CN113304241A

CN113304241A - Application of radix stephaniae tetrandrae and radix astragali decoction and components thereof in preparation of anti-myocardial hypertrophy medicines

Info

Publication number: CN113304241A
Application number: CN202110731543.0A
Authority: CN
Inventors: 赵筱萍; 杨振中; 王毅
Original assignee: Zhejiang Chinese Medicine University ZCMU
Current assignee: Zhejiang Chinese Medicine University ZCMU
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2021-08-27
Anticipated expiration: 2041-06-30
Also published as: CN113304241B

Abstract

The invention relates to the technical field of traditional Chinese medicines, and discloses a radix stephaniae tetrandrae and astragalus decoction and application of components thereof in preparing a medicament for resisting myocardial hypertrophy, wherein the radix stephaniae tetrandrae and astragalus decoction comprises the following raw material components: 50-70g of radix stephaniae tetrandrae, 60-90g of astragalus membranaceus, 35-55g of bighead atractylodes rhizome, 20-40g of liquorice, 35-55g of ginger and 25-45g of Chinese date, and the radix stephaniae tetrandrae and astragalus membranaceus decoction is obtained by decocting the raw materials in water, filtering and concentrating under reduced pressure. Dispersing radix Stephaniae Tetrandrae and radix astragali decoction with water, sequentially eluting with water, 15-22% ethanol, 35-45% ethanol and 90-100% ethanol, wherein the 35-45% ethanol and 90-100% ethanol eluates are component A and component B of radix Stephaniae Tetrandrae and radix astragali decoction; animal pharmacodynamic studies prove that the radix stephaniae tetrandrae and radix astragali decoction, the component A and the component B can obviously improve the cardiac function level of a heart failure model mouse, reduce serum heart failure markers, relieve inflammatory reaction and improve the pathological change of heart tissues, play an obvious role in resisting the heart failure of the mouse, and have certain development potential in the aspect of preparing medicaments for treating myocardial hypertrophy.

Description

Application of radix stephaniae tetrandrae and radix astragali decoction and components thereof in preparation of anti-myocardial hypertrophy medicines

Technical Field

The invention relates to the technical field of traditional Chinese medicines, in particular to application of fangji astragalus decoction and components thereof in preparing a medicament for resisting myocardial hypertrophy.

Background

Heart Failure (HF) refers to a group of clinical syndromes in which the function of pumping blood of the Heart is impaired due to structural or functional abnormalities of the Heart caused by various reasons, and is mainly manifested by dyspnea, fluid retention, physical weakness, and the like. Ventricular remodeling is an important process for the occurrence and development of heart failure, and myocardial hypertrophy is one of the key pathological links in the process of ventricular remodeling. Myocardial hypertrophy (myocardiac hypertrophy), which is a decompensated response caused by excessive activation or long-term overload of the heart, is an important stage in the development of many heart diseases, and is manifested by enlargement of cardiomyocytes, increase of protein synthesis, promotion of interstitial fibrosis, and the like. Persistent pathological myocardial hypertrophy leads to ventricular dilation, decreased contractile function, and imbalance in energy metabolism, ultimately leading to heart failure. Therefore, improvement of myocardial cell hypertrophy plays an important role in delaying the worsening of heart failure caused by ventricular remodeling. The drug effect substance with the effect of resisting myocardial hypertrophy is researched and screened in the field of traditional Chinese medicines, and has great practical significance and clinical reference value for preventing and treating and improving heart failure diseases.

The active substance of the traditional Chinese medicine is the drug effect basis of the traditional Chinese medicine for preventing and treating various diseases, and how to screen and discover the active component with the effect of resisting myocardial hypertrophy and the preparation thereof from the traditional Chinese medicine and the compound is one of the important research contents of the modernization research of the traditional Chinese medicine.

Fangji Huangqi Tang, as a classic and famous prescription, was recorded in the book of jin Kui Yao L ü e, which is written by Zhang Zhongjing of famous physicians. The traditional Chinese medicine compound consists of four traditional Chinese medicinal materials of radix stephaniae tetrandrae, astragalus, bighead atractylodes rhizome and honey-fried licorice root, and ginger and Chinese date are often added for compatibility in clinic, so that the traditional Chinese medicine compound has the effects of tonifying qi, dispelling wind, strengthening spleen and inducing diuresis. It is clinically used for treating diseases such as cardiogenic edema, hypertension, chronic glomerulonephritis and the like. At present, few studies on myocardial hypertrophy resistance of the medicinal taste of the formula in the tetrandra and astragalus decoction are carried out, and the astragalus extract can obviously inhibit myocardial cell hypertrophy induced by Ang II by down-regulating the expression of PKD1 mRNA (Yang Lei, hair Reynaud, Xuguo Chang. the astragalus extract has the influence on the expression of PKD1 mRNA of mastoid mouse hypertrophic myocardial cells [ J ]. China journal of modern medicine, 2013(08): 12-16.); the astragalus injection can obviously reverse the ATP and calcium ion content in myocardial hypertrophy cells and improve the mitochondrial function (in Yan, Wang Shuoren, Niibo, and the like.) the astragalus injection reverses the influence on the mitochondrial structure and function of myocardial cells in the process of myocardial hypertrophy [ J ].

Chinese traditional medicine J2012, 37(007): 979-984). Cui et al (Cui X H, Wang H L, Wu R, et al. Effect of orthoclades macroperoenols-induced ventricular remodeling in rats [ J ]. Molecular Medicine Reports,2018,17(2).) found that administration of 2.4g/kg/d aqueous decoction of Atractylodis rhizoma inhibited myocardial hypertrophy and myocardial fibrosis by anti-oxidation and inhibition of RAAS activation and thereby reversed ventricular remodeling in mice. Shishirr et al (Shishirr U, oil K.M, Monosha D.Glycyrrhiza glabra (Licorice) root extract obtained from both cardiac activity assays and static the cardiac health in H9c2 cardiac cells [ J ] Journal of Ethnopharmacology,2020,258:112690.) found that aqueous extracts of licorice root had some antioxidant effect and protected cardiomyocytes from DOX-induced toxicity. However, pharmacological research on the anti-heart failure of the compound of the radix stephaniae tetrandrae and astragalus decoction and components thereof is only reported, and no relevant basic research report on the aspect of resisting myocardial hypertrophy is found.

Disclosure of Invention

The invention aims to provide the application of the radix stephaniae tetrandrae and astragalus decoction in preparing the myocardial hypertrophy resistant medicament and provide a brand-new treatment method for heart failure diseases by evaluating the heart failure resistant effect of the radix stephaniae tetrandrae and astragalus decoction and screening and researching drug effect substances for resisting myocardial hypertrophy. Provides scientific basis for the development and utilization of the clinical medicine for treating the heart failure.

In order to achieve the purpose, the invention adopts the technical scheme that:

application of Fangji Huangqi decoction in preparing medicine for resisting myocardial hypertrophy is provided.

In the invention, myocardial hypertrophy refers to the pathological phenomena that cardiac muscle cells are enlarged, protein synthesis is increased, interstitial fibrosis is promoted and the like due to decompensation reaction of the heart caused by various external factors including excessive activation of neuroendocrine factors, long-term overload of the heart, apoptosis, energy metabolism disorder and the like. The invention discloses the application of the radix stephaniae tetrandrae and astragalus decoction in the preparation of the anti-myocardial hypertrophy medicament for the first time, and researches show that the radix stephaniae tetrandrae and astragalus decoction can improve the heart function level of heart failure mice, reduce the relative area of primary myocardial cells and the like, so that the radix stephaniae tetrandrae and astragalus decoction can play the role of resisting myocardial hypertrophy, and can be applied to the preparation of the medicament for treating myocardial hypertrophy.

The radix stephaniae tetrandrae and radix astragali decoction comprises the following raw material components: 50-70g of radix stephaniae tetrandrae, 60-90g of astragalus, 35-55g of bighead atractylodes rhizome, 20-40g of liquorice, 35-55g of ginger and 25-45g of Chinese date.

Preferably, the raw material components of the tetrandra and astragalus decoction comprise: 60g of radix stephaniae tetrandrae, 75g of astragalus, 45g of bighead atractylodes rhizome, 30g of liquorice, 45g of ginger and 36g of Chinese date.

The preparation method of the radix stephaniae tetrandrae and radix astragali decoction comprises the following steps:

a. taking 50-70g of radix stephaniae tetrandrae, 60-90g of astragalus, 35-55g of bighead atractylodes rhizome, 20-40g of liquorice, 35-55g of ginger and 25-45g of Chinese date, and adding water for decoction;

b. filtering the decoction obtained in the step (a), and concentrating under reduced pressure to obtain a paste, namely the tetrandra and astragalus decoction.

The myocardial hypertrophy refers to heart failure. The inventor confirms that the tetrandra and astragalus decoction can obviously improve the heart function level of a heart failure model mouse, reduce serum heart failure markers, relieve inflammatory reaction and improve the pathological change of heart tissues through animal pharmacodynamic research, and plays an obvious role in resisting the heart failure of the mouse.

The dosage of the tetrandra and astragalus decoction for the administration of the myocardial cells is 300-.

The radix stephaniae tetrandrae and astragalus decoction comprises pharmaceutically acceptable medicinal auxiliary materials in traditional Chinese medicine pharmacy, and is prepared into clinical medicament formulations, including tablets, oral liquid, pills, granules, capsules, injections and sustained and controlled release agents.

The invention also provides a component of the radix stephaniae tetrandrae and astragalus membranaceus decoction, which comprises a component A or a component B, and the preparation method of the component A comprises the following steps: dispersing the radix Stephaniae Tetrandrae and radix astragali decoction with water, separating with macroporous adsorbent resin column, sequentially eluting with water, 15-22% ethanol, 35-45% ethanol and 90-100% ethanol, collecting eluates, concentrating the eluates under reduced pressure, and vacuum drying, wherein the eluate of 35-45% ethanol is dried to obtain component A of radix Stephaniae Tetrandrae and radix astragali decoction;

the preparation method of the component B comprises the following steps: dispersing the radix Stephaniae Tetrandrae and radix astragali decoction with water, separating with macroporous adsorbent resin column, sequentially eluting with water, 15-22% ethanol, 35-45% ethanol and 90-100% ethanol, collecting eluates, concentrating the eluates under reduced pressure, and vacuum drying, wherein the 90-100% ethanol eluate is dried to obtain component B of radix Stephaniae Tetrandrae and radix astragali decoction.

The invention also provides application of the tetrandra and astragalus decoction component in preparing a medicament for resisting myocardial hypertrophy. The inventor finds that the radix stephaniae tetrandrae and radix astragali decoction can improve the heart function level of heart failure mice and reduce the relative area of primary myocardial cells, and the like, and the component A and the component B in the eluent also have the effect of reducing the relative area of the primary myocardial cells, which shows that the component of the radix stephaniae tetrandrae and radix astragali decoction can also play the role of resisting myocardial cell hypertrophy, so that the radix stephaniae tetrandrae and radix astragali decoction can be applied to the preparation of the medicament for treating myocardial hypertrophy.

The myocardial hypertrophy is also referred to as heart failure. The inventor also confirms that the components of the tetrandra and astragalus decoction can obviously improve the heart function level of a heart failure model mouse, reduce serum heart failure markers, relieve inflammatory reaction and improve the pathological change of heart tissues through animal pharmacodynamic research, and play an obvious role in resisting the heart failure of the mouse.

Preferably, the preparation method of the component A comprises the following steps: dispersing the radix Stephaniae Tetrandrae and radix astragali decoction with water, separating with macroporous adsorbent resin column, sequentially eluting with water, 20% ethanol, 40% ethanol and 95% ethanol, collecting eluates, concentrating the eluates under reduced pressure, and vacuum drying, wherein the 40% ethanol eluate is dried to obtain component A of radix Stephaniae Tetrandrae and radix astragali decoction;

preferably, the preparation method of the component B comprises the following steps: dispersing the radix Stephaniae Tetrandrae and radix astragali decoction with water, separating with macroporous adsorbent resin column, sequentially eluting with water, 20% ethanol, 40% ethanol and 95% ethanol, concentrating the eluate under reduced pressure, and vacuum drying, wherein the 95% ethanol eluate is dried to obtain component B of radix Stephaniae Tetrandrae and radix astragali decoction.

The dosage of the component A is 6.0-12.5 mug/mL; the dosage of the component B is 15-25 mug/mL.

Similarly, the components of the radix stephaniae tetrandrae and astragalus decoction can comprise pharmaceutically acceptable pharmaceutic adjuvants in traditional Chinese medicine pharmacy, and can be prepared into clinically applicable pharmaceutical formulations including tablets, oral liquid, pills, granules, capsules, injections and sustained and controlled release agents, so that the preparation is convenient to use practically.

The preparation method of the components of the tetrandra and astragalus decoction comprises the following steps:

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

(1) the invention provides the application of the radix stephaniae tetrandrae and astragalus membranaceus decoction and the components thereof in the preparation of the anti-myocardial hypertrophy medicines for the first time, and the inventor confirms that the radix stephaniae tetrandrae and astragalus membranaceus decoction can obviously improve the heart function level of a heart failure model mouse, reduce serum heart failure markers, relieve inflammatory reaction, improve pathological changes of heart tissues and play an obvious role in resisting the heart failure of the mouse through animal pharmacodynamics research.

(2) The invention discovers a new application of the tetrandra and astragalus decoction and the components thereof, has a remarkable inhibiting effect on myocardial cell hypertrophy, and the 35-45% ethanol extract and the 90-100% ethanol extract can also be effective components for resisting myocardial cell hypertrophy, and have certain development potential in the aspect of preparing medicaments for treating myocardial hypertrophy.

(3) The preparation method of the radix stephaniae tetrandrae and astragalus membranaceus decoction and the components thereof are simple, the extraction process cost is low, the raw materials are all extracted from traditional Chinese medicinal materials, the traditional Chinese medicinal materials are used as natural medicinal resources, the source is wide, the safety is high, the toxicity is low, the decoction is used as a new raw material for preparing the anti-myocardial hypertrophy medicine, the dosage is low, the curative effect is obvious, and the toxic and side effects are low.

(4) In the animal pharmacodynamic test, the lowest administration doses of the single medicines of the radix stephaniae tetrandrae, the astragalus membranaceus, the bighead atractylodes rhizome and the liquorice are respectively 4g/kg/d, 5g/kg/d, 3g/kg/d and 2g/kg/d, and the administration time of the radix stephaniae tetrandrae and the astragalus membranaceus decoction and the effective components thereof in the cell pharmacodynamic test is only 48h, which are respectively lower than the administration dose and time of the existing research, so that the potential medical value and the good social benefit are achieved.

Drawings

FIG. 1 is an echocardiogram of mice of the control group and the model group in application example 1.

FIG. 2 is the ejection fraction, left ventricular short axis shortening rate and cardiac output of mice to which the control group and the model group of example 1 were applied.

Fig. 3 shows the posterior wall thickness, the ventricular septal thickness, the inner diameter and the volume of the left ventricle at the end systole and the end diastole of the mice of the control group and the model group in the application example 1.

Fig. 4 is an echocardiogram of mice in the control group, model group, low dose group and high dose group in application example 1.

Fig. 5 is a graph showing the ejection fraction, left ventricular short axis shortening rate and cardiac output of the mice in the control group, model group, low dose group and high dose group in application example 1.

Fig. 6 shows the pathological morphology of the myocardial tissues and the deposition of collagen fibers of the mice in the control group, the model group, the low dose group and the high dose group of application example 1.

Fig. 7 shows the area ratio of collagen fibers in the myocardial tissue of mice in the control group, model group, low dose group, and high dose group in application example 1.

FIG. 8 shows the ALT, AST, BUN and Crea contents in blood of mice in the control group, model group, low dose group and high dose group in application example 1.

FIG. 9 shows the control of the concentrations of NT-proBNP and Ang II in the serum of mice in the control group, model group, low dose group and high dose group in application example 1.

FIG. 10 shows the control of the levels of NO, IL-6, IL-1. beta. and TNF-. alpha.in the serum of mice in the control group, model group, low dose group and high dose group of application example 1.

FIG. 11 shows the relative area ratios of cardiomyocytes in primary suckling mice in the control group, model group, positive drug group, and different dosages of Fangji Huangqi Tang group of application example 2.

FIG. 12 is the relative area ratio of the myocardial cells of primary suckling mice of the control group, the model group, the positive drug group, the tetrandra and astragalus decoction component A and the component B in the application example 2.

FIG. 13 shows the relative expression levels of PGC-1. alpha. and PPAR. gamma. proteins in the cardiomyocytes of primary mice in the control group, model group and Fangji Huangqi Tang group of application example 3.

FIG. 14 shows the relative expression levels of PGC-1. alpha. and PPAR. gamma. proteins in the primary mice cardiomyocytes of the control group, the model group, the Fangji Huangqi decoction component A and the component B in application example 3.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Those skilled in the art should understand that they can make modifications and equivalents without departing from the spirit and scope of the present invention, and all such modifications and equivalents are intended to be included within the scope of the present invention.

The raw materials used in the following embodiments are all commercially available.

Example 1 preparation of Menispermaceae and Astragalus decoction and Components thereof

60g of radix stephaniae tetrandrae, 75g of astragalus, 45g of bighead atractylodes rhizome, 30g of liquorice, 45g of ginger and 36g of Chinese date are taken and decocted with water; filtering the obtained decoction, and concentrating under reduced pressure to obtain paste, i.e. radix Stephaniae Tetrandrae and radix astragali decoction;

dispersing the paste with water, separating with macroporous adsorbent resin column, sequentially eluting with water, 20% ethanol, 40% ethanol and 95% ethanol, collecting eluates, concentrating the eluates under reduced pressure, and vacuum drying to obtain radix Stephaniae Tetrandrae and radix astragali decoction component A and component B after drying 40% and 95% ethanol eluates.

Application example 1 pharmacodynamic application study of tetrandra and astragalus decoction in resisting heart failure of mice

1. Establishment of Isoproterenol (ISO) induced mouse heart failure model

60C 57 mice are randomly divided into a control group and a model group, the model group is continuously injected with ISO (5mg/kg/d) for 3 weeks to construct a heart failure model, and the control group is injected with 0.9% sodium chloride injection with the same volume subcutaneously. After the molding is finished, performing echocardiography detection on all mice, and regarding the mice with ejection fraction (EF%) < 55% as successful molding.

As shown in fig. 1-3, compared with the control group, after 3 weeks of ISO subcutaneous injection, the heart ejection fraction (EF%), left ventricular short axis shortening rate (FS%) and Cardiac Output (CO) of the mice in the heart failure model group were significantly decreased according to echocardiography (P < 0.01); the posterior wall thickness (LVPW, d; LVPW, s) of the left ventricle at the end stage of systole and diastole is thinned, the ventricular septal thickness (IVS, d; IVS, s) of the left ventricle at the end stage of systole and diastole, the inner diameter (LVID, d; LVID, s) of the left ventricle and the volume (LV Vol, d; LV Vol, s) of the left ventricle at the end stage of systole and diastole are increased to different degrees, so that the structural and functional changes of the left ventricle of the mice in the model group caused by ISO induction are shown, and the successful construction of the heart failure model of the mice is shown.

2. Action of fangji Huangqi decoction on anti-heart failure of mice

The 30 mice in the model group were randomly and evenly divided into three groups, including model group, low dose group and high dose group of Fangji Huangqi Tang. The tetrandra and astragalus decoction (FJHQT) is administrated to mice of a model group by intragastric administration according to clinical equivalent dose of the tetrandra and astragalus decoction, purified water is prepared into suspension, wherein the intragastric administration of a low dose group is 19.4g/kg/d, the intragastric administration of a high dose group is 58.2g/kg/d, the intragastric volume is 0.2mL/10g, and the intragastric administration is continuously carried out for 2 weeks. The control group and the model group were administered with purified water of the same volume by gavage. After the administration, all mice are subjected to echocardiogram detection and data acquisition, and the change of the animal cardiac function before and after the administration is analyzed.

As shown in fig. 4 and 5, after the administration of the tetrandra and astragalus decoction through gastric lavage for 2 weeks, according to the echocardiogram detection result, the heart ejection fraction (EF%), left ventricular minor axis shortening rate (FS%) and Cardiac Output (CO) of the mice in the high dose group and the low dose group are found to have significant return rise, and have statistical difference (P <0.01), which indicates that the tetrandra and astragalus decoction can obviously improve the cardiac function level of the mice with heart failure caused by ISO.

Obtaining animal materials: at the end of the experiment, mice in each group were weighed and recorded and anesthetized by intraperitoneal injection of 15mg/mL sodium pentobarbital (0.7mL/100 g). The mice were fixed in the supine position, and the eyeball was removed to draw blood. The chest is opened, and a small opening is cut in the liver for perfusion. One part of heart for extracting RNA and protein is perfused from the heart to the white liver by using 0.9 percent sodium chloride injection, the heart is cut, blood vessels and redundant tissues on the surface of the heart are removed, the atrium part is quickly cut after water is absorbed by filter paper, the ventricle part is left, the atrium part is immediately placed in dry ice for quick freezing, and then the heart is transferred to a refrigerator at the temperature of minus 80 ℃ for storage; and the other part of the heart is used for pathological tissue section observation, after the heart is perfused by 4 percent paraformaldehyde, the heart is cut, blood vessels and redundant tissues on the surface of the heart are removed, and the heart is fixed in the 4 percent paraformaldehyde.

3. Influence of Fangji Huangqi decoction on heart histopathology of heart failure mice

Staining hearts fixed in 4% paraformaldehyde with hematoxylin and eosin (H.E) to observe pathological morphological changes of mouse myocardial tissues; collagen fiber deposition in myocardial tissue was observed using Masson staining. As shown in fig. 6 and 7, the h.e staining results showed that the control group had well-aligned and compact normal cardiomyocytes, while the heart failure model group mice had disorganized cardiomyocytes and cardiomyocyte fibers in the cardiomyocyte tissue, and enlarged cardiomyocyte gaps; after the administration of the fangji Huangqi decoction, the arrangement disorder of the myocardial cells of the mice in the low-dose group and the high-dose group is improved, and the arrangement disorder of the myocardial cells of the mice in the high-dose group is normal. Masson staining results show that no obvious collagen fiber hyperplasia is seen in the normal myocardial tissues of the control group, the myocardial cell gaps in the myocardial tissues of the model mice with heart failure are increased, the arrangement of the myocardial fibers is disordered, the collagen fibers are obviously proliferated, and the collagen fiber deposition of the low-dose group and the high-dose group is reduced to different degrees after the administration of the radix stephaniae tetrandrae and astragalus decoction.

4. Influence of the decoction of radix Stephaniae Tetrandrae and radix astragali on the serum ALT, AST, Urea and Crea content of heart failure mice

Standing the blood of the mouse at room temperature for 30min, centrifuging at 4 ℃ and 16000g for 10min, sucking the supernatant into a new centrifugal tube, centrifuging at 4 ℃ and 16000g for 10min again, sucking the supernatant into the new centrifugal tube, and freezing the obtained serum at-80 ℃ for later use. Detecting a serum sample by adopting a full-automatic biochemical analyzer, and inspecting the influence of the radix stephaniae tetrandrae and radix astragali decoction on the content of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), urea nitrogen (BUN) and creatinine (Crea) in a mouse.

As shown in fig. 8, the ALT, AST, Urea indices of each group of mice showed normal, and there was no significant difference between groups. Compared with the control group, the Crea content in the serum of the model group mouse is increased, and the tetrandra and astragalus decoction is reduced in different degrees after administration and tends to be a normal value. The experiment shows that the mice of each group do not have drug-induced liver and kidney injury in the experiment process, the administration dosage is safe, and scientific basis is provided for the safety of the formula for treating the chronic heart failure.

5. Influence of Fangji Huangqi decoction on serum central failure marker NT-proBNP and Ang II content

Thawing frozen serum on ice, and balancing the N-terminal B-type pro-natriuretic peptide (NT-proBNP) enzyme-linked immunosorbent assay kit and the angiotensin II (Ang II) enzyme-linked immunosorbent assay kit for 15-30min at room temperature. And (3) detecting the contents of NT-proBNP and Ang II in the serum according to the steps of the kit specification.

As shown in FIG. 9, the serum concentrations of NT-proBNP and Ang II in the mice in the model group are significantly increased compared with the control group, which indicates that ISO-induced heart failure can cause ventricular dilatation and left ventricular wall pressure increase in the mice, and stimulates the heart to secrete BNP and Ang II, resulting in impaired cardiac function. The concentrations of NT-pro BNP and Ang II in the serum of mice in the high-dose group and the low-dose group of the radix stephaniae tetrandrae and astragalus membranaceus decoction are obviously reduced, which shows that the radix stephaniae tetrandrae and astragalus membranaceus decoction can lower heart failure markers NT-pro BNP and Ang II and improve the cardiac function.

6. The influence of the decoction of radix Stephaniae Tetrandrae and radix astragali on the content of NO, inflammatory factors IL-6, IL-1 beta and TNF-alpha in blood serum

Frozen serum is unfrozen on ice, the content of Nitric Oxide (NO) is detected by adopting a greens method, and the content of interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) in the serum is detected by adopting an enzyme linked immunosorbent assay (Elisa).

As shown in FIG. 10, compared with the control group, the serum concentrations of NO, IL-6, IL-1 beta and TNF-alpha in the model group mice are all significantly increased, which indicates that the heart failure of the ISO-induced mice is closely related to the inflammatory reaction. After the high and low doses of the fangji Huangqi decoction are administrated, the concentrations of NO, IL-6, IL-1 beta and TNF-alpha are reduced to different degrees and have obvious differences, which shows that the fangji Huangqi decoction can relieve the inflammatory reaction caused in the process of heart failure.

Extraction and culture of primary suckling mouse myocardial Cells (CMs)

Sterilizing newborn SD suckling mice with 75% alcohol, opening chest, clipping heart, placing in a culture dish containing 1% double-antibody Phosphate Buffered Saline (PBS), cleaning for many times to remove blood, and clipping blood vessel and other redundant tissues. The apex of the heart was excised into an empty petri dish, with one batch of 10 suckling mice hearts each. Cutting heart tissue to 1mm³Size, add a small amount of 1% double antibody in PBS, transfer the small pieces of heart tissue to a 50mL centrifuge tube, gently rest and aspirate as much PBS as possible. Adding 2.5mL of the uniformly mixed digestive juice into a 50mL centrifuge tube containing the cardiac tissue small blocks, placing the centrifuge tube in a 37 ℃ shaking table, oscillating at 200rpm for 45min, and blowing and beating the mixture by using a bent tube for multiple times and uniformly mixing every 15min on average. Digesting until no obvious tissue blocks exist, adding an equal amount of neutralizing solution to stop digestion, uniformly mixing, filtering by a 70-micrometer filter screen, washing the filter screen by using an appropriate amount of neutralizing solution, and collecting filtrate containing cells in a 50mL centrifugal tube. The centrifuge tube containing the filtrate was placed at 4 ℃ and centrifuged at 600g for 5min, and the supernatant was discarded. Adding high-sugar DMEM (Dulbecco's modified eagle medium) into the cell sediment to resuspend the cells, mixing, and inoculating to 75cm²In a culture flask. And (3) after the culture flask is placed in the culture box and the wall is differentially adhered for 30min, the fibroblasts are adhered to the wall in the culture flask, and the cell suspension without adhesion is the myocardial cell suspension. The cardiomyocyte suspension in the culture bottle is sucked into a centrifugal tube, 0.1 percent Brdu (0.1mM) is added to inhibit the growth of fibroblasts, and the plate is placed in a constant-temperature incubator to be incubated until the cardiomyocytes adhere to the wall and have spontaneous pulsation for subsequent experiments. To make the cells adhere more stably, 0.5% gelatin was uniformly pre-plated on the bottom of each well plate before plating, and the gelatin was aspirated after incubation in an incubator at 37 ℃ for 1h for cell plating.

Application example 2 Effect of Fangji Huangqi decoction and its Components on hypertrophic myocardial cells

Primary cardiomyocytes were seeded in 60 wells of a 96-well black board at 5000 cells/well density, and 100. mu.L PBS was added to the remaining wells to avoid edge effects. Placing 96-hole blackboard at 37 deg.C and 5% CO₂And incubating for 48h in the cell culture box. Taking out 9Removing culture solution from 6-well blackboard, adding 100 μ L of high-sugar DMEM medium containing 0.1% Brdu into each well, synchronously culturing for 24h, and sucking out the culture medium for later use. The experiment is provided with a control group, a model group, a positive drug group and a sample group, wherein the model group is added with a high-glucose DMEM culture medium containing 100 mu M PE, the positive drug group is added with the high-glucose DMEM culture medium containing 100 mu M PE and 10 mu M captopril, the sample group is respectively added with the high-glucose DMEM culture medium containing 100 mu M PE, different concentrations of radix Stephaniae Tetrandrae and radix astragali decoction (100, 200, 300 and 400 mu g/mL) and different components (12.5 mu g/mL for the component A and 25 mu g/mL for the component B), and after treatment, the pore plate is placed in a cell culture box for incubation for 48 h.

The 96-well blackboard is taken out, the culture solution is sucked off, and PBS is added for rinsing for 3 times and 5 min/time. Add 100. mu.L paraformaldehyde to each well and fix at room temperature for 20 min. The paraformaldehyde is aspirated, and the mixture is rinsed 3 times for 5 min/time with PBS. mu.L of Phalloidin (Alexa Fluor 488Phalloidin-FITC) labeled cardiomyocyte actin F-actin (1:20) was added to each well in the dark at room temperature and incubated for 30 min. After recovering the blotted phalloidin, 50. mu.L of Hoechst (1:10000) labeled cell nucleus is added into each well for incubation for 15min, and PBS is added for rinsing for 3 times for 10 min/time after the Hoechst is discarded. And adding a proper amount of PBS into each hole to keep the cells moist, and storing in dark place. Placing a 96-hole blackboard in

The Pico personal high content imaging analysis system is adopted to automatically collect the fluorescence image

And (4) carrying out automatic analysis on the picture by software, and counting the area of the myocardial cells.

As shown in figure 11, the radix stephaniae tetrandrae and radix astragali decoction remarkably reduces the myocardial cell area under the concentration of 300 and 400 mug/mL, and the relative areas are respectively reduced by 24.99 percent and 22.82 percent;

as shown in fig. 12, fraction a and fraction B also significantly reduced cardiomyocyte area by 20.54% and 35.27%, respectively.

Application example 3 the effects of Fangji Huangqi decoction and its components on PGC-1 alpha and PPAR gamma proteins in hypertrophic myocardial cells

Primary cardiomyocytes at 4 × 10⁵Inoculating to 6-well plate at a density of one well and placing at 37 deg.C and 5% CO₂The cells are grown in the incubator for 48 h. Taking out the 6-hole plate, discarding the culture solution, adding PBS into each hole, rinsing and completely sucking, adding 2mL of high-glucose DMEM medium containing 0.1% Brdu into each hole, synchronously culturing for 24h, and completely sucking the medium for later use. The experiment is divided into a control group, a model group and different sample groups, wherein the model group is added with a high-sugar DMEM culture medium containing 100 mu M PE, the radix Stephaniae Tetrandrae and radix astragali decoction sample group is added with a high-sugar DMEM culture medium containing 100 mu M PE and 400 mu g/mL radix Stephaniae Tetrandrae and radix astragali decoction, the effective component sample group is respectively added with a high-sugar DMEM culture medium containing 100 mu M PE and 12.5 mu g/mL component A or 25 mu g/mL component B, and after uniform mixing, the 6-pore plate is placed in a cell culture box for incubation for 48 h.

The 6-well plate was removed, the culture medium was discarded, rinsed with an appropriate amount of PBS and aspirated. Add 70. mu.L of protein lysate containing 1% PMSF, 1% protease inhibitor and 1% phosphatase inhibitor to each well, lyse on ice for about 5min, lyse the cells thoroughly with a cell scraper, and draw the cell lysate suspension into a 1.5mL centrifuge tube. The cells were centrifuged at 12000rpm for 10min at 4 ℃ to aspirate the supernatant, and protein quantification was performed by BCA method. Adding a certain amount of loading buffer solution 4X Laemmli Sample buffer (containing 1% beta-mercaptoethanol, 9:1) according to the concentration of a protein Sample, uniformly mixing, putting the protein Sample into a water bath kettle at 100 ℃ to boil for 10min to denature the protein Sample, and putting the protein Sample on ice for cooling. Protein immunoblotting Western blot method was used to detect the expression levels of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 alpha), peroxisome proliferator-activated receptor gamma (PPAR gamma), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins.

As shown in fig. 13, the fangji Huangqi decoction can significantly up-regulate the expression of PGC-1 α and PPAR γ proteins in the hypertrophic cardiac muscle cells;

as shown in FIG. 14, component A of the Fangji Huangqi decoction can significantly increase the expression of PGC-1 alpha protein in cardiac muscle cells, and component B can simultaneously increase the expression of PGC-1 alpha and PPAR gamma protein in cardiac muscle cells;

experimental results show that the radix stephaniae tetrandrae and astragalus decoction and the effective components thereof have obvious effect of resisting myocardial hypertrophy. Therefore, the tetrandra and astragalus decoction and the effective components thereof have certain potential in the aspect of treating heart failure diseases.

Claims

1. Application of Fangji Huangqi decoction in preparing medicine for resisting myocardial hypertrophy is provided.

2. The application of the radix stephaniae tetrandrae and radix astragali decoction in preparing the anti-myocardial hypertrophy medicine according to claim 1, wherein the radix stephaniae tetrandrae and radix astragali decoction is prepared from the following raw material components: 50-70g of radix stephaniae tetrandrae, 60-90g of astragalus, 35-55g of bighead atractylodes rhizome, 20-40g of liquorice, 35-55g of ginger and 25-45g of Chinese date.

3. The application of the radix stephaniae tetrandrae and radix astragali decoction in preparing the anti-myocardial hypertrophy medicine according to claim 1 is characterized in that the preparation method of the radix stephaniae tetrandrae and radix astragali decoction comprises the following steps:

4. The use of the tetrandra and astragalus decoction of claim 1 in the preparation of a medicament for resisting myocardial hypertrophy, wherein the myocardial hypertrophy refers to heart failure.

5. The application of Fangji Huangqi Tang in preparing anti-myocardial hypertrophy medicament as claimed in claim 1, wherein the dosage of Fangji Huangqi Tang is 300-400 μ g/mL.

6. The application of the radix stephaniae tetrandrae and radix astragali decoction in preparing the anti-myocardial hypertrophy medicines according to claim 1, wherein the radix stephaniae tetrandrae and radix astragali decoction comprises pharmaceutically acceptable medicinal auxiliary materials in traditional Chinese medicine pharmacy, and is prepared into clinical medicine dosage forms, including tablets, oral liquid, pills, granules, capsules, injections and sustained and controlled release agents.

7. The radix stephaniae tetrandrae and radix astragali soup component is characterized by comprising a component A or a component B, and the preparation method of the component A comprises the following steps: dispersing the radix Stephaniae Tetrandrae and radix astragali decoction with water, separating with macroporous adsorbent resin column, sequentially eluting with water, 15-22% ethanol, 35-45% ethanol and 90-100% ethanol, collecting eluates, concentrating the eluates under reduced pressure, and vacuum drying, wherein the eluate of 35-45% ethanol is dried to obtain component A of radix Stephaniae Tetrandrae and radix astragali decoction;

8. The use of the tetrandra and astragalus decoction component of claim 7 in the preparation of a medicament for resisting myocardial hypertrophy.

9. The use of the tetrandra and astragalus decoction component of claim 8 in the preparation of a medicament for resisting myocardial hypertrophy, wherein the dosage of the component A is 6.0-12.5 μ g/mL; the dosage of the component B is 15-25 mug/mL.

10. The application of the radix stephaniae tetrandrae and radix astragali decoction component in preparing the anti-myocardial hypertrophy medicine according to claim 8, wherein the radix stephaniae tetrandrae and radix astragali decoction component comprises pharmaceutically acceptable medicinal auxiliary materials in traditional Chinese medicine pharmacy, and is prepared into clinical medicine dosage forms, including tablets, oral liquid, pills, granules, capsules, injections and slow-release and controlled-release agents.