Effects Of Dingjifumai Decoction On Electrocardiogram And Sodium Potassium Pump Of Rats With Ventricular Arrhythmia

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Ling Fu, Hui-Ling Liao, Yan Zhou, Fei-Hu Zou, Ling Luo

1 Chongqing Traditional Chinese Medicine Hospital, Chongqing, China.

2 Southwest Medical University, Luzhou, Sichuan, China. 3Traditional Chinese Medicine Hospital, TongLiang, Chongqing, China.

Highlights

In this study, the effects of Dingjifumai Decoction and cistanche (DJFM) on Electrocardiogram and sodium-potassium pump of rats with ventricular arrhythmia were observed, and the effects and mechanism of DJFM on ventricular arrhythmia were discussed. We found that DJFM can prolong the occurrence time of cardiac arrhythmias caused by aconitine in rats, such as VP, VT, VF, et al. The mechanism may be related to fast Na+ channel, and it may prevent and control arrhythmias by inhibiting Na+ influx and reducing the fast response cellular self-discipline. DJFM can protect the myocardial tissue sodium-potassium pump, which can protect the myocardial cells and improve the myocardial metabolism.

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Abstract

Objective: To investigate the effect of Dingjifumai Decoctionand cistanche(DJFM) on Electrocardiogram (ECG) and sodium-potassium pump in rats with ventricular arrhythmia. Methods: Forty healthy male SD rats (200  20g) were randomly divided into blank group, model group, Metoprolol group, and DJFM group. Ten rats in each group were fed with a normal diet and free drinking water. Each group was given gavage, and the amount of gavage in each group was calculated according to body weight. In the model group, 0.001% Aconitine was injected into the tail vein at 30ug/kg. In the Metoprolol group, Metoprolol suspension was given according to the standard of 5.2mg/kg per day. In the DJFM group, DJFM was given at 17.6g/kg per day. After 2 weeks of administration, the biologic experiment system BL-420F was used to monitor the II lead ECG curve, and the ECG changes were observed and recorded. Then, the left ventricle of the rat was taken, and part of the heart tissue sodium-potassium pump was detected. Results: (1) The effect of DJFM(Dingjifumai Decoction and cistanche) on ECG of rats with ventricular arrhythmia: After intravenous injection of aconitine, the incidence of Ventricular Premature beat (VP), Ventricular Tachycardia (VT), Ventricular Fibrillation (VF) in the model group was 100%, suggesting that the model building of rats with ventricular arrhythmia was successful. (2) VP, VT, and VF time: Compared with the model group, the DJFM group, and Metoprolol group can significantly delay the VP, VT, and VF, the difference was statistically significant (P < 0.05). The effect of the DJFM group and Metoprolol group on delaying the appearance of VP, VT and VF was the same, there was no significant difference (P > 0.05). (3) The effect of DJFM on the sodium-potassium pump in rat ventricular arrhythmia heart tissues: Compared with the blank group, the sodium-potassium pump value in the model group was significantly decreased, and the difference was statistically significant (P < 0.05). Compared with the model group, the sodium-potassium pump value of the tissues in the Metoprolol group and the DJFM group increased, and the difference was statistically significant (P < 0.05). There was no significant difference in the sodium-potassium pump between the Metoprolol group and the DJFM group (P > 0.05). Conclusion: 1. The rat model of ventricular arrhythmia can be successfully prepared by intravenous injection of Aconitine. 2. DJFM(Dingjifumai Decoction and cistanche) can prolong the occurrence time of cardiac arrhythmias caused by aconitine in rats, such as VP, VT, VF, et al. The mechanism may be related to fast Na+ channel, and it may prevent and control arrhythmias by inhibiting Na+ influx and reducing the fast response cellular self-discipline. 3. DJFM(Dingjifumai Decoctionand cistanche) can protect the myocardial tissue sodium-potassium pump, which can protect the myocardial cells and improve the myocardial metabolism.

Keywords: Dingjifumai Decoction, Ventricular Arrhythmia, Rats, Electrocardiogram, sodium-potassium pump

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Background

Ventricular Arrhythmia (VA) is a type of the common clinical cardiovascular disease emergency, Ventricular Premature beat (VP), Ventricular Tachycardia (VT), Ventricular Fibrillation (VF) and others all belong to VA [1] the category, Common causes include various organic heart diseases, diseases other than cardiac causes, electrolyte acid-base balance disorders, etc. [2]. The unpredictability and potentially catastrophic consequences of VA are forcing doctors to seek treatment to prevent and treat VA. At present, the four types of anti-arrhythmic drugs commonly used in clinical practice have significant side effects causing new arrhythmias, while traditional Chinese medicine has fewer side effects and stable efficacy, and can reduce the side effects of western medicine anti-arrhythmias [3]. Therefore, it is of great significance to develop effective Chinese herbal preparations for the prevention and treatment of ventricular arrhythmia. In the clinical diagnosis and treatment, it was found that (Dingjifumai Decoctionand cistanche) (DJFM) had a better effect in anti-ventricular arrhythmia, so this experiment was designed for research.

Materials and methods

Experimental materials and instruments

Experimental animals Forty healthy male SD rats of clean grade, weighting (200  20) g (provided by Animal Experiment Center of Southwest Medical University, license number: SCXK (Chuan) 2013-17). Grouping and processing The rats were weighed and given adaptive feeding for 3 days, and then randomly divided into 4 groups and numbered, 10 rats in each group, respectively the blank group (normal saline 2ml/200g), model group (normal saline 2ml/200g), Metoprolol group and (Dingjifumai Decoctionand cistanche) group. The Metoprolol group was administered a Metoprolol suspension at a daily dose of 5.2mg/kg. DJFM is composed of Suanzaoren (Ziziphus jujuba Mill.var.Spinosa), Fusion (Poria cocos Schw.), Long gu (OsDraconis), Muli (Oyster), Guizhi (CassiaTwig), Yuanzhi (Polygala tenuifolia Willd.), Dazao (Ziziphus zizyphus), Hehuanpi (Silk tree Albizia Bark), Zhigancao (Glycyrrhizae.), et al. It is purchased from Sichuan New Green Pharmaceutical Technology Development Co. LTD. Each rat was gavaged once a day with a dose of 17.6 g/kg per day for a total of 14 days.

Model establishment

The rats were weighed before the last gavage on 14 days. Ten rats in each group were injected with 1% pentobarbital sodium at 30mg/kg and anesthetized. The anesthetized rats were placed in a prone position on a wooden board. Insert the acupuncture needle into the rat, connect the electrode and the electrocardiogram (ECG) [12]. Tracing the II lead ECG, recording the normal ECG, zeroing THE baseline, waiting for the rat’s ECG to stabilize, the model group immediately injected 0.001% Aconitine 30 ug/kg solution through the tail vein, and the blank group injected the volume 0.9% saline. The bolus is completed within 5 seconds, and began to record ECG for 20 minutes, recording the VP, VT, and VF in time. After the observation, cut the chest skin and frame with scissors, immediately open the chest, cut all blood vessels connected to the heart tissue eat the bottom of the heart and take out the whole heart, squeeze out the blood in the heart cavity, and dry the residual blood on the surface of the heart gauze. After that, the left ventricle cut was excised, and a part of the cut was placed in a cryotube, stored in a - 80 C refrigerator, and the heart tissue sodium-potassium pump detection was performed according to the kit instruction.

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Results

Model establishment

In this study, aconitine was not used to induce arrhythmias in the blank group, so VP, VT, VF, and other VA did not occur in the blank group, so the pair-based comparison of experimental results did not involve the blank group. After intravenous injection of Aconitine, the incidence of VP, VT, and VF in the model group was 100%, indicating that the VA rat model was successfully constructed. (Figure 1)

The effects of DJFM(Dingjifumai Decoctionand cistanche) on ECG of rats with VA

① The incidence of VP: Compared with the model group, the time of VP was significantly delayed in the DJFM(Dingjifumai Decoction and cistanche) group and the metoprolol group, and the difference was statistically significant (P < 0.05), while the difference between the Metoprolol group and DJFM group was not statistically significant (P > 0.05). ② Onset time of VT: Compared with the model group, the onset time of VT was significantly delayed in the DJFM group and the Metoprolol group, and the difference was statistically significant (P < 0.05), while the difference between the Metoprolol group and DJFM group was not statistically significant (P > 0.05). ③ Time of occurrence of VF: Compared with the model group, the time of occurrence of VF was significantly delayed in the DJFM group and the Metoprolol group, and the difference was statistically significant (P < 0.05), while the difference between the metoprolol group and DJFM group was not statistically significant (P > 0.05). (Table 1)

Effect of DJFM(Dingjifumai Decoction and cistanche) on the sodium-potassium pump in VA heart tissue of rats

Compared with the blank group, the sodium-potassium pump value in the model group was significantly decreased, and the difference was statistically significant (P < 0.05). Compared with the model group, the sodium-potassium pump value of the tissues in the Metoprolol group and the DJFM(Dingjifumai Decoction and cistanche) group increased, and the difference was statistically significant (P < 0.05). There was no significant difference in the sodium-potassium pump between the metoprolol group and the dDJFM group (P>0.05). (Table 2, Figure 2).

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Discussion

The Animal models of VA are mainly made by rabbits, guinea pigs, rats, mice, cats, dogs or goats, et al. Rats are easy to obtain and widely used, with physiological and anatomical structures similar to humans, so they are widely used in VA models [4]. The establishment of the VA model has several methods such as drug-induced, electrical stimulation-induced, coronary artery ligation, and stress-induced VA. The drug-induced method is easier operation, controllable condition, stability, relatively short building period, and high success rate. So this experiment method is induced by drugs.

Aconitine is a well-known alkaloid causing arrhythmia. Its mechanism of action to induce ventricular arrhythmia is able to make the myocardial cell membrane of the high voltage-gated sodium channels be trigger activation. After activation of the Na + channel, it is in an open state, and the influx of Na+ in myocardial cell increases, which accelerates the depolarization of the cell membrane, thus increasing the self-discipline of a pacemaker, which induces ectopic rhythm point [4-6]. Aconitine can also increase Ca2+ in myocardial cells by increasing the expression of ICa-L, induced intracellular Ca2+ elevation triggering activity, delayed depolarization [8]. It can improve the self-regulation of fast-responding cells, thus forming one source or multiple sources of heterotopic rhythm, shortening the refractory period of the myocardium and leading to arrhythmia.

Myocardial cells almost all rely on aerobic metabolism. Sodium potassium pump is a biofilm enzyme system ubiquitous on the cell membrane of the body [8-10]. Its main function is to transport Na+ and K+ across the membrane. It can at resisting electrochemical gradient on either side of the cell membrane while hydrolyzing ATP and keep the ion concentration difference inside and outside the membrane stable, keeping the balance of electrolyte and fluid, maintaining the activity of the sodium-potassium pump is beneficial to the normal physiological function of cells. Myocardial membrane Sodium potassium pump is also a cardiac glycoside receptor, which plays a regulatory role in the metabolism and function of the heart, and improves the ability of anti-arrhythmia from multiple perspectives. Sodium potassium pump can hydrolyze ATP to release energy and realize the continuous activity of the sodium-potassium pump through the reverse electrochemical gradient. Only maintaining the activity of the sodium-potassium pump can maintain the difference in ion concentration and ensure the normal electrical activity of tissue cells. When myocardial cell sodium-potassium pump is inhibited, a series of changes in ion flow will occur, making extracellular Na+ flow into the cell, and intracellular Na+ increase indirectly activates Na+-Ca2+ exchange protein, making intracellular Ca2+ excessive and causing Ca2+ overload to damage myocardial contractility [11], thus resulting in ventricular arrhythmia. Therefore, the content of the sodium-potassium pumps in cardiac myocytes is an important indicator to monitor the occurrence of ventricular arrhythmias.

DJFM(Dingjifumai Decoction and cistanche) consists of ten kinds of Chinese herbal medicines such as Suanzaoren, Fushen, Chuanxiong, Longgu, Muli, Guizhi, Yuanzhi, Dazao, Hehuanpi, Zhigancao. This prescription mainly uses methods of enriching blood, nourishing blood, tranquilization, and nourishing Yin. In this prescription, Suanzaoren plays the role of the monarch in traditional Chinese medicine formulation and has flavors of sweet and sour. It acts on the heart meridian and liver meridian, and has functions of nourishing heart yin, supplementing liver blood, and tranquilizing the mind. Fushen plays the role of the minister in traditional Chinese medicine formulation and has the nature of calm and flavors of sweet and tasteless. It acts on the heart meridian and spleen meridian and has the function of supplementing the heart and spleen and tranquilizing the mind. Chuang Xiong plays the role of the assistant in traditional Chinese medicine formulation and has the function of tonifying Qi and activating blood. Guizhi plays the role of the assistant in traditional Chinese medicine formulation and has functions of warming the heart-Yang, promoting heart-blood circulation, warming the spleen and stomach, and improving symptoms such as palpitation. The functions of compatibility of medicines between Chuangxiong, Guizhi, and Suanzaoren are nourishing blood, activating blood, and improving the ability of integration of enriching blood and promoting blood circulation through combining of acrid taste for dispersing, pungent-warm and sour for astringbg. Zhiyuan plays the role of the assistant in traditional Chinese medicine formulation and has the function of tranquilizing the mind and eliminating phlegm. Hehuanpi plays the role of the assistant in traditional Chinese medicine formulation and has functions of tranquilizing the mind and harmonizing qi and blood. Longgu and Muli play the role of the assistant in traditional Chinese medicine formulation and have functions of tranquilizing, invigorating Yin, and conferencing dissipation of heart Qi. Separately, Longgu has the effect of tranquilizing on the liver meridian and Muli has the effect of anchoring the mind on the lung meridian. Zhigancao and Suanzaoren play the role of the guide in traditional Chinese medicine formulation. Baked Licorice has the effect of Yin-enriching, Qi-boosting, dredging Yang, and recovering the pulse. Fructus Ziziphi Jujubae has the effect of invigorating the spleen to replenish Qi, nourishing blood, and tranquilizing the mind.

This prescription combines Suanzaoren with Longgu, Muli, et al. for the purpose of increasing synergy and curative effect, avoiding the disadvantages of ineffective efficacy and unstable efficacy of single medicinal materials. This prescription combines these medicines has the effect of enriching blood, nourishing blood, tranquilizing the mind, and nourishing Yin. Clinical studies suggest that Suanzaoren’s nature is calm, flavors are sour and sweet, and the main components of Wild jujube are saponins, flavonoids, alkaloids, and the like. Related studies have shown that Suanzaoren has sedative, anticonvulsant, anti-arrhythmia, anti-atherosclerosis, improved microcirculation, anti-ischemia, and immune enhancement, and has a positive cardiovascular pharmacological effect [12]. Huang Yisheng et al. confirmed that jujube saponin A can inhibit the decrease of Bcl2 and Bax expression in rat myocardial tissue after reperfusion injury, and can resist arrhythmia by significantly reducing serum LDH and myocardial MDA content [13-14], increasing myocardial SOD activity and the ability to resist oxygen-free radicals. The Longgu and Muli have good sedative, hypnotic and anticonvulsant effects [15].

Oxygen-free radicals may play an important role in the induction of potentially fatal arrhythmias, and Muli aqueous extracts can enhance the activity of SOD and reduce the content of MDA [16], thus playing a role in the prevention and treatment of ventricular arrhythmias. Fushen has a variety of biologically active ingredients such as Lycium polysaccharides, tetracyclic triterpenoids, et al. It has the functions of calming hypnosis, nourishing the heart, calming the nerves, resisting tumors, enhancing immunity, and lowering blood glucose. Lin Xiaoming et al. confirmed that Fuling can enhance SOD activity [17], inhibit MDA production, and have the effect of scavenging oxygen free radicals. The main chemical components of Guizhi are volatile oils, phenols, organic acids, et al. Guizhi has the functions of dilating blood vessels, anti-oxidation, lowering blood lipid, lowering blood pressure, promoting blood circulation, and anti-platelet aggregation, anti-inflammatory and anti-allergic effects [18]. For example, cinnamaldehyde can dilate blood vessels, thereby enhancing blood circulation [19-20], increasing coronary blood flow, enhancing myocardial contractility, and slow heart rate abnormalities, thereby maintaining the normal function of the heart; such as Guizhi liquid extracted by hydro alcohol method can reduce blood lipids [21], improve myocardial ischemia and reduce the incidence of arrhythmia in rats with coronary heart disease.

Licorice mainly contains triterpenoids, flavonoids, alkaloids, organic acids, licorice polysaccharides, and other chemical components. The most important active substances are triterpenoid saponins and flavonoids. Licorice has many effects such as anti-oxidation, lowering blood lipids and anti-atherosclerosis, anti-arrhythmia, anti-myocardial ischemia, and immune regulation [22-23]. Many studies have confirmed that Zhigancao can exert anti-arrhythmia effects in various ways. Xie Shirong et al. found that glycyrrhetinic acid can inhibit aconitine and coronary artery ligation-induced reperfusion ventricular arrhythmia [23]. Total flavonoids from licorice can delay the occurrence of arrhythmia in aconitine model rats and reduce the incidence of ventricular fibrillation in chloroform model rats, indicating that it has an anti-ventricular arrhythmia effect [25].

The results of this study suggest that compared with the model group, the DJFM(Dingjifumai Decoction and cistanche) group, and metoprolol group can significantly delay the occurrence of VP, VT, and VF, the difference is statistically significant (P < 0.05). The DJFM group and the metoprolol group had the same effect in delaying the appearance of VP, VT, and VF, and there was no significant difference (P > 0.05). It is indicated that DJFM can prolong the occurrence of ventricular arrhythmias such as VP, VT, and VF in aconitine model rats. The mechanism may be related to the inward rectifier potassium channel (IK+ ), which can prevent arrhythmia by promoting K+ efflux to reduce intracellular positive potential and reduce self-discipline. On the other hand, it may also be related to fast Na+ channels, which may reduce the self-discipline of fast-reacting cells by inhibiting Na+ influx, thereby achieving the effect of preventing and treating arrhythmia. It is indicated that DJFM may have the function of preventing and treating ventricular arrhythmia models of different ion channels.

The experiment also found that the sodium-potassium pump in the tissues of the model group was significantly lower than the blank group, and the difference was statistically significant (P < 0.05). The metoprolol group and DJFM(Dingjifumai Decoction and cistanche) group can increase the sodium-potassium pump in the tissues of rats with ventricular arrhythmia (P < 0.05). There was no significant difference in the sodium-potassium pump between the DJFM group and the metoprolol group (P > 0.05). It was concluded that DJFM can protect the sodium-potassium pumps. The mechanism may be to reduce intracellular Ca2+ concentration indirectly by reducing intracellular Na+ concentration, thereby preventing myocardial contractility damage caused by Ca2+ concentration overload, and thus preventing ventricular arrhythmia.

In summary, the intravenous injection of aconitine can successfully prepare a rat model of ventricular arrhythmia. DJFM(Dingjifumai Decoction and cistanche) is similar to metoprolol in preventing and treating arrhythmia. The mechanism may be related to the protection of myocardial tissue sodium-potassium pump, and the inhibition of Na+ influx reduces the self-discipline of fast-reacting cells, thereby protecting the cardiomyocytes and improving myocardial metabolism. However, there are few side effects of traditional Chinese medicine. If the syndrome type is consistent, there is no obvious contraindication. Therefore, we should promote the use of traditional Chinese medicine in clinical practice and provide treatment services for more patients with arrhythmia.

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