cells

AIM: To study the changes of zinc transporter gene expression in MCF-7 cell line exposed to ZnCl₂ and TPEN. METHODS: Human breast cancer cell line MCF-7 was exposed to different concentrations of ZnCl₂ (0, 50, 100, 150, 200 μmol/L) and TPEN (0, 5, 10, 15 μmol/L), respectively. Twelve hours later, the cell viability was measured by MTT and levels of zinc transporter mRNA by RT-PCR. Zinquin was used to estimate the intracellular zinc concentrations. RESULTS: MCF-7 cells viability rate was significantly decreased when exposed to ZnCl₂ (with 150 μmol/L and 200 μmol/L) and TPEN. The intracellular zinc concentration was significantly increased when exposed to ZnCl₂ and decreased when exposed to TPEN. ZnT-1 mRNA level was increased along with the increasing concentration of ZnCl₂ but decreased when exposed to TPEN. The expressions of ZIP2 and ZIP10 were increased along with the increasing concentration of TPEN. CONCLUSION: ZnT-1 gene expression is induced by zinc supplement and repressed by zinc deficiency. ZIP2 and ZIP10 gene expressions are induced by zinc deficiency.     

Role of mitochondrial calcium uniporter in hypoxic preconditioning

AIM: To investigate the role of mitochondrial calcium uniporter (MCU) in the cardioprotection by hypoxic preconditioning (HPC) and its relationship to mitochondrial permeability transition pore (MPTP). METHODS: Intraventricular balloon technique was employed to measure the left ventricular developed pressure (LVDP), the maximum rise/fall rate of left ventricular pressure (±dp/dtmax), and the left ventricular end-diastolic pressure (LVEDP) in Langendorff isolated rat heart. The hypoxia was achieved by ligation of left anterior coronary artery for 30 min followed by release of ligation for 120 min as reoxygenation. Hypoxic preconditioning was set as two episodes of 5 min global hypoxia and 5 min reoxygenation. RESULTS: Both HPC and treatment with ruthenium red (5 μmol/L) during the first 10 min reoxygenation improved recovery of LVDP, ±dp/dtmax and decreased LVEDP, which was associated with reduced infarct size and lactate dyhydrogenase release. These protective effects were attenuated by treatment with spermine (20 μmol/L) during the first 10 min reoxygenation. Administration of cyclosporin A (0.2 μmol/L) during the last 5 min of hypoxia period and first 15 min of reoxygenation period reduced the injury effect by spermine. CONCLUSION: These results indicate that inhibition of MCU is involved in the cardioprotection of HPC via inhibiting MPTP.     

Effect of formononetin on viability,migration and invasion of ovarian cancer cells in vitro

AIM To observe the effect of formononetin on the viability, migration and invasion of ovarian cancer cells, and to explore its mechanism. METHODS Human ovarian serous cystadenocarcinoma SKOV-3 cells were cultured in vitro. The cells were treated with formononetin at 0, 25, 50 and 100 μmol/L for 48 h. The cell viability was measured by MTS assay. The migration and invasion abilities of the SKOV-3 cells were detected by scratch wound assay and Transwell assay. RT-qPCR and Western blot were used to detect the mRNA and protein levels of E-cadherin and matrix metalloproteinase-9 (MMP-9). RESULTS The viability of SKOV-3 cells was decreased with the increase in the formononetin concentration compared with control group (P<0.01). The wound migration distance of the cells in 50 μmol/L formononetin group was less than that in control group (P<0.01). The number of invasive SKOV-3 cells across the Transwell sub-compartment was significantly decreased in 50 μmol/L formononetin group compared with control group (P<0.01). The mRNA and protein levels of E-cadherin in 50 μmol/L formononetin group were significantly higher than those in control group (P<0.01), while the mRNA and protein levels of MMP-9 in 50 μmol/L formononetin group were significantly lower than those in control group (P<0.01). CONCLUSION Formononetin inhibits the migration and invasion abilities of ovarian cancer SKOV-3 cells by increasing expression of E-cadherin and decreasing expression of MMP-9.     

Effect of platelet activating factor on the action potential and potassium currents in guinea-pig ventricular myocytes

AIM: To investigate the effects of platelet activating factor (PAF) on the action potential and potassium currents in guinea-pig ventricular myocytes. METHODS: By using whole-cell patch clamp technique, the effects of PAF on APD₉₀, I_K1 and I_K were investigated in enzymatically dispersed single guinea-pig ventricular myocytes. RESULTS: With 5 mmol/L ATP in the pipette electrode, 1 μmol/L PAF increased APD₉₀ from (225.8±23.3) ms to (352.8±29.8) ms (n=5, P<0.05), decreased I_K1 and I_K tail currents from (-6.1±1.3) nA to (-5.6±1.1) nA (n=5, P<0.05) at -120 mV and from (173.5±16.7) pA to (152.1±11.5) pA (P<0.05, n=4) at +30 mV, respectively. In contract, without ATP in the pipette electrode, 1 μmol/L PAF shortened APD₉₀ from (153.0±24.6) ms to (88.2±19.4) ms (n=5, P<0.01). Incubation of myocytes with 1 μmol/L glibenclamide, a blocker of I_KATP restored prolongation of APD induced by PAF. CONCLUSION: In guinea-pig ventricular myocytes, with 5 mmol/L ATP in the pipette, PAF prolonged APD partly due to the inhibition of I_K and I_K1, while with 0 mmol/L ATP in the pipette, PAF induced an activation of I_KATP, hence a decrease in APD was observed. Therefore, PAF might amplify the heterogeneity between ischemia and normal cardiac myocytes during ischemic reperfusion, which might play a vital role in the pathogenesis of the arrhythmias induced by ischemia/reperfusion.     

Ligustilide inhibits angiogenesis and epithelial-mesenchymal transition in human hemangioendothelial cells by reducing VEGF levels

AIM To investigate the effect of ligustilide on human hemangioendothelial cells (HemECs) and to analyze its mechanism. METHODS The effect of ligustilide at different concentrations on the viability of HemECs was measured by CCK-8 assay. The HemECs were divided into control group and ligustilide （10, 25 and 50 μmol/L） treatment groups, and the proliferation of HemECs was detected by EdU staining. The effects of ligustilide on the angiogenesis of HemECs was tested by microtubule formation experiment. The protein expression of vascular endothelial growth factor (VEGF) and epithelial-mesenchymal transition (EMT)-related markers in HemECs cells was determined by Western blot. RESULTS There was no significant difference in the viability of the cells treated with ligustilide at the concentrations between 0.1~50 μmol/L compared with control cells. Compared with control group, ligustilide at 25 and 50 μmol/L significantly reduced the number of EdU-positive cells and microtubule-like structures (P<0.05), reduced the protein expression level of VEGF (P<0.05), increased the protein expression of E-cadherin, and decreased the protein expression of vimentin and β-catenin (P<0.05). Compared with control group, the expression of VEGF and vimentin was significantly up-regulated, and the protein expression of E-cadherin was significantly down-regulated in VEGF overexpression group (P<0.05). Compared with VEGF overexpression group, the expression of VEGF and vimentin in 50 μmol/L ligustilide-treated VEGF-overexpressing cells were significantly reduced (P<0.05), and the protein expression of E-cadherin was significantly increased (P<0.05). CONCLUSION Ligulide inhibits the proliferation of HemECs, and also inhibits the angiogenesis and EMT process of HemECs by reducing the level of VEGF.     

Effect of preconditioning with pioglitazone on ischemia reperfusion/hypoxia reoxygenation-induced mitochondrial structure and membrane potential in rats

AIM: To observe the effect of preconditioning with pioglitazone on ischemia reperfusion/hypoxia reoxygenation-induced mitochondrial ultramicro-structure and membrane potential in rats. METHODS: Sprague-Dawley rats were randomly divided into four groups: sham-operated (SO) group, ischemia reperfusion (IR) group, pioglitazone preconditioning group (Pio-P) and 5-HD+pioglitazone (5-HD+Pio) group. Apart from the SO group, IR, Pio-P and 5-HD+Pio groups were subjected to 30 min ischemia and 4 h reperfusion. The heart was quickly removed for observing the structure of mitochondria and measurement of the apoptosis index (AI) by TUNEL. Primary cultured cardiomyocytes of Sprague-Dawley rats were divided into control, hypoxic reoxygenation (HR) and different concentrations of Pio-P group. JC-1 staining flowcytometry was adopted to examine mitochondrial membrane potential (ΔΨm). RESULTS: The injury of mitochondrial structure in IR group was severer than that in Pio-P group, while the difference between 5-HD+Pio group and IR group was not evident. Flameng score in Pio-P group(1.62±0.60) was significantly lower than that in IR group (2.75±1.09), P<0.01. AI in Pio-P group (28.19%±4.93%) was lower than that in IR group (55.44%±6.63%),P<0.05. The rates of low ΔΨm cells in (5 μmol/L,10 μmol/L and 15 μmol/L) Pio-P group were (45.89±3.63)%, (17.13±1.37)% and (18.43±2.44)%, significantly lower than that in HR group (56.52%±2.87%),P<0.05, while the difference between 10 μmol/L group and 15 μmol/L group was not significant (P>0.05). CONCLUSION: Pioglitazone protects the heart from ischemia reperfusion/ hypoxia reoxygenation injury evidenced by improving mitochondrial ultrastructure and lessening the loss of mitochondrial membrane potential, and decreasing apoptosis. The cardioprotective effects can be inhibited by the blocker of mitochondrial ATP-sensitive potassium channels.     

Nitric oxide-mediated the cardioprotection of tumor necrosis factor-alpha on cultured neonatal rat cardiomyocytes during hypoxia/reoxygenation

AIM: To investigate the role of nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC) and protein kinase C (PKC) signaling in tumor necrosis factor-α (TNF-α)-induced cardioprotection against hypoxia/reoxygenation (H/R) injury. METHODS: Neonatal rat ventricular myocytes were pretreated with TNF-α or sodium nitroprusside (SNP) or L-arginine (L-Arg), respectively, for 12 h and then subjected to continuous hypoxia for 12 h, followed by reoxygenation for 6 h. The manganese superoxide dismutase (Mn-SOD) activity of the cells was measured after H/R. Myocyte injury was determined by the release of lactic dehydrogenase (LDH). RESULTS: TNF-α (10⁵ U/L) significantly increased the Mn-SOD activity and decreased release of LDH from ventricular myocytes. The cardioprotection against H/R injury was induced by the pretreatment with SNP (5 μmol/L) or L-Arg (5 mmol/L), which was blocked by ODQ (10 μmol/L), the specific sGC inhibitor, and Chel (5 μmol/L), the specific PKC inhibitor. Pretreatment with L-NAME (100 μmol/L), ODQ, Chel, antoxidant 2-MPG (400 μmol/L) or tyrosine kinase inhibitor genistein (50 μmol/L) attenuated the increased Mn-SOD activity and reduced LDH level induced by TNF-α. CONCLUSION: The results suggest that NO may play a role in TNF-α-induced cardioprotection, which is mediated by sGC and PKC.     

Effects of DHEA and G6PD antisense oligodeoxynucleotides on Raji cells

AIM:To investigate the suppressive effects of dehydroepiandrosterone (DHEA) and glucose-6-phosphate dehydrogenase (G6PD) antisense oligodeoxynucleotides on Raji cells. METHODS:Raji cell line was cultured in vitro in the presence of DHEA at different concentrations ranged from 0.05 μmol/L to 500 μmol/L or G6PD antisense oligodeoxynucleotides. The viability and proliferation of the cells pretreated with dehydroepiandrosterone or G6PD antisense oligodeoxynucleotides were evaluated. Meanwhile, intracellular activities and mRNA expression of G6PD were analyzed. RESULTS:DHEA and G6PD antisense oligodeoxynucleotides does not influence the viability of cells in culture. Raji cells treated with DHEA at concentration of 50 μmol/L or 500 μmol/L for 72 h or with 10.0 μmol/L G6PD antisense oligodeoxynucleotides for 48 h had significant lower cell numbers compared with control (P<0.01). Raji cells treated with DHEA at concentration more than 5.0 μmol/L for 72 h had significant decreased G6PD activities (P<0.01) but no change in mRNA expression levels was observed. With 10.0 μmol/L G6PD antisense oligodeoxynucleotides pretreatment for 48 h, the G6PD mRNA expression levels and activities were significantly decreased (P<0.01). CONCLUSION:DHEA or G6PD antisense oligodeoxynucleotides at specific concentration have suppressive effects on G6PD activities and proliferation in Raji cells to a certain extent, but the suppressive mechanisms are different.     

Zacopride enhances inward rectifier potassium current (IK1) to antagonize arrhythmias

AIM: To investigate the effects of zacopride on aconitine or BaCl2-induced arrhythmias and involved ionic mechanisms. METHODS: The whole-cell patch-clamp technique was used to record the inward rectifier K+ current (IK1), Ca+ current (ICa-L), Na+ current (INa), transient outward K+ current (Ito), the resting membrane potential (RMP) and action potential (AP) in the single cell of rat ventricular myocardium. Two arrhythmic models were elicited by injection of 30 μg/kg aconitine or 4 mg/kg BaCl2 intravenously. Zacopride at dose of 15 μg/kg was administered immediately after the development of first sinus rhythm disorders to observe its effects on arrhythmias. The ECGs were recorded simultaneously. RESULTS: In voltage clamp mode, 0.1-10.0 μmol/L zacopride dose-dependently enhanced IK1 with no significant effects on ICa-L, INa and Ito (P>0.05). Zacopride at concentration of 1.0 μmol/L showed the most potent activity on IK1 with approximately 30% increment both in inward current and outward current (P<0.01). Correspondingly, 0.1-10.0 μmol/L zacopride hyperpolarized RMP and shortened the action potential duration (APD) in a concentration-dependent manner (P<0.01). Furthermore, 1.0 μmol/L zacopride abolished aconitine-induced delayed afterdepolarization (DAD) and triggered activity (TA). In vivo, zacopride (15 μg/kg) significantly shortened the duration of arrhythmias elicited by aconitine ［from (57.58±3.21) min to (38.25±2.59) min］ or BaCl2 ［from (49.31±2.46) min to (30.94±1.73) min］. CONCLUSION: As an agonist of IK1, zacopride enhanced IK1, hyperpolarized RMP and shortened APD, which may be the fundamental mechanisms underlying its antiarrhythmic effect. Enhancing IK1 might be a new antiarrhythmic strategy.     

Stable reversal of multidrug resistance of colon cancer cells by RNAi targeting MDR1 gene

AIM: To investigate the reversal effect of RNAi targeting MDR1 gene on MDR1/P-gp-dependent multidrug resistance of colon cancer cells. METHODS: Plasmid vectors pSilencer-#4029 encoding #4029 MDR1 siRNA and pSilencer-#4123 encoding #4123 MDR1 siRNA were constructed, respectively. Plasmid vectors were transfected into COLO 320DM cell line, a colon cancer multidrug resistance cell line. Clone cells were screened by G418 and identified by real time RT-PCR and Western blotting. Cell viabilities were measured by MTT assay. Cell cycle analysis was performed with flow cytometry. Intracellular adriamycin accumulations were measured by flow cytometry. RESULTS: The expression of MDR1 mRNA and P-gp in positive clone cells (clone #4029 and clone #4123) were all inhibited. IC50 of adriamycin and vincristine for COLO 320DM parent cells were 9.616 μmol/L and 0.358 μmol/L, respectively. However, for clone #4029, they were decreased to 1.094 μmol/L and 0.023 μmol/L, respectively (P<0.01). For clone #4123, they were decreased to 0.780 μmol/L and 0.035 μmol/L, respectively (P<0.01). The PI/AI values of COLO 320DM parent cells treated with adriamycin and vincristine were 5.68 and 9.59, respectively. However, they were decreased to 2.74 and 3.59 respectively for clone #4029 (P<0.01). They also decreased to 2.75 and 3.24 respectively for clone #4123 (P<0.01). The intracellular adriamycin accumulation concentrations of COLO 320DM parent cells treated with 10 μmol/L adriamycin were 27.92. However, they were increased to 187.24 and 215.57 for clone #4029 and clone #4123, respectively (P<0.01). CONCLUSION: Stable transfection of plasmid vector encoding MDR1 siRNA stably reverses MDR1/P-gp-dependent multidrug resistance of colon cancer cells.     

Effect of nuclear factor kappa B inhibitor on the responsiveness of pulmonary artery rings to protein kinase C in rats with hypoxia-induced pulmonary hypertension

AIM: To investigate the role of nuclear factor kappa B (NF-κB) inhibitor in the responsiveness of isolated pulmonary artery rings to protein kinase C (PKC) in rats with hypoxia-induced pulmonary hypertension. METHODS: The pulmonary artery rings removed endothelium were prepared from model rats with hypoxia-induced pulmonary hypertension and control rats. The effects of PKC activator PMA (0.5 μmol/L) time-response cures and NF-κB inhibitor PDTC (0-1 000 μmol/L) concentration-response cures on pulmonary artery rings were observed. The responsiveness of each ring was tested by applying a maximally effective concentration of phenylephrine (10 μmol/L). Data were calculated as relative ratio by the maximally responseness ( P₀ ) setting at 100%, and the relative responseness tensions to PMA and PDTC were derived by dividing by the counts in P₀. t_1/2 and T show the time achieving half-maximal response and lasting maxima response to 0.5 μmol/L PMA, respectively. RESULTS: mPAP and RV/(LV+S)in hypoxia group were greater than those in control group(P<0.05).For the responseness of the artery rings to PMA of 0.5 mol/L,the relat ive tensions of hypoxia group were significantly higher(P<0.05)as compared with respective controls;mean t_1/2 in hypoxia group was shorter than that in control group(P<0.05).Mean T in hypoxia group was longer than that in control group(P<0.05).For the relative tensions of the artery rings to PDTC and PMA,hypoxia group were higher than those of controls in the range of PDTC 0-100 mol/L(P<0.05);the relative tensions of two group significantly decreased beyond PDTC of 500 mol/L(P<0.05). CONCLUSIONS: The responsiveness of pulmonary artery rings to PMA was increased during hypoxia and decreased to PDTC in concentration-dependent manner. These results further suggest that changes of PKC－NF-κB signaling pathway of pulmonary artery smooth muscle cells may be involved in vasoconstriction of hypoxia-induced pulmonary hypertension.     

Effects of glucagon-like peptide-1 on myocardial ischemia-reperfusion/hypoxia-reoxygenation injury in rats

AIM: To investigate the effects of glucagon-like peptide-1 (GLP-1) on myocardial ischemia-reperfusion (IR)/hypoxia-reoxygenation (HR) injury in rats. METHODS: Sprague-Dawley rats were randomly divided into 5 groups: sham group, IR group and IR+GLP-1 (0.03 nmol/L, 0.16 nmol/L and 0.30 nmol/L) groups. IR group and IR+GLP-1 group were subject to 30 min of ischemia and 3 h of reperfusion. The myocardial infarct size, the ultrastructural changes of the myocardial tissues, the apoptosis of the cardiomyocytes, the activity of superoxide dismutase (SOD) and the concentration of malondialdehyde (MDA) were detected. Primarily cultured cardiomyocytes were divided into 5 groups at random: control group, HR group and HR+GLP-1 (1 μmol/L, 5 μmol/L and 10 μmol/L) groups. The morphology and apoptosis of the cardiomyocytes were observed. The levels of lactate dehydrogenase (LDH),MDA,SOD,reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in different groups were detected. RESULTS: Compared with IR group, the myocardial infarct size and cardiomyocyte apoptosis were remarkably reduced, mitochondrial ultrastructures were improved, the activity of SOD was increased and the concentration of MDA was decreased in IR+GLP-1 (0.03 nmol/L, 0.16 nmol/L and 0.30 nmol/L) groups. Compared with HR group, GLP-1 (1 μmol/L, 5 μmol/L and 10 μmol/L) preconditioning significantly decreased the myocardial injury, increased SOD activity, decreased MDA concentration and ROS production, and heightened MMP in a dose-dependent manner. CONCLUSION: GLP-1 protects cardiomyocytes from IR/HR injury, which may be partially due to the effects of anti-oxidative mechanism and the function of mitochondrial protection.