Application of traditional Chinese medicine compound preparation in preparation of medicine for repairing myocardial injury caused by sepsis

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and discloses application of a traditional Chinese medicine compound preparation in preparation of a medicine for repairing myocardial injury caused by sepsis. The traditional Chinese medicine compound preparation is prepared from the following raw materials in parts by weight: 3-20 parts of red ginseng, 3-25 parts of radix aconiti carmichaeli, 5-30 parts of semen coicis and 2-20 parts of exocarpium citri grandis. The traditional Chinese medicine compound preparation acts on sepsis cardiomyopathy in a multi-link and multi-target mode, the treatment mode is converted from improvement of a short-term hemodynamic state to a long-term repair strategy, the re-hospitalization rate and the case fatality rate of patients with sepsis cardiomyopathy are further reduced, the life quality of the patients is improved, and the defects of western medicines are overcome. The medicine for repairing myocardial injury caused by sepsis is a medicine for resisting sepsis cardiomyopathy by inhibiting immune response generated by macrophages, reducing NO release and inhibiting generation of IL-1beta, IL-6 and TNF-alpha cell factors.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to use of a traditional Chinese medicine compound preparation in preparing a medicine for repairing myocardial damage caused by sepsis. Background Art

[0002] Sepsis is a disorder of the immune response caused by infection, and even life-threatening multiple organ failure. Its mortality rate is close to 25-30%, and the in-hospital mortality rate of septic shock is as high as 40-60%. As the power source of the circulatory system, the heart's dysfunction will lead to a decrease in effective blood flow, which will in turn aggravate tissue ischemia and hypoxia, causing mitochondrial dysfunction, oxidative stress, energy metabolism disorders, cell death, etc. Sepsis-induced cardiomyopathy is a serious complication of severe sepsis and septic shock, and the severity of myocardial damage is closely related to the prognosis of sepsis.

[0003] The most common trigger of sepsis is bacterial or viral infection. After infection, innate immune cells (including neutrophils, macrophages, dendritic cells, natural killer cells, and T cells) are key mediators not only of the initial host response, but also of the dysregulated immune response. In particular, macrophages play a crucial role in regulating cardiac dysfunction, as cardiac stress is significantly associated with an expansion of the cardiac macrophage population. In addition to the local proliferation of cardiac resident macrophages, circulating monocytes are recruited and differentiated into macrophages. When cells are damaged or die after bacterial and viral infections, endogenous molecules (DAMPs) are produced. DAMPs, as danger signals, can stimulate these recruited macrophages to produce an immune response, thereby affecting the occurrence of septic cardiomyopathy.

[0004] Once PAMPs or DAMPs are recognized by pattern recognition receptors (including toll-like receptors expressed on the cell surface of cardiomyocytes), intracellular signaling cascades induce the activation and translocation of nuclear factor κB (NF-κB), and subsequently release immune cytokines such as TNF-α, IL-1β, etc. These cytokines can affect cardiomyocyte homeostasis and, in severe cases, lead to the occurrence of septic cardiomyopathy.

[0005] At present, the cost of treating septic cardiomyopathy is high. For example, the use of EMCO treatment is complicated. The existing treatment of septic cardiomyopathy focuses on stabilizing organ function, controlling infection and fluid resuscitation. Drugs for stabilizing organ function include esmolol, vasoactive drugs, etc. Drugs for controlling infection are mainly antibiotics or antiviral drugs. However, existing therapeutic drugs may bring some side effects. For example, norepinephrine among vasoactive drugs is the first-line drug for treatment. Early use can reduce the cardiac preload of septic cardiomyopathy and prevent pulmonary edema. However, excessive afterload will reduce CO and lead to abnormal cardiac function. Summary of the invention

[0006] In order to solve the deficiencies and shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a use of a Chinese medicine compound preparation in the preparation of a drug for repairing myocardial damage caused by sepsis. The Chinese medicine compound preparation can better repair myocardial damage caused by sepsis, has the effects of promoting blood circulation and removing blood stasis, strengthening the body and consolidating the foundation, can better inhibit the immune response in septic cardiomyopathy, and can better make up for the shortcomings of Western medicine.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A use of a Chinese medicine compound preparation in preparing a drug for repairing myocardial damage caused by sepsis. The Chinese medicine compound preparation is prepared from the following raw materials in parts by weight: 3-20 parts of red ginseng, 3-25 parts of aconite root, 5-30 parts of coix seeds, and 2-20 parts of tangerine peel.

[0009] Preferably, the Chinese medicine compound preparation is composed of the following raw materials in parts by weight: 10 parts of red ginseng, 10 parts of aconite, 5 parts of coix seeds, and 6 parts of tangerine peel.

[0010] The traditional Chinese medicine compound preparation comprises the following ingredients in percentage by mass: ginsenoside Rg1 0.2%-5%, ginsenoside Rb1 0.10%-5%, naringin 4%-12%, aconitine components 0.2%-5%, and triolein 0.2%-5%; the aconitine components include benzoylneaconitine, benzoylaconitine and benzoylhyaconitine.

[0011] The Chinese medicine compound preparation is prepared according to the following water extraction process steps: weighing red ginseng, tangerine peel, coix seed and aconite, crushing to obtain micropowder, adding water 4-8 times the volume of the micropowder, soaking overnight, ultrasonic extraction twice, each extraction time 50-120 minutes, filtering to obtain the Chinese medicine compound extract, and then spray drying to prepare the Chinese medicine compound preparation.

[0012] The Chinese medicine compound preparation is prepared according to the following alcohol extraction process steps: weighing red ginseng, tangerine peel, coix seed and aconite, crushing them to obtain micro powder, adding ethanol with a volume of 6-8 times that of the micro powder, the concentration of ethanol is 50-90% by volume, ultrasonic extraction is performed twice, each extraction takes 20-40 minutes, filtering, combining the filtrate, recovering ethanol under reduced pressure, obtaining a concentrated extract, adding auxiliary materials to mix, sieving, spray drying, and preparing the Chinese medicine compound preparation.

[0013] The drug for repairing myocardial damage caused by sepsis is a drug for preventing and treating septic cardiomyopathy.

[0014] The traditional Chinese medicine compound preparation is made into capsules, oral liquids, tablets or granules.

[0015] The drug for repairing myocardial damage caused by sepsis is a drug for resisting sepsis cardiomyopathy by inhibiting the immune response produced by macrophages, reducing the release of NO, and inhibiting the production of IL-1β, IL-6, and TNF-α cytokines.

[0016] Compared with the prior art, the present invention has the following advantages and effects:

[0017] (1) Sepsis cardiomyopathy has a high mortality rate, and there is currently no clear treatment method. The treatment cost is high and the treatment is complicated. In view of the above shortcomings, the present invention proposes to use a Chinese medicine compound preparation to repair myocardial damage caused by sepsis. The present invention is safe and effective, has a good repair effect, is low in cost, has few side effects, has a wide range of indications, and is easy to use.

[0018] (2) The Chinese medicine compound preparation of the present invention can be made into capsules, oral liquids, tablets, granules, etc., which is convenient for treatment, can be carried around, is easy to use, and has low cost.

[0019] (3) The Chinese medicine compound preparation of the present invention is prepared from four Chinese medicines, namely, red ginseng, aconite, coix seed and tangerine peel, and has the effects of promoting blood circulation and removing blood stasis, strengthening the body and consolidating the foundation. Its bioactive substances are mostly saponins, alkaloids, flavonoids and polysaccharides, etc., which can reduce immune response, scavenge free radicals, improve blood circulation, and thus improve cardiomyopathy caused by sepsis. Among them, red ginseng and aconite have the effects of promoting blood circulation and removing blood stasis, restoring yang and rescuing adverse conditions, dissipating blood stasis in the body, increasing blood flow, reducing the production of immune factors, and thus reducing myocardial damage caused by sepsis; coix seed has the effects of clearing away heat and purging fire, improving microcirculation, regulating immunity, etc., and tangerine peel can eliminate stagnation, regulate qi, reduce platelet aggregation, increase blood flow, and reduce immune response. Based on the above, the present invention uses it to treat septic cardiomyopathy, can inhibit the excessive production of immune factors, improve cell apoptosis, and has a good repair effect on septic cardiomyopathy.

[0020] (4) The present invention uses LPS to induce inflammation in RAW264.7 cells, and uses the Chinese herbal compound preparation to act on the cells, and it is found that it can reduce the production of NO and effectively inhibit the reduction of the production of immune factors such as IL-1β, TNF-α, IL-6, etc., indicating that the present invention can exert its effect by inhibiting the activity of macrophages; then the present invention establishes an in vitro macrophage-cardiomyocyte crosstalk model, and uses the Chinese herbal compound preparation to act on LPS-induced RAW264.7 cells for 24 hours, extracts the supernatant, and uses macrophage culture medium to induce H9C2 cardiomyocytes to produce an immune response. The CCK8 results show that the macrophage culture medium containing the Chinese herbal compound extract (raw drug concentration 4, 8 mg / mL) can significantly improve the cell viability after acting on cardiomyocytes. The in vitro experiment proves that the Chinese herbal preparation can protect cardiomyocytes by inhibiting the immune response produced by macrophages, thereby playing a repairing role in septic cardiomyopathy.

[0021] (5) The Chinese medicine compound preparation of the present invention is used to repair myocardial damage caused by sepsis. It acts on septic cardiomyopathy through multiple links and multiple targets. The treatment method is changed from improving short-term hemodynamic status to a long-term restorative strategy, further reducing the re-hospitalization rate and mortality rate of patients with septic cardiomyopathy, improving the quality of life of patients, and making up for the shortcomings of Western medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the safe concentration of the Chinese medicine compound extract of the present invention for acting on RAW264.7 cells.

[0023] Figure 2 This is the safe concentration of the Chinese medicine compound extract of the present invention for acting on H9C2 cells.

[0024] Figure 3 The invention discloses the effect of the Chinese herbal compound extract on NO produced by LPS-induced RAW264.7 cells.

[0025] Figure 4 The invention discloses the effect of the Chinese medicinal compound extract on the mRNA expression level of immune cytokines (IL-1β, IL-6 and TNF-α) in RAW264.7 cells induced by LPS.

[0026] Figure 5 The invention discloses the effect of the Chinese herbal compound extract on the viability of cardiomyocytes H9C2 exposed to LPS-stimulated macrophage RAW264.7 conditioned medium.

[0027] Figure 6The results show that the extracts of Chinese herbal compound 1-5 have an effect on the mRNA expression levels of immune cytokines (IL-1β, IL-6 and TNF-α) in LPS-induced RAW264.7 cells, where compound 1, compound 2, compound 3, compound 4 and compound 5 are abbreviations of Chinese herbal compound 1 extract, Chinese herbal compound 2 extract, Chinese herbal compound 3 extract, Chinese herbal compound 4 extract and Chinese herbal compound 5 extract, respectively.

[0028] Figure 7 The above graph represents the effects of Chinese herbal compound 1-5 extracts on the viability of cardiomyocytes H9C2 exposed to the conditioned medium of macrophages RAW264.7 stimulated by LPS, wherein Compound 1, Compound 2, Compound 3, Compound 4 and Compound 5 are abbreviations of Chinese herbal compound 1 extract, Chinese herbal compound 2 extract, Chinese herbal compound 3 extract, Chinese herbal compound 4 extract and Chinese herbal compound 5 extract, respectively. DETAILED DESCRIPTION

[0029] The present invention is further described below with reference to specific embodiments, but they should not be construed as limiting the present invention.

[0030] Example 1

[0031] A traditional Chinese medicine compound extract is prepared according to the following steps: 10 parts by weight of red ginseng, 10 parts by weight of aconite root, 5 parts by weight of coix seed and 6 parts by weight of tangerine peel are weighed, the above-mentioned amounts of red ginseng, tangerine peel, coix seed and aconite root are crushed to obtain micro powder, 4-8 times the volume of water of the micro powder is added, soaked overnight, ultrasonically extracted twice, each extraction lasting 50-120 minutes, filtered, and prepared into the traditional Chinese medicine compound extract.

[0032] Example 2

[0033] The CCK8 method was used to screen the safe concentration of the Chinese medicine compound extract obtained in Example 1 for RAW264.7 cells and H9C2 cardiomyocytes. The specific implementation scheme is as follows:

[0034] H9C2 and RAW264.7 cells were seeded onto 96-well plates and incubated with the Chinese herbal compound extract obtained in Example 1 (the crude drug concentration was 0-20 mg / mL, and the crude drug concentration was the total mass of the Chinese herbal raw materials used in the preparation process / the total volume of the extraction water) for 24 h; then, the absorbance at 450 nm was measured using a CCK8 kit (CCK8, Dojindo, Kumamoto, Japan) on a microplate reader (Perkin-Elmer, Waltham, MA, USA) to evaluate cell viability. Figure 1 and Figure 2As shown, the concentration of the Chinese herbal compound extract is 0-20 mg / mL, which can be used as a safe concentration for acting on RAW264.7 cells and H9C2 cardiomyocytes. In Examples 3-6, we acted on RAW264.7 cells and H9C2 cells with Chinese herbal extracts (raw drug concentrations of 1, 4, and 8 mg / mL).

[0035] Example 3

[0036] The effect of the Chinese herbal compound extract obtained in Example 1 on the NO production of RAW264.7 cells induced by LPS was detected. The specific implementation scheme is as follows:

[0037] After RAW264.7 cells were removed from the culture flask, the final cell concentration was adjusted to 5 × 10 5 / mL, add 100μL of cell suspension to each well of a 96-well plate. After 24 hours of culture, pour out the supernatant and add 50μl of the basic culture medium of the Chinese medicine compound extract obtained in Example 1 (raw drug concentration of 1, 4, and 8mg / mL) to each well. After 1 hour of culture, add LPS to make its final concentration of 1μg / mL, and set up a blank group and an LPS model group. After continuing to act for 24 hours, take 50μL of the upper supernatant and transfer it to another 96-well plate, add 50μL of Griess Reagent I and Griess Reagent II respectively, and measure the absorbance at a wavelength of 540nm. According to Figure 3 Based on the CCK8 results, 1, 4, and 8 mg / mL of the Chinese herbal compound extract were selected to compare their effects on the NO produced by LPS-induced RAW264.7 cells. Figure 3 It can be seen that compared with the LPS model group, the Chinese herbal compound extracts at crude drug concentrations of 1, 4, and 8 mg / mL can reduce the production of cellular NO in a concentration gradient-dependent manner, indicating that the Chinese herbal compound extracts of the present invention have an inhibitory effect on LPS-induced NO production in RAW264.7 cells.

[0038] Example 4

[0039] The effect of the Chinese medicine compound extract obtained in Example 1 on the expression levels of IL-1β, IL-6, and TNF-α mRNA in RAW264.7 cells induced by LPS was detected. The specific implementation scheme is as follows:

[0040] After RAW264.7 cells were pipetted off the culture flask, the final cell concentration was adjusted to 5 × 10 5 / mL, add 2ml of cell suspension to each well of a 6-well plate. After 24 hours of culture, pour out the supernatant and add 2ml of the basic culture medium containing the Chinese medicine compound extract obtained in Example 1 (raw drug concentration of 1, 4, and 8mg / mL) to each well. After 1 hour of culture, add LPS to a final concentration of 1μg / mL, and set up a blank group and an LPS model group. After a further 24 hours of action, add 1ml of TRIzol reagent to each well to extract RNA from the cells, then use a reverse transcription kit to synthesize cDNA, and use real-time quantitative fluorescence PCR to detect the mRNA expression levels of IL-1β, IL-6, and TNF-α in the cells. By Figure 4 It can be seen that the Chinese herbal compound extract of the present invention can significantly inhibit the activation of LPS-induced RAW264.7 mouse peritoneal macrophage cell line and significantly reduce the production of TNF-α, IL-6 and IL-1β immune cytokines.

[0041] Example 5

[0042] In order to verify the effect of the Chinese herbal compound extract obtained in Example 1 on repairing myocardial damage caused by sepsis, this example established an in vitro macrophage-cardiomyocyte crosstalk model, in which RAW264.7 cells treated with LPS and the Chinese herbal compound extract were indirectly co-cultured with H9C2 cardiomyocytes, and the cell viability of H9C2 was detected by CCK8. The specific implementation plan is as follows:

[0043] After RAW264.7 cells were pipetted off the culture flask, the final cell concentration was adjusted to 5 × 10 5 / mL, add 2ml of cell suspension to each well of a 6-well plate. After culturing for 24 hours, pour off the supernatant, and add 2mL of the basic culture medium of the Chinese medicine compound extract obtained in Example 1 (1, 4, 8mg / mL crude drug concentration) to each well. After culturing for 1 hour, add LPS to a final concentration of 1μg / mL, and set up a blank group and an LPS model group. After continuing to act for 24 hours, take the supernatant and centrifuge it at 300g for 10min.

[0044] At the same time, H9C2 cells were inoculated into 96-well plates, and the above-obtained culture medium and 1640 complete culture medium were mixed at a ratio of 1:1, and 100 μL was added to each well. After culturing for 24 h, the absorbance at 450 nm was measured using a CCK8 kit on a microplate reader to evaluate cell viability. Figure 5 It can be seen that the Chinese herbal compound extract with a crude drug concentration of 4 and 8 mg / mL can improve the cell viability of cardiomyocytes exposed to LPS-stimulated macrophage conditioned medium, indicating that the Chinese herbal compound extract in the present invention can protect cardiomyocytes by inhibiting the immune response in macrophages, thereby alleviating septic cardiomyopathy.

[0045] Example 6

[0046] Select red ginseng, aconite root, coix seed and red tangerine peel according to 10 parts, 10 parts, 5 parts and 6 parts by weight, and prepare the extract of Chinese medicine compound 1 according to the method described in Example 1;

[0047] Aconite root, coix seed and red orange peel were selected and matched according to 10 parts, 5 parts and 6 parts by weight, and the extract of Chinese medicine compound 2 was prepared according to the method described in Example 1;

[0048] Select red ginseng, coix seed, and red tangerine peel according to 10 parts, 5 parts, and 6 parts by weight, and prepare the extract of Chinese medicine compound 3 according to the method described in Example 1;

[0049] Select red ginseng, aconite root, and red tangerine peel according to 10 parts, 10 parts, and 6 parts by weight, and prepare the Chinese medicine compound 4 extract according to the method described in Example 1;

[0050] Red ginseng, aconite root and coix seed were selected and matched according to 10 parts, 10 parts and 5 parts by weight, and the extract of Chinese medicine compound 5 was prepared according to the method described in Example 1.

[0051] (1) Detect the effect of the above-mentioned Chinese herbal compound extracts on the expression levels of IL-1β, IL-6, and TNF-α mRNA in RAW264.7 cells induced by LPS, and establish an in vitro macrophage-cardiomyocyte crosstalk model to evaluate the therapeutic efficacy of the above-mentioned Chinese herbal compound extracts 1-5 on septic cardiomyopathy. The specific implementation plan is as follows:

[0052] After RAW264.7 cells were pipetted off the culture flask, the final cell concentration was adjusted to 5 × 10 5 / mL, 2mL of cell suspension was added to each well of a 6-well plate. After 24 hours of culture, the supernatant was discarded, and the extract of Chinese herbal compound 1-5 was used to act on RAW264.7 cells at a crude drug concentration of 4mg / mL. After 1 hour of culture, LPS was added to a final concentration of 1μg / mL, and a blank group and an LPS model group were set up at the same time. After continuing to act for 24 hours, 1mL of TRIzol reagent was added to each well to extract RNA from the cells, and then a reverse transcription kit was used to synthesize cDNA, and real-time quantitative fluorescence PCR was used to detect the mRNA expression levels of IL-1β, IL-6, and TNF-α in the cells.

[0053] from Figure 6 The experimental results show that the extract of Chinese herbal compound 1 can more significantly reduce the mRNA expression levels of IL-1β, IL-6 and TNF-α, that is, the use of four Chinese herbal medicines including red ginseng, aconite, coix seed and tangerine peel has a better therapeutic effect than the compound composed of three of them. The four Chinese herbal raw materials of red ginseng, aconite, coix seed and tangerine peel are indispensable, and the lack of any one of them will affect the efficacy of treating septic cardiomyopathy.

[0054] (2) After RAW264.7 cells were removed from the culture flask, the final cell concentration was adjusted to 5×10 5 / mL, add 2mL of cell suspension to each well of a 6-well plate. After 24 hours of culture, pour out the supernatant, select the extract of Chinese herbal medicine compound 1-5 to act on RAW264.7 cells at a crude drug concentration of 4mg / mL. After 1 hour of culture, add LPS to a final concentration of 1μg / mL, and set up a blank group and LPS model group. After continuing to act for 24 hours, take the supernatant and centrifuge it at 300g for 10min.

[0055] At the same time, H9C2 cells were inoculated into 96-well plates, and the above-obtained culture medium and 1640 complete culture medium were mixed at a ratio of 1:1, and 100 μl was added to each well. After culturing for 24 h, the absorbance at 450 nm was measured using a CCK8 kit on a microplate reader to evaluate cell viability. Figure 7 It can be seen that the extract of Chinese herbal compound 1 can significantly improve the cell viability of cardiomyocytes exposed to LPS-stimulated macrophage conditioned medium compared with other Chinese herbal compound extracts, indicating that the use of four Chinese medicines including red ginseng, aconite, coix seed and tangerine peel can better inhibit the immune response in macrophages to protect cardiomyocytes than the use of a compound composed of three of them, and has a better therapeutic effect on septic cardiomyopathy.

[0056] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of a Chinese medicine compound preparation in the preparation of a drug for repairing myocardial damage caused by sepsis, characterized in that: The traditional Chinese medicine compound preparation is prepared from the following raw materials in parts by weight: 3-20 parts of red ginseng, 3-25 parts of aconite root, 5-30 parts of coix seeds, and 2-20 parts of tangerine peel.

2. The use according to claim 1, characterized in that: The traditional Chinese medicine compound preparation is prepared from the following raw materials by weight: 10 parts of red ginseng, 10 parts of aconite root, 5 parts of coix seeds and 6 parts of tangerine peel.

3. The use according to claim 1, characterized in that: The traditional Chinese medicine compound preparation includes the following ingredients by mass percentage: ginsenoside Rg1 0.2%-5%, ginsenoside Rb1 0.10%-5%, naringin 4%-12%, aconitine components 0.2%-5%, and triolein 0.2%-5%; the aconitine components include benzoylmeaconitine, benzoylaconitine and benzoylhyaconitine.

4. The use according to claim 1, characterized in that: The Chinese medicine compound preparation is prepared according to the following water extraction process steps: weighing red ginseng, tangerine peel, coix seed and aconite, crushing to obtain micropowder, adding water 4-8 times the volume of the micropowder, soaking overnight, ultrasonic extraction twice, each extraction lasting 50-120 minutes, filtering, spray drying, and preparing the Chinese medicine compound preparation.

5. The use according to claim 1, characterized in that: The Chinese medicine compound preparation is prepared according to the following alcohol extraction process steps: weighing red ginseng, tangerine peel, coix seed and aconite, crushing them to obtain micro powder, adding ethanol with a volume of 6-8 times that of the micro powder, the concentration of ethanol is 50-90% by volume, ultrasonic extraction is performed twice, each extraction takes 20-40 minutes, filtering, combining the filtrate, recovering ethanol under reduced pressure, obtaining a concentrated extract, adding auxiliary materials to mix, sieving, spray drying, and preparing the Chinese medicine compound preparation.

6. The use according to claim 1, characterized in that: The drug for repairing myocardial damage caused by sepsis is a drug for preventing and treating septic cardiomyopathy.

7. The use according to claim 1, characterized in that: The drug for repairing myocardial damage caused by sepsis is a drug for resisting sepsis cardiomyopathy by inhibiting the immune response produced by macrophages, reducing the release of NO, and inhibiting the production of IL-1β, IL-6, and TNF-α cytokines.

8. The use according to claim 1, characterized in that: The traditional Chinese medicine compound preparation is made into capsules, oral liquids, tablets or granules.

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