CN111617088A

CN111617088A - Pharmaceutical composition for treating myocardial injury caused by Kawasaki disease and application thereof

Info

Publication number: CN111617088A
Application number: CN202010667169.8A
Authority: CN
Inventors: 魏兵; 齐双辉
Original assignee: General Hospital of Shenyang Military Region
Current assignee: General Hospital of Shenyang Military Region; General Hospital of Northern Theater Command of PLA
Priority date: 2020-07-13
Filing date: 2020-07-13
Publication date: 2020-09-04

Abstract

The invention relates to the technical field of medicines, and particularly relates to a medicinal composition for treating myocardial damage caused by Kawasaki disease, which comprises tanshinone IIA and salvianolic acid B, wherein the tanshinone IIA and the salvianolic acid B are mixed in a weight ratio of 1: 10-10: 1. The purity of the tanshinone IIA is more than or equal to 98 percent; the purity of the salvianolic acid B is more than or equal to 98 percent. The application of the pharmaceutical composition for treating the myocardial damage of Kawasaki disease in preventing or treating the myocardial damage diseases. The myocardial damage is myocardial damage caused by Kawasaki disease. Experimental results show that compared with a model group and a positive medicine group, the tanshinone IIA + salvianolic acid B group (1:5) compatible composition can obviously reduce the activity of CK, CK-MB, LDH, alpha-HBDH and AST, the difference has statistical significance (P is less than 0.05), and the tanshinone IIA + salvianolic acid B group (1:5) compatible composition can reduce the activity of a myocardial zymogram and obviously improve the damage condition of myocardial tissues of KD mice.

Description

Pharmaceutical composition for treating myocardial injury caused by Kawasaki disease and application thereof

Technical Field

The invention belongs to the technical field of medicines, particularly relates to a compatible composition of a traditional Chinese medicine monomeric compound for treating myocardial damage of Kawasaki disease, and particularly relates to an effect of tanshinone IIA and salvianolic acid B in preparation of a medicine for treating the myocardial damage of Kawasaki disease.

Background

Kawasaki Disease (KD) is an acute systemic autoimmune vasculitis, mainly characterized by inflammation of small and medium blood vessels and myocardial damage, with a high incidence of juvenile and young diseases in northeast asia, especially infants under 5 years old. With the significant increase in the incidence of KD in recent years, it has become one of the important causes of acquired heart disease in infants. Myocardial damage is one of the main complications of KD, and data show that 50-70% of KD children suffer from myocarditis in the acute stage to further cause myocardial damage, but scholars have few researches on the myocardial damage induced by KD and related action mechanisms, and clinical treatment methods and medicines for treating the myocardial damage caused by KD are very limited.

Clinically, aspirin, gamma globulin for intravenous injection, glucocorticoid and the like are taken by western medicine to control inflammation of KD and reduce the risk of complications. Aspirin and gamma globulin are common medicine compositions for treating KD, and the aspirin has the effects of clearing heat and relieving pain, can effectively inhibit inflammatory reaction in bodies of children patients, also has the effects of resisting platelet aggregation and preventing thrombosis, and can reduce risks of diseases such as myocardial injury, myocardial infarction and the like of the children patients with KD to a certain extent. The gamma globulin contains various antibodies, can strengthen the body resistance of children patients, inhibit inflammation diffusion, reduce the level of inflammatory factors of the body, adjust the colony balance of the body, control the disease development and have good immune protection effect on KD children patients. However, aspirin orally taken by children with KD (KD) easily causes gastrointestinal hemorrhage or Reye syndrome and the like, but the cost of applying gamma globulin is high, and drug resistance appears in 10-15% of children, which is the bottleneck of treating KD at present. In conclusion, no literature reports on the research and development of medicaments for myocardial damage caused by child KD at present. Therefore, the search for the medicine for preventing and treating KD myocardial damage has important clinical significance.

The basic theory of traditional Chinese medicine puts KD into the category of epidemic febrile disease, and scholars think that KD should belong to the category of epidemic rash or macula. Most modern Chinese medical scientists consider that the pathogenesis of blood stasis exists all the time from clinical diagnosis to clinical treatment of KD, so that the traditional Chinese medicine has positive guiding effect on preventing and treating KD by 'activating blood and dissolving stasis' throughout treatment when treating KD. Therefore, the medicines with the effects of promoting blood circulation and removing blood stasis, such as the salvia miltiorrhiza, the red paeony root, the ligusticum wallichii, the pseudo-ginseng, the peach kernel, the safflower, and the like, are clinically applied to the prescription for treating KD. According to 212 documents in the systematic evaluation and Meta analysis of the clinical efficacy of treating KD by traditional Chinese medicines, a plurality of traditional Chinese medicines with the effects of promoting blood circulation to remove blood stasis in a prescription are sequenced, and the salvia miltiorrhiza with the highest application frequency is screened out from the traditional Chinese medicines to serve as a research object for exploring and preventing KD medicines.

Salvia miltiorrhiza, which is the dried root and rhizome of Salvia miltiorrhiza Bunge of Salvia of Labiatae, is one of the most commonly used Chinese medicines for promoting blood circulation and removing blood stasis, and is first reported in Shen nong Ben Cao Jing and listed as a top grade of the grass. It is bitter in taste and slightly cold in nature, enters heart and liver channels, and has effects of promoting blood circulation, regulating menstruation, removing blood stasis, relieving pain, cooling blood, resolving carbuncle, clearing heart fire, relieving restlessness, nourishing blood, and tranquilizing mind. Ancient times, there is a statement that "one herb of red sage, with the same effect as four herbs": to enrich blood and produce blood, radix Angelicae sinensis and rehmanniae radix are used; regulating blood and astringing blood, and making the peony root; dispel blood stasis and promote tissue regeneration, which is twice as strong as Chuan Xiong. Modern medical research shows that the main chemical components of the salvia miltiorrhiza are fat-soluble diterpenoid quinone compounds and water-soluble phenolic acid compounds, and can effectively improve the rheological indexes of blood, reduce the viscosity of blood, reduce plasma fibrinogen, inhibit platelet aggregation and activation, prevent and treat thrombosis, improve microcirculation and the like.

Tanshinone IIA is one of fat-soluble components in salvia miltiorrhiza, and is widely applied to treatment of adult cardiovascular and cerebrovascular diseases. Tanshinone IIA contains an o-quinone structure, and the compound is easily reduced to a diphenol derivative and further oxidized to quinone. Tanshinone IIA has antiinflammatory, platelet aggregation inhibiting, antioxidant, myocardial cell protecting and coronary circulation improving effects. Can reduce the expression of inflammatory cytokines in serum of children with KD to a certain extent, thereby reducing inflammatory reaction. At present, the observation and research of tanshinone IIA for treating KD are clinically available.

Salvianolic acid B is one of water soluble components of Saviae Miltiorrhizae radix, and has high content and strong bioactivity. A plurality of basic researches show that the salvianolic acid B can improve the survival rate of endothelial cells and myocardial cells by reducing the release of plasma thromboxane, endothelin and NO by the endothelial cells. Salvianolic acid B can also protect vascular endothelium by increasing superoxide dismutase (SOD) activity and reducing Lactate Dehydrogenase (LDK) activity. Previous studies have also found that salvianolic acid B counteracts myocardial damage caused by myocardial infarction and ischemia-reperfusion and inhibits the formation of atherosclerotic plaques by reducing tissue oxidative stress levels. The document reports that the salvianolic acid B has the effect of improving myocardial damage induced by a diabetes model mouse, and the action mechanism of the salvianolic acid B is that the salvianolic acid B reduces the myocardial tissue apoptosis and the oxidative stress level of the diabetes model mouse by activating the expression of the sirtuin 1, thereby relieving the myocardial cell damage of the diabetes model mouse and playing the role of myocardial protection. In addition, researchers study that salvianolic acid B has a protective effect on myocardial damage of rats with sepsis, and the action mechanism of salvianolic acid B probably reduces oxidative stress of rats with sepsis and inhibits myocardial cell apoptosis by influencing autophagy protein, so that myocardial damage of rats with sepsis is relieved. Both myocardial injuries may be therapeutic in that salvianolic acid B regulates the relevant oxidative stress pathways, while the mechanism of action of salvianolic acid B in regulating KD myocardial injury may be more relevant to inflammation.

Disclosure of Invention

There are also numerous theories on the pathogenesis of KD-mediated myocardial injury in children. At present, the acknowledged view is that systemic immunity of KD in the acute phase has an obvious activation state, so that the immune system is unbalanced, and children with KD can have wide inflammatory lesions such as coronary artery, cardiac muscle, pericardium and endocardium, so as to cause myocardial ischemia and hypoxia, accumulate acidic substances in cells, cause energy metabolism disorder of myocardial cells, and combine troponin with expansion competition to destroy an excitation-contraction coupling mechanism, so as to cause myocardial injury. First, due to the damage caused by immunity, researches in recent years find that the mediation of certain cytokines and intercellular adhesion molecules, certain inflammatory mediators and the degeneration of genes are also related to the occurrence and development of the disease. The pathogenesis of myocardial injury of adults is also dispute 32429, while the main patients of KD are children and the metabolic systems of children, especially the liver function development is incomplete, and no relevant report is reported at present to study the correlation between the pathogenesis of myocardial injury of children patients with KD and the pathogenesis of adults.

From the histopathological point of view, the essence of KD myocardial injury refers to inflammatory lesions caused by edema, degeneration, necrosis, myofibril breakage and lysis of myocardial cells, and severe myocardial injury can lead to myocarditis and heart failure. The structure and function of the cardiac muscle of children are not mature, and the compensatory ability of the cardiac muscle is worse, so the incidence of cardiac muscle damage is higher. Reports of myocardial infarction caused by myocardial injury are also rare, and diagnosis faces serious challenges. Myocardial injury leads to hyperemia, edema, degeneration and even apoptosis of myocardial cells, resulting in changes in serum levels of the relevant biomarkers. The common biochemical indexes for clinically diagnosing KD myocardial injury comprise serum creatine kinase, serum creatine kinase isozyme, serum alpha-hydroxybutyrate dehydrogenase, serum glutamic-oxaloacetic transaminase, TNF-alpha, IL-6, IL-1 beta and the like. The therapeutic application of the invention is proved by detecting the biochemical indexes through specific experiments.

The invention aims to solve the problem of providing application of a tanshinone IIA and salvianolic acid B in preparation of a medicine for treating myocardial damage caused by Kawasaki disease.

The technical scheme of the invention is as follows: a pharmaceutical composition for treating myocardial damage caused by Kawasaki disease comprises tanshinone IIA and salvianolic acid B, wherein the tanshinone IIA and the salvianolic acid B are mixed in a weight ratio of 1: 10-10: 1.

Preferably, in the pharmaceutical composition for treating myocardial damage of kawasaki disease, the weight ratio of tanshinone IIA to salvianolic acid B is 1: 5.

Preferably, in the pharmaceutical composition for treating myocardial damage of Kawasaki disease, the purity of the tanshinone IIA is more than or equal to 98%; the purity of the salvianolic acid B is more than or equal to 98 percent.

The medicinal composition for treating the Kawasaki disease myocardial damage is applied to preventing or treating myocardial damage diseases.

Preferably, for the above use, the myocardial damage is myocardial damage caused by kawasaki disease.

The tanshinone IIA and the salvianolic acid B can be purchased from the market, and can also be prepared from the traditional Chinese medicine salvia miltiorrhiza according to the prior art.

The invention has the beneficial effects that: at present, no medicine with definite curative effect for treating KD myocardial injury exists at home and abroad. When KD infant presents myocardial injury, mainly rely on western medicines such as creatine phosphate sodium, vitamin C, compound coenzyme, levocarnitine to control the symptom, the effect is not good and the side effect is great, and some infant can present the drug resistance to make gamma globulin unable to use. Glucocorticoid reduces the immune function of children patients, affects the recovery of damaged cardiac muscle cells and is easy to induce sudden death caused by myocardial infarction; gamma globulin increases blood viscosity and contributes to thrombosis, thereby exacerbating myocardial damage. The tanshinone IIA and salvianolic acid B compounds have a long medicinal history and high safety. According to the anti-inflammatory, anti-oxidation and anti-myocardial injury related biological activities of the tanshinone IIA and the salvianolic acid B, the two are combined in a compatible manner, and scientific experimental data prove that the composition is helpful for preventing and treating KD myocardial injury pathological changes. The formula of the invention has reasonable compatibility, and can be used for symptomatic treatment, pathological section results and biochemical indexes aiming at myocardial injury of children patients with KD to prove that the traditional Chinese medicine composition can obviously inhibit the myocardial injury. Meanwhile, the cost is low, and various dosage forms which can be accepted by KD children patients can be easily prepared.

Drawings

FIG. 1 Effect of tanshinone IIA and salvianolic acid B composition on myocardial tissue morphology of KD mice (HE staining, x 400) (A: blank group; B: model group; C: positive drug group; D: tanshinone IIA + salvianolic acid B (1: 1); E: tanshinone IIA + salvianolic acid B (1: 5); F: tanshinone IIA + salvianolic acid B (5: 1))

FIG. 2 the difference in change in CK activity in mice from each group (Tan2A: tanshinone IIA; SanB: salvianolic acid B). P < 0.05,. P < 0.01 and. P < 0.001, indicating comparison with the blank group;^#P＜0.05，^##p < 0.01 and^###p < 0.001, representing comparison to the model set. (tanshinone IIA: Tan 2A; salvianolic acid B: SanB)

FIG. 3 shows the difference in the change in CK-MB activity among mice in each group (Tan2A: tanshinone IIA; SanB: salvianolic acid B) < 0.05, < 0.01, and < 0.001, as shown in the tableCompare with blank set;^#P＜0.05，^##p < 0.01 and^###p < 0.001, representing comparison to the model set.

FIG. 4 differences in LDH activity changes (Tan2A: tanshinone IIA; SanB: salvianolic acid B) < 0.05, < 0.01, and < 0.001) among mice in each group, indicating comparison to the blank group;^#P＜0.05，^##p < 0.01 and^###p < 0.001, representing comparison to the model set.

FIG. 5 the difference in α -HBDH activity change in mice from group to group (Tan2A: tanshinone IIA; SanB: salvianolic acid B) < 0.05, < 0.01 and < 0.001, indicating comparison with the blank group;^#P＜0.05，^##p < 0.01 and^###p < 0.001, representing comparison to the model set.

FIG. 6 the difference in AST activity change in mice of each group (Tan2A: tanshinone IIA; SanB: salvianolic acid B) < 0.05, < 0.01 and < 0.001, indicating comparison with the blank group;^#P＜0.05，^##p < 0.01 and^###p < 0.001, representing comparison to the model set.

FIG. 7 differences in changes in TNF- α (Tan2A: tanshinone IIA; SanB: salvianolic acid B) P < 0.05, P < 0.01 and P < 0.001 in the groups of mice, indicating comparison with the blank group;^#P＜0.05，^##p < 0.01 and^###p < 0.001, representing comparison to the model set.

FIG. 8 differences in IL-6 changes in mice from group to group (Tan2A: tanshinone IIA; SanB: salvianolic acid B) P < 0.05, P < 0.01 and P < 0.001, indicating comparison to the blank group;^#P＜0.05，^##p < 0.01 and^###p < 0.001, representing comparison to the model set.

FIG. 9 the difference in IL-1 β changes in mice from group to group (Tan2A: tanshinone IIA; SanB: salvianolic acid B) P < 0.05, P < 0.01 and P < 0.001, indicating comparison to the blank group;^#P＜0.05，^##p < 0.01 and^###p < 0.001, representing comparison to the model set.

Detailed Description

1. Experimental Material

1.1 Experimental animals

60 SPF male BALB/C mice with 5-6 weeks old are healthy and 16-25 g in weight. The feed is divided into 6 groups at random, each group comprises 12 feed, the feed is fed in cages, the feed is self-fed, the state is good, the activity is normal, the temperature (22 +/-2) DEG C, the humidity (45-55 percent), the ventilation is carried out, the light and dark light are alternated for 12 hours, and the adaptive feed observation is carried out for one week.

1.2 major experimental drugs

1.3 Primary reagents

1.3.1 Elisa kit composition (taking TNF-alpha as an example)

(1) A micropore enzyme label plate: 1 block of 96 wells (coated with anti-mouse TNF- α mab); (2) and (3) standard substance: 6 bottles, 0.3mL (concentration of 640pg/mL, 320pg/mL, 160pg/mL, 80pg/mL, 40pg/mL, 20pg/mL respectively); (3) blank control: 1 bottle, 1.0 mL; (4) standard dilution buffer: 1 bottle, 5 mL; (5) biomarker anti-TNF- α antibodies: 1 bottle, 6 mL; (6) streptavidin-HRP: 1 bottle, 10 mL; (7)20 × washing buffer: 25 mL; (8) sample diluent: 6 mL; (9) substrate A: 6 mL; (10) substrate B: 6 mL; (11) stopping liquid: 6 mL; (12) and (3) covering with a plastic film plate: 1 block.

1.3.2 preparation of Primary reagents

10% bovine serum albumin: adding 100mL of normal saline into 10g of normal saline as a solvent, fully dissolving to prepare 10% bovine serum albumin, subpackaging, and storing at-20 ℃. 0.5% sodium hydroxymethyl cellulose: adding 100mL of physiological saline into 0.5g of physiological saline as a solvent, adding the physiological saline for 3 times, and stirring to prepare 0.5% of sodium carboxymethylcellulose for uniform suspension.

2. Experimental methods

2.1 raising, grouping and modeling of laboratory animals

The mice were randomly divided into 6 groups, 10 mice each, namely a blank control group, a model group, a positive drug group, a tanshinone IIA + salvianolic acid B group (1:1), a tanshinone IIA + salvianolic acid B group (1:5), and a tanshinone IIA + salvianolic acid B group (5: 1). Except the blank control group, the other groups are respectively subjected to modeling of KD myocardial injury by a method of intraperitoneal injection of 2.5g/kg bovine serum albumin on

days

1, 3, 5, 7, 9 and 11, and the blank control group is simultaneously subjected to intraperitoneal injection of 2.5g/kg physiological saline on

days

1, 3, 5, 7, 9 and 11 respectively to observe changes of the activity, appetite, reaction and the like of the mice.

2.2 animal administration

After the molding is finished, the medicine is administered to each group. The positive medicine group aspirin is respectively prepared into 10mg/mL and 1mg/mL, the first 3 days are intragastrically filled with 455mg/(kg d), the last 7 days are intragastrically filled with 45.5mg/(kg d), and the times of each day are 1. Respectively applying 0.5 percent of hydroxymethyl cellulose sodium aqueous solution in mass-volume ratio to respectively prepare 1mg/mL tanshinone IIA and salvianolic acid B solutions, and then preparing the composition according to the volume ratio: tanshinone IIA and salvianolic acid B group (1:1), tanshinone IIA and salvianolic acid B group (1:5), and tanshinone IIA and salvianolic acid B group (5: 1). The three groups of compatible compositions are respectively administrated to experimental mice by intragastric administration for 10 days according to the dosage of 40 mg/(kg. d), and 1 time per day. The blank control group and the model group were each subjected to intragastric administration with an equal amount of physiological saline for 10 days, 1 time per day.

2.3 specimen Collection

After the last administration for 24 hours, two mice are randomly left in each group for perfusion, heart tissues are taken for preparing histopathological HE staining sections, and eyeball blood taking is carried out on the remaining mice in each group.

Collecting heart tissues: the method comprises the steps of carrying out intraperitoneal injection of 10% chloral hydrate for anesthesia on each group of mice for histopathological examination, fixing the mice in a supine position on a sterile operating table, disinfecting the skin at the chest cavity with alcohol, operating with a sterile special instrument, opening the chest cavity, penetrating a needle of an infusion apparatus connected with normal saline into the left ventricle, cutting the right auricle at the same time, enabling blood to flow out, after about 10min, observing the mice, drawing out the needle after the liver becomes white, rapidly picking the heart, and placing the heart in a 4% paraformaldehyde solution for fixing for 12 h.

Blood collection: the left thumb and forefinger grasp the skin of the mouse behind the ear and neck, and the little finger and ring finger fix the tail. The eye skin on the blood side is slightly pressed to make the eyeball congestion and protrude. The eyeball is clamped by an elbow forceps, and the direction of the thumb and the forefinger is twisted according to the requirement, so that the blood vertically flows into the EP tube from the orbit at different speeds. When the blood dripping speed is slow, the heart of the mouse can be lightly pressed to accelerate the blood pumping speed of the heart so as to obtain more blood. And placing the taken whole blood into a 1.5mL centrifuge tube, standing overnight at 4 ℃, centrifuging all the taken whole blood by a centrifuge at 3000r/min for 10min, taking transparent supernatant, namely serum, into a new centrifuge tube after centrifugation, and placing the centrifuge tube at-80 ℃ for storage for detecting various indexes in the serum.

2.4 index detection

2.4.1 pathological assays

(1) Washing: the fixed tissue was rinsed with distilled water. (2) Material taking: the left ventricle part of myocardial tissue and part of coronary artery are taken.

(3) And (3) dehydrating: an ethanol dehydration method is adopted. The mouse myocardial tissues are dehydrated in a gradient way by using 70 percent, 80 percent, 90 percent and 95 percent of absolute ethyl alcohol in sequence, and each time is about 30 min. (4) And (3) transparency: the transparency was carried out with xylene. Xylene I is transparent for 15min, and xylene II is transparent for 15 min. (5) Wax dipping and paraffin embedding: the transparent tissue is transferred into melted paraffin for dipping and embedded by a paraffin embedding machine. (6) Slicing: the embedded tissue was sectioned with a paraffin microtome, which cut approximately 4 μm thick.

(7) Unfolding, sticking and baking: and (4) spreading the slices in a constant-temperature water bath box, taking the spread slices out at 1/3 by using a glass slide, inserting the glass slide into a slice rack, and putting the slices into the constant-temperature box for baking. (8) Dewaxing: the oven-dried slices were removed from the incubator and dewaxed using xylene. Sequentially adding xylene I for 10min and xylene II for 10 min. (9) Hydration: placing the slices in 100% ethanol I for 10min, 100% ethanol II for 10min, 95% ethanol for 5min, 90% ethanol for 5min, 80% ethanol for 3min, 70% ethanol for 3min, and distilled water for 5 min. (10) Dyeing: hematoxylin-eosin staining method is adopted. Placing the slices into hematoxylin solution for 8min, washing with tap water for 5min, differentiating with 1% ethanol hydrochloride for 15s, washing with tap water until the slices are blue, and sequentially placing 70% ethanol for 3min, 80% ethanol for 3min, 90% ethanol for 5min, 95% ethanol for 5min, 0.5% eosin solution for 5min, 95% ethanol for 5min, 100% ethanol for 5min, and 100% ethanol for 5 min.

(11) And (3) transparency: the slices are sequentially placed in xylene I for 5min and xylene II for 5 min. (12) Sealing: mounting was performed using neutral gum. Neutral gum was dropped onto the slide tissue and the coverslip was slowly covered from one side to prevent the appearance of air bubbles. (13) And (4) observation: the morphology of the mouse coronary artery and myocardial tissue was observed using a microscope.

2.4.2 ELISA detection

The activity of CK, CK-MB, LDH, alpha-HBDH and AST and the levels of TNF-alpha, IL-6 and IL-1 beta in the serum of each group of mice are measured by Enzyme-linked immunosorbent assay (ELISA), and the operation is strictly carried out according to the specific steps of the kit specification (taking TNF-alpha as an example). Preparing a reagent: washing buffer solution: distilled water 1:20 dilution, i.e. 1 part 20 × washing buffer plus 19 parts distilled water.

The method comprises the following operation steps:

(1) sample adding of the standard: setting 6 standard product holes, and respectively adding 50 mu L of standard products with different concentrations; (concentration was 640pg/mL, 320pg/mL, 160pg/mL, 80pg/mL, 40pg/mL, 20pg/mL in this order)

(2) Sample adding: and blank holes and sample holes to be detected are respectively arranged, and the blank holes are not added with samples and enzyme labeling reagents. 40 mu L of sample diluent is added into sample holes to be detected on the enzyme-labeled coated plate, and then 10 mu L of sample to be detected is added (the final dilution of the sample is 5 times). Adding sample to the bottom of the plate hole of the enzyme label, keeping the sample from touching the hole wall as much as possible, and gently shaking and mixing the sample and the hole wall. Adding 50 mu L of biotin-labeled TNF-alpha antibody into each of the standard wells and the sample wells except for blank wells;

(3) and (3) incubation: covering the sealing plate with a membrane plate, slightly shaking and uniformly mixing, and incubating at 37 ℃ for 1 hour;

(4) washing: carefully uncovering the membrane plate cover, discarding liquid, spin-drying, filling washing liquid into each hole, vibrating for 30 seconds, discarding the washing liquid, patting the holes dry by absorbent paper, and repeating for 3 times;

(5) adding 80 mu L of streptavidin-HRP into each hole, and lightly shaking and uniformly mixing;

(6) and (3) incubation: covering the sealing plate with a membrane plate again, and then placing the sealing plate at 37 ℃ for incubation for 30 minutes;

(7) washing: uncovering the membrane plate cover again, discarding liquid, drying, filling cleaning solution into each hole, vibrating for 30 seconds, discarding the cleaning solution, drying by absorbent paper, and repeating for 3 times;

(8) color development: adding 50 mu L of each of the substrate A and the substrate B into each hole, shaking gently and mixing uniformly, and developing for 10 minutes at 37 ℃ in a dark place;

(9) and (4) terminating: taking out the enzyme label plate, quickly adding 50 mu L of stop solution into each hole, uniformly mixing, and stopping reaction;

(10) and (3) determination: after the addition of the stop solution, the wells were zeroed with blank wells within 15 minutes, and the absorbance (OD value) of each well was measured in order at a wavelength of 450 nm.

(11) And (3) taking the OD value of the measured standard substance as an abscissa (X) and the concentration value of the standard substance as an ordinate (Y), drawing a standard curve by using relevant software, obtaining a linear regression equation, substituting the OD value of the sample into the equation, and calculating the concentration of the sample.

2.5 statistical methods

The SPSS25.0 software is adopted to process and analyze the experimental results of each group, the data are represented by mean +/-standard deviation (mean +/-SD), the multi-sample comparison is carried out by adopting one-factor variance analysis, the multi-group comparison is carried out by adopting a Tukey method, the p is less than 0.05 to represent that the difference is significant, the p is less than 0.001 to represent that the difference is extremely significant, and the statistical significance is achieved.

3. Results of the experiment

3.1 general status of mice

In the molding and treatment process, the blank control group mice have normal mobility, sensitive response to stimulation, normal fur glossiness and normal eating and drinking water amount. After the bovine serum albumin is injected into the abdominal cavity of the mouse, the activity is obviously reduced, the stimulation response is poor, the fur is disordered, the glossiness is poor, the feeding and drinking amount is reduced, the eyeball of the mouse is reddened, and the red and swollen expression appears at the nose and around the mouth. In the treatment process, the food intake and the water intake of the mice in the model group are slightly increased, the fur is still dull, and the symptoms are obviously improved after 10 days of treatment in each treatment group.

3.2 morphological changes in mouse Heart tissue (HE X400)

As shown in FIG. 1, A-F, the blank group had smooth and intact myocardial fibers and aligned cells; compared with the blank group, the myocardial tissues of the mice in the model group have inflammatory cell infiltration and are accompanied by large-area myocardial rupture; compared with the model group, the positive drug group mouse has local myocardial rupture and has unobvious symptoms; compared with the model group, the mice of the tanshinone IIA + salvianolic acid B (1:1) group occasionally have local myocardial rupture; compared with a model group, the mouse myocardial tissues of the tanshinone IIA + salvianolic acid B (1:5) group have no abnormality basically, the structural arrangement is orderly and compact, and the fiber breakage condition is remarkably relieved; the myocardial rupture of mice in the tanshinone IIA + salvianolic acid B (5:1) group is obvious and almost not improved.

3.3 serum Creatine Kinase (CK) Activity in groups of mice

The CK activity detection results of the groups are shown in figure 2, compared with a blank control group, the model group, the positive medicine group, the tanshinone IIA + salvianolic acid B group (1:1) and the tanshinone IIA + salvianolic acid B group (5:1) are obviously increased, the differences have statistical significance (P is less than 0.05), and the tanshinone IIA + salvianolic acid B group (1:5) has no statistical significance (P is more than 0.05) compared with the blank control group; compared with the model group, the positive medicine group, the tanshinone IIA + salvianolic acid B group (1:1), the tanshinone IIA + salvianolic acid B group (1:5) and the tanshinone IIA + salvianolic acid B group (5:1) are obviously reduced, and the differences have statistical significance (P is less than 0.05); compared with the positive medicine group, the tanshinone IIA + salvianolic acid B group (1:5) is obviously reduced, and the differences have statistical significance (P is less than 0.05).

3.4 serum creatine kinase-isozyme (CK-MB) Activity in groups of mice

The CK-MB activity detection results of each group are shown in a figure 3, compared with a blank control group, the model group, the tanshinone IIA + salvianolic acid B group (1:1), the tanshinone IIA + salvianolic acid B group (1:5) and the tanshinone IIA + salvianolic acid B group (5:1) are obviously increased, the differences have statistical significance (P is less than 0.05), and the positive medicine group and the blank control group have no statistical significance (P is more than 0.05) in comparison; compared with the model group, the positive medicine group, the tanshinone IIA + salvianolic acid B group (1:1) and the tanshinone IIA + salvianolic acid B group (1:5) are obviously reduced, the differences have statistical significance (P is less than 0.05), and the tanshinone IIA + salvianolic acid B group (5:1) has no statistical significance (P is more than 0.05) compared with the model group; compared with the positive medicine group, the differences of the tanshinone IIA + salvianolic acid B group (1:1), the tanshinone IIA + salvianolic acid B group (1:5) and the tanshinone IIA + salvianolic acid B group (5:1) have no statistical significance (P is more than 0.05).

3.5 serum Lactate Dehydrogenase (LDH) Activity in groups of mice

The detection results of LDH activity of each group are shown in figure 4, compared with the blank control group, the model group is obviously increased, the difference has statistical significance (P is less than 0.05), and the differences of the positive medicine group, the tanshinone IIA + salvianolic acid B group (1:1), the tanshinone IIA + salvianolic acid B group (1:5), and the tanshinone IIA + salvianolic acid B group (5:1) and the blank control group have no statistical significance (P is more than 0.05); compared with the model group, the positive medicine group, the tanshinone IIA + salvianolic acid B group (1:1), the tanshinone IIA + salvianolic acid B group (1:5) and the tanshinone IIA + salvianolic acid B group (5:1) are obviously reduced, and the differences have statistical significance (P is less than 0.05); compared with the positive medicine group, the differences of the tanshinone IIA + salvianolic acid B group (1:1), the tanshinone IIA + salvianolic acid B group (1:5) and the tanshinone IIA + salvianolic acid B group (5:1) have no statistical significance (P is more than 0.05).

3.6 serum alpha-hydroxybutyrate dehydrogenase (alpha-HBDH) Activity in groups of mice

The detection results of the alpha-HBDH activities of all groups are shown in figure 5, compared with a blank control group, the model group is obviously increased, the differences have statistical significance (P is less than 0.05), and compared with the positive medicine group, the tanshinone IIA + salvianolic acid B group (1:1), the tanshinone IIA + salvianolic acid B group (1:5) and the tanshinone IIA + salvianolic acid B group (5:1) and the blank control group, the differences have no statistical significance (P is more than 0.05); compared with the model group, the positive medicine group, the tanshinone IIA + salvianolic acid B group (1:1), the tanshinone IIA + salvianolic acid B group (1:5) and the tanshinone IIA + salvianolic acid B group (5:1) are obviously reduced, and the difference has statistical significance (P is less than 0.05); compared with the positive medicine group, the differences of the tanshinone IIA + salvianolic acid B group (1:1), the tanshinone IIA + salvianolic acid B group (1:5) and the tanshinone IIA + salvianolic acid B group (5:1) have no statistical significance (P is more than 0.05)

3.7 serum glutamic-oxaloacetic transaminase (AST) of each group of mice

The AST activity detection results of the groups are shown in figure 6, compared with the blank control group, the model group, the positive medicine group, the tanshinone IIA + salvianolic acid B group (1:1) and the tanshinone IIA + salvianolic acid B group (5:1) are obviously increased, the differences have statistical significance (P is less than 0.05), and the differences between the tanshinone IIA + salvianolic acid B group (1:5) and the blank control group have no statistical significance (P is more than 0.05); compared with the model group, the positive medicine group, the tanshinone IIA + salvianolic acid B group (1:1) and the tanshinone IIA + salvianolic acid B group (1:5) are obviously reduced, the differences have statistical significance (P is less than 0.05), and the tanshinone IIA + salvianolic acid B group (5:1) has no statistical significance (P is more than 0.05) compared with the model group; compared with the positive medicine group, the tanshinone IIA + salvianolic acid B group (1:5) is obviously reduced, and the difference has statistical significance (P is less than 0.05).

3.8 serum tumor necrosis factor alpha (TNF-alpha) levels in groups of mice

The TNF-alpha level detection results of each group are shown in figure 7, compared with a blank control group, the levels of the model group, the tanshinone IIA + salvianolic acid B group (1:1) and the tanshinone IIA + salvianolic acid B group (5:1) are higher, the differences have statistical significance (P is less than 0.05), and the differences of the positive medicine group, the tanshinone IIA + salvianolic acid B group (1:5) and the blank control group have no statistical significance (P is more than 0.05); compared with the model group, the levels of the positive medicine group and the tanshinone IIA and salvianolic acid B group (1:5) are lower, the differences have statistical significance (P is less than 0.05), and the differences of the tanshinone IIA and salvianolic acid B group (1:1) and the tanshinone IIA and salvianolic acid B group (5:1) and the model group have no statistical significance (P is more than 0.05); compared with the positive medicine group, the differences of the tanshinone IIA + salvianolic acid B group (1:1), the tanshinone IIA + salvianolic acid B group (1:5) and the tanshinone IIA + salvianolic acid B group (5:1) have no statistical significance (P is more than 0.05).

3.9 Interleukin 6(IL-6) levels in groups of mice

The IL-6 level detection results of each group are shown in FIG. 8, compared with the blank control group, the levels of the model group, the tanshinone IIA + salvianolic acid B group (1:1), the tanshinone IIA + salvianolic acid B group (1:5) and the tanshinone IIA + salvianolic acid B group (5:1) are higher, the differences have statistical significance (P is less than 0.05), and the differences between the positive medicine group and the blank control group have no statistical significance (P is more than 0.05); compared with the model group, the levels of the positive medicine group, the tanshinone IIA + salvianolic acid B group (1:5) and the tanshinone IIA + salvianolic acid B group (5:1) are lower, the differences have statistical significance (P is less than 0.05), and the differences of the tanshinone IIA + salvianolic acid B group (1:1) and the model group have no statistical significance (P is more than 0.05); compared with the positive medicine group, the levels of the tanshinone IIA + salvianolic acid B group (1:1) and the tanshinone IIA + salvianolic acid B group (5:1) are higher, the difference has statistical significance (P is less than 0.05), and the difference of the tanshinone IIA + salvianolic acid B group (1:5) and the positive medicine group has no statistical significance (P is more than 0.05).

3.10 Interleukin 1 beta (IL-1 beta) levels in groups of mice

The detection results of IL-1 beta levels of each group are shown in figure 9, compared with the blank control group, the levels of the model group, the tanshinone IIA + salvianolic acid B group (1:1), the tanshinone IIA + salvianolic acid B group (1:5) and the tanshinone IIA + salvianolic acid B group (5:1) are higher, the differences have statistical significance (P is less than 0.05), and the differences between the positive medicine group and the blank control group have no statistical significance (P is more than 0.05); compared with the model group, the levels of the positive medicine group, the tanshinone IIA + salvianolic acid B group (1:5) and the tanshinone IIA + salvianolic acid B group (5:1) are lower, the differences have statistical significance (P is less than 0.05), and the differences of the tanshinone IIA + salvianolic acid B group (1:1) and the model group have no statistical significance (P is more than 0.05); compared with the positive medicine group, the levels of the tanshinone IIA + salvianolic acid B group (1:1) and the tanshinone IIA + salvianolic acid B group (5:1) are higher, the difference has statistical significance (P is less than 0.05), and the difference between the tanshinone IIA + salvianolic acid B group (1:5) and the positive medicine group has no statistical significance (P is more than 0.05).

4. Conclusion

4.1 myocardial enzymes

The myocardial enzyme is important evidence for clinically evaluating myocardial damage degree of a patient, and common detection indexes comprise Creatine Kinase (CK), creatine kinase isoenzyme (CK-MB), Lactate Dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (alpha-HBDH), serum glutamic-oxaloacetic transaminase (AST) and the like. The myocardial enzymes are mainly distributed in skeletal muscle, cardiac muscle, smooth muscle and the like. Muscle disorders can lead to an increase in the activity of many myocardial enzymes, particularly inflammatory myopathies, and are commonly used as indicators for the diagnosis of myocardial and skeletal muscle diseases. Therefore, an increase in serum central myogenases generally indicates the possibility of increased permeability or cell destruction of CK-containing tissue cells, particularly abnormal or impaired membrane permeability of muscle fibers, and can represent the degree of myocardial cell damage due to its abundant presence in myocardial cells. Experimental results show that compared with a model group and a positive medicine group, the tanshinone IIA and salvianolic acid B group (1:5) compatible composition can obviously reduce the activity of CK, CK-MB, LDH, alpha-HBDH and AST, the difference has statistical significance (P is less than 0.05), and the tanshinone IIA and salvianolic acid B group (1:5) compatible composition can reduce the activity of a myocardial zymogram and obviously improve the damage condition of myocardial tissues of KD mice.

4.2 TNF-alpha, IL-6 and IL-1 beta

TNF- α is a pleiotropic inflammatory cytokine secreted primarily by activated macrophages and plays an important role in body defense, infection, and immune response. In healthy individuals, TNF- α is not usually detectable, whereas elevated serum and tissue levels of the protein are detectable under infectious and inflammatory conditions, with the severity of the infection being correlated with TNF- α serum levels. TNF-alpha is involved in the pathophysiological processes of various cardiovascular diseases, such as myocardial infarction, heart failure and the like, and has also been shown to be an important cytokine capable of inducing KD in children, and the level of the TNF-alpha is remarkably increased in the acute phase of KD.

IL-6 and IL-1 beta are typical cytokines with functions and pleiotropic effects, and have effects on inflammation, immune response, hematopoiesis and the like. When infection or tissue damage occurs, monocytes and macrophages immediately produce IL-6 and IL-1 β, etc. through stimulation pattern recognition receptors and help to remove the source of infection and restore damaged tissue by activating the acute phase and immune response. Studies have also demonstrated that IL-6 levels are significantly increased during the acute phase of KD, particularly in patients with incomplete KD or IVIG resistant KD, suggesting that IL-6 levels may be a candidate biomarker for patients with KD and IVIG resistant KD.

The experimental results show that the levels of TNF-alpha, IL-6 and IL-1 beta of the mice in the model group are higher than those of the mice in the blank control group on average, which indicates that pathogenesis of myocardial injury of the mice with KD is possibly related to the three inflammatory factors, and after the tanshinone IIA + salvianolic acid B group (1:5) is given with the compatible composition, the levels of TNF-alpha, IL-6 and IL-1 beta are all obviously lower than those of the mice in the model group, and the differences have statistical significance (P is less than 0.05). Particularly, the results of the pathological section of the myocardial tissue indicate that the myocardial rupture degree is obviously reduced compared with that of a model group, and the mechanism that the tanshinone IIA + salvianolic acid B group (1:5) is compatible with the composition for treating the myocardial injury of KD mice is probably related to the down-regulation of TNF-alpha, IL-6 and IL-1 beta levels.

Claims

1. A pharmaceutical composition for treating myocardial damage caused by Kawasaki disease is characterized by comprising tanshinone IIA and salvianolic acid B, wherein the tanshinone IIA and the salvianolic acid B are mixed in a weight ratio of 1: 10-10: 1.

2. The pharmaceutical composition for treating Kawasaki disease myocardial injury as claimed in claim 1, wherein the weight ratio of tanshinone IIA to salvianolic acid B is 1: 5.

3. The pharmaceutical composition for treating Kawasaki disease myocardial injury as claimed in claim 1 or 2, wherein the tanshinone IIA has a purity of greater than or equal to 98%; the purity of the salvianolic acid B is more than or equal to 98 percent.

4. The pharmaceutical composition for treating Kawasaki disease myocardial damage according to claim 1, wherein the pharmaceutical composition is used for preventing or treating myocardial damage diseases.

5. The use of claim 4, wherein the myocardial damage is myocardial damage caused by Kawasaki disease.