Study On The Mechanism Of Anti Toxicity Of Ginseng And Accessory Compatibility From CaN-NFAT3 Pathway

Aconitum is a common Chinese medicine in China and belongs to the Ranunculus.The Aconitum is the lateral root of the plant.The main effect of the Fuzi is to return to the sun to save the counter,to supplement the fire to help the Yang,and to relieve the cold and relieve the pain.Aconitine is the main component of the Aconitum,which is both the effect component and the toxic component.When aconitine is used as an effective ingredient,it has strong heart,antiarrhythmic,anti shock,anti thrombotic and central inhibitory effects.When aconitine is used as a toxic component,it can lead to slow heart rate,conduction block and ventricular tachycardia.Panax Araliaceae,has Dabu strength,solid Fumai off,spleen and lung function.The main components of ginseng are ginsenoside A,ginsenoside Rg1,ginsenoside Re and so on.The laboratory toxicity experiments show that the main component of aconitine in aconitumcarmichaeli induced arrhythmia can be antagonistic and ginseng,according to reports in the literature,aconitine and ginsenoside Re combined inhibitory effects of L type calcium channel than the separate use of ginsenoside Re effect.In this paper,the main components of aconitine and ginsenoside Re,the main component of Radix Aconiti,were studied in this paper.It is known that after the stimulation of cardiomyocytes,the intracellular Ca²⁺is increased,and the elevated Ca²⁺can bind to CAM,which activates CaN and CAMK II.After activation,CaN and CAMK II can transfer NFAT3 and NF-kB into the nucleus,resulting in arrhythmia.Aconitine can promote the sodium influx and change the concentration of sodium ions in the cells.The calcium ion changes through the Na⁺/Ca²⁺exchanger.When the calcium ions in the cardiac cells change,the CaN-NFAT3 signaling pathway is activated.The calcium ion in myocardial cells increased the activation of CaN,followed by the activation of CaN NFAT3 gene transfer to the nucleus to the phosphorylation of NFAT3 was activated,combined with the GATA4 gene induced by c-fos oncogene expression,then fetal gene expression of ANP increased,resulting in arrhythmias of congestive heart failure at last.The previous work of the research group showed that aconitine could increase the phosphorylation level of CAMK II.Can aconitine induce the activation of CaN,thus transferring NFAT3 to the nucleus and arrhythmia?Therefore,this study should be based on the CaN-NFAT3 signaling pathway to explore the mechanism of combining compatibility and reducing toxicity.In the first part of this paper,we first discussed whether aconitine can regulate calcium ion through the Na⁺/Ca²⁺exchange body through the excitation contraction coupling system of cardiomyocytes,and finally lead to arrhythmia.Using laser scanning confocal microscope,RT-PCR and Western blot technology,with different concentrations（1μM,5μM,10μM）of aconitine stimulated cardiomyocytes,activity of aconitine on myocardial cells in Ca²⁺-ATP and Ca²⁺-Mg²⁺-ATP enzyme and Na⁺-K⁺-ATP enzyme,Na⁺,Ca²⁺,K⁺content and effect of calcium overload;The transient changes of calcium ion were detected after stimulation of aconitine with different concentrations.The changes of above indicators were detected by Na⁺,Ca²⁺,K⁺detection kit,Ca²⁺-ATP enzyme,Ca²⁺-Mg²⁺-ATP enzyme and Na⁺-K⁺-ATP enzyme detection kit;Then the changes of mRNA and protein of SERCA₂ and RyR₂ were observed.The results show that the aconitine stimulated cells,compared with the control group,different concentration of aconitine can inhibit calcium transient concentration,high concentration of aconitine can significantly increase the calcium transient frequency,and the increase of calcium concentration;intracellular Na⁺,Ca²⁺concentration increased,while the concentration of K⁺decreased,and decreased Ca²⁺Ca²⁺-Mg²⁺-ATP enzyme and Na⁺-K⁺-ATP enzyme activity;The expression of mRNA and protein in SERCA2 was significantly down-regulated,and the expression of mRNA and protein in RyR2 was significantly up-regulated.When the concentration of aconitine is high,the results have significant difference.Research suggests that aconitine can cause obvious myocardial intracellular ion disorder at high doses,reducing the activity of ATP,decreased the ion transport capacity,the increase of Na⁺and Ca²⁺concentration;also significantly related to regulation of calcium channels and SERCA2 RyR2 mRNA expression and protein level,the intracellular calcium disorder.Calcium transients.Studies have shown that aconitine can change the calcium ion in cardiac cells through the Na⁺/Ca²⁺exchanger and the mechanism of myocardial contraction coupling,thereby inducing the occurrence of ionic signaling pathway,causing a variety of cardiovascular diseases,eventually leading to the occurrence of heart failure.In the second part of this paper,the effects of aconitine,aconitine and ginsenoside Re on the CaN-NFAT3 signaling pathway were investigated from two aspects in vitro and in vivo.First,at the cell level,after stimulating the cardiac myocytes,the mRNA and protein level of ANP,CaN,c-NFAT3 and n-NFAT3 protein levels were detected by real-time quantitative PCR and Western blot.The results showed that ANP mRNA expression level increased,the expression level of ANP protein increased;the expression level of CaN protein increased,c-NFAT3 protein expression decreased,n-NFAT3 protein expression level increased,compared with the control group,when aconitine as high concentration,the above results have significant difference.The concomitant use of aconitine and ginsenoside Re after administration of ginsenoside Re has myocardial protective effect,the use of Western blot technology,c-NFAT3,n-NFAT3,CaN to detect the expression of GATA4 protein levels.The results showed that ginsenoside Re and aconitine after compatibility with aconitine alone compared to treatment,the expression level of CaN protein decreased,c-NFAT3 the protein levels,the expression level of n-NFAT3 protein decreased,decreased expression level of GATA4 protein in the cell nucleus,and the results have significant difference;In order to further CaN-NFAT3 signal pathway and arrhythmia is directly related to verification,using FK506 FK506 as stimulating myocardial cells,inhibitors of CaN,using Western blot technology to detect ANP and c-NFAT3,the expression level of n-NFAT3 protein,the results show that compared with aconitine alone group,can be combined with FK506 decreased the expression level of ANP protein,c-NFAT3protein expression level increased,the expression level of n-NFAT3 protein decreased,the result has the difference.The results suggest that ginsenoside Re has protective effects on myocardial cells,can inhibit the calcium ion disorder caused by aconitine,causing the protein level change;after adding CaN inhibitor FK506 can significantly reduce the expression level of ANP protein,and c-NFAT3 protein expression level increased,the expression level of n-NFAT3 protein decreased,no the impact on the downstream pathway,further proof of aconitine does affect the signal pathway of CaN-NFAT3.At the animal level,the proportion of three（Radix Aconiti:Ginseng=2:1,Radix Aconiti:Ginseng=1:1,Radix Aconiti:Ginseng=1:2）were given to mice orally for 14 days,take heart tissue,tissue protein extraction,using Western blot technology,c-NFAT3,CaN and ANP detection n-NFAT3 and the expression of n-GATA4 protein levels,results showed that the compatibility of aconite with ginseng and aconite alone after administration,compared to treatment,can inhibit ANP and CaN protein expression,NFAT3 protein can antagonize into cells,the NFAT3 protein in the cytoplasm increased in the nucleus of NFAT3 protein decreased in the nucleus of GATA4 protein increased.When the ratio of aconite and ginseng was 1:1,the above results have significant difference.The experimental results indicate that the compatibility of ginseng and aconite seed is 1:1,which has the best protective effect on the heart,and can also inhibit the toxicity of monkshood when used alone.This paper explores the aconitine can cause disorders of calcium ion on myocardial cells,the effects of calcium homeostasis,regulation of calmodulin pathway CaN-NFAT3 ions play a role;at the same time reveal the ginsenoside Re can inhibit aconitine caused by calcium ion channel disorders and changes play antagonism.In animal levels,it is further confirmed that human involvement in the compatibility of aconite can protect the heart by regulating the CaN-NFAT3 ion pathway.This study provides evidence for the systematic study of the compatibility of Shenfu side and provides an experimental basis for the prevention and treatment of arrhythmia in Shenfu prescription.