Mechanism Of The Effect Of Shenfu Compatibility Attenuation On Cardiomyocytes

Traditional Chinese medicine is the main form of TCM drugs, through the combination and interaction between the components, the effect was single drugs can not be reached. But because of the complexity of components, resulting in the target to treat disease is variety, mechanism is not clear, become one of the resistance of TCM to the world. This topic mainly Shenfu Decoction as the object to research, to explore the ways and mechanism of Fuzi toxic effects, and research on the mechanism of Shenfu Decoction compatibility attenuation.In this article H9c2 cells were exposed to Fu Zi decoction 6.25, 12.5, 25, 50, 100 g L-1 for 24 h,fluorescence staining CCK-8 assay cell viability;H9c2 cells were exposed to Fu Zi decoction 6.25, 12.5, 25 g L-1 for 24 h,the effect on mitochondrial membrane potential and reactive oxygen species(ROS)was detected by flow cytometry; fluorescence molecular probe was used,and laser scanning confocal microscope to observe the effect on Ca2+ in cells, Ca2+ and superoxide in mitochondria; detect the effect on ATP concentration in cells; expression of expression of Pgc-1α, Bcl-2 and Bax m RNA evaluated by real-time PCR while the expression of PGC-1α protein was measure by Western blot. We found that H9c2 cell viability was significantly inhibited by Fuzi decoction 12.5~100 g·L-1(P <0.05, P <0.01); the IC50 value was 47.4669g·L-1,95% confidence limit was 32.5997~69.1145 g·L-1. After the treatment of Fuzi decoction 25 g·L-1, the fluorescence intensity of ROS in the normal control group is 203.7±66.8 increased to 453.5 ± 78.3(P<0.05), the fluorescence intensity of mitochondrial superoxide from 5.35 ±1.77 increased to 26.82±8.52(P<0.01); mitochondrial membrane potential decreased to 0.836±0.39 from 1.72±0.46(P<0.05), the fluorescence intensity of intracellular Ca2+ increased to 22.07±0.51 from 7.82±0.79(P<0.05) and the fluorescence intensity of mitochondrial Ca2+ from 37.96±4.31 decreased to 9.24±1.65(P<0.01), intracellular ATP concentration from 10.56±0.39 μmol·g-1 protein decreased to 5.27±1.06μmol·g-1 protein(P<0.05). Q-PCR and Western blotting test results showed that, compared with the normal control group, Fuzi decoction 25 g·L-1 group Pgc-1α and Bcl-2 m RNA relative expression level respectively by the normal control group 1±0.10 and 1±0.10 decreased to 0.086±0.062(P<0.01) and 0.429±0.059(P<0.01), the relative expression of Bax m RNA from 1±0.031 to 1.173±0.06 increased(P<0.05); the expression of Pgc-1α protein in the normal control group 0.906±0.034 decreased to 0.541±0.003(P<0.01). The result showed that Fu Zi have a certain mitochondrial toxicity to cardiomyocytes, the affection is the result of combined action of kinds of ways. Mitochondrial toxicity of myocardial may be for this reason that Fu Zi cause cardiac toxicity.Secondly, through explore the effects of aconitine, ginsenoside Re and KN-93(Ca MKII inhibitor) on myocardial cell calcium homeostasis, and the impact on Ca MKII and related calcium regulatory protein gene and protein expression levels, to research the mechanism of aconitine lead to arrhythmia and mechanism of ginsenoside Re anti-arrhythmia induced by. After stained with Fluo-4AM, the cells in each group treated with drugs, dynamic real-time monitoring of Ca2+. The expression of Ry R2 m RNA was detected by Q-PCR, and the expression of Ca MKII, P-Ca MKII, Ry R2 and SERCA2 protein detected by Western blot. We find that aconitine can cause cardiomyocytes calcium transient increase in the frequency and the relative content of Ca2+ was increased, which leads to disorder of calcium homeostasis in cardiomyocytes. However, this effect can be inhibited by verapamil(a calcium channel blocker) and KN93(Ca MKII inhibitor), suggest that aconitine induced arrhythmia may be associated with Ca MKII. At the same time, ginsenoside Re can reduce the aconitine induced calcium transients frequency increases, make the calcium transient amplitude recovery. Gene detection results show that aconitine can cause the increase of sarcoplasmic reticulum calcium channel Ry R2 m RNA transcription level and protein level showed that aconitine can cause of Ca MK II phosphorylation and Ry R2 protein is expressed at high levels, this phenomenon can by KN-93 and ginsenoside Re reversed. Experimental results show that the aconitine induced arrhythmia may be associated with increased Ca MK II phosphorylation. Experimental results show that the aconitine induced arrhythmia may be associated with increased Ca MK II phosphorylation; and ginsenoside Re can induce Ca MK II phosphorylation by reducing against aconitine arrhythmia.This paper originally discloses the mitochondrial toxicity effect of Radix Aconiti Lateralis Praeparata; by the effective components compatibility in Shenfu Decoction to study the attenuating mechanism of Shenfu Decoction. Discovered a possible new antiarrhythmic mechanism which aconitine induced and aiso find the protective effect of ginsenoside Re. This study offers the basis for the research of attenuating mechanism of Shenfu Decoction and compatibility regulation of Chinese Traditional Medicine.