Effects of acupuncture combined with Kaijingtongmai Decoction on ATP sensitive potassium channel related proteins Kir6.1 and Kir6.2 in myocardial infarction rats

Kun Jian, Lan-Shi Li, Ying Wang

College of acupuncture and massage, Liaoning University of traditional Chinese medicine, Shenyang 110847, China

ABSTRACT Objective: To study the effect of combination of acupuncture and medicine on the expression of ATP sensitive potassium channel related proteins Kir6.1 and Kir6.2 in rats with myocardial infarction, and to study the possible mechanism of combination of acupuncture and medicine on the improvement of myocardial infarction, so as to provide experimental data basis for the development of new treatment methods for myocardial infarction. Methods: 65 healthy male SD rats were randomly selected as the control group. The other rats were fed with high-fat food for three weeks. The rats in the control group were injected with normal saline subcutaneously,and the other rats were injected with isoproterenol hydrochloride in the same way. Through ECG comparison, 40 successful Mi rats were randomly divided into model group, acupuncture group, western medicine group and acupuncture drug combination group, with 10 rats in each group. After the corresponding treatment, the ECG changes of rats in each group were observed, the pathological changes of rat cardiomyocytes were observed by HE staining, and the expression of ATP sensitive potassium channel (Kir6.1, Kir6.2) protein was detected by Western blot. Results: compared with the control group, 40 experimental specimens in the experimental group showed significant changes in cardiomyocyte protein The expression of Kir6.1 and Kir6.2 increased, and the difference was statistically significant. After treatment,compared with the model group, the protein expression of Kir6.1 and Kir6.2 in cardiomyocytes of Western medicine group, acupuncture group and acupuncture drug combination group showed a downward trend, among which the decline degree of acupuncture drug combination group was the most obvious, and the difference was statistically significant. The decline degree of acupuncture group and Western medicine group was not significant, and there was no significant difference Conclusion: acupuncture combined with medicine has a significant effect on improving myocardial infarction in rats, which may be related to the expression of ATP sensitive potassium channel related proteins Kir6.1 and Kir6.2 in rat cardiomyocytes.

1. Introduction

Acute myocardial infarction (AMI) is a myocardial necrotic disease caused by acute ischemia and hypoxia of coronary artery based on coronary atherosclerosis [1]. Acute ischemia and hypoxia of cardiomyocytes is the mechanism of AMI. Therefore, it is the center of treatment to effectively curb acute ischemia and hypoxia of cardiomyocytes. ATP sensitive potassium channel (KATP channel) It is an inward rectifier potassium channel affected by ATP concentration [2], which is called the terminal effect channel of myocardial ischemia, hypoxia ischemia-reperfusion injury and ischemic preconditioning. Now it has become a new research direction for the protection of cardiomyocytes [3]. KATP channel is composed of inward rectifier potassium channel subunit (Kir6.1/ Kir6.2) and sulfonylurea receptor subunit (SUR1 / sur2) is composed of two parts. Kir6.1 / Kir6.2 form a small hole on the channel, and SUR1 / sur2 plays a regulatory role from the side.

KATP channel is closed under physiological conditions, and will be activated and opened by stimulation in case of ischemia or necrosis,with a greatly increased expression, which strengthens the density of ikatp current and mediates the protection of myocardium [4].

In the previous related fields, the regulatory effect of acupuncture at Neiguan point on transient outward potassium channel and inward rectifier potassium channel related proteins has been confirmed[5-6]. This experiment will further explore the effect of acupuncture and drug combination on the expression of KATP channel related proteins (Kir6.1 and Kir6.2) in AMI rats, so as to provide experimental basis for clinical treatment of myocardial infarction.

2. Materials and methods

2.1 Experimental animals and groups

65 healthy male SD rats were purchased from Liaoning Changsheng Biotechnology Co., Ltd. [license No.: syxk (Liao) 2013-0009],body weight (200 ± 20) g. The ethical code of experimental animals was observed during the experimental operation. The ethical batch number was 21000092018010. All rats were adaptively fed in the animal feeding room under the same conditions of room temperature 23-25 ℃ and relative humidity 45-55%. After one week, 10 SD rats were randomly selected as the blank control group. The other rats were induced into myocardial infarction model by MI. 40 surviving rats were randomly divided into 4 groups, It includes model group,acupuncture group, western medicine group and acupuncture drug combination group.

2.2 Experimental drugs and instruments

Chloral hydrate (solai company t8590); isoproterenol hydrochloride(sigma company of the United States); normal saline (Chenxin Pharmaceutical Co., Ltd.); aspirin enteric coated tablets (Bayer, gyzz j20171021); isosorbide mononitrate (Lu Nan, gyzz h10940039).High fat feed [Ke'ao cooperation, license No.: scxk (Beijing)

2019-0001. The path of Tongmai Decoction was composed of centipede, Achyranthes bidentata, Poria cocos, leech, woodlouse worm, Dalbergia, Bombyx Silkworm, Salvia miltiorrhiza, Acorus calamus, Radix Ophiopogon, licorice, Ligusticum chuanxiong and Angelica sinensis. It was originated from the First Affiliated Hospital of Liaoning University of Traditional Chinese Medicine and made into decoction.

Biological function experiment system (taimeng technology,bl-420s); 6805-d electroacupuncture instrument (Shantou Medical Equipment Co., Ltd.); Huatuo brand acupuncture needle(specification: 0.18mm * 25MM) (Suzhou Medical Supplies Factory Co., Ltd.); - 80 ℃ ultra-low temperature refrigerator (Sanyo Electric Appliance Group of Japan); eps600 electrophoresis instrument(tanon); cryogenic centrifuge (jouan SA); multifunctional enzyme labeling instrument (TECAN); Ultramicro nucleic acid protein analyzer (biodrop), etc.

2.3 Model preparation

The rats in the control group were fed with ordinary diet (15g/ animal) for 3 weeks, and the other rats were fed with the same amount of high-fat food for 3 weeks. There was no limit on drinking water. ECG was recorded after 3 weeks. The rats in the control group were injected with normal saline (85mg / kg) at multiple points subcutaneously, and the rats in other groups were injected with a relatively equal amount of isoproterenol hydrochloride (85mg/ kg) in the same way, once every 24 hours, twice in total, and the ECG was recorded. When there are other arrhythmias such as R wave downward shift, ST segment elevation, pathological Q wave and QRS wave widening in ECG, the modeling can be considered successful [7].

2.4 Intervention methods

In the combined acupuncture and medicine group, Neiguan,Xinshu and Zusanli points were acupuncture once a day, and Xinjing Tongmai Decoction 19.35g / (kg·d) was administered once a day;In the acupuncture group, the same acupoints were punctured once a day; The western medicine group was given aspirin 9.00 mg / kg and isosorbide mononitrate 3.6 mg once a day; The control group and the model group were given the same amount of normal saline once a day. The acupoints used are located according to the rat acupuncture positioning method in experimental acupuncture [8], and the model is 0.18mm × 25 mm "Huatuo brand" acupuncture filiform needle was obliquely inserted into the subcutaneous 2 mm of each group of acupoints along the meridian. Connect 6805-d electroacupuncture instrument to apply density wave with the frequency of 2-20hz.Take the slight tremor of rat limbs as the degree, and keep the needle for 15min. Determine the intragastric dose of rats according to experimental zoology. Refer to the formula: adult one-day effective dose / adult body weight (calculated according to 70kg) * 6.3(conversion coefficient) = x (rat one-day effective dose) * rat body weight [9].

2.5 Sampling

After 15 days of intervention treatment, the experimental rats were killed by cutting off the spine the next day, the heart tissue was quickly separated and placed on an ice bag. After washing with normal saline, part of the apical tissue was cut, fixed with 4%paraformaldehyde for 1 day, and transferred to - 80 ℃ ultra-low temperature refrigerator for freezing.

2.6 index detection

2.6.1 ECG detectionECG was recorded before and after modeling and after intervention treatment. The rats were anesthetized with 10% chloral hydrate,and then fixed on the console in an abdominal upward posture.According to the connection mode of human standard lead (II),the positive electrode was connected to the left lower limb and the negative electrode was connected to the right upper limb. The ECG was recorded with the help of the biological function experimental system to observe the T-wave voltage change of the rats.

2.6.2 HE staining of myocardial tissue of rats in each groupCut 1cm ² The rat apical tissue was fixed with 4% paraformaldehyde for 1 day. It was embedded in paraffin and dewaxed with xylene for HE staining. Prepare four concentrations of alcohol: 70%, 80%,90%, 95% and 100%, conduct gradient dehydration step by step, and then shake it dry. The xylene is transparent and observed under the microscope.

2.6.3 Western blot analysisThe myocardium was smashed into a glass homogenate container and added 10% lysate. After being lysated, the centrifuge was centrifuged (4 12000r/min, centrifuge) and centrifugated (10min).The supernatant was taken in a total of BCA. The concentration was determined strictly according to the concentration of the protein kit, and the gel was electrophoretic. Wet transfer to the membrane and seal for 1H. Prepare the primary antibody, put the target protein together into the hybridization bag, shake in a 37 ℃ water bath for 1h, incubate at 4 ℃ overnight, wash the membrane with tbst for 3 times (5min / time), prepare the secondary antibody in the same way,and wash the membrane with tbst for 4 times (5min / time). The results were observed using tanon5200 chemiluminescence imager.

2.7 Statistical analysis

SPSS 24.0 software was used for statistical analysis. The measurement data were expressed in (` C ± s). When p < 0.05, the difference was statistically significant.

3. Results

3.1 Analysis of T wave voltage changes in ECG of rats

Intra group comparison: compared with that before modeling,the ECG T-wave voltage in the control group had no significant change (P > 0.05), and the ECG T-wave voltage in the other four groups decreased significantly (P < 0.05); Compared with that after modeling, the T-wave voltage in the control group, model group and acupuncture group had no significant change after treatment (P >0.05), but the T-wave voltage in the western medicine group and the acupuncture drug combination group increased significantly (P <0.05).

Comparison between groups: before modeling, there was no significant change in ECG T-wave voltage in each group (P >0.05); After modeling, compared with the control group, T wave voltage in model group, western medicine group, acupuncture group and acupuncture drug combination group decreased significantly (P < 0.05); Compared with the model group, there was no significant change in western medicine group, acupuncture group and acupuncture drug combination group (P > 0.05). After treatment, compared with the control group, there were significant differences among the model group, western medicine group and acupuncture group (P < 0.05), and there was no significant change in T-wave voltage in the acupuncture drug combination group(P > 0.05); Compared with the model group, T wave voltage in western medicine group, acupuncture group and acupuncture drug combination group increased significantly (P < 0.05); Compared with western medicine group, T wave voltage in acupuncture group and acupuncture drug combination group had no significant change(P > 0.05).

3.2 Morphological changes of myocardium in each group

As shown in Figure 1, he stained cardiomyocytes were observed by microscope. By comparing the cells of acupuncture group, western medicine group and acupuncture drug combination group with those of blank control group and model group, it was found that the myocardial tissue structure of blank group was normal, arranged neatly, closely and orderly, and there was no obvious degenerationand inflammatory cell infiltration in the tissue. However, the myocardial tissue structure of the model group was abnormal, a large number of cardiomyocytes were arranged irregularly and disorderly,some cardiomyocytes shrank significantly compared with the blank group, a small number of inflammatory cells infiltrated into the myocardial space, and some cardiomyocytes were hypertrophic. In the acupuncture group, the myocardial tissue structure was slightly abnormal, the arrangement of myocardial cells was relatively neat,and a small amount of inflammatory reaction cells could be seen in the myocardial space. The western medicine group was slightly abnormal, the myocardial cells in the visual field were cross cut, and the gap in some areas was large, which may be caused by sample preparation, and a small amount of connective tissue can be seen.Myocardial tissue contains parallel collagen fiber bundles and inflammatory factors. Compared with other groups, the myocardial tissue structure of rats in the acupuncture drug combination group was basically normal, the arrangement level was clear, and there was no obvious inflammatory cell infiltration.

Table 1 T wave voltage changes of rats in each group (x±s,mV)

Figure 1 morphological changes of myocardial tissue of rats in each group after he staining(× 200)

3.3 Comparison of the expression of KATP channel related proteins Kir6.1 and Kir6.2 in each group

The results of Western blot showed that compared with the control group, the expressions of KATP channel related proteins Kir6.1 and Kir6.2 in model group, western medicine group, acupuncture group and acupuncture drug combination group increased significantly (P< 0.05); Compared with the model group, the expression of Kir6.1 and Kir6.2 in acupuncture group, western medicine group and acupuncture drug combination group decreased significantly (P <0.05); Compared with western medicine group, the change of protein expression in acupuncture group was not significant (P > 0.05), but decreased significantly in acupuncture drug combination group (P <0.05). See Figure 2 and table 2:

Figure 2 Kir6.1 and Kir6.2 protein expression bands in myocardial tissue of rats in each group

4. Discussion

With the continuous improvement of people's living standard,the increase of high-fat food intake has caused the high incidence rate of cardiovascular diseases. It has become one of the three fatal diseases in the world. Therefore, it is of great clinical significance to find effective treatment for AMI. [10] ATP is the most effective endogenous blocker of KATP channel [11]. In most cases, there is enough ATP content in cells to bind to the corresponding sites on KATP channel to achieve saturation, so as to close KATP channel [12]. Under the condition of myocardial ischemia, ATP content decreased compared with physiological state, ATP / ADP ratio decreased, ATP binding on KATP channel was insufficient,KATP channel was activated and opened, K + outflow increased,cardiomyocyte repolarization or hyperpolarization, and action potential time decreased. At the same time, Na + - Ca2 + channel is inhibited and the concentration of Ca2 + in cells decreases, resultingin decreased contractility and oxygen consumption, which protects myocardium [13]. The results showed that the protein expression of Kir6.1 and Kir6.2 changed significantly in AMI model, and its mechanism may be related to the opening of ATP sensitive potassium channel.

Table 2 Changes of Kir6.1 and Kir6.2 protein expression in myocardial tissue of rats in each group(x±s)

The TCM disease of AMI is called "true heartache" [14], and the syndrome belongs to the combination of phlegm and blood stasis.Due to the loss of healthy movement of the spleen and the decline of gastric harmony, phlegm is endogenous, resulting in meridian stagnation, suffocation and pain in the heart and chest. The treatment principle is to remove blood stasis and phlegm and dredge the blocked Qi in the chest. In this experiment, bilateral Neiguan point,Zusanli point and Xinshu point were selected as three stimulation points, combined with Yijing Tongmai Decoction prepared by Professor Wang Ying. Xinjing Tongmai Decoction takes Salvia miltiorrhiza as its king, promoting blood circulation, removing blood stasis and relieving pain, which can be used for first aid in case of crisis. Professor Wang Ying takes leeches, centipedes and other "sentient things" as his officials. He can search, pick and pull them out, and reach the whole body. The prescription is combined with Ophiopogon japonicus to nourish yin, generate fluid and moisten dryness, so as to restrict the severity and dryness of insect drugs. With Ligusticum chuanxiong and Dalbergia odorifera as the assistant, one rise and one fall, Xuanchang Qi machine can regulate qi, activate blood circulation and relieve pain. Acorus tatarinowii and poria cocos can strengthen the spleen, remove phlegm and open the orifices. It is reported that Salvia miltiorrhiza has the effective pharmacological effect of vasodilation, so as to enhance blood circulation [15]. Four insect drugs can reduce blood viscosity and reduce total cholesterol and triacylglycerol to the standard range [16].Ophiopogon japonicus can enhance the contraction of myocardial fiber, improve cardiac output and reduce myocardial oxygen consumption [17]. Achyranthes bidentata is a recognized drug for nervous system protection and effectively control the infarct area,Reduce cell withering, suppress inflammatory response and improve myocardial function injury [18].

"Acupuncture and moxibustion A and B classics" contains: "the heart is calm and good, panic, sadness, and the master of internal customs.". Neiguan, as the collateral point and eight vessel intersection point of the hand Jueyin pericardial meridian, is the first choice for the treatment of heart disease [19], which is similar to the effects of Salvia miltiorrhiza, Angelica sinensis, Dalbergia odorifera and Ligusticum chuanxiong in Xinjing Tongmai Decoction. It has the functions of calming the mind, regulating qi, activating blood circulation and relieving pain. Xinshu point is the back Shu point of the heart. It is a place where heart qi gathers. It can directly regulate the patient's heart function, nourish blood and calm the heart,cooperate with the use of insect drugs, not only activate blood and blood, but also dredge meridians and collaterals. According to Yu Long Fu, "heart palpitation is weak and annoying, and it stabs three li". Zusanli, as a combined point under the stomach, has the effect of supporting righteousness and removing evil. It can regulate the spleen and stomach, activate meridians, remove phlegm, remove arthralgia and relieve pain [20]. It helps to properly raise and lower the Qi mechanism of the whole body, and synergistically enhance the effects of Neiguan, Xinshu and decoction. Modern social medical research has proved that acupuncture at Zusanli can not only improve the activities of various gastric digestive enzymes, but also increase blood volume and promote blood circulation [21]. Du Huijing et al. [22] found that electroacupuncture at Neiguan and Xinshu points can regulate the release of cytokines, growth factors and inflammatory factors in myocardial tissue and regulate the microenvironment around tissue, so as to protect myocardial tissue in ischemic infarct area and improve cardiac function. The three points are shared, promoting blood circulation and removing blood stasis,harmonizing the stomach and resolving phlegm, dredging collaterals and relieving pain.

Acupuncture and medicine must be combined to treat, and play the work of "promoting blood circulation and removing blood stasis,resolving phlegm and dredging collaterals". The synergistic effect of acupuncture and medicine can enhance the function and relieve the chest tightness and pain symptoms of myocardial infarction.Therefore, this experimental study shows that acupuncture combined with medicine, simple acupuncture and clinical conventional western medicine can effectively improve the state of MI rats, but the curative effect of acupuncture combined with medicine is the best.The mechanism may be realized by reducing the protein expression of Kir6.1 and Kir6.2 and affecting the function of ATP sensitive potassium channel. Kir6.1 and Kir6.2 are expressed in the heart, It shows that it may have some effects on cardiac function, but there are still few studies on it, and its specific mechanism needs to be further studied.