Application of traditional Chinese medicine composition in preparation of sepsis cardiomyopathy resisting medicine

Abstract

The invention discloses application of a traditional Chinese medicine composition in preparation of a sepsis cardiomyopathy resisting medicine. The traditional Chinese medicine composition comprises astragalus membranaceus and radix aconiti carmichaeli. The traditional Chinese medicine composition disclosed by the invention is applied to preparation of medicines for resisting sepsis and cardiomyopathy, can be used for remarkably improving heart functions, relieving myocardial damage and reducing inflammatory response, effectively inhibits sepsis and cardiomyopathy, and has a good medicinal prospect.

Description

Technical Field

[0001] The present invention relates to the use of a traditional Chinese medicine composition in the preparation of a drug for treating sepsis-induced cardiomyopathy. Background Art

[0002] Sepsis-induced cardiomyopathy (SIC) is a cardiac dysfunction caused by sepsis. Approximately 50% of sepsis patients show signs of myocardial dysfunction, with a high mortality rate and complex pathological mechanisms, mainly including myocardial depression, excessive inflammation, mitochondrial dysfunction, cardiomyocyte apoptosis, oxidative stress, and calcium ion imbalance, etc. In traditional Chinese medicine, the cause of the disease is mostly considered to be related to the invasion of exogenous pathogens. The pathogens enter the body, transform into heat over time, and accumulate excessive heat-toxicity, resulting in the blockage of qi and blood in the zang-fu organs and meridians. Over time, it depletes the healthy qi of the body, leading to a complex situation of deficiency and excess, with deficiency of healthy qi and excess of pathogenic factors (Traditional Chinese Medicine, 13(4): 738 - 743, 2024). Currently, there is no treatment measure supported by evidence-based medicine for sepsis-induced cardiomyopathy. Clinically, the existing treatment for sepsis-induced cardiomyopathy mainly focuses on stabilizing organ function, such as using EMCO treatment, controlling infection, fluid resuscitation, and symptomatic treatment, etc. In terms of medication, anti-infective drugs, vasoactive drugs, β-blockers, and anti-inflammatory drugs, etc. are mostly used for basic treatment. It is worth noting that there have been some research reports that traditional Chinese medicine compounds can be used to prevent and treat SIC, such as Huanglian Jiedu Decoction (Lishizhen Guoyao, 34(9): 2080 - 2083, 2023), Sini Decoction (Western Journal of Traditional Chinese Medicine, 36(11): 19 - 23, 2023), etc. Chinese Patent CN117959362 A discloses a traditional Chinese medicine composition for treating sepsis-induced cardiomyopathy, which is composed of ginseng, rhubarb, aconite, coptis, scutellaria, and safflower, and lists various dosage ratios, and can improve the circulation, cardiac function, etc. of patients with yang deficiency and toxin stasis syndrome of sepsis.

[0003] Currently, there is no treatment measure supported by evidence-based medicine. Clinically, vasoactive drugs, β-blockers, etc. are mostly used for basic treatment, but it is often difficult to achieve satisfactory effects, with large side effects and high treatment costs. Therefore, finding safe and effective drugs for treating sepsis-induced cardiomyopathy can improve the prognosis of sepsis patients and reduce their mortality rate, which has important clinical application value. Summary of the Invention

[0004] Object of the Invention: The object of the present invention is to provide the use of a traditional Chinese medicine composition in the preparation of a drug for treating sepsis-induced cardiomyopathy.

[0005] Technical Solution: The present invention discloses the use of a traditional Chinese medicine composition in the preparation of a drug for treating sepsis-induced cardiomyopathy, characterized in that the traditional Chinese medicine composition includes astragalus membranaceus and aconite.

[0006] Among them, in the traditional Chinese medicine composition, the mass ratio of Astragalus membranaceus and Aconiti Lateralis Radix Praeparata is 1-3:1; preferably 2:1.

[0007] Among them, the septic cardiomyopathy is cardiac function injury caused by sepsis.

[0008] Among them, the cardiac function injury is cardiac diastolic and / or systolic dysfunction.

[0009] Among them, the traditional Chinese medicine composition alleviates the disorder and rupture of myocardial fiber arrangement caused by sepsis.

[0010] Among them, the traditional Chinese medicine composition reduces the contents of CK-MB, LDH, IL-1β and TNF-α in the serum elevated by sepsis.

[0011] Among them, the preparation method of the traditional Chinese medicine composition includes the following steps: taking the raw Chinese medicinal materials Astragalus membranaceus and Aconiti Lateralis Radix Praeparata, adding 10-15 times the volume of deionized water and soaking for 1-2 hours, extracting 2 times by boiling water method, extracting for 2-3 hours for the first time, pouring out the extract, adding 8-10 times the volume of deionized water again, extracting by boiling water method for 1-2 hours, combining the two extracts, filtering, and concentrating under reduced pressure to obtain; preferably, taking the raw Chinese medicinal materials Astragalus membranaceus and Aconiti Lateralis Radix Praeparata, adding 10 times the volume of deionized water and soaking for 1 hour, extracting 2 times by boiling water method, extracting for 2 hours for the first time, pouring out the extract, adding 8 times the volume of deionized water again, extracting by boiling water method for 1 hour, combining the two extracts, filtering, and concentrating under reduced pressure to obtain.

[0012] Among them, the administration route of the traditional Chinese medicine composition is intragastric administration, the administration dose is 3-6 g / kg, preferably 6 g / kg, and it is applied in the anti-septic cardiomyopathy drug in the form of an oral preparation.

[0013] Principle of the Invention: The traditional Chinese medicine composition used in the present invention comprises astragalus membranaceus and aconite root. In the ancient medical book "Jisheng Fang", it is stated that "when pathogenic qi attacks healthy qi, the pathogenic and healthy qi clash, the qi passage is blocked, stagnated in the middle jiao, and then angina pectoris occurs". In "Plain Questions - Treatise on Pain Caused by Various Factors", it is said that "when cold qi enters the channels and lingers, it stagnates and does not move. When it lodges outside the vessels, there is less blood; when it lodges in the vessels, qi cannot flow through, so sudden pain occurs". If the heart yang fails to thrive and is invaded by cold pathogen, resulting in excessive yin-cold in the chest and heart, the yang qi fails to spread, cold congeals the heart vessels, the nutritive blood circulation becomes sluggish, or if the body is inherently deficient in yang qi, or if damp-cold yin pathogen damages the heart yang, leading to deficiency of heart yang, loss of warming and promoting function, and the heart vessels losing nourishment, then "angina pectoris" can occur. The traditional Chinese medicine composition used in the present invention is composed of astragalus membranaceus and aconite root, and the dosage ratio is preferably 2:1, which is mainly used for treating qi deficiency and yang weakness, limb fatigue, and incessant sweating. In the formula, astragalus membranaceus is sweet and warm in nature, tonifying qi, consolidating the exterior and lifting yang, containing flavonoids and saponins, and has a good therapeutic effect on heart failure; aconite root is pungent and hot, restoring yang and rescuing from reversal, tonifying fire and assisting yang, rich in various alkaloids, and has the effects of strengthening the heart and relieving oxidative stress injury. The so-called "assisting yang must first tonify qi", the combination of astragalus membranaceus and aconite root can not only lift yang and consolidate the exterior but also warm yang and assist fire, relieve "angina pectoris", and "angina pectoris" is equivalent to myocardial ischemia and heart failure in Western medicine. Sepsis cardiomyopathy is an acute cardiac dysfunction syndrome that occurs when sepsis worsens, which may cause problems such as arrhythmia and heart failure. The main pathogenesis includes excessive inflammation, oxidative stress, myocardial energy metabolism disorder, etc. When it occurs, it is accompanied by symptoms such as left ventricular dilation, decreased left ventricular ejection fraction, elevated levels of inflammatory factors (TNF-α, IL-1β, etc.) and myocardial injury markers (CK-MB, LDH). Qifu Decoction has the potential to be applied in cardiac protection and is expected to be used for treating sepsis cardiomyopathy.

[0014] Beneficial Effects: Compared with the prior art, the present invention has the following remarkable advantages: When the traditional Chinese medicine composition of the present invention is applied in the preparation of drugs for treating sepsis cardiomyopathy, it can increase the abnormally decreased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) caused by LPS by 38.2% and 53.1% respectively, significantly enhancing cardiac function; improve the disordered and broken arrangement of myocardial fibers caused by sepsis, and down-regulate the abnormally elevated serum CK-MB and LDH levels caused by LPS by 28.7% and 32.3% respectively, effectively reducing myocardial injury; reduce the proportion of CD68-positive cells in the myocardium, and down-regulate the abnormally elevated serum IL-1β and TNF-α levels caused by LPS by 38.6% and 36.7% respectively, significantly reducing the inflammatory response, effectively inhibiting sepsis cardiomyopathy, and having good medicinal prospects. Description of the Drawings

[0015] Figure 1 It is the echocardiogram result of mice;

[0016] Figure 2 It is the left ventricular ejection fraction result graph of mice; * in the graph represents p < 0.05, ** represents p < 0.01;

[0017] Figure 3 It is the result graph of the left ventricular shortening fraction of mice; in the graph, * represents p < 0.05, and ** represents p < 0.01;

[0018] Figure 4 It is the HE staining result graph of the heart tissues of mice in each group;

[0019] Figure 5 It is the staining result graph of CD68-positive cells in the heart tissues of mice in each group; among them, A is the staining graph of CD68-positive cells, B is the statistical graph of the proportion of CD68-positive cells, in the graph, * represents p < 0.05, *** represents p < 0.001, and **** represents p < 0.0001;

[0020] Figure 6 It is the result graph of the serum LDH and CK-MB levels of mice; among them, A is LDH, B is CK-MB, in the graph, * represents p < 0.05, *** represents p < 0.001, and **** represents p < 0.0001;

[0021] Figure 7 It is the result graph of the serum IL-1β and TNF-α levels of mice; among them, A is IL-1β, B is TNF-α, in the graph, *** represents p < 0.001, and **** represents p < 0.0001. Specific implementation manners

[0022] The technical solution of the present invention will be further described below in combination with embodiments. The test materials used in the embodiments can be obtained through conventional channels.

[0023] Experimental materials:

[0024] Experimental animals: Healthy ICR mice (male, body weight 24 - 26 g), clean grade, provided by the Comparative Medicine Center of Yangzhou University.

[0025] Experimental drugs: Astragalus membranaceus (produced in Hebei) and Aconitum carmichaelii Debx. (produced in Sichuan) were purchased from Beijing Tongrentang Pharmacy (Xi'an).

[0026] Experimental reagents: Normal saline: purchased from Anhui Shuanghe Pharmaceutical Co., Ltd.; Lipopolysaccharide (LPS): Sigma Company, USA; PBS and paraformaldehyde: Jiangsu KeyGen Biotech Co., Ltd.; LDH kit: Nanjing Jiancheng Bioengineering Institute; Mouse CK-MB, IL-1β, TNF-α detection kits: Hunan Aifang Biotechnology Co., Ltd.

[0027] Experimental instruments: Ultra-high resolution small animal ultrasound imaging system Vevo 3100LT: FUJIFILM VisualSonics; SpectraMax M2e microplate reader: Molecular Devices, USA.

[0028] Example 1

[0029] The traditional Chinese medicine composition of the present invention: 30 g of Astragalus membranaceus and 15 g of Aconitum carmichaelii Debx.

[0030] Its preparation method is as follows: Take 30 g of Astragalus membranaceus and 15 g of Aconitum carmichaelii Debx. as raw Chinese medicinal materials, add 10 times the volume of water and soak for 1 hour, decoct for 2 hours, pour out the decoction, add 8 times the volume of water again, decoct for 1 hour, combine the two extraction liquids, filter, and concentrate under reduced pressure to 90 mL (the crude drug concentration is 0.5 g / mL) to obtain.

[0031] Example 2

[0032] Establishment and grouping of mouse sepsis cardiomyopathy model

[0033] Forty-six healthy ICR mice (male, body weight 24 - 26 g), clean grade, free access to food and water, 12-hour light exposure per day, and adaptively fed for 7 days before the formal experiment. The mice were randomly divided into a control group (Control, 10 mice), a model group (LPS, 12 mice), a low-dose group of the traditional Chinese medicine composition of the present invention (QFD-L, 12 mice), and a high-dose group of the traditional Chinese medicine composition of the present invention (QFD-H, 12 mice). The control group and the model group were continuously gavaged with normal saline (0.15 mL / 10 g) for 7 days; the low-dose group of the traditional Chinese medicine composition of the present invention was continuously gavaged at a dose of 3 g / kg for 7 days; the high-dose group of the traditional Chinese medicine composition of the present invention was continuously gavaged at a dose of 6 g / kg for 7 days. One hour after the last administration, the control group was intraperitoneally injected with PBS, and the remaining groups were intraperitoneally injected with LPS (10 mg / kg) per mouse and treated for 12 hours.

[0034] Determination of cardiac function indexes:

[0035] After treating the above groups of mice for 12 hours, an appropriate amount of depilatory cream was evenly applied to the heart area of the mice to depilate them, exposing the skin of the heart area completely. After anesthetizing the mice with 1% isoflurane, they were fixed on the operating board with their chests facing up. An appropriate amount of coupling agent for detection was applied to the heart, and the probe was placed on the chest of the mice. Using the ultra-high resolution small animal ultrasonic imaging system Vevo 3100LT, two-dimensional M-mode ultrasound was selected for echocardiography examination. Three consecutive cardiac cycles were measured, and the data were processed with VevoLAB 3.2.6 software to obtain the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS).

[0036] From Figures 1 to 3 It can be seen that compared with the control group (Control), the LVEF and LVFS of the model group (LPS) were significantly decreased (p<0.01); compared with the LPS group, the LVEF and LVFS of the low-dose group of the traditional Chinese medicine composition of the present invention (QFD-L) and the high-dose group of the traditional Chinese medicine composition of the present invention (QFD-H) were significantly increased (p<0.05, p<0.01), and the cardiac function indexes of the mice in the QFD-H group increased more significantly than those in the QFD-L group, indicating that the pretreatment of the traditional Chinese medicine composition of the present invention can improve the impaired cardiac function of septic cardiomyopathy mice, and the improvement effect of the high-dose group of the traditional Chinese medicine composition of the present invention is better than that of the low-dose group of the traditional Chinese medicine composition of the present invention.

[0037] Example 3

[0038] HE staining:

[0039] After treating the above groups of mice for 12 hours, the hearts of the mice were collected and fixed in 4% formalin for 24h. After dehydration, tissue clearing, paraffin embedding, sectioning, dewaxing, hematoxylin-eosin (HE) staining, dehydration and sealing, the morphology of cardiomyocytes was observed with an optical microscope.

[0040] From Figure 4 It can be seen that the myocardial texture of the mice in the Control group was clear, and the myocardial tissue cells were arranged neatly and regularly. However, obvious myocardial damage was visible in the LPS group, with disordered arrangement of myocardial fibers, breakage in many parts, showing extensive cytoplasmic vacuolization, interstitial edema and partial nuclear deletion (nuclear lysis). After pretreatment with the traditional Chinese medicine composition of the present invention, compared with the LPS group, the cell texture tended to be clear in the QFD-L and QFD-H groups, the arrangement of cardiomyocytes gradually became neat, the degree of myocardial damage improved significantly, and the phenomena of vacuoles and nuclear deletion decreased.

[0041] Example 4

[0042] Immunohistochemical staining:

[0043] Deparaffinize the paraffin-embedded heart tissue, incubate it in a 3% hydrogen peroxide solution in the dark for 25 minutes, and then rinse it 3 times with 1×PBS. Then, drip 3% bovine serum albumin into the histochemical circle and seal it for 30 minutes. Incubate the sections with the anti-CD68 primary antibody overnight at 4°C, and then incubate them with goat anti-rabbit IgG(H+L) HRP at room temperature for 50 minutes, followed by washing 3 times. Then, drip the 3,3'-diaminobenzidine (DAB) chromogenic solution, control the chromogenic time under the microscope, the positive color is brownish-yellow, and rinse the sections with tap water to terminate the chromogenesis. Re-stain the cell nuclei with hematoxylin, then dehydrate and seal the sections, and observe them under an upright microscope.

[0044] CD68 is highly expressed in macrophages and is a signal of inflammation in the body. Figure 5 This is the staining result diagram of CD68-positive cells in the heart tissue of mice in each group of the present invention. As shown in the figure, the proportion of CD68-positive cells in the LPS group increased significantly (p<0.0001), while the levels in the QFD-L and QFD-H groups were significantly decreased (p values were both less than 0.001), indicating that the pretreatment with the traditional Chinese medicine composition of the present invention can alleviate myocardial inflammation in mice with septic cardiomyopathy, and the improvement effect of the high-dose group of the traditional Chinese medicine composition of the present invention is better than that of the low-dose group.

[0045] Example 5

[0046] Determination of serum CK-MB, LDH, IL-1β, and TNF-α levels:

[0047] After treating the mice in each group above for 12 hours, collect blood by eye socket puncture, collect the serum, and determine the levels of lactate dehydrogenase (LDH), creatine kinase isoenzyme (CK-MB), interleukin 1β (IL-1β), and tumor necrosis factor (TNF-α) in the mouse serum according to the kit instructions.

[0048] Serum LDH and CK-MB levels are reliable indicators for judging myocardial injury. As can be seen from Figure 6 , compared with the Control group, the serum LDH and CK-MB levels in the LPS group of mice increased significantly (p<0.0001); while compared with the LPS group, the LDH and CK-MB levels in the QFD-L and QFD-H groups of mice were significantly decreased (p values were both less than 0.05), and the decrease in the QFD-H group was more obvious than that in the QFD-L group, indicating that the pretreatment with the traditional Chinese medicine composition of the present invention can alleviate myocardial injury in mice with septic cardiomyopathy, and the improvement effect of the high-dose group of the traditional Chinese medicine composition of the present invention is better than that of the low-dose group.

[0049] IL-1β and TNF-α are typical pro-inflammatory cytokines. As can be seen from Figure 7It can be seen that compared with the Control group, the levels of IL-1β and TNF-α in the LPS group of mice were significantly increased (p < 0.0001); compared with the LPS group, the levels of IL-1β and TNF-α in the QFD-L and QFD-H groups of mice were significantly decreased (p values were both less than 0.001), and the decrease in the QFD-H group was more obvious than that in the QFD-L group, indicating that the pretreatment with the traditional Chinese medicine composition of the present invention can significantly reduce the levels of serum inflammatory factors in mice with septic cardiomyopathy, reduce the inflammatory response, and the high-dose group of the traditional Chinese medicine composition of the present invention has a better effect than the low-dose group of the traditional Chinese medicine composition of the present invention.

[0050] Statistical analysis: One-way analysis of variance (ANOVA) and Tukey's test were used to examine the statistical differences in the samples through IBM SPSS Statistics 26.0. A p value < 0.05 was considered statistically significant.

[0051] The present invention investigated the effect of the traditional Chinese medicine composition of the present invention against septic cardiomyopathy based on ICR mice. The main investigation indicators included the cardiac function of mice, the morphology of cardiomyocytes, and the levels of serum LDH, CK-MB, IL-1β, and TNF-α. The results showed that the traditional Chinese medicine composition of the present invention can significantly improve cardiac function, reduce myocardial injury, and reduce the inflammatory response, effectively inhibiting septic cardiomyopathy, having good medicinal prospects, and providing a new drug for the prevention and treatment of septic cardiomyopathy.

Claims

1. Use of a Chinese medicine composition in the preparation of an anti-septic cardiomyopathy drug, characterized in that: The traditional Chinese medicine composition comprises astragalus and aconite.

2. The use according to claim 1, characterized in that: In the traditional Chinese medicine composition, the mass ratio of astragalus to aconite is 1 to 3:

1.

3. The use according to claim 1, characterized in that: The septic cardiomyopathy is the cardiac function damage caused by sepsis.

4. The use according to claim 3, characterized in that: The cardiac function damage is cardiac diastolic and / or systolic dysfunction.

5. The use according to claim 1, characterized in that: The traditional Chinese medicine composition reduces the disordered arrangement and rupture of myocardial fibers caused by sepsis.

6. The use according to claim 1, characterized in that: The traditional Chinese medicine composition reduces the contents of CK-MB, LDH, IL-1β and TNF-α in serum which are elevated due to sepsis.

7. The use according to claim 1, characterized in that: The preparation method of the traditional Chinese medicine composition comprises the following steps: taking raw Chinese medicinal materials astragalus and aconite, adding 10 to 15 times the volume of deionized water to soak for 1 to 2 hours, extracting twice by boiling water method, the first extraction is 2 to 3 hours, pouring out the extract, adding 8 to 10 times the volume of deionized water, extracting by boiling water method for 1 to 2 hours, combining the two extracts, filtering, and concentrating under reduced pressure to obtain the composition.

8. The use according to claim 1, characterized in that: The Chinese medicine composition is administered by intragastric administration, with a dosage of 3 to 6 g / kg, and is used in anti-septic cardiomyopathy drugs in the form of oral preparations.

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