CN113082190A

CN113082190A - Application of cardiac mixture in preparation of medicine for blocking DCM through DCM myocardial cell autophagy mechanism

Info

Publication number: CN113082190A
Application number: CN202110499735.3A
Authority: CN
Inventors: 宋春晖; 谢正侠; 李林; 龚志刚; 陈洪涛
Original assignee: Jiangxi Normal University
Current assignee: Jiangxi Normal University
Priority date: 2021-05-08
Filing date: 2021-05-08
Publication date: 2021-07-09

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a cardiac mixture in preparation of a drug for blocking DCM through a DCM myocardial cell autophagy mechanism.

Description

Application of cardiac mixture in preparation of medicine for blocking DCM through DCM myocardial cell autophagy mechanism

Technical Field

Background

Diabetic Cardiomyopathy (DCM) is one of the leading causes of death in diabetic patients, particularly in type 2 diabetic patients. The diabetic cardiomyopathy refers to changes such as microvascular injury in the myocardial wall, fibrosis around blood vessels and interstitium on the basis of metabolic disorder and microangiopathy, so that subclinical cardiac dysfunction appears, and finally, the diabetic patients progress to arrhythmia, cardiac origin restriction and heart failure, and even sudden death occurs to severe patients. Currently, diabetic cardiomyopathy has been demonstrated and considered to be an independent complication of diabetes. Some clinical data show that hyperglycemia promotes cardiovascular disease before diabetes is diagnosed, and diabetic patients have poor prognosis ability and extremely high fatality rate. Diabetic complications have increased rapidly in recent years and have become a worldwide public health problem that seriously threatens human health.

Diabetic cardiomyopathy is the most serious complication of diabetes and the mechanism is unknown. Hyperglycemia is one of the major causes of myocardial damage in diabetes. Recent literature indicates that myocardial damage caused by hyperglycemia and hyperlipidemia is related to autophagy level, and long-term chronic hyperglycemia and hyperlipidemia have significant damaging effects on the mitochondrial function of myocardial cells, namely, the conditions are called 'sugar toxicity' and 'lipid toxicity'. Mitochondria are the main energy supply site of cell activities, are the central station of substance metabolism and energy conversion, and all oxidative phosphorylation processes of organisms are carried out in mitochondria. Disorders of carbohydrate metabolism result in changes in the energy metabolic pathways in the heart, accumulation of lipid substances in the blood, rapid increase in the formation of peroxides and superoxides beyond the body's ability to clear, a series of reactions resulting from imbalance between oxidation and antioxidation in the body, often accompanied by increased production of ROS and/or impaired function of the antioxidant defense system, and excess ROS can induce opening of mitochondrial Membrane Permeability Transition Pore (MPTP), causing mitochondrial swelling, rupture, release of cytochrome c (eytochromic), destruction of mitochondrial structure, and mitochondrial damage. Mitochondria synthesize ATP by tricarboxylic acid cycle and oxidative phosphorylation to provide direct energy for life activities, and mitochondria are damaged and ATP synthesis is insufficient.

AMPK (adenosine monophosphate activated protein kinase) is widely present in eukaryotic cells and can regulate the decomposition and synthesis of carbohydrates, lipids and the like, and AMPK is a protein kinase which can sense energy states and regulate metabolism in cells and plays a role in autophagy regulation. As ATP synthesis decreases, AMP/ATP ratio increases and serine/threonine kinase LKB1 phosphorylates directly to activate AMPK, phosphorylates TSC2, and in turn inhibits mTORC1 activity, causing autophagy to occur. Also, AMPK can also be directly phosphorylated without TSC to inhibit mTORC1 complex subunit Raptor to enhance autophagy. During the autophagy of cardiac muscle cells, Beclin1 is an essential molecule for forming autophagosome, and serves as a molecular reaction 'platform', so that autophagy-related proteins are positioned in phagocytic vesicles and react with various proteins to regulate the formation and maturation of autophagosome. Cardiomyocyte autophagy is also controlled by autophagy-related genes Atg, Atg12-Atg5, Atg8 (tubulin-related protein light chain 3, LC3) are two protein binding systems, which are coupled to and interact with each other. LC3 is a homologous protein of Atg8, when autophagy occurs in cells, 22 amino acids are removed from the carbon terminal of Atg8 to form LC3-II, and the LC3-II is combined with cell membrane phospholipid, so that the process only occurs in newly generated autophagosomes, and the conversion rate of LC3-I to LC3-II is positively correlated with the autophagy activity to form autophagosomes. The pathophysiological mechanism of the development of DM (diabetes) into DCM is complex, and oxidative stress, mitochondrial damage, activation of cellular pathways, cardiomyocyte autophagy and its related target drug development are the current research hotspots. How to perfect the autophagy of the myocardial cells and the pathogenesis of DCM thereof has great significance for the treatment of DCM.

In the theory of traditional Chinese medicine, scholars believe that once cells start autophagy program, the cells must be caused by the fact that the body receives external pathogenic factors of external environment, or sudden stress is generated inside the body, such as iron-blood hypoxia, emotional stimulation, stress or crisis and other factors which need to be washed. The occurrence of autophagy is closely related to qi deficiency and phlegm stasis in traditional Chinese medicine. The middle-qi deficiency is consistent with the hunger state that the cell clip can supply ATP, amino look askance at and the like, for example, the Chinese medicine' valley is not entered, half day is qi deficiency, and day is few. The accumulation of pathological metabolites with excessive cellular microenvironment is closely related to the phlegm stasis of damp turbidity in traditional Chinese medicine. In the later stage of diabetic cardiomyopathy, excessive deposition of pathological metabolites is caused by relative or absolute deficiency of autophagy. The autophagy is consistent with the balance of yin and yang by removing and preventing pathological products accumulated in cells and reutilizing wastes, which is the function of 'refined qi' for promoting the conversion of wastes into energy in traditional Chinese medicine, and taking a way for abusing yang to eliminate qi, promoting circulation of water, eliminating phlegm and removing blood stasis to eliminate pathogenic qi. Therefore, the application considers that the yang warming and blood circulation promoting diuresis method can block the formation of DCM by coordinating the autophagy of the myocardial cells.

However, the prior art does not find out which specific drugs can block the formation of DCM, nor is the principle of blocking the formation of DCM clear.

Disclosure of Invention

In order to solve the technical problems, the invention provides application of a cardiac mixture in preparing a medicament for blocking DCM through a DCM myocardial cell autophagy mechanism.

The invention aims to provide an application of a cardiac mixture in preparing a medicament for blocking DCM through a DCM myocardial cell autophagy mechanism, wherein the cardiac mixture is prepared from the following components in parts by weight: 10-15g of prepared aconite accessory slice, 20-25g of prepared astragalus root, 10-15g of cassia twig, 20-25g of salvia miltiorrhiza, 10-15g of peach kernel, 10-15g of safflower, 20-25g of tuckahoe, 15-20g of motherwort, 10-15g of hiraute shiny bugleweed herb, 10-15g of cortex acanthopanacis, 10-15g of semen lepidii, 20-25g of north hawthorn, 20-25g of white paeony root, 6-12g of ginger and 10-18g of Chinese date.

Preferably, the use of the cardiac mixture in the preparation of a medicament for blocking DCM by a DCM cardiomyocyte autophagy mechanism is as follows: 10g of prepared aconite accessory slice, 20g of roasted astragalus, 10g of cassia twig, 20g of salvia miltiorrhiza, 10g of peach kernel, 10g of safflower, 20g of tuckahoe, 15g of motherwort, 10g of herba lycopi, 10g of cortex acanthopanacis, 10g of semen lepidii, 20g of north hawthorn, 20g of white paeony root, 6g of ginger and 15g of Chinese date.

Preferably, the application of the cardiac mixture in the preparation of the medicine for blocking DCM by the DCM myocardial cell autophagy mechanism is used for preparing the medicine for reducing blood sugar and triglyceride.

Preferably, the application of the cardiac mixture in the preparation of the medicine for blocking DCM by the DCM myocardial cell autophagy mechanism is used for preparing the medicine for reducing total cholesterol and low-density lipoprotein cholesterol.

Preferably, the application of the cardiac mixture in the preparation of the medicine for blocking DCM by the DCM myocardial cell autophagy mechanism is used for preparing the medicine for increasing high-density lipoprotein cholesterol.

Preferably, the application of the cardiac mixture in the preparation of the medicine for blocking DCM by the DCM myocardial cell autophagy mechanism is used for preparing the serum insulin increasing medicine.

Preferably, the application of the cardiac mixture in preparing the medicine for blocking DCM by the DCM myocardial cell autophagy mechanism is used for preparing AMPK, Beclin1, TSC2 and LC3-II expression promoters.

Preferably, the application of the cardiac mixture in the preparation of the medicine for blocking DCM by the DCM myocardial cell autophagy mechanism is used for preparing the RHEB and LC3-I, mTOR expression inhibitor.

Compared with the prior art, the invention has the following beneficial effects:

1. the cardiac mixture used in the present invention has been used clinically for over 20 years. In the theory of materia medica, Su Wen Zhi Zhen Yao Da Lun, the prescription has the effect of treating cold syndrome in the interior, with sweet and hot syndrome, bitter and pungent syndrome as well as salty and purging syndrome, pungent and moist syndrome and bitter and hard syndrome. Being predominant in the pathogenic cold, it is pungent and hot, bitter and sweet with salty flavor and purgative. The pure yang warm aconite root is used for greatly vibrating yang qi, the prepared astragalus root is used for tonifying qi and raising yang, and the two are combined to be monarch drug; ramulus Cinnamomi warms and releases qi to assist yang and transform qi; the red sage root, the peach kernel and the safflower have the functions of promoting blood circulation and removing blood stasis so as to promote new blood generation; motherwort and herba lycopi have the effects of promoting blood circulation and inducing diuresis; ting Li Zi, Fu Ling and Wu Jia Pi have the actions of inducing diuresis and excreting dampness, and they are also indicated for the actions of regulating and tonifying lung, spleen and kidney, which are closely related to water metabolism, and each of them is emphasized; the hawthorn can remove stasis and promote digestion, and strengthen stomach qi of middle jiao; the herbs with toxic heat property such as Bai Shao, Fu Zi and Gui Zhi can nourish the middle-jiao by sour-sweet and transforming yin; fresh ginger and Chinese date regulate the spleen and stomach. In the recipe, Fu Zi is combined with gan tonify and strengthen the body resistance of Huang Qi, which can strengthen the body resistance to control its toxicity. Radix Aconiti lateralis has effects of warming channels, strengthening yang, dispelling cold, relieving pain, and strengthening exterior; gui Zhi has the synergistic effect of warming yang, restoring qi, expelling pathogenic factors from muscles and skin, dredging meridians and warming yang, restoring menstrual flow and dispelling cold. Radix astragali is sweet and warm and has the effects of tonifying qi, and strengthening the exterior by excess defense; the pungent and warm-natured cassia twig, ramulus Cinnamomi, with the property of dispersing pathogenic heat and penetrating, reaches ying and wei, warms and unblocks meridians, helps yang and regulates qi, and runs transversely on the limbs to warm meridians: the two complement each other, and the functions of invigorating the vital function, dredging the channels and collaterals, treating both the principal and secondary aspects, eliminating pathogenic factors without damaging the vital qi, dredging the channels and collaterals and benefiting the blood and the pulse are achieved together. Gui Zhi warms and transforms water-dampness and warms qi of bladder, and is combined with Fu Ling to warm and activate spleen yang, transform dampness and induce diuresis to help qi transformation of bladder. Safflower is light in weight and long in floating, runs up externally, passes through meridians and collaterals, and is good at removing blood stasis in meridians and collaterals, while peach kernel is heavy in weight and descends, runs well in the interior and runs down the lower energizer, and is good at breaking blood stasis in viscera. The combination of the above herbs, including Zhenwu Tang, Wuling san, Ting Li Zi Zao Xie Fei Tang, is used as an agent for warming yang, activating blood and inducing diuresis.

2. The invention proves that the cardiotonic mixture prepared by the yang-warming, blood-activating and diuresis-promoting method has good clinical curative effect on DCM. According to the invention, a DCM rat model is constructed, the blood sugar and blood fat change of a rat is detected, the AMPK-mTOR signal pathway and the expression conditions of downstream mitochondrial autophagy specific proteins TSC2 and RHEB thereof, myocardial cell autophagy related proteins Beclin1 and LC3-I, LC3-II and the like are detected, and the heart-strengthening mixture is proved to play a role in blocking the DCM process by exciting the AMPK-mTOR mediated DCM myocardial cell autophagy mechanism, so that a new way is provided for the treatment of DCM.

Drawings

FIG. 1 shows the results of HE staining (400X);

A-F are samples of a normal control group, a DCM model group, a cardiac mixture high-dose group, a cardiac mixture medium-dose group, a cardiac mixture low-dose group and a captopril control group respectively;

FIG. 2 shows the observation results of a transmission electron microscope;

a-F are normal control (20000 ×), DCM model (20000 ×), cardiac cocktail high dose (20000 ×), cardiac cocktail medium dose (20000 ×), cardiac cocktail low dose (10000 ×), captopril control (20000 ×), respectively;

FIG. 3 shows the expression of AMPK protein by immunohistochemical staining (100 ×);

FIG. 4 is the expression of mTOR protein by immunohistochemical staining (100X);

FIG. 5 shows the expression of TSC2 protein by immunohistochemical staining (100X);

FIG. 6 shows the expression of RHEB protein by immunohistochemical staining (100X);

FIG. 7 shows the expression of Beclin1 protein by immunohistochemical staining (100 ×)

FIG. 8 shows the expression of LC3-I protein by immunohistochemical staining (100X);

FIG. 9 shows the expression of LC3-II protein by immunohistochemical staining (100X);

in FIGS. 3-9, A-F are normal control, DCM, cardiac mixture high dose, cardiac mixture medium dose, cardiac mixture low dose, and captopril control samples, respectively;

FIG. 10 is a Western Blot of rat heart tissue AMPK, LC3-I, LC 3-II;

FIG. 11 is a graph of Beclin1, mTOR, TSC2, Western Blot of rat heart tissue;

in fig. 10 and 11, 1 to 6 represent samples of the normal control group, DCM model group, cardiac mixture high dose group, cardiac mixture medium dose group, cardiac mixture low dose group, and captopril control group, respectively;

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present invention to be implemented, the present invention will be further described with reference to the following specific embodiments and accompanying drawings.

In the description of the present invention, reagents used are commercially available and methods used are conventional in the art, unless otherwise specified.

1 materials and methods

1.1 animals

120 Sprague-Dawley rats with the age of 8 weeks, half male and female and 140 +/-20 g of body weight are selected and purchased from the center of experimental animals of Jiangxi Master university, the qualification number of the experimental animals is SCXK gan 2009-. Raising in cages with 5 cages each. The room temperature is 18-22 ℃, and the relative humidity is 45-65%. The related guidelines of the management and protection of experimental animals of the university of Master and West in Jiangxi are obeyed in the process of word culture and experiment of the animals involved in the experiment.

1.2 model replication and animal grouping

After one week of adaptive feeding of 120 rats, 20 normal control groups were randomly assigned, and the normal control groups were given standard rat feed (purchased from the center of laboratory animals at Nanchang university) with caloric ratios of 60% carbohydrate caloric, 11% fat caloric and 29% protein caloric. The rest 100 rats in the experimental group are fed with high-fat and high-calorie feed, and the calorie and nutrient component ratio is as follows: carbohydrate calorie accounts for 40%, fat calorie accounts for 42%, and protein calorie accounts for 18%; the high-fat high-calorie feed contains 10% of lard stearin, 20% of cane sugar, 2.5% of cholesterol, 1% of cholate and 66.5% of conventional feed. Food intake and water intake were recorded daily, body weight was measured weekly, and bedding was changed. All rats had free access to food and water during the experiment.

After 8 weeks of feeding, the rats were fasted for 12 hours, and the experimental group rats were administered a single intraperitoneal injection of 30mg/kg Streptozotocin (STZ) dissolved in sterilized 0.1mmol/L citric acid buffer pH 4.2 at a concentration of 2% (w/v). Normal control group rats were given the same dose of 0.1mmol/L pH 4.2 citrate buffer for intraperitoneal injection. After 2 weeks, a glucose tolerance test is performed, glucose is perfused into stomach 2.0g/kg of body weight, blood is collected from inner canthus vein for 0.5-1m before perfusing stomach and for 120min before perfusing stomach, and blood sugar is measured. The diabetes model animal is the animal with fasting blood sugar of more than or equal to 7.8mmol/L and/or the animal with postprandial blood sugar of more than or equal to 11.1 mmol/L. After 2 weeks of adaptive feeding, rats successfully constructed for diabetes were randomly divided into a DCM model group, a cardiac cocktail high dose group, a medium dose group, a low dose group, and a captopril control group.

1.3 administration of drugs

Cardiac mixture: 10g of prepared aconite accessory slice, 20g of roasted astragalus, 10g of cassia twig, 20g of salvia miltiorrhiza, 10g of peach kernel, 10g of safflower, 20g of tuckahoe, 15g of motherwort, 10g of herba lycopi, 10g of cortex acanthopanacis, 10g of semen lepidii, 20g of north hawthorn, 20g of white paeony root, 10g of ginger and 5g of Chinese date.

The high, medium and low doses of cardiac mixture corresponding to the high, medium and low dose cardiac mixture groups were: the high dose of cardiac mixture is 3 g/kg body weight^-1·d^-1The dose of the cardiac mixture is 2 g/kg body weight^-1·d^-1The low dose of cardiac mixture is 1 g/kg body weight^-1·d^-1. The control group of western medicine captopril adopts captopril tablet with dosage of 4 mg/kg body weight^-1·d^-1. The normal control group and the DCM model group were drenched with the same amount of distilled water.

Each group was treated for 8 weeks.

1.4 specimen Collection

The weights were weighed once a week, and each group of animals was bled at the inner canthus vein at the end of the experiment at the end of 12 weeks. After 12 hours from the beginning of the night when food is forbidden, blood is collected from the canthus veins in the eyes, EDTA is used for anticoagulation, and serum is separated and used for measuring the fasting blood sugar, fasting serum insulin and triglyceride level.

The results are shown in the following table 1, and show that the blood sugar level of the DCM model group is obviously increased, and the fasting serum insulin level is obviously reduced; the cardiotonic treatment groups and the captopril treatment group can improve blood sugar and fasting serum insulin changes caused by DCM, and the curative effect of the cardiotonic high-dose group is superior to that of the western captopril group.

TABLE 1 blood glucose, fasting insulin and blood lipid levels of the groups

Note that P is < 0.01 compared with normal control group; comparison with DCM model set, P^△＜0.05，P^△△Less than 0.01; comparison with cardiotonic high dose group, P^☆＜0.05。

1.5 drugs and general Agents

Streptozotocin is produced by Sigma in the United states, and blood sugar (BG), Cholesterol (CH), Triglyceride (TG), High Density Lipoprotein (HDL) and Low Density Lipoprotein (LDL) determination kits are purchased from Shenzhen Merrill biomedical electronic shares, Inc.; the kit for measuring Lactate Dehydrogenase (LDH) and Creatine Kinase (CK) is a product of Nanjing institute of bioengineering. Anti- β -actin, AMPK, mTOR, TSC2, RHEB, Beclin1, LC3-I, LC3-II antibodies and corresponding secondary antibodies are all available from Sigma, USA. The captopril tablet can be obtained from Shanghai Shi Guibao pharmaceutical Co., Ltd.

1.6 primer nucleotide sequence

The primers are related to nucleotide sequences of beta-actin, AMPK, mTOR, Beclin1 and LC3 registered by GenBank, and are synthesized by Shanghai Biotechnology engineering service company Limited.

TABLE 2 primer nucleotide sequences

1.7 data statistics method

Statistical analysis was performed using SPSS17.0 software, and data were measured as mean + standard deviation (x + S).

2 results of the experiment

2.1 Biochemical index detection energy determination

Blood biochemical index determination: after blood is collected from the heart, Total Cholesterol (TC), Triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) are measured. The results are shown in the following table 3, and show that the cardiac mixture can inhibit the increase of TG, TC and LDL-C levels caused in a DCM model, can relieve the decrease of HDL-C level caused in the DCM model, and the curative effect of the cardiac mixture high-dose group is superior to that of the western medicine captopril.

TABLE 3 TG, TC, HDL-C, LDL-C levels for each group: (

mmol/L)

Group of	Number of examples	TG	TC	HDL-C	LDL-C
						Normal control group	30	0.72±0.28	0.88±0.23	1.55±0.15	0.38±0.68
DCM model group	24	1.49±0.65^**	1.75±0.34^**	0.59±0.16^**	0.84±0.20^**
						Cardiotonic high dose group	26	0.92±0.36^△☆	0.98±0.36^△☆	1.39±0.31^△☆	0.42±0.79^△☆
Cardiotonic medium dose group	25	1.12±0.33^*△	1.24±0.33^*△	1.11±0.22^*△	0.56±0.90^*△
						Cardiac mixture low dose group	25	1.36±0.85^*	1.56±0.47^*	0.70±0.36^*	0.78±0.11^*
Captopril control group	25	1.19±1.49^*△	1.23±0.30^*△	1.16±0.33^*△	0.52±0.14^*△

Note that P is compared with the normal control group^*＜0.05，P^**Less than 0.01; comparison with DCM model set, P^△Less than 0.05; compared to the cardiac cocktail low dose group: p^☆＜0.05。

2.2 histopathological examination

Taking out the myocardial specimen fixed by 10% neutral formalin solution for 24h, dehydrating, transparentizing, and paraffin embedding. Soaking the glass slide in a potassium dichromate concentrated sulfuric acid cleaning solution for 24h, sequentially washing the glass slide with tap water and distilled water, drying the glass slide at 37 ℃, and carrying out gelatinization treatment for later use. The embedded paraffin blocks are sliced continuously at the normal size of 4 mu m, fished and dried by a 60 ℃ chip drier for later use. Dewaxing the slices by 100% xylene for 10min multiplied by 2 times, rinsing the slices by absolute ethyl alcohol for 5min, sequentially soaking the slices by 95%, 80% and 70% alcohol for 3min, and rinsing the slices by PBS and distilled water for 2min multiplied by 3 times respectively; the tissue sections prepared above were subjected to conventional HE staining. The results are shown in fig. 1, and the HE staining results show that the normal control rats have normal cardiac muscle structure, regular arrangement of muscle fibers, uniform cytoplasm staining, uniform nucleus size, circular or elliptical shape and are positioned in the center of cells; the DCM model group shows that the arrangement of the myocardial fibers is disordered and irregular, the myocardial structures of the high-dose group and the medium-dose group of the cardiac mixture are improved, and the arrangement of the myocardial fibers is compact and regular. The arrangement of the myocardial fibers of rats in the western medicine captopril control group and the cardiac mixture low-dose group is irregular, and part of the myocardial fibers are broken.

2.3 Transmission Electron microscope observation of myocardial cell ultrastructure

Preparing an electron microscope specimen: firstly, fixing: rinsing with 0.2M PBS for 15min for 4 times, fixing with 1% 0504 for 1.5h, and rinsing with 0.2M PBS for 15 min; and (2) dehydrating: dehydrating in gradient acetone 50%, 70%, 90%, 100% for 15min × 2 respectively; (iii) infiltration: 100% acetone epoxy (1: I, v/v) for 2h, then 100% acetone: epoxy resin (1: I, v/v) for 2h, then pure epoxy resin for more than 3h, and the mixture can stay overnight; embedding: dripping 1 drop of epoxy resin to a plastic capsule, then picking a sample into the capsule bottom, inserting a label, filling the epoxy resin, adjusting the sample to a proper position by using a needle point, and putting the sample into a constant temperature box for polymerization for 12 hours at 60 ℃; slicing: semi-thin sections, 1.0-2.0mm thick, 2% toluidine blue staining, and under light microscopy, cardiomyocytes were identified. Sixthly, observing and photographing by a transmission electron microscope. The results are shown in FIG. 2. The microscopic observation of the myocardial ultrastructure shows that: the normal control group rat has normal myocardial cell ultrastructure, neat and compact arrangement of muscle fibers, complete intercalated disc structure, regular arrangement, clear muscle segments and abundant mitochondria; the DCM model group rat has disordered myocardial fiber arrangement, partial muscle fiber damage, irregular arrangement of muscle segments, invisible intercalated disc structure, damaged muscle segments, fewer mitochondria and cavities in cytoplasm. The cardiotonic agent has the advantages of orderly arrangement of myocardial fibers in a high-dose group, clear intercalated disc structure, abundant mitochondria and visible autophagosomes. The myocardial fibers of rats in the cardiotonic mixture are arranged neatly, occasionally the myocardial fibers are arranged loosely, mitochondria are rich, and a small amount of autophagosomes can be seen. The arrangement disorder of myocardial fibers, the destruction of sarcomere, the destruction of muscle fibers, fewer mitochondria, the destruction of part of mitochondria and the structure of part of area of rats in the cardiac mixture low-dose group and the captopril control group are proved. No autophagosomes were found.

2.4 immunohistochemical method for detecting the expression of AMPK, mTOR, TSC2, RHEB, Beclin1 and LC3-I, LC3-II proteins in myocardial tissues

Dewaxing and dehydrating the anti-drop slices by a conventional method; repairing at high temperature with 2% EDTA for 20min, cooling, and washing with PBS; 3% H₂O₂Blocking for 10min, and washing with PB 8; respectively dropwise adding AMPK, mTOR, TSC2, RHEB, Beclin1 and LC3-I, LC3-II polyclonal antibody (1:200, v/v); after 1h, washing with PBS; respectively dripping corresponding secondary antibodies, and washing with PBS (phosphate buffer solution) after 15 min; DAB color development; hematoxylin counterstaining, conventional dehydration, transparency, neutral gum sealing, and observation under light lens. Known positive sections were used as positive controls and PBS was used as a negative control instead of primary antibody. The staining results of AMPK, mTOR, TSC2, RHEB, Beclin1 and LC3-I, LC3-II are respectively shown in figures 3-9, wherein figure 3 shows that AMPK protein expression condition is observed in light brown in normal control group staining, light brown in DCM model group staining, small staining range, obviously increased interstitial space and cardiotonicThe group with high dose stained dark brown, the interstitial space was normal, the group with middle dose of cardiac mixture stained weakly positive, and the group with middle dose of cardiac mixture and the captopril control group stained light brown. FIG. 4 shows the mTOR protein expression, negative staining in the normal control group, dark brown staining in the DCM model group, light brown staining in the cardiac mixture high dose group, normal interstitial staining, light brown staining in the cardiac mixture medium dose group, smaller staining range, increased interstitial staining, light brown staining in the cardiac mixture low dose group and the captopril control group, and increased interstitial staining. FIG. 5 shows the TSC2 protein expression, which shows negative staining in the normal control group, light brown staining in the DCM model group, dark brown staining in the cardiac mixture high dose group, wide staining range and strong positive staining. The cardiac medium dose group stained dark brown, the captopril control group and the cardiac low dose group stained light brown, and the tissue gap of the captopril control group increased. FIG. 6 shows RHEB protein expression, negative staining in normal control group, dark brown staining in DCM model group, and obvious tissue gap broadening. Cardiotonic high dose groups stained negative. The cardiotonic medium-dose group stained light brown, the cardiotonic low-dose group stained slightly dark brown, the captopril group stained negatively, and interstitial spaces increased. FIG. 7 shows the Beclin1 protein expression, wherein the staining of the normal control group is weakly positive, the staining of the DCM model group is negative, the staining of the cardiac mixture high-dose group is dark brown, the staining range is wide, and the staining is strongly positive. The cardiac medium dose group stained slightly dark brown, the chartopril control and cardiac low dose group stained light brown. FIG. 8 shows the expression of LC3-I protein, which was negative in the normal control group, dark brown in the DCM model group, light brown in the cardiac mixture high dose group, normal in interstitial space, light brown in the cardiac mixture medium dose group, light brown in the cardiac mixture low dose group and captopril control group, and increased interstitial space in the cardiac mixture low dose group. FIG. 9 shows the expression of LC3-II protein, which shows negative staining in the normal control group, light brown staining in the DCM model group, dark brown staining in the cardiac mixture high dose group, wide staining range and strong positive staining. The cardiac medium dose group stained dark brown, the captopril control group and the cardiac low dose group stained light brown, and the interstitial space between the captopril groups increased.

2.5ELISA method for detecting serum AMPK (adenosine activated protein kinase, AMPK), TSC2, RHEB, Beclin1, LC3-I, LC3-II expression

3ml of peripheral blood is extracted at the end of 6 weeks after the treatment of the experimental rat, centrifuged at 3000r/min for 10min, and separated plasma is placed in a refrigerator with the temperature of 20 ℃ below zero for standby inspection. All samples were collected and assayed once by ELISA. The detection process is carried out according to the kit instructions to obtain the concentrations of AMPK, TSC2, RHEB, Beclin1 and LC3-I, LC 3-II.

The experimental operations are all strictly operated according to the kit specification and comprise the following steps:

taking out the enzyme label plate, and respectively adding 100 mul of standard substance into the blank micropores according to the sequence;

secondly, marking sample numbers respectively, and adding 100 mul of sample into a blank micropore;

thirdly, adding 50 mul of enzyme labeling solution into the standard sample hole and the sample hole;

fourthly, incubating and reacting for 60min at 37 ℃;

fifthly, washing the plate washer for 5 times, and standing for 10-20s each time;

sixthly, adding 50 mu l of substrate A, B solution into each hole;

seventhly, incubating for 15min at 37 ℃ in a dark place;

eighth, 50. mu.l of stop buffer was added to each well to stop the reaction.

Then the sample is placed into a microplate reader for automatic monitoring, data is read out, and the data is recorded and analyzed. The results are shown in the following table 4, and show that the DCM model groups AMPK, TSC2, Beclin1 and LC3-II are obviously increased in level, and RHEB and LC3-I are obviously reduced in level; cardiotonic and captopril-treated groups improved AMPK, TSC2, RHEB, Beclin1, LC3-I, LC3-II changes caused by DCM, and the cardiotonic high dose group was due to the western drug captopril. Wherein, three traditional Chinese medicines and one western medicine group can improve the expression quantity of Beclin1, which is beneficial to the treatment of the illness state of patients.

TABLE 4 levels of AMPK, TSC2, RHEB, Beclin1, LC3-I, LC3-II for each group (II) ((III))

ng/ml)

Group of	AMPK	TSC2	RHEB
				Normal control group	1.87±0.22	2.27±0.8	3.8±0.32
DCM model group	3.42±0.2*	3.13±1**	2.7±0.22*
				Cardiotonic high dose group	7.28±0.56*^※	9.41±1.12*^※	0.98±0.13*^※
Cardiotonic medium dose group	6.91±0.32*^※△	8.38±1.49*^※△	1.26±0.36*^※△
				Cardiac mixture low dose group	3.69±0.37*^#≠▲	3.47±0.78*^≠▲	2.51±0.42*^≠▲
Captopril control group	6.88±0.43*^※△	7.79±1.09*^※≠	1.31±0.13*^※△
				Group of	Beclin1	LC3-I	LC3-II
Normal control group	0.63±0.06	39.05±5.21	4.89±0.48
				DCM model group	0.93±0.07*	26.46±7.91*	8.18±0.28*
Cardiotonic high dose group	2.81±0.45*^※	12.29±6.18*^※	16.72±1.09*^※
				Cardiotonic medium dose group	2.44±0.39*^※△	15.11±5.06*^※	14.71±1.12*^※△
Cardiac mixture low dose group	1.23±0.47*^#≠▲	19.22±7.9*^△▲	9.61±3.01*^△▲
				Captopril control group	2.32±0.33*^※≠	15.24±5.42*^※△	13.84±1.14*^※△

Note: in the above table, P is < 0.01, compared to the normal control group; p < 0.05; comparison with DCM model set, P^※＜0.01，P^#Less than 0.05; comparison with cardiac Admixture high dose group P^△＜0.05，P^≠Less than 0.01; comparison with cardiotonic dose group, P^▲＜0.01。

2.6Western Blot to detect the content of AMPK, mTOR, TSC2, Beclin1 and LC3-I, LC3-II protein in myocardial tissues

Cell protein is extracted after cell lysis by cell protein lysate, and the protein content is determined by a Bio-Rad method. SDS-PAGE was performed on 30. mu.g of the protein, and the protein sample was transferred to a polyvinylidene fluoride (PVDF) membrane. After membrane transfer, the membrane is sealed by 5% skimmed milk powder for 1-1.5 h, washed by PBST for 3 times, and added with anti-beta-actin, AMPK, mTOR, TSC2, RHEB, Beclin1, LC3-I and LC3-II polyclonal antibodies for overnight at 4 ℃. Adding peroxidase-labeled corresponding secondary antibodies, decolorizing and shaking for 1h, and washing with PBST for 3 times, each for 5 min. DAB color development and gel imaging system imaging photo analysis. Meanwhile, beta-actin is used as an internal reference, and the experiment is repeated for 3 times. Electrophoretic band density value-grayscale × area/reference value. See table 5, table 6, fig. 10, fig. 11.

TABLE 5 relative expression of rat myocardium AMPK, LC3-I, LC3-II

Note: p < 0.05 compared to normal control group; comparison with DCM model set, P^#Less than 0.05; comparison with cardiac Admixture high dose group, P^△Less than 0.05; comparison with the captopril control group, P^▲＜0.05。

TABLE 6 relative expression levels of mTOR, TSC2, and Beclin1 in rat myocardium of each group

2.7Real-Time PCR detection of mRNA expression of AMPK, mTOR, Beclin1, LC3-I

1.0 cm. times.1.0 cm. times.0.5 cm of myocardial cell tissue was taken, placed in a 0.1% DEPC water-treated cryopreservation tube, and 1mL of TRIzol solution was quickly added. And sealing with a sealing film, and storing in liquid nitrogen.

Extracting total RNA and reverse transcription reaction, namely extracting the total RNA by a TRIZOL one-step method.

Reverse transcription reaction system: 2 μ g of total RNA, 2 μ L, dNTP (10mol/L)3 μ L, DTT (0.1 μmol/L)2 μ L of OligodT (0.5kg/L), 1 μ L, MMLV-RT (200U/μ L)1 μ L of RNase inhibitor (40U/μ L), 6 μ L of 5 XMMLV-RT Buffer, and adding DEPC-treated sterile water to 30 μ L; the reaction conditions are that the reaction is carried out for 1h at 45 ℃ and is stopped at 95 ℃ for 5 min.

Real-time PCR reaction 20. mu.L of PCR reaction system contained: 10 μ L of 2 XSSYBR Green PCR Master Mix, 5.4 μ L of RNase Free Water, 1.8 μ L each of upstream and downstream primers (0.05g/L), and 1 μ L each of cDNA; reaction conditions are as follows: pre-denaturation at 94 deg.C for 5 min; denaturation, at 95 ℃ for 30 s; annealing at 58 ℃ for 30s, extending at 72 ℃ for 30 s; a total of 40 cycles of amplification were performed and extension was carried out for 5min at 72 ℃. In the experiment, the quantitative detection of the reference gene beta-actin is synchronously carried out, and the relative concentration ratio of the target gene and the reference gene is taken as the relative expression quantity of the target gene. The amplification reaction is carried out on an ABI7000 fluorescence quantitative PCR instrument, and the fluorescence signal value of each sample is generated in real time and automatically calculated by ABIP prism 7000SDS software supporting the fluorescence quantitative PCR instrument.

Taking 10 mu L of PCR product, performing electrophoresis with 1.5% agarose gel (containing 5 mu g/ml ethidium bromide) and 5V/cm, taking 100bp Ladder as DNAmkers, taking 499, 462, 529, 271 and 300bp as positive results of beta-actin, AMPK, mTOR, Beclin1 and LC3-I, mRNA respectively, observing the results under an ultraviolet lamp, performing scanning analysis on a gel electrophoresis chart by using Alphalmager2200 analysis software, taking peak areas under curves as the content of the PCR product, and taking the ratio of the integral values of AMPK mRNA, mTORmRNA, Beclin1mRNA and LC3-I mRNA and the mRNA of the beta-actin to represent the mRNA expression level of the gene. See table 7. The result shows that the expression level of AMPK and Beclin1 in the DCM model group is obviously increased, and the level of mTOR and LC3-I is obviously reduced; cardiotonic and captopril-treated groups improved AMPK, mTOR, Beclin1, LC3-I changes caused by DCM.

TABLE 7 comparison of the expression levels of Beclin1mRNA, mTORmRNA, AMPKmRNA, LC3-ImRNA in rat myocardium

Note: p is < 0.01, compared with normal control group^★Less than 0.05; comparison with model groups, P^※＜0.01，P^#Less than 0.05; comparison with cardiotonic dose group P^△＜0.05，P^≠Less than 0.01; comparison with cardiotonic dose group, P^▲＜0.01。

It should be noted that, the connection relation of the components not specifically mentioned in the present invention is the default of the prior art, and the connection relation of the structures is not described in detail since it does not relate to the invention point and is a common application of the prior art.

It should be noted that, when the present invention relates to a numerical range, it should be understood that two endpoints of each numerical range and any value between the two endpoints can be selected, and since the steps and methods adopted are the same as those in the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Sequence listing

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Claims

1. Use of a cardiac cocktail for the manufacture of a medicament for blocking DCM by the autophagy mechanism of DCM cardiomyocytes, wherein the formulation of the cardiac cocktail is as follows: 10-15g of prepared aconite accessory slice, 20-25g of prepared astragalus root, 10-15g of cassia twig, 20-25g of salvia miltiorrhiza, 10-15g of peach kernel, 10-15g of safflower, 20-25g of tuckahoe, 15-20g of motherwort, 10-15g of hiraute shiny bugleweed herb, 10-15g of cortex acanthopanacis, 10-15g of semen lepidii, 20-25g of north hawthorn, 20-25g of white paeony root, 6-12g of ginger and 10-18g of Chinese date.

2. The use of a cardiac mixture as claimed in claim 1 for the manufacture of a medicament for blocking DCM by a DCM cardiomyocyte autophagy mechanism, the formulation of the cardiac mixture being as follows: 10g of prepared aconite accessory slice, 20g of roasted astragalus, 10g of cassia twig, 20g of salvia miltiorrhiza, 10g of peach kernel, 10g of safflower, 20g of tuckahoe, 15g of motherwort, 10g of herba lycopi, 10g of cortex acanthopanacis, 10g of semen lepidii, 20g of north hawthorn, 20g of white paeony root, 6g of ginger and 15g of Chinese date.

3. Use of a cardiac mixture as claimed in claim 2 for the manufacture of a medicament for blocking DCM by a DCM cardiomyocyte autophagy mechanism, wherein the cardiac mixture is used for the manufacture of a hypoglycemic, triglyceride-lowering medicament.

4. Use of a cardiac mixture as claimed in claim 3 for the manufacture of a medicament for blocking DCM by the mechanism of DCM cardiomyocyte autophagy, wherein the cardiac mixture is used for the manufacture of a medicament for lowering total cholesterol, lowering low density lipoprotein cholesterol.

5. Use of a cardiac mixture as claimed in claim 3 for the manufacture of a medicament for blocking DCM by the mechanism of DCM cardiomyocyte autophagy, wherein the cardiac mixture is used for the manufacture of a medicament for elevating high density lipoprotein cholesterol.

6. Use of a cardiac mixture as claimed in claim 2 for the manufacture of a medicament for blocking DCM by a DCM cardiomyocyte autophagy mechanism, wherein the cardiac mixture is used for the manufacture of a serum-increasing insulin medicament.

7. Use of a cardiac mixture as claimed in claim 2 for the preparation of a medicament for blocking DCM by DCM cardiomyocyte autophagy mechanism, wherein the cardiac mixture is used for the preparation of AMPK, Beclin1, TSC2, LC3-II expression promoters.

8. Use of a cardiac mixture as claimed in claim 2 for the preparation of a medicament for blocking DCM by DCM cardiomyocyte autophagy mechanism, wherein the cardiac mixture is used for the preparation of an inhibitor of RHEB, LC3-I, mTOR expression.