Traditional Chinese medicine composition for treating dilated cardiomyopathy with reduced ejection fraction heart failure, preparation method and application thereof

Abstract

The present invention relates to a Chinese medicine composition for treating dilated cardiomyopathy with reduced ejection fraction heart failure, as well as a preparation method and application. Currently, there is no Chinese medicine formula specifically targeting dilated cardiomyopathy combined with reduced ejection fraction heart failure. The Chinese medicine composition comprises ginseng, salvia miltiorrhiza, angelica sinensis, cinnamon twig, bombyx batryticatus, and corn silk, and is used to prepare a medicine for treating dilated cardiomyopathy with qi deficiency, blood stasis, and yang deficiency combined with reduced ejection fraction heart failure. The present invention is used to treat dilated cardiomyopathy combined with reduced ejection fraction heart failure, and has a significant effect on such patients with qi deficiency, blood stasis, and yang deficiency, filling the gap in Chinese medicine for this aspect of the disease. The effective ingredients are extracted according to modern extraction technology of Chinese medicine, and have the advantages of strong pertinence, good efficacy, short cycle, and no toxic side effects, and can quickly relieve the clinical symptoms of patients.

Description

Technical Field

[0001] The invention relates to a traditional Chinese medicine composition, in particular to a traditional Chinese medicine composition for treating dilated cardiomyopathy-induced heart failure with reduced ejection fraction, a preparation method and application thereof. Background Art

[0002] Dilated cardiomyopathy (DCM) is a heterogeneous cardiomyopathy that is difficult to cure. The 5-year survival rate is usually less than 50%. Heart failure (hereinafter referred to as HF), especially HF with reduced ejection fraction, is a very common complication with a poor prognosis. It is difficult to cure and recurrent, which seriously affects the prognosis and quality of life of patients. Therefore, early diagnosis and early treatment, and effective improvement of ventricular remodeling are of great significance to reduce the incidence of adverse cardiovascular events, improve the quality of life, and increase the survival rate of patients.

[0003] In terms of western medicine, the efficacy of the "golden triangle" treatment of heart failure for DCM with reduced ejection fraction is not optimistic. Although the new anti-heart failure drug sacubitril valsartan can reduce the risk of cardiovascular death or re-hospitalization for heart failure in patients, the safety of the drug needs to be further confirmed. In addition, a large number of clinical examples have shown that ultra-high-dose ARB (Angiotensin Receptor Blocker) treatment can reduce the mortality and re-hospitalization rate of patients of different ages compared with conventional treatment doses, but it is prone to adverse reactions such as hypotension and liver and kidney damage.

[0004] In traditional Chinese medicine, heart failure belongs to the category of "heart failure" and "heart paralysis" in traditional Chinese medicine. For example, Wang Shuhe said in "Pulse Classic": "When the heart fails, it is hidden, and when the liver is weak, it is deep, so the pulse is hidden and deep. The best doctor comes to diagnose, and he turns the acupoints to facilitate urination and defecation, so that the water channel is unblocked, the sweet liquid flows down, and the yin and yang are balanced, the panting is light, and the sweating is normal. The liver is at its root, the heart qi rises, the yang flows to the limbs, the lung qi is balanced, and the panting is relieved." At present, traditional Chinese medicine has a consistent understanding of the etiology and pathogenesis of this disease, that is, it belongs to the deficiency of the root and the excess of the symptoms. The deficiency of the root is mostly attributed to qi deficiency, qi and yin deficiency, and yang deficiency, and the excess of the symptoms is mostly attributed to blood stasis, phlegm, and water retention. However, the views held by various schools on the syndrome differentiation and classification of this disease are different. Although some scholars believe that this disease can be divided into four types: qi and yin deficiency, qi and blood deficiency, yang deficiency and water flooding, and heart pulse obstruction, there is still a lack of unified syndrome differentiation and classification, and doctors mostly use their own clinical experience for syndrome differentiation, lacking objective and quantitative basis. Although traditional Chinese medicine has unique advantages in alleviating symptoms, improving heart function and enhancing quality of life, there is a phenomenon of irregular use of drugs during the treatment process, and there is currently a lack of traditional Chinese medicine prescriptions for dilated cardiomyopathy combined with heart failure with reduced ejection fraction. Summary of the invention

[0005] The purpose of the present invention is to provide a Chinese medicine composition for treating dilated cardiomyopathy with reduced ejection fraction heart failure and a preparation method and application thereof, so as to make up for the defect that there is currently no clear Chinese medicine formula for dilated cardiomyopathy combined with reduced ejection fraction heart failure, and effectively improve the clinical symptoms of such patients with qi deficiency, blood stasis and yang deficiency.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A Chinese medicine composition for treating dilated cardiomyopathy with reduced ejection fraction heart failure, the Chinese medicine composition comprising the following components in parts by weight:

[0008] 5-10 portions of ginseng;

[0009] 10-30 parts of Salvia miltiorrhiza;

[0010] 10-20 portions of Chinese Angelica;

[0011] 6-9 parts of cinnamon twig;

[0012] Bombyx batryticatus 6-9 parts;

[0013] 10 to 30 portions of corn silk.

[0014] Furthermore, the Chinese medicine composition comprises the following components in parts by weight:

[0015] 10 portions of ginseng;

[0016] 30 parts of Salvia miltiorrhiza;

[0017] 10 parts of Chinese Angelica;

[0018] 6 parts of cinnamon twig;

[0019] Bombyx batryticatus 9 parts;

[0020] 30 servings of corn silk.

[0021] Furthermore, the dosage form of the Chinese medicine composition is granules.

[0022] On the other hand, a method for preparing the Chinese medicine composition as described is provided, wherein the method for preparing the granules comprises the following steps:

[0023] Step 1: Mix ginseng, salvia miltiorrhiza, angelica, cinnamon twig, silkworm and corn silk, add water and boil twice, each time for 2 hours, filter, combine the filtrate and concentrate;

[0024] Step 2: Add ethanol, let stand, filter, recover ethanol from the filtrate, let stand, filter, concentrate under reduced pressure to form a thick paste, make granules, and dry.

[0025] Furthermore, in step 2, the alcohol content of ethanol is 50%.

[0026] Furthermore, in step 2, the mixture is concentrated under reduced pressure to a thick paste until the relative density reaches 1.30-1.35, and the temperature is maintained at 60-70°C during the concentrated process.

[0027] Furthermore, in step 2, after the mixture is concentrated under reduced pressure into a thick paste, sucrose is added and mixed, and then granules are prepared.

[0028] On the other hand, the invention provides the use of the Chinese medicine composition as described above in the preparation of a drug for treating dilated cardiomyopathy of qi deficiency, blood stasis and yang deficiency type combined with heart failure of reduced ejection fraction.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The Chinese medicine composition of the present invention is used to treat dilated cardiomyopathy combined with heart failure with reduced ejection fraction, and has a significant effect on patients of this type with qi deficiency, blood stasis and yang deficiency. The effective ingredients are extracted according to modern Chinese medicine extraction technology, and have the advantages of strong pertinence, good efficacy, short cycle and no toxic side effects. It can quickly relieve the clinical symptoms of patients, shorten the course of treatment for patients, reduce the risk of relapse, save time and cost, and reduce mortality.

[0031] The present invention proposes that Qi deficiency and blood stasis are the basic syndromes through clinical research on dilated cardiomyopathy with reduced ejection fraction heart failure, and at the same time considers Yang deficiency constitution. Traditional Chinese medicine mainly improves chest pain and heart pain caused by Qi deficiency and blood stasis and Yang deficiency and water retention. Through clinical observation and statistics, it is proved that the Chinese medicine composition of the present invention has the effect of significantly improving the patient's symptoms such as fatigue, shortness of breath and lower limb edema, as well as reducing the risk of hypotension and increasing the LVEF value to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, drawings of other embodiments can be obtained based on these drawings without paying creative work.

[0033] Figure 1 The figure is a comparison of cardiac ultrasound images of experimental rats in each group.

[0034] Figure 2 It is a comparison of NT-proBNP, CK, and cTn-T levels in each group of experimental rats.

[0035] Figure 3 This is the comparison of HE staining (×200) of the myocardial tissue of the experimental rats in each group.

[0036] Figure 4 This is a comparison of Masson staining (×200) images of cardiac tissues of experimental rats in each group.

[0037] Figure 5 Figure 3 is a comparison of the degree of myocardial fibrosis (%) in each group of experimental rats.

[0038] Figure 6 It is the comparison of the detection results of cardiomyocyte apoptosis in each group of experimental rats.

[0039] Figure 7 The expression levels of PI3K, AKT, Bal / Bax, Caspase-3, and Caspase-9 proteins in the myocardial tissue of each group of experimental rats were compared. DETAILED DESCRIPTION

[0040] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0041] In the description of the application, it is to be understood that all the techniques and scientific terms used have the same meanings as those generally understood by those skilled in the art to which the application belongs. When there is a contradiction, the definition in this specification shall prevail. If not otherwise specified, the technical means used in the embodiment are conventional means well known to those skilled in the art, the reagents used in the embodiment are commercially available goods, and the devices used in the embodiment are existing devices, and the limitation of means, reagents or devices cannot be understood as limitations to the application, and the means, reagents or devices for solving the same technical problems of the same type are within the protection scope of the application.

[0042] In the description of the present application, it is to be understood that when an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range limited by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end value and all integers and fractions within the range.

[0043] In the description of the present application, it should be understood that multiple steps are involved in describing the method, which should not be construed as a limitation on the order of the method steps. Technical solutions obtained by merely changing the order of the steps when solving the same technical problem are also within the scope of protection of the present application.

[0044] The present invention provides a Chinese medicine composition for treating dilated cardiomyopathy with reduced ejection fraction heart failure. In western medicine, dilated cardiomyopathy combined with reduced ejection fraction heart failure is considered, and in traditional Chinese medicine, qi deficiency, blood stasis and yang deficiency are considered. The Chinese medicine composition is mainly used to treat qi deficiency, blood stasis and yang deficiency type dilated cardiomyopathy combined with reduced ejection fraction heart failure. The Chinese medicine composition comprises the following components in parts by weight:

[0045] 5-10 portions of ginseng;

[0046] 10-30 parts of Salvia miltiorrhiza;

[0047] 10-20 portions of Chinese Angelica;

[0048] 6-9 parts of cinnamon twig;

[0049] Bombyx batryticatus 6-9 parts;

[0050] 10 to 30 portions of corn silk.

[0051] Embodiment 1:

[0052] The Chinese medicine composition comprises the following components in parts by weight:

[0053] 5 parts of ginseng;

[0054] 10 parts of Salvia miltiorrhiza;

[0055] 10 parts of Chinese Angelica;

[0056] 6 parts of cinnamon twig;

[0057] Bombyx batryticatus 6 parts;

[0058] 10 portions of corn silk.

[0059] Embodiment 2:

[0060] The Chinese medicine composition comprises the following components in parts by weight:

[0061] 7 parts of ginseng;

[0062] Salvia miltiorrhiza 20 parts;

[0063] 15 parts of Chinese Angelica;

[0064] 7 parts of cinnamon twig;

[0065] Bombyx batryticatus 7 parts;

[0066] 20 portions of corn silk.

[0067] Embodiment 3:

[0068] The Chinese medicine composition comprises the following components in parts by weight:

[0069] 10 portions of ginseng;

[0070] 30 parts of Salvia miltiorrhiza;

[0071] Angelica 20 parts;

[0072] 9 parts of cinnamon twig;

[0073] Bombyx batryticatus 9 parts;

[0074] 30 servings of corn silk.

[0075] The Chinese medicine composition can be in the form of granules, and the preparation method comprises the following steps:

[0076] Step 1: Mix ginseng, salvia miltiorrhiza, angelica, cinnamon twig, silkworm and corn silk, add water and boil twice, each time for 2 hours, filter, combine the filtrate and concentrate;

[0077] Step 2: add ethanol (the alcohol content of ethanol is 50%), let stand, filter, recover ethanol from the filtrate, let stand, filter, and concentrate under reduced pressure to form a thick paste until the relative density reaches 1.30-1.35. During the process of reduced pressure concentration, maintain the temperature at 60-70°C, make granules, and dry to obtain.

[0078] In step 2, after the reduced pressure is concentrated into a thick paste, sucrose may be added and mixed to form granules.

[0079] Of course, the Chinese medicine composition of the present invention is not limited to this dosage form, and other dosage forms with mature preparation methods are also within the protection scope of the present invention.

[0080] The Chinese medicine composition of the present invention has the following characteristics:

[0081] The Chinese medicine composition involved in the present invention is mainly used to treat dilated cardiomyopathy of qi deficiency and blood stasis combined with yang deficiency type heart failure with reduced ejection fraction. With qi deficiency and blood stasis as the basic syndrome, this syndrome is characterized by insufficient vital energy, inability to push, and obstruction of the heart vessels, so the patient suffers from fatigue, chest pain, and pain in a certain place, mainly stabbing pain; the qi loses its warmth, the heart yang is not vigorous, and the pulse is weak, and the pulse is knotted and intermittent; qi deficiency and blood stasis last for a long time, the water flow is not smooth, and water retention occurs, resulting in facial edema, and even limb edema. The treatment is suitable for invigorating qi and activating blood circulation, warming the meridians and unblocking the meridians, and promoting diuresis and reducing swelling.

[0082] The Chinese medicine composition of the present invention is named Shendan Recipe, which is mainly used for chest pain and heartache caused by qi deficiency and blood stasis, yang deficiency and water retention. The recipe mechanism is different from the traditional medication idea. It is obtained by improving and combining the classic medication, especially the use of silkworm. In the recipe:

[0083] Ginseng tastes sweet, slightly bitter and slightly warm. It enters the lung and spleen meridians and has the functions of replenishing vital energy, tonifying the spleen and benefiting the lungs. Shennong's Herbal Classic lists it as a "top-grade product" with the saying that it "tonifies the five internal organs, calms the spirit, settles the soul,..., lightens the body and prolongs life". Qi is the commander of blood, and blood is its mother. Qi deficiency is unable to promote blood circulation and leads to blood stasis. Therefore, the ginseng in the prescription has the power to replenish qi and promote blood circulation; Salvia miltiorrhiza tastes bitter and slightly cold. It mainly enters the heart and liver meridians and has the effects of promoting blood circulation, removing blood stasis, dredging menstruation and relieving pain. Compendium of Materia Medica says that it "promotes blood circulation and unblocks the pericardium". When blood stasis is removed, the blood vessels are unobstructed, and when new blood is produced, the blood vessels are full. Therefore, the blood vessels are unobstructed and chest pain heals itself. The two medicines are both the main medicines and work together to replenish qi and promote blood circulation.

[0084] Angelica and cinnamon twig are auxiliary drugs. Angelica is pungent, sweet and warm. It enters the heart, spleen and liver meridians, and can nourish blood, promote blood circulation and relieve pain. "Jingyue Complete Works" says, "... its taste is sweet and heavy, so it can only nourish blood; its qi is light and pungent, so it can also promote blood circulation...", so the angelica in the prescription can help salvia miltiorrhiza to promote blood circulation and remove blood stasis. Because this syndrome has always been characterized by qi deficiency, which will inevitably lead to blood deficiency over a long period of time, ginseng and angelica are used together to replenish qi and blood, and help the whole prescription to remove blood stasis; cinnamon twig is sweet and pungent in taste, warm in nature, and enters the heart, lung and bladder meridians. "Benjing Shuzheng" records that "harmony of camp, unblocking yang, promoting diuresis, descending qi, promoting qi circulation, and tonifying the middle are the six major effects of cinnamon twig". Cassia twig enters the heart meridian to unblock heart yang, runs through the limbs and enters the meridians, is pungent and warm to dispel cold, warms the meridians and dredges the collaterals, and is used together with angelica to strengthen the blood circulation of the whole prescription, enters the bladder meridian to help yang and transform qi to achieve the effect of warming yang and promoting diuresis.

[0085] Bombyx batryticatus tastes pungent and salty, is neutral in nature, and enters the liver, lung, and stomach meridians. Its name was first seen in the "Shennong Bencao Jing". It has the effects of dispelling wind and relieving spasms, resolving phlegm and dispersing knots. "Bencao Qiuzhen" says "Bombyx batryticatus, dispels wind and cold, dries dampness and resolves phlegm, and warms the blood vessels". "Yuzhou Yaojie" records "activates collaterals and dredges meridians, dispels wind and relieves numbness. Bombyx batryticatus expels wind evil, treats aphasia caused by stroke, headache and chest pain,...", and "Huangdi Neijing Suwen" says "If the heart wants to be soft, eat salty food to soften it." Pungent to move it, salty to soften it. Therefore, the Bombyx batryticatus in the prescription not only warms and dredges the blood vessels, but also helps the three medicines of Dangui Gui to activate blood circulation and dredge collaterals. Corn silk tastes sweet and light, is neutral in nature, and enters the bladder, liver, and gallbladder meridians. It has the functions of penetrating water and reducing swelling, and leads the medicine into the bladder meridian, and together with cinnamon twigs, it warms the yang, promotes water and reduces swelling. The two medicines are adjuvants. Looking at the whole prescription, the combination of various medicines has the effects of invigorating Qi and activating blood circulation, warming the meridians and dredge meridians, and promoting diuresis and reducing swelling.

[0086] The Chinese medicine composition of the present invention is aimed at the characteristics of patients with dilated cardiomyopathy and heart failure with reduced ejection fraction who suffer from qi deficiency, blood stasis and yang deficiency. The compatibility is rigorous and sophisticated. After a large number of clinical verifications, it has a significant therapeutic effect on dilated cardiomyopathy combined with heart failure with reduced ejection fraction (qi deficiency, blood stasis and yang deficiency type), has no obvious toxic and side effects, has a short treatment course, and is quick to take effect, with an effective rate of more than 90%.

[0087] The following is the statistical analysis of clinical effects:

[0088] 1. Case selection:

[0089] The Western medicine diagnosis conforms to the diagnostic criteria for dilated cardiomyopathy with reduced ejection fraction heart failure in the 9th edition of Internal Medicine in 2018, and the TCM diagnosis conforms to the diagnostic criteria for "heart distension" with qi deficiency, blood stasis and yang deficiency in the TCM Disease Diagnosis and Efficacy Criteria issued by the State Administration of Traditional Chinese Medicine in 2002. The clinical symptoms are: chest tightness, palpitations, shortness of breath, fatigue, lower limb edema, pale or pale complexion, pale red tongue, moist white fur, and smooth pulse.

[0090] A total of 100 patients aged 60-80 years were selected and randomly divided into two treatment groups, 50 cases in each group. There was no significant difference in the samples.

[0091] 2. Treatment method: Both treatment groups selected the above-mentioned Chinese medicine formula granules. The method of use was: take 15g of the above-mentioned formula granules, soak in water and drink, 3 times / day.

[0092] The Chinese medicine formula is: 10g ginseng, 30g salvia miltiorrhiza, 10g angelica, 6g cinnamon twig, 9g bombyx batryticatus, and 30g corn silk.

[0093] 3. Criteria for determining efficacy: refer to the Guidelines for Diagnosis and Treatment of Common Diseases in Traditional Chinese Medicine

[0094] Clinical cure: Occasionally, chest tightness and palpitations occur, but they can be relieved without medication;

[0095] Markedly effective: Clinical symptoms are significantly alleviated, chest tightness and palpitations still occur regularly, but the frequency is significantly reduced;

[0096] Effective: chest tightness and palpitations still occur, but the frequency is relatively reduced;

[0097] Ineffective: The frequency of chest tightness and palpitations did not decrease, and the clinical symptoms even worsened.

[0098] 4. Results:

[0099] After one course of treatment, the results were evaluated according to the above-mentioned efficacy evaluation criteria. The statistical results and total effective rate are as follows:

[0100] Group Clinical cure Significant effect efficient invalid Total Effectiveness Treatment Group 1 20 21 5 4 92％ Treatment Group 2 18 21 6 5 90％

[0101] It can be seen from the above table that the embodiment has a significant effect on dilated cardiomyopathy combined with heart failure with reduced ejection fraction (Qi deficiency, blood stasis and Yang deficiency), with an effective rate of more than 90%.

[0102] The following further experimental detection and analysis proves the effect of the Chinese medicine composition of the present invention on dilated cardiomyopathy combined with heart failure with reduced ejection fraction (Qi deficiency, blood stasis and Yang deficiency):

[0103] 1. Experimental materials:

[0104] 1.1 Animals:

[0105] 40 male SD rats, 6 weeks old, SPF grade, weight 200±20g, were purchased from the Experimental Animal Center of the Air Force Medical University of the Chinese People's Liberation Army, license number: SCXK(Shaanxi)2019-001. Standard experimental animals and water were used for feeding, the indoor temperature was controlled at (25±1)℃, and adaptive feeding was performed for 1 week.

[0106] 1.2 Main drugs and reagents:

[0107] Shendan Recipe is composed of 10g ginseng, 30g salvia miltiorrhiza, 10g angelica, 6g cinnamon twig, 9g bombyx batryticatus, and 30g corn silk. Adriamycin hydrochloride (Aladdin Biochemical Technology Co., Ltd., catalog number A183027); sodium pentobarbital (Sewell Biotechnology Co., Ltd.); NT-proBNP, CK, cTn-T ELISA kits (Shanghai Guyan Industrial Co., Ltd.); anhydrous ethanol, xylene and neutral gum (Sinopharm Group, batch numbers: 100092683, 10023418 and 10004160); HE staining and Masson staining kits (Beijing Solebold Technology Co., Ltd., catalog numbers G1120 and G1340); BCA protein concentration determination kit (Biyuntian, catalog number P0012); PI3K antibody AKT antibody (CST, USA); protein pre-stained marker (Fermentas, Canada, batch number SM1811).

[0108] 1.3 Experimental equipment:

[0109] Ultra-high-resolution small animal ultrasound imaging system (VisualSonics, Vevo2100); microplate reader (Tecan, Australia); grinder (Carl Zeiss, Germany); microscopic imaging system (Carl Zeiss, Germany); rotating paraffin slicer (Thermofisher, USA); protein electrophoresis and membrane transfer instrument (model 1658033, BioRad, USA).

[0110] 2. Methods:

[0111] 2.1 Model preparation and grouping:

[0112] After 1 week of adaptive feeding, the rats were randomly divided into five groups: normal group, model group, Shendanfang low-dose group, Shendanfang medium-dose group, and Shendanfang high-dose group, with 8 rats in each group. The model group and the treatment group (Shendanfang low-dose group, Shendanfang medium-dose group, and Shendanfang high-dose group) were given intraperitoneal injection of doxorubicin hydrochloride 4 mg / kg according to the body weight of the rats, once a week for 4 consecutive weeks. After 4 weeks, echocardiography was performed. When the weight of the rats decreased, LVIDd and LVIDs increased, and EF and FS decreased and were statistically significant, it indicated that the DCM rat model was successfully established. According to the human and rat body surface area conversion method, the Shendanfang low-dose, medium-dose, and high-dose groups were gavaged with 1.46g, 2.92g, and 5.84g / kg / day, respectively (here: 0.5 times the clinical dose, clinical equivalent dose, and 2 times the clinical dose, respectively). The normal group and the model group were gavaged with an equal amount of 0.9% sodium chloride solution, once a day, for 28 consecutive days.

[0113] 2.2 Animal specimen collection:

[0114] Rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, and then fixed on a surgical operating board in a supine position to fully expose the chest and abdomen. After blood was drawn from the abdominal aorta, heart tissue was removed and rinsed with saline, and part of it was placed in a sterile enzyme-free cryopreservation tube and stored at -80°C for Western blot detection. The remaining part was placed in 4% paraformaldehyde fixative and subsequently prepared for paraffin sections.

[0115] 2.3 Index observation and testing:

[0116] 2.3.1 Observation indicators:

[0117] General conditions of experimental rats.

[0118] 2.3.2 Echocardiography of experimental rats:

[0119] One day after the last administration, the left chest of the rats was depilated and wiped clean in advance. After isoflurane inhalation anesthesia, the rats were fixed in the supine position, and the probe was placed in the precordial area. The ultrasound frequency was 6-12MHz, and the left ventricular ejection fraction (LVEF), short-axis shortening (FS), left ventricular end-diastolic diameter (LVIDd) and left ventricular end-systolic diameter (LVIDs) were recorded.

[0120] 2.3.3ELISA method to detect serum NT-ProBNP, CK, and cTn-T levels in experimental rats:

[0121] Collect 5ml of blood from each group of experimental rats, centrifuge, take the supernatant and set aside; add different concentrations of 50uL / well standard diluent to the 96-well plate, then add 50uL / well serum; add 50uL / well working solution A, shake well, cover and incubate; discard the solution in the well, add 350uL / well washing solution, pat dry with filter paper; add 100uL / well working solution B and 90uL / well substrate solution, incubate in a constant temperature box; add 50uL stop solution to each well, and measure the OD value immediately after mixing; calculate the concentration of the sample according to the drawn standard curve.

[0122] 2.3.4 HE staining of myocardial tissue:

[0123] Myocardial tissue was dehydrated, waxed and embedded, and the slice thickness was 5 μm; xylene was transparent, and after dewaxing, it was immersed in gradient alcohols with concentrations of 100%, 95%, 85%, 75%, and distilled water for 5 minutes each; hematoxylin for 5 minutes, deionized water for 3 minutes, hydrochloric acid solution for differentiation for 3 seconds, deionized water for 3 minutes, eosin for 3 minutes, and washed with running water for 30 seconds; immersed in 75%, 85%, 95%, and 100% alcohol for 3 minutes each, xylene for transparent 10 minutes, and sealed.

[0124] 2.3.5 Masson staining of myocardial tissue:

[0125] The sections were dewaxed and hydrated (same as HE staining (1)); fixed with Bouin's solution, incubated for 2 hours, and washed with water for 15 minutes; stained with lapis lazuli blue staining solution and Mayer's hematoxylin staining solution for 3 minutes each, then washed with water; differentiated with acidic ethanol for 10 seconds, then washed with water for 10 minutes; stained with Ponceau fuchsin and tungstolyl molybdic acid solution for 10 minutes each, then treated with aniline blue staining solution for 5 minutes, weak acid for 1 minute, and then dehydrated with graded alcohol; finally, transparentized with xylene and sealed.

[0126] 2.3.6 TUNEL staining to detect cell apoptosis:

[0127] Dewax and rehydrate the sections (same as HE staining (1)). 90% ethanol, 70% ethanol, distilled water, 2 min each; add protease (refer to the instructions for dosage), 25°C, 3 min. Wash repeatedly with PBS to remove surface protease. Add sodium citrate and incubate on ice for 2 min, rinse with PBS; add TUNEL stain, incubate in the dark, wash 3 times, and take pictures under a microscope.

[0128] 2.3.7 Western-Blot method to detect PI3K, AKT, Bal / Bax, Caspase-3, Caspase-9, protein expression levels:

[0129] Cut the tissue into small pieces, add the lysis solution and homogenize the tissue at -4℃, place it in an ice box for 15min, centrifuge at 2000g, 4℃ for 5min, and take the supernatant. Use the BCA protein concentration determination kit to measure the protein concentration (according to the instructions), heat at 95℃ for 10min, and set aside at -80℃; use the SDS-PAGE gel preparation kit (the ratio table is as follows), check whether the two glass plates are exposed to gel, add the separation gel, add the concentrated gel after solidification, and insert the comb into the concentrated gel. The voltage of the concentrated gel is 90V, and the separation gel is 150V. The electrophoresis can be terminated when it reaches about 1cm from the bottom of the bromophenol blue, and the membrane is transferred. Activate the PVDF membrane with 100% methanol for 2min; 300mA constant current, 30min, in ice. Wash once with TBST, add 5% skim milk and place on a decolorization shaker, and block at room temperature for 30min;

[0130] Dilute the primary antibody according to the instructions and incubate at 4°C overnight; wash with TBST 4 times, 5 min each time; place on a shaker and incubate with secondary antibody at room temperature for 1 hour; wash with TBST 4 times, 5 min each time, add ECL luminescent solution, and save the image after exposure.

[0131] 2.4 Statistical analysis

[0132] SPSS 20.0 was used for statistical analysis, and the mean ± standard deviation was used. The experimental results were processed by expression. When the groups were compared, the one-way analysis of variance (LSD-ANOVA) was used for testing. When P < 0.05, it means that the difference is statistically significant.

[0133] 3. Results:

[0134] 3.1 General conditions of experimental rats:

[0135] The rats in the normal group had shiny hair, agile activities, normal food and water intake and the color of the extremities of the limbs, and their weight gradually increased; the rats in the model group had sparse hair, lethargy, reduced food and water intake, weight loss, diarrhea, abdominal ascites and mild limb edema; after administration of the low, medium and high dose groups of Shendan Fang, the mental state of the rats improved slightly, diarrhea was alleviated, weight increased steadily, and the diet and activity increased.

[0136] 3.2 Experimental rat echocardiographic test results, such as Figure 1 :

[0137] LVIDd: The model group was significantly higher than the normal group; compared with the model group, the LVIDd of the rats in the treatment group decreased; the low-dose group of Shendan Fang P>0.05, no statistical difference; the medium and high-dose groups of Shendan Fang, P values ​​were 0.033, 0.034, P<0.05, the difference was statistically significant. LVIDs: The model group was significantly higher than the normal group; compared with the model group, the LVIDs of all Shendan Fang groups decreased, the low-dose group of Shendan Fang P>0.05, no statistical difference; the medium and high-dose groups of Shendan Fang, P values ​​were 0.027, 0.033, P<0.05, the difference was statistically significant. LVEF: The model group was significantly lower than the normal group; compared with the model group, the LVEF of all Shendan Fang groups increased, the low-dose group of Shendan Fang, the medium-dose group, the high-dose group of Shendan Fang P values ​​were 0.039, 0.005, 0.0002, P<0.05, the difference was statistically significant. FS: The model group was significantly lower than the normal group. Compared with the model group, the FS of each Shendan prescription group increased. The P values ​​of the low, medium and high dose groups of Shendan prescription were 0.028, 0.006 and 0.003, respectively, P < 0.05, and the difference was statistically significant.

[0138] Table 1 Results of cardiac ultrasound examination of rats in each group

[0139]

[0140] Note: Compared with the model group, *P<0.05, **P<0.01

[0141] 3.3 Serum NT-proBNP, CK, and cTn-T levels in experimental rats:

[0142] The serum NT-proBNP, CK and cTn-T levels of rats in the model group were significantly increased, which was statistically different from that in the normal group (P<0.05); compared with the model group, the serum NT-proBNP, CK and cTn-T levels of rats in the low, medium and high dose groups of Shendan Fang were decreased to varying degrees (P<0.05). The results showed that Shendan Fang could significantly reduce the serum NT-proBNP, CK and cTn-T levels of DCM rats ( Figure 2 , Table 2).

[0143] Table 2 NT-proBNP, CK and cTn-T levels in rats in each group

[0144]

[0145] Note: Compared with the normal group, # P<0.05, ## P<0.01, compared with the Model group * P<0.05, ** P<0.01

[0146] 3.4 Experimental rat heart tissue HE staining (such as Figure 3 ):

[0147] Normal group: The myocardial tissue morphology and structure were normal, the cell nucleus was centered, the myocardial fibers were neatly arranged, the continuity was good, and no obvious histological changes were observed. Model group: The myocardial tissue was damaged, showing pathological changes, myocardial cell hypertrophy, cell nucleus deformation or even disappearance, myocardial fiber rupture, and widened intercellular space. Shendanfang low-dose group: The lesion area showed a slight improvement in cell nucleus morphology compared with the model group, but disordered myocardial cell arrangement, muscle fiber rupture and widening were still seen. Shendanfang medium-dose group: The lesion area showed a significant improvement in cell nucleus morphology compared with the model group, the cell arrangement was slightly disordered, the degree of fibrosis was reduced, and there were still a few myocardial fiber ruptures. Shendanfang high-dose group: The lesion area showed a significant improvement compared with the model group, the myocardial cell arrangement was acceptable, the cell nucleus was centered, and only a small part of the myocardial tissue was fibrotic.

[0148] 3.5 Experimental Masson staining of rat heart tissue (eg Figure 4 ):

[0149] Normal group: Most of the normal myocardial tissue is visible, with a few collagen fibers. Model group: A large number of blue collagen fibers are visible, separating the myocardial tissue into blocks. The extracellular matrix around the blood vessels increases. Low, medium and high dose groups of Shendan Fang: The collagen fiber area gradually becomes smaller, the muscle fibers are arranged more neatly, and the myocardial fibrosis gradually decreases. Figure 5 . (Compared with the normal group, # P<0.05, ## P<0.01, compared with the Model group * P<0.05, ** P<0.01)

[0150] 3.6 Cell apoptosis was observed by TUNEL staining under a microscope (eg Figure 6 ):

[0151] DAPI is used for nucleus staining, and apoptotic cells show green fluorescence. There was basically no positive signal detected in the normal group, and almost no apoptotic cells were found; the number of TUNEL-positive myocardial cells in the myocardial tissue of rats in the model group increased significantly, and the apoptotic rate of myocardial cells increased significantly; compared with the model group, the apoptotic rate of myocardial cells in each Shendan prescription group was significantly reduced, and the green fluorescence marked by TUNEL method was significantly reduced, which reduced the number of apoptotic myocardial cells, and the apoptotic cells in the high-dose Shendan group were the least.

[0152] 3.7 Western-Blot detection of rat PI3K, AKT, Bal / Bax, Caspase-3, Caspase-9, protein expression levels (such as Figure 7 ):

[0153] like Figure 7 It can be seen that compared with the normal group, the expression of PI3K / AKT protein in the model group was significantly decreased (P < 0.05); after applying the low, medium and high dose groups of Shendan Fang, the expression of PI3K / AKT protein was significantly increased compared with the model group, especially in the high dose group (P < 0.05). Compared with the normal group, the protein expression and Bcl-2 / Bax ratio in the model group were significantly reduced, while Bax was significantly increased (P < 0.05); compared with the model group, the expression of Bcl-2 protein and Bcl-2 / Bax levels in the low, medium and high dose groups of Shendan Fang were significantly increased (P < 0.05) and were positively correlated with the dose; while the Bax levels were significantly offset (P < 0.05) and were negatively correlated with the dose. Compared with the normal group, the expression of Caspase-3 and Caspase-9 signaling pathway proteins in the model group was increased; compared with the model group, the expression of Caspase-3 and Caspase-9 signaling pathway proteins in the low, medium and high dose groups of Shendan prescription were significantly decreased (P<0.05), and were negatively correlated with the drug concentration. Figure 7 A: normal group B: model group C: low-dose group D: medium-dose group E: high-dose group (compared with the normal group, # P<0.05, ## P<0.01, compared with the Model group * P<0.05, ** P<0.01)

[0154] The above experimental test analysis shows that:

[0155] 1. Echocardiography can observe the functional structure of the heart, conduct real-time dynamic scanning in multiple directions and angles, evaluate ventricular wall motion, the opening and closing of valves, and measure cardiac function. It is also an important and reliable evaluation method in animal experimental research. The echocardiography detection indicators of the above experiments include: LVIDd, LVIDs, LVEF, and EF. The results showed that after the DCM rats were treated with Shendan Fang: the LVIDd and LVIDs values ​​of the low, medium, and high dose groups of Shendan Fang decreased, and the LVEF and EF values ​​increased. The rats in the treatment group were significantly better than those in the model group, and the inner diameter of the heart cavity decreased. It was determined that Shendan Fang was effective in treating DCM and significantly improved the cardiac function of DCM rats.

[0156] NT-pro BNP is an inactive and stable amino acid fragment that has been found to be an important biomarker for left ventricular systolic dysfunction and left ventricular stress, and has a longer half-life and higher serum stability. CK and cTnT are the most sensitive indicators for judging myocardial cell necrosis, and their levels are positively correlated with the degree of myocardial cell necrosis. In this experiment, after drug treatment, the levels of NT-proBNP, CK, and cTn-T in the serum of DCM rats were reduced (P < 0.05), and the reduction level was more obvious as the dose of Shendan Fang increased.

[0157] 2. The effect of Shendan prescription on improving the heart function of DCM rats may be related to the regulation of PI3K / AKT signaling pathway:

[0158] The above results show that compared with the model group, with the increase of the concentration of Shendan Fang, the expression of PI3K and AKT proteins gradually increased. It suggests that Shendan Fang may be related to the regulation of PI3K and AKT signaling pathways in improving the cardiac function of DCM rats. In recent years, studies have shown that the activation of PI3K and AKT signaling pathways can regulate energy production, slow down heart failure, and inhibit cell apoptosis. The PI3K / AKT pathway affects the changes of myocardial cells in the pathogenesis of myocardial fibroblasts, the abnormal ratio of type I and III collagen, and the synthesis and deposition of extracellular matrix in the heart failure pathway. Therefore, the above experiments show that doxorubicin hydrochloride can significantly reduce the expression of PI3K / AKT proteins in myocardial tissue, while the application of Shendan Fang can upregulate the expression level of PI3K / AKT proteins. It shows that Shendan Fang has a protective effect on DCM induced by doxorubicin hydrochloride, and its mechanism of action may be related to regulating the expression level of PI3K / AKT proteins, activating the PI3K and AKT signaling pathways, and promoting it to increase the anti-apoptosis ability of myocardial tissue in DCM rats and protect myocardial tissue.

[0159] In the occurrence and development of DCM, the activation of the PI3K / AKT signaling pathway can promote the proliferation and hypertrophy of cardiomyocytes, thereby leading to the expansion and hypertrophy of myocardial tissue. Studies have shown that the activation of the PI3K / AKT signaling pathway may not only promote the proliferation and growth of cardiomyocytes by regulating the expression of genes such as myocardial cell cyclin D1 and Bcl-2, but also affect the structure and function of myocardial tissue by regulating processes such as cell apoptosis, fibrosis and hypertrophy. The Caspase family is a group of proteases with similar structures that exist in the cytoplasm. They are closely related to the apoptosis of eukaryotic cells and are involved in the regulation of cell growth, differentiation and apoptosis. Among them, Caspase 9 is the initiator of cell apoptosis, and Caspase 3 is the main executor of cell apoptosis. In this experiment, Shendan Recipe can improve the apoptosis of cardiomyocytes in DCM rats, and can upregulate the expression of PI3K, AKT, and Bcl-2 proteins, and downregulate the expression of Caspase-3, Caspase-9, and Bax proteins.

[0160] Therefore, the above experiments show that Shendan prescription can improve doxorubicin-induced cardiac enlargement and cardiac fibrosis in DCM rats, reduce myocardial tissue damage, improve LVEF and cardiomyocyte apoptosis. Its mechanism of action may be related to regulating Bcl-2 / Bax through PI3K / AKT and ultimately affecting Caspase-3 / Caspase-9 expression.

[0161] The above specific examples are used to illustrate the present invention, which is only used to help understand the present invention and is not intended to limit the present invention. For those skilled in the art, according to the concept of the present invention, some simple deductions, modifications or substitutions can be made.

Claims

1. A Chinese medicine composition for treating dilated cardiomyopathy with reduced ejection fraction heart failure, Features: The Chinese medicine composition is made of the following components in parts by weight: 5-10 portions of ginseng; 10-30 parts of Salvia miltiorrhiza; 10-20 portions of Chinese Angelica; 6-9 parts of cinnamon twig; Bombyx batryticatus 6-9 parts; 10 to 30 portions of corn silk.

2. The Chinese medicine composition according to claim 1, Features: The Chinese medicine composition is made of the following components in parts by weight: 10 portions of ginseng; 30 parts of Salvia miltiorrhiza; 10 parts of Chinese Angelica; 6 parts of cinnamon twig; Bombyx batryticatus 9 parts; 30 servings of corn silk.

3. The Chinese medicine composition according to claim 1, Features: The dosage form of the Chinese medicine composition is granules.

4. The method for preparing the Chinese medicine composition according to claim 3, Features: The preparation method of the granules comprises the following steps: Step 1: Mix ginseng, salvia miltiorrhiza, angelica, cinnamon twig, silkworm and corn silk, add water and boil twice, each time for 2 hours, filter, combine the filtrate and concentrate; Step 2: Add ethanol, let stand, filter, recover ethanol from the filtrate, let stand, filter, concentrate under reduced pressure to form a thick paste, make granules, and dry.

5. The preparation method according to claim 4, Features: In step 2, the ethanol content is 50%.

6. The preparation method according to claim 5, Features: In step 2, the mixture is concentrated under reduced pressure to a thick paste until the relative density reaches 1.30-1.35, and the temperature is maintained at 60-70°C during the concentrated process.

7. The preparation method according to claim 6, Features: In step 2, after the mixture is concentrated under reduced pressure into a thick paste, sucrose is added and mixed, and then granules are formed.

8. Use of the Chinese medicine composition according to claim 1 in the preparation of a medicament for treating dilated cardiomyopathy of qi deficiency and blood stasis combined with yang deficiency type combined with heart failure with reduced ejection fraction.

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