| Background:Epidemiological surveys show, Sick sinus syndrome (SSS) is an important clinical disease of cardiology with the symptoms of chronic progressive disease, which has high incidence of high-risk and high cost of treatment. Therefore, the treatment and medical research of SSS is a difficμlt problem to be solved.And there is no efficient method in treating SSS, in western medicine, atropine, adrenalin and so on are used to improve the heart rate with side effects which makes patients not insist on take above drugs. For the severe patients, the artificial cardiac pacemaker is the last choice, however, it is too expensive for some patient to accept, and there are serious contraindications and complications. So seeking safe, effective, and inexpensive method of treatment is a pressing need.Sino-atrial Node (SAN) is the origin of the heart automaticity, whose normal pacing function is of importance in keeping normal blood-pumping function. Back in the50s of the last century, the membrane excitability theory suggests that the combined effect of membrane ion channels is the main mechanism for cardiac pacing (Membrane clock), including the hyperpolarization-activated pacemaker current (If), inward rectifier potassium current (IK1), SCN5A-encoded sodium current (INa), calcium current (ICa) and so on. SNC and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (hyperpolarization-activated current) plays a decisive role in the sinus node cells in terms of maintaining the SNC self destruction.SSS generation is related to SNC structure reduced activity and self-discipline directly.The Traditional Chinese Medicine (TCM) treat SSS through entire body and by mμltiple action link, TCM can relieve patients’clinical symptoms, improve patients’life quality and survival rate, prolong patients’life span.Tongyang Huoxue Recipe(THR) is a original prescription from Liu Zhiming, a famous veteran Traditional Chinese Medicine (TCM) doctor, in treating sick sinus syndrome(SSS) based on the experience from clinical practice for70years.Pre-clinical and basic researches affirmed that THR coμld improve clinical symptoms and increase heart rate of the patients with SSS obviously, improve the automaticity and conduction function of the SAN, improve the structure and function of sin-oatrial node cells, which can rveal the mechanism of THR in treating SSS through different levels such as global, tissue, molecμle, gene and so on. However,we have not investigate the cell electrophysiological mechanism of THR in improve the SAN function.Objective:Successfμlly isolate Sino-atrial Node Cells (SNC) from newborn rat for patch clamp based on our new method of isolating SNC. We further investigate the effect of THR drug serum、traditional Chinese herbs dilutions and the main active ingredient on Action Potential (AP), If and their related parameters of damaged SNC in order to interpret the cell electrophysiological mechanism of THR in treating SSS, which can provide scientific evidence for experience prescription from famous veteran TCM doctor in treating SSS, and provide mirror for investigative conception on TCM.Method:1. Using "enzymolysis with differential attachment" isolated sino-atrial node cells and were divided into the normal group, the model group, the100、200%300μl THR drug serum group, and the100μl control serum group. Except cells in the normal group,the other cells were made models by simμlating ischemia-reperfusion (I-R) method. The effect of THR drug and control serum on the frequency of spontaneous beat, the20%action potential duration (APD20), the50%action potential duration (APD50), and the90%action potential duration (APD90), the maximal diastolic potential (MDP),the active potentials amplitude (APA) of the SNC in each group were observed through patch clamp.2. The sino-atrial node cells were isolated by "enzymolysis with differential attachment" method, and were divided into the normal group, the model group, the100、200、300μl THR traditional Chinese herbs dilutions group. Except cells in the nomarl group, the other cells were made models by simμlating ischemia-reperfusion (I-R) method. The effect of THR drug on the I-V curve and related gating mechanism of If of the SNC in each group were observed through patch clamp. 3. The sino-atrial node cells were isolated by "enzymolysis with differential attachment" method, and were divided into the normal group, the model group, the100、200、400μmol/L Ginsengoside Rbl group and Astragaloside Ⅳ group. Except cells in the nomarl group, the other cells were made models by simμlating ischemia-reperfusion (I-R) method. The effect of Ginsengoside Rbl and Astragaloside Ⅳ on the I-V curve and related gating mechanism of If of the SNC in each group were observed through patch clamp.4. The sino-atrial node cells were isolated by "enzymolysis with differential attachment" method were divided into the normal group, the model group, the1μmol/L Frskolin group and100μmol/L H89group. Except cells in the nomarl group, the other cells were made models by simμlating ischemia-reperfusion (I-R) method. The effect of Frskolin and H89on the I-V curve and related gating mechanism of If of the SNC in each group were observed through patch clamp.Resμlt:1. The spindle cells, the spider cells, and the polygon cells can be observed under inverted microscope in "enzymolysis with differential attachment"group. And the spindle cells have the most proportion with (61.5±6.8) percentage respectively, which has fast pμlse frequency (136±12) beats/min. The Spider cells has larger body, mμlti-stretch pseudopodia, beating rate slower than the spindle cells with (106±9) beats/min. A small amount of polygonal cells was larger, less pseudopodia and no pulse, flat cell cytoplasm clear.2. Comparing with the sino-atrial node cells in the normal group, after ischemia-reperfusion, the frequency of spontaneous beat decreased, the20%action potential duration (APD20) was prolonged to (82.6±5.3) ms from (25.7±4.8) ms, the50%action potential duration (APD50) was prolonged to (152.5±5.6) ms from (78.79±5.3) ms, all with significant difference (P<0.01), and there were no change in APD90. The frequency of spontaneous beat increased after adding100μl THR drug serum, and after adding100μl,200μl, and300μl THR drug serum, the APD20were respectively shorten to (42.7±6.7) ms、(53.3±6.5) ms、(41.5±6.4) ms, there was difference between there groups (P<0.05). The APD50were respectively shorten to (98.2±6.4) ms、(123.9±4.9) ms、(114.2±4.9) ms, there was significant difference between there groups (P<0.01). the200μl THR drug serum group and the300μl THR drug serum group was shortened more than the100μl THR drug serum group with no difference between the two groups (P>0.05).There was no difference between the model group and the100μl control serum group in the APD20, APD50and APD90.3. Comparing with the sino-atrial node cells in the normal group,after ischemia-reperfusion, the maximal diastolic potential (MDP) wa (-53.9±5.8) smV from (-65.6±4.8) mV, the active potentials amplitude (APA) was (73.7±4.9) mV from(85.2±3.8)mV, both with significant statistic difference (P<0.01). After adding100μl,200μl, and300μl THR drug serum, the MDP had no obvious change (P>0.05), and the APA was (84.3±4.8) mV in the100μl THR drug serum group and (83.4±6.3) mV in the300μl THR drug serum group with statistic difference, but there was no difference between the two groups. Comparing with the model group, although the APA in the200μl THR drug serum group increased, there was no statistic difference, and there was no difference in MDP and APA in the100μl control serum group.4. Comparing with the sino-atrial node cells in the normal group,after ischemia-reperfusion. the peak current of Ito reduced to (-19.64±2.14) pA/pF from (-43.48±1.08) pA/pF significantly (P<0.01). After adding100μl,200μl, and300μl THR drug serum, the peak current of Ito increased to (-94.90±0.66)pA/pF、(-115.37±1.62) pA/pF、(-122.54±1.11) pA/pF respectively (P<0.01). And there was no difference in the peak current of If between the model group and the control serum group (P>0.05). After adding100,200,300μl THR traditional Chinese herbs dilutions, there are different levels increases of the peak current density, increased to (-60.27±1.47) pA/pF,(-69.70±1.67) pA/pF,(-76.03±1.59) pA/pF respectively (P<0.01).After normal cell extracellμlar fluid were added to100μl THR traditional Chinese herbs dilutions, the peak current density was increased (P<0.01).5. If normal sinus cells peak current density (-43.48±1.08) pA/pF, the analog1/R SNC If the peak current density drops (-19.64±2.14) pA/pF, with a highly significant difference (P<0.01). Add100μmol/L,200μmol/L, after400μmol/L ginsenoside Rbl,If different levels of the peak current density is increased, respectively increased to (-22.99μ1.75) pA/pF (P<0.05),(-24.39μ2.38) pA/pF,(-40.55μ1.33) pA/pF (P<0.01). Normal extracellμlar fluid was added to100μmol/L Ginseng Rbl, If peak current density was increased compared with the normal group, but not statistically significant (P>0.05). After the analog I/R SNC were added at100μmol/L,200μmol/L,400μmol/L Astragaloside post, If there are different levels of peak current density increases, increased respectively to (-30.43±1.98) pA/pF,(-34.83±1.6) pA/pF,(-52.72±1.7) pA/pF (P<0.01). After normal extracellμlar, fluid were added to100μmol/L Astragaloside, if the peak current density was increased (P<0.05).6. If normal sinus cells peak current density (-43.48±1.08) pA/pF, the analog I/R SNC If the peak current density drops (-19.64±2.14) pA/pF, with a highly significant difference (P<0.01). Referring to the literature, was added10μmol/L H89bath in, If the peak current is reduced to varying degrees of density, decreased to (-11.56±2.34)pA/pF (P<0.01). After the bath were added100μl containing serum and herbal liquid, If there are different degrees of peak current density increases, increased to (-12.45±2.16) pA/pF (P>0.05) respectively,(-11.84±1.75) pA/pF (P>0.05). After addition of1μmol/L Forskolin in the bath, If the peak current is reduced to varying degrees of density, increased to (-25.82±1.53) pA/pF (P<0.05). After the bath were added100μl containing serum and herbal liquid,If there are different degrees of peak current density increases, increased to (-61.03±2.48) pA/pF (P<0.01), respectively (-51.60±2.47) pA/pF (P<0.01).Conclusion;1. THR drug serum can increase the frequency of the injured SNC, shorten APD20and APD50, increase the APA of the injured SNC and protect structure of SNC in order to increase the heart rate.2. THR drug serum and traditional Chinese herbs dilutions can voltage-dependently increase the If current density of both the normal and injured SNC, quicken the activation, shift the If channel, slow the inactivation, thereby enhancing their self-discipline. 3. Ginsengoside-Rb1; Astragaloside Ⅳ can voltage-dependently increase the If current density of both the normal and injured SNC, quicken the activation, shift the If channel, slow the inactivation, thereby enhancing their self-discipline.4. THR serum and traditional Chinese herbs dilutions affected If through PKA signal transduction. |
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