| Fuzi is the root of Aconitum carmichaeli Debx, which belongs to ranunculaceae plants. It can enhance heart function and has antiarrhythmic,antiinflammatory,antinociceptive and antitumor effects, so it has a wide range of clinical applications. However, due to its strong toxicity on cardiovascular sysytem and central nervous sysytem, Fuzi lead to death easily, thereby its applications and development were limintied in clinical. The Aconitine, Mesaconitine and Hypaconitine were thought to be the important factors in leading to arrhythmia.Our previous studies have show that, cardiotoxicity was observed in Beagle dogs after administered with Fuzi decoction, and both Fuzi constituents-containing serum and aconitine inhibited hERG tail current obviously. Aconitine inhibited hERG tail currents in a dose-dependent manner, with an IC50 of 20μM. Since hERG channel is the molecular basis of cardiac Ikr current, Ikr current play an important role in the 3-phase repolarization of myocardium, so hERG channel is a one of the target of Fuzi causes arrhythmia. But later results showed that Aconitine (3×10-7mol/L) affects action potential and cause arrhythmia in papillary muscle of guinea pig.The concentration is less nearly 100 times than that inhibits hERG current, so we speculated that hERG channel is not the most sensitive target of aconitine, Aconitine only inhibit the hERG channel at high concentrations leading to QT interval prolongation. So which kind of alkaloid is more sensitive to QT interval prolongation effect of Fuzi? Does Fuzi serum have impact effect on APD as its effect on hERG channel?Since previous studies only focused on the effects of aconitine, and rare study was done on that of meaconitine, we observed the effects of Fuzi serum and Mesaconitine on APD and analyse its mechanism further. The study are very important to the development and utilization of Fuzi.In this study, Fuzi decoction we administered to SD rats by i.g., ECG was detected at different period, blood was taken after obvious ventricular arrhythmia was appeared. By using serum pharmacology and electrophysiology methods, we observed the effects of Fuzi serum on the action potentials in papillary muscle of guinea pig, and analysis the components of Fuzi serum by liquid chromatography - mass spectrometry technique. Further study was done on the effects of mesaconitine in APD, intracellular calcium concentration [Ca2+]i and hERG channel current to analysis the underlying toxic mechanism of Fuzi.1 The effect of Fuzi on rats'ECGObjective: ECG changes were observed in rats with or without Fuzi decoction administered by i.g., and the blood was got when ventricular arrhythmias were appeared and the serum were prerared for further study on APD and the anlysis the compound in serum.Methods: 20 SD rats were randomly divided into two groups. Fuzi decoction were administrated by i.g., administration twice a day for 5 days, and control group was given sterilized water at the same volume. The parameter of ECG were recorded in anesthetized rats by RM-6240 CD in the day before administration, 1 day administration,3 days administration and 5 days administration.Results: No significant changes in ECG were observed in control group after anesthesia compared with that before anesthesia (p> 0.05). The amplititude of T wave was increased in Fuzi group after administration one day later, compared with that before administration or control group at the same period(p <0.01), no significant changes were observed in other ECG indexes. after administration 3 days, Paroxysmal ventricular heart premature beats were observed in 30% rats, bigemineies and trigeminies were observed in 70% rats, HR was accelerated significantly (p <0.01), QRS wave appeared large deformity (p <0.05), T wave was increased significantly (p <0.01), QT interval was significantly prolonged (p<0.01), QTc was prolonged significantly (p<0.01), PR interval was significantly shorter (p <0.05). the incidence of ventricular arrhythmias further increased after administration 5 days, parts of the animal by the ventricular premature beat-discrete or in salvos, ventricular premature beat-bigemineies and trigeminies to the development of ventricular tachycardia and ventricular fibrillation, the incidence was 10%, 40%, 30%, 20% respectively, the significance of each parameter was more obvious. Na+, K+, Cl- concentrations in serum were elevated, the most significant increase of K+ were significantly higher than control, blood creatine kinase isoenzyme (CK-MB) levels tended to increase, but there was no significant difference (p> 0.05).Conclusions: Fuzi decoction prolonged leads to QT interval prolongation, Premature ventricular contractions, triple and ventricular tachycardia and ventricular fibrillation when rats were ministrated i.g.2 Effects of containing-Fuzi compound serum on the action potential of papillary muscle in guinea pig and its compound analysisObjective: To investigate the effect of Fuzi serum on the action potential of papillary muscle in guinea pig, and the compounds analysis of Fuzi in serum by liquid chromatography - mass spectrometry technique.Methods: Healthy adult female guinea pigs, weighing 250 350g, papillary muscle experiment samples taken, with O2 saturated Tyrode's solution constant speed (4 ml / min) perfusion, temperature 36℃,with intracellular microelectrode technique was recorded in guinea pig papillary muscles under the stimulation frequency of 1Hz. The action potential profile was collected before and 20 min later in the differences of proportions were added to dilute the drug serum respectively.Results: (1) Blank serum(diluted 30 times) has no effect on APD30, APD50, APD90 and Vmax(P>0.05). While 3 days Fuzi serum diluted 102 times and 30 times, the APD90 was prolonged from 203±6ms to 225±9ms and 238±12ms;the APD90-30 was prolonged from 103±4ms to 119±6ms and 133±5ms .When the serum diluted 3×102 times, it has no effect on APD. 5 days Fuzi serum (diluted 104,103 times) has no effect on graphic and APD. While the serum diluted 102,it was significantly broadened the action potential graph and the serum could prolong the APD90 from 170±8ms to 221±19ms;increase the APD90-30 from 106±6ms to 143±4ms. Compared with 3 days Fuzi serum and 5 days Fuzi serum, the rate of change [(after-before)/before×100%] with 5 days Fuzi serum was higher than the rate of 3 days Fuzi serum. (2) Fuzi serum by HPLC-MS detection, which mainly detected Aconitine, Mesaconitine, Hypaconitine and Bulleyaconitine A and Lappaconite were not detected.Conclusions: Blank serum has no effect on AP. Fuzi serum (diluted 100 times) can increase APs. The effect of serum which was extended of administration in vitro was more obvious.3 Effects of mesaconitine on the action potential of papillary muscle in guinea pig and intracellular calcium of cardiomyocytesObjective: To investigate the effects and mechanism of mesaconitine on the action potential of papillary muscle in guinea pig, intracellular calcium of cardiomyocytes and hERG currents.Methods: (1) The conventional glass electrode technique was used to record the action potential of papillary muscle in guinea pig under the stimulation frequency of 1Hz. The action potential profile was collected before and 20 min later in the presence of Meaconitine(10-10,10-9,10-8and 3×10-8mol/L)respectively.(2) The action potential was collected before and 20 min later in the presence of Meaconitine under different stimulation frequency (0.3, 1.0, and 3.0Hz).Observed the APD90 of control and MA 3×10-8mol/L.(3) The guinea pig ventricular myocytes were obtained by enzymatic dissociation technique, then were incubated with Fluo4-AM. The Fluo4-AM fluorescent signal was detected with confocal laser scanning microscopy. The change of [Ca2+]i was represented by the percentage of fluorescence intensity change(FI -FI0)/FI0(%).Treated with different concentrations of Mesaconitine, observe the effect and mechanism of meaconitine on [Ca2+]i. (4) hERG-HEK293 cells were resuscitated, administered with meaconitine. The changes in hERG currents were observed.Results: (1) MA has no effect on AP in the range of 10-10 mol/L and 10-9 mol/L at the stimulatory frequencies of 1.0 Hz. While MA increase 10-8mol/L, it can shorten the APD90 from190.6±4.6ms to 177.2±6.9ms (P<0.05). And with the MA concentration increased further reduce APD and APA. While MA 3×10-8mol/L caused arrhythmia ,it reduced the APD90 from190.6±4.6ms to 166±4.7ms,but the APD90-30 was prolonged from 111.5±6.3ms to 133.4±6.5ms.(2) The changces of APD90-30 at the stimulating frequencies of 0.3, 1.0, 3.0 Hz were shorted with MA 3×10-8mol/L concertration.(4) In normal Tyrode's solution and Ca2+-free Tyrode's solution, MA (10-10,10-9,10-8mol/L) elevated [Ca2+]i in a concentration dependent manner, in normal Tyrode's solution by 24.3%, 54.8%, 129.2%, however, the level of [Ca2+]i elevation in Ca2+-free Tyrode's solution was lower than that in normal Tyrode's solution by 18.6%,49.5%,91.5% respectively. Ryanodine(10-5mol/L) remarkably blocked the effects of MA(10-9mol/L) elevation in Ca2+-free Tyrode's solution from 41.5% to 13.5%, however, while blocked the effects of MA(10-9mol/L) elevation in normal Tyrode's solution from 53.0% to 45.3%.(4) MA(10-5mol/) has no effect on hERG current.Conclusions: (1) MA(3×10-8mol/L) can cause arrhythmia on the action potential of papillary muscle in guinea pig (2) Ca2+elevation induced by MA depends on both intracellular and extracellular Ca2+stores.Summary: Mesaconitine, one composition of FUZI,could induce arrhythmia, especially the phenomenon of long QT, whereby the methods which maybe inhance the ventricular myocyte [Ca2+]i not by the effects on hERG channel.
|
PDF Full Text Request