CN110693889B - Traditional Chinese medicine composition based on compatibility of tanshinone IIA and puerarin and application - Google Patents

Traditional Chinese medicine composition based on compatibility of tanshinone IIA and puerarin and application Download PDF

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CN110693889B
CN110693889B CN201910976809.0A CN201910976809A CN110693889B CN 110693889 B CN110693889 B CN 110693889B CN 201910976809 A CN201910976809 A CN 201910976809A CN 110693889 B CN110693889 B CN 110693889B
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puerarin
tanshinone iia
myocardial infarction
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樊官伟
高珊
李玲燕
李澜
高秀梅
毛静远
李丹
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FIRST AFFILIATED HOSPITAL OF TIANJIN UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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Abstract

The invention discloses a traditional Chinese medicine composition based on compatibility of tanshinone IIA and puerarin, which can inhibit the immersion of inflammatory cells, reduce the damage of myocardial cells, relieve the synthesis of collagen, inhibit myocardial fibrosis and ventricular remodeling by improving cardiac function, improving hemodynamics and confirming that the tanshinone IIA: the ratio of puerarin is 1. The invention also discloses application of the traditional Chinese medicine composition based on compatibility of tanshinone IIA and puerarin.

Description

Traditional Chinese medicine composition based on compatibility of tanshinone IIA and puerarin and application
Technical Field
The invention relates to the field of traditional Chinese medicines, and in particular relates to a traditional Chinese medicine composition based on compatibility of tanshinone IIA and puerarin and application thereof.
Background
Myocardial Infarction (MI) is an extremely critical manifestation of coronary heart disease. Cardiac remodeling after myocardial infarction can lead to serious consequences such as heart failure, heart rupture and the like, and is the most common death cause after myocardial infarction. Ventricular remodeling is manifested by destruction of the original structure of the myocardium and the formation of non-functional collagenous connective tissue. In the heart, apoptosis, myocardial fibrosis and changes in collagen composition are typically manifested. Early remodeling is mainly scarring of the infarct area and changes in the extracellular matrix at later stages. Ventricular remodeling has now been found to be caused by inflammatory responses, oxidative stress, cytokine increase and neuroendocrine system activation.
In recent years it has been discovered that inflammatory responses are involved in the pathological process of ventricular remodeling, with macrophages being an important component. Under different inflammatory microenvironments, macrophages can be classified into the classical activated (M1) and the alternative activated (M2) types. The partial molecular markers of M1 are iNOS, IL-1, TNF-alpha and the like, and the partial molecular markers of M2 are Arg1, CD206 and IL-10. After myocardial infarction, inflammatory reaction occurs, and macrophage infiltration is gradually increased. Early inflammation is dominated by M1 and late inflammation is dominated by M2. The secretion of inflammatory factors is gradually increased, and the formation of alpha-SMA positive myofibroblasts is promoted, so that the later-stage reconstruction of the heart is caused.
The compatibility of the traditional Chinese medicines is treasure of the traditional Chinese medicines. Since the advent of the Shen nong Ben Cao Jing, the compatibility of Chinese herbs has been regulated and advocated clinically. With the development of traditional Chinese medicine, the application of the compatibility of traditional Chinese medicines is more and more emphasized.
Disclosure of Invention
The invention designs and develops a traditional Chinese medicine composition based on the compatibility of tanshinone IIA and puerarin, and aims to provide a compatibility medicine of tanshinone IIA and puerarin, so that the traditional Chinese medicine composition can improve the cardiac function, improve the hemodynamics, reduce the damage of myocardial cells, relieve the synthesis of collagen and inhibit the myocardial fibrosis and ventricular remodeling.
The invention designs and develops application of a traditional Chinese medicine composition based on compatibility of tanshinone IIA and puerarin, and the traditional Chinese medicine composition is used for preparing a myocardial infarction medicine.
The invention designs and develops an application of a traditional Chinese medicine composition based on compatibility of tanshinone IIA and puerarin for preparing a medicine for inhibiting CD11b monocyte infiltration and F4/80 + Ly6C + Macrophage-expressed drug, CD11b inhibition + Ly6C + Monocyte expressed medicine and medicine for inhibiting myocardial tissue proinflammatory inflammatory cytokine expressionThe use of (1).
The invention designs and develops application of a traditional Chinese medicine composition based on compatibility of tanshinone IIA and puerarin for preparing F4/80 promoter + Ly6C - The application of macrophage expressing medicine and medicine for promoting the expression of myocardial tissue anti-inflammatory cell factor.
The technical scheme provided by the invention is as follows:
a traditional Chinese medicine composition based on compatibility of tanshinone IIA and puerarin is composed of the following raw materials in parts by mass:
1 part of tanshinone IIA and 1-2 parts of puerarin.
Application of a Chinese medicinal composition based on compatibility of tanshinone IIA and puerarin in preparing myocardial infarction medicine is provided.
An application of a Chinese medicinal composition based on the compatibility of tanshinone IIA and puerarin in preparing medicine for inhibiting CD11b monocyte infiltration is provided.
A Chinese medicinal composition for inhibiting F4/80 based on combination of tanshinone IIA and puerarin + Ly6C + Macrophages and/or CD11b + Ly6C + Application of monocyte expression medicine is disclosed.
A Chinese medicinal composition for promoting F4/80 preparation based on compatibility of tanshinone IIA and puerarin + Ly6C - Use of macrophage expressing drugs.
An application of a Chinese medicinal composition based on combination of tanshinone IIA and puerarin in preparing medicine for inhibiting expression of proinflammatory inflammatory cytokine in myocardial tissue is provided.
Preferably, the proinflammatory inflammatory cytokines comprise: IL-6, IL-1. Beta. And/or iNOS.
An application of a Chinese medicinal composition based on the compatibility of tanshinone IIA and puerarin in preparing medicine for promoting the expression of myocardial tissue anti-inflammatory cytokines is provided.
Preferably, the anti-inflammatory cytokines include: IL-10 and/or Arg1.
Compared with the prior art, the invention has the following beneficial effects:
1. the echocardiogram results show that the structure of the ventricle is remarkably changed along with the remarkable decrease of the ejection fraction, the left ventricular short axis shortening rate and the outflow tract blood flow speed of the model group with the prolongation of the ischemia time; the hemodynamics results show that the maximum ascending rate and the maximum descending rate of the model group after ischemia are obviously reduced, the volume at the end of systole and diastole is increased, the pressure difference is reduced, and when the medicine is used for treating, each group is improved, namely, the tanshinone IIA: puerarin =1 group can significantly improve the above indexes after 28 days of administration, and can effectively relieve cardiac function deterioration caused by acute myocardial infarction;
2. model mice were examined by flow cytometry at 3d, 7d after administration for monocytes of inflammatory cells CD11b and macrophages of F4/80 phenotype in heart tissue, and as a result, tanshinone iia: puerarin =1 group is able to inhibit the release of inflammatory cells. The tanshinone IIA is detected by RT-PCR: puerarin =1 group can significantly improve the expression of inflammatory cytokines IL-1 beta, IL-6, IL-10 and iNOS in heart tissues. Immunohistochemical results show that tanshinone IIA: the puerarin =1 dose group can inhibit the expression of M1 type macrophages in early inflammatory stages (iNOS) and promote the expression of (Arg-1) M2 type macrophages;
3. the heart index of the myocardial 28d model mouse is obviously increased, TGF-beta in serum is obviously increased, and the expression level of TGF-beta can be obviously reduced by the combined application of tanshinone IIA and puerarin after the medicine is given. In HE, masson, tianlang scarlet and alpha-SMA immunohistochemical staining, the combined application of tanshinone IIA and puerarin is found to remarkably reduce the damage of myocardial cell structure and interstitial edema in acute ischemia, reduce the synthesis of collagen fiber and the release of fibroblast, thereby inhibiting myocardial fibrosis and ventricular remodeling.
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FIG. 1a shows CK values of 3d myocardial enzymes of mice after myocardial infarction. ( * P <0.01 compared to control; p <0.01 compared to model group; comparison of p <0.01 with model group )
FIG. 1b shows CK-MB values of myocardial enzyme 3d after myocardial infarction in mice according to the present invention. ( * P <0.01 compared to control; p <0.01 compared to model group; comparison of p <0.01 with model group )
FIG. 1c shows the LDH values of the groups of myocardial enzymes 3d after myocardial infarction in mice according to the present invention. ( * P <0.01 compared to control; p <0.01 compared to model group; comparison of p <0.01 with model group )
FIG. 1d is a wall motion diagram of an ultrasound chamber 24h after myocardial infarction in a mouse according to the present invention.
FIG. 1e is the EF values of each group of the sham-operated group and the model group 24h after the myocardial infarction of the mice.
FIG. 1f shows EF values of groups 28d after myocardial infarction in mice according to the present invention.
FIG. 1g shows the FS values of the groups 28d after myocardial infarction of mice according to the present invention.
FIG. 2a is an echocardiogram evaluation of 28d cardiac function after myocardial infarction in mice with tanshinone IIA and puerarin combination according to the present invention.
Fig. 2b to 2c show the effect of the combined medication of tanshinone IIA and puerarin on the inner diameter of ventricles of each group 28d after myocardial infarction of mice. (p <0.05vs control group, p <0.01vs control group, p <0.05vs model group, p <0.01vs model group, p <0.05vs pue group, n = 9)
FIG. 2d is a graph showing the effect of the combined administration of tanshinone IIA and puerarin according to the present invention on the posterior wall of ventricles of each group at 28d after myocardial infarction of mice. (p <0.05vs control group, p <0.01vs control group, p <0.05vs model group, p <0.01vs model group, p <0.05vs pue group, n = 9)
Fig. 2 e-2 f show the effect of combined administration of tanshinone IIA and puerarin according to the present invention on the left ventricular volume of each group 28d after myocardial infarction of mice. (p <0.05vs control group, <0.01vs control group, p <0.05vs model group, p <0.01vs model group, p <0.05vs pue group, n = 9)
Fig. 2g shows the effect of combined administration of tanshinone IIA and puerarin on the left ventricular quality of each group 28 days after myocardial infarction of mice. (p <0.05vs control group, p <0.01vs control group, p <0.05vs model group, p <0.01vs model group, p <0.05vs pue group, n = 9)
Fig. 2 h-2 i show the effect of combined administration of tanshinone IIA and puerarin on the change of left ventricular structure of each group 28d after myocardial infarction of mice.
Fig. 3 a-3 b show the effect of the combined medication of tanshinone IIA and puerarin in the invention on the blood flow of aortic outflow tracts of each group 28d after myocardial infarction of mice. (p <0.05vs control group, <0.01vs control group, p <0.05vs model group, p <0.01vs model group, p <0.05vs pue group, n = 9)
Fig. 3c to 3e show the effect of combined administration of tanshinone IIA and puerarin on the ejection time of each group at 28d after myocardial infarction of mice on isovolumetric contraction/relaxation interval. (p <0.05vs control group, p <0.01vs control group, p <0.05vs model group, p <0.01vs model group, p <0.05vs pue group, n = 9)
Fig. 3 f-3 g show the effect of combined administration of tanshinone IIA and puerarin on left ventricular end contraction pressure of each group 28d after myocardial infarction of mice. (p <0.05vs control group, <0.01vs control group, p <0.05vs model group, p <0.01vs model group, p <0.05vs pue group, n = 9)
Fig. 3 h-3 i show the effect of combined administration of tanshinone IIA and puerarin on the maximum rate of left ventricular rise and decrease in 28d after myocardial infarction of mice. (p <0.05vs control group, <0.01vs control group, p <0.05vs model group, p <0.01vs model group, p <0.05vs pue group, n = 9)
FIG. 3j is a 28d cardiac weight index of mice post myocardial infarction treated by tanshinone IIA and puerarin according to the present invention.
FIG. 3k shows the effect of tanshinone IIA and puerarin according to the present invention on TGF-beta concentration in plasma 28d after myocardial infarction in mice.
FIGS. 4 a-4 b are graphs showing the changes in CD11b expression in 3d cardiac monocytes after myocardial infarction in groups of mice according to the present invention. (comparing p <0.01 with control group, p <0.01 with model group)
FIGS. 4 c-4 d are graphs showing the change in CD11b expression in 7d cardiac monocytes after myocardial infarction in groups of mice according to the present invention. (. X.p <0.01 compared to control group, p <0.01 compared to model group)
FIG. 4e shows HE staining of cardiac tissues of 3, 7, 14 and 28d groups after myocardial infarction in mice according to the present invention.
FIGS. 5 a-5 e are graphs showing the changes in 3d cardiac macrophages after myocardial infarction in each group of mice according to the present invention. ( * P <0.01vs control group; p <0.01vs model group, p <0.01vs model group )
FIGS. 5 f-5 j are graphs showing the 7d cardiac macrophage changes after myocardial infarction in various groups of mice according to the present invention. ( * P <0.01vs control group; p <0.01vs model group, p <0.01vs model group )
FIG. 5k shows Masson staining of myocardial tissues in groups 3, 7, 14 and 28d after myocardial infarction in mice according to the present invention. ( * P <0.01vs control group; p <0.01vs model group, p <0.01vs model group )
FIG. 6a shows immunohistochemistry for M1 (iNOS positive macrophages) and M2 (Arg 1 positive macrophages) in 3d or 7d cardiac tissues after myocardial infarction in accordance with the present invention. (Scale: 50 μm)
FIG. 6b shows the results of quantifying M1 (iNOS positive macrophages) macrophages in 3d or 7d cardiac tissues after myocardial infarction. ( * P <0.05 compared to control, p <0.01 compared to control, p <0.001 compared to control; p <0.05vs model group, p <0.01vs model group, p <0.001vs model group )
FIG. 6c is a graph showing the results of quantifying M2 (Arg 1 positive macrophages) macrophages in 3d or 7d groups of heart tissues after myocardial infarction according to the present invention. ( * P <0.05 compared to control, p <0.01 compared to control, p <0.001 compared to control; p <0.05vs model group, p <0.01vs model group, p <0.001vs model group )
FIG. 6d is a sirius red stain of 28d groups of myocardial tissues following myocardial infarction in mice according to the present invention.
FIG. 7a shows the concentration of serum inflammatory cytokine IL-1 β in each group 3d after myocardial infarction due to the combined application of tanshinone IIA and puerarin according to the present invention. ( * p <0.05 to control group,. P <0.01 to control group; p <0.05 to model group, p <0.01 to model group; & p <0.05, & p <0.01, $ p <0.01 )
FIG. 7b shows the concentration of TNF- α in each group of serum inflammatory cytokines 3d after myocardial infarction by the combined application of tanshinone IIA and puerarin according to the present invention. ( * p <0.05 to control group,. P <0.01 to control group; p <0.05 to model group, p <0.01 to model group; & p <0.05, & p <0.01, $ p <0.01 )
FIG. 7c shows the expression of serum inflammatory cytokine IL-6 in each group 3d after myocardial infarction by the combined application of tanshinone IIA and puerarin according to the present invention. ( * p <0.05 to control group,. P <0.01 to control group; p <0.05 to model group, p <0.01 to model group; & p <0.05, & p <0.01, $ p <0.01 )
FIG. 7d shows the expression of serum inflammatory cytokine IL-1 β in each group 3d after myocardial infarction by the combined application of tanshinone IIA and puerarin according to the present invention. ( * p <0.05 to control group,. P <0.01 to control group; p <0.05 to model group, p <0.01 to model group; & p <0.05, & p <0.01, $ p <0.01 )
Fig. 7e shows the expression of serum inflammatory cytokine iNOS in each group 3d after myocardial infarction by combined application of tanshinone iia and puerarin according to the present invention. ( * p <0.05 to control group,. P <0.01 to control group; p <0.05 to model group, p <0.01 to model group; & p <0.05, & p <0.01, $ p <0.01 )
FIG. 7f is a graph showing the expression of serum inflammatory cytokine IL-10 in each group 3d after myocardial infarction by combined application of tanshinone IIA and puerarin according to the present invention. ( * p <0.05 to control group,. P <0.01 to control group; p <0.05 to model group, p <0.01 to model group; & p <0.05, & p <0.01, $ p <0.01 )
Fig. 8a is a panoramic view of a 28d Masson stained heart after myocardial ischemia by combined application of tanshinone iia and puerarin in accordance with the present invention. (scale bar: 50 μm.)
Fig. 8b is a diagram illustrating quantitative analysis of 28d myocardial collagen after myocardial ischemia by combined application of tanshinone IIA and puerarin according to the present invention.
Fig. 8c to 8d show immunohistochemical staining and quantitative analysis of 28d alpha-SMA in myocardial ischemia by combined application of tanshinone IIA and puerarin according to the present invention. ( * P <0.01vs control; p <0.05vs model set; p <0.01vs model group; & P <0.05; $ 0.05 )
Fig. 9a is MTT measurement of RAW264.7 cells treated with different drugs for 24 hours by combined application of tanshinone iia and puerarin in the present invention. ( * p <0.05vs control; * P <0.01vs control group; * P <0.001vs control group; and & & p <0.001; $ p <0.001 )
FIG. 9b is a MTT assay of H9C2 cells treated with different drugs for 24 hours by the combined application of tanshinone IIA and puerarin according to the present invention. ( * p <0.05vs control; * P <0.01vs control group; * P <0.001vs control group; and & & p <0.001; $ p <0.001 )
FIG. 9c is a graph showing the tube formation rate of human umbilical vein endothelial cells treated with different drugs for 4 hours after combined application of tanshinone IIA and puerarin in accordance with the present invention. ( Scale bar: 100 μm. (d-e) quantitative analysis of grid and node counts. )
Fig. 9d to 9e are the quantitative analysis of the grid and node counts after the combined application of tanshinone IIA and puerarin according to the present invention. ( * p <0.05vs control; * P <0.01vs control group; * P <0.001vs control group; and & & p <0.001; $ p <0.001 )
FIG. 10a is a representative immunoblot of tanshinone IIA and puerarin of the present invention on TLR4 and C/EBP-beta, key proteins in the inflammatory pathway mediated in RAW264.7 cells.
Fig. 10b is a bar graph showing the corresponding quantitative data of the key protein TLR4 of the tanshinone iia and puerarin in the present invention on the mediated inflammatory pathway in RAW264.7 cells (n = 3). ( * P <0.01vs control group; * P <0.001vs control group; $ p <0.01; $ p <0.001 )
Fig. 10C is a bar graph showing the corresponding quantitative data for key protein C/EBP- β of tanshinone iia and puerarin in RAW264.7 mediated inflammatory pathway described in the present invention (n = 3). ( * P <0.01vs control group; * P <0.001vs control group; $ p <0.01; $ p <0.001 )
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
Examples
Step one, preparing materials and experimental method description:
1. animal model of MI and dosing: all animal experiments and procedures were approved by the ethical committee on laboratory animals of Tianjin Chinese medicine university. For the mouse myocardial infarction model, the Left Anterior Descending (LAD) coronary artery was ligated according to previous studies. The operation group judges success when the EF value is within the range of 38-50 percent through ultrasonic examination. After the myocardial infarction model is established, the myocardial infarction model is randomly divided into 7 groups: (1) sham group (control group, n = 9); (2) myocardial infarction group (model group, n = 9); (3) Metoprolol tartrate group (meta, n =9,0.3mg/20 g/d); (4) puerarin group (pue, n =9,1.2mg/20 g/d); (5) Tanshinone iia group (TanIIA, n =9,0.3mg/20 g/d); (6) tanshinone IIA: puerarin =1 group (t: p = 1; (7) tanshinone IIA: puerarin =1 for group 2 (t: p = 1. The sham operation group and the model group were given 0.2ml/20g/d0.5% sodium carboxymethylcellulose. The above groups were gavaged at 3d, 7d, 14d, and 28d, respectively.
2. Echocardiographic assessment of cardiac function: at a predetermined myocardial infarction time, left ventricular function was echocardiographically examined using an MS-250 ultrasound scanning transducer.
3. Detection of myocardial enzymology: each group of abdominal aorta samples was taken 3 days after the operation. After centrifugation at 3000g serum in a 37 ℃ water bath for 30min, 1.5 ml EP tubes were repackaged to 80 ℃ for storage for later use. The enzyme indexes of myocardial Lactate Dehydrogenase (LDH), creatine Kinase (CK) and creatine kinase isoenzyme (CK-MB) are measured by a full-automatic biochemical analyzer.
4. Hemodynamic examination: hemodynamics analysis was performed 3, 7, 14, 28 days after myocardial infarction using the biofunctional test system MP100-CE (Biopac Systems, inc., santa Barbara, CA, USA) to obtain Heart Rate (HR), left Ventricular Diastolic Pressure (LVDP), left Ventricular Systolic Pressure (LVSP), left ventricular myocardial diastolic time (LV MRT), left ventricular maximum. A maximum rise rate (+ dp/dt max) and a left chamber maximum fall rate (-dp/dtmax).
5. ELISA: serum from the patient was collected, assayed by ELISA for IL-1 β, TNF- α levels, and quantified immediately at 450nm using a VieloChan-Chan-Mead microplate reader.
6. Flow cytometry: selecting myocardial infarction mice 3d and 7d, firstly anesthetizing with tribromoethanol, taking abdominal aorta blood again, then perfusing with precooled 5ml PBS, cutting heart tissue in a culture dish, cutting about 1mm to digest at 37 ℃ for 1h, filtering with 74 μm mesh, adding 1ml of whole culture solution, centrifuging at 4 ℃ 1500r for 5min, discarding supernatant, adding 1ml of erythrocyte lysate for each sample, then centrifuging at 4 ℃ 1500r for 5min, discarding supernatant, adding 500 μ l PBS, adding 2.5 μ l CD11b antibody, 1.25 μ l F4/80 antibody, 1.25 μ l Ly6C antibody in dark place, and mixing cells and antibodies. Incubate at 4 ℃ for 30 minutes and wash twice with 2ml PBS. Add 500. Mu.l of 1% paraformaldehyde for fixation at 4 ℃ and analyze the next day with flow cytometry.
7. Immunohistochemistry: mouse heart tissue was fixed with 4% paraformaldehyde and paraffin embedding. Sections (5 μm thick) were cut under a microscope and immunohistochemical analysis was performed with rabbit anti-iNOS (1. Sections were dewaxed in PBS, rehydrated, permeabilized, and incubated overnight at 4 ℃ with 0.1% (v/v) triton x-100/0.25% BSA. After primary antibody incubation, HRP was used in combination with anti-rabbit secondary antibody, and visualization was performed with the chromogenic peroxidase substrate DAB. Sections were counterstained with hematoxylin and examined under optical microscope (come card).
8. Histopathological evaluation and RT-PCR: histopathological evaluation and quantitative real-time RT-PCR were performed. The primer sequences used in the present invention are shown in Table 1.
TABLE 1 primer sequence Listing for qRT-PCR
Figure BDA0002233904530000091
9. Cell culture: RAW264.7 mouse macrophages and H9C2 were derived from the cell culture center of the academy of chinese medical sciences (beijing). Cells were found to contain 10% FBS, 100. MuThe penicillin/ml and streptomycin 100. Mu.g/ml in DMEM (high sugar) in a humidified incubator at 37 ℃. HUVEC originated from cell culture center of Chinese academy of medical sciences (Beijing). Cells are cultured in ECM medium at 37 deg.C, with 5% CO 2 The wet incubator of (2) is used for culture.
10. Cell microtubule formation assay: in the HUVEC tube formation experiment, 50. Mu.l of cold matrix gel was added to each well of a 96-well plate, incubated at 37 ℃ for 45min, and after gelation, 1X 10 cells were added 4 Individual HUVECs were seeded into matrix gel-coated wells and treated with different drug compositions and concentrations. The cells were incubated for 4h and the tube formation process was observed under a light microscope. The tube nodes and grids formed by the HUVEC in each image were counted using ImageJ software.
11. Western blotting: protein samples were extracted from lysates of the cultured cells and protein concentrations were determined using the BCA protein detection system. Then denatured in 5 Xloading buffer at 100 ℃ for 5 minutes and separated by 12% SDS-PAGE. Proteins were transferred from the gel to a teflon membrane. The membrane was then incubated with the appropriate primary antibody at 4 ℃: TLR4 antibody (Boster Biotechnology, ba 1717), anti-C/EBP-beta antibody (Abcam, ab 53138), beta-actin (cell signaling technology, 4967). After washing 3 times with TBST, incubation with secondary antibody was performed for 2 hours at room temperature. Protein samples were visualized with ECL-western blotting substrates and immediately imaged with C-digit 3600 (li cor, USA).
Step two, expressing the experimental result by using an average standard deviation(s), and performing single-factor variance analysis by using SPSS 22.0 statistical software; p <0.05 was statistically significant.
Step three, analyzing the experimental result
1. Influence of tanshinone IIA and puerarin on the activity of the enzyme of myocardium:
as shown in fig. 1 a-1 b, the ejection fraction of mice at 1d after surgery is significantly lower than that of the sham surgery group (p <0.01, n =9 for the control group, and n =20 for the model group), the anterior wall of the left ventricle is significantly changed, the movement is reduced, and the wall of ventricle is thinned. The ejection fraction and fractional shortening rate were significantly increased after each group administration compared to the model group (p < 0.01).
As shown in fig. 1a to 1c, LDH, CK-MB values were significantly higher in each group (model group, metoprolol group, puerarin group, tanshinone iia group: puerarin = 1. In the administration group, the positive drugs metoprolol, puerarin and tanshinone IIA: puerarin =1, compared with the model group, there is no significant difference between the 2 groups, the tanshinone group ia: puerarin =1, group 1 was significantly lower than the model group. Wherein the tanshinone IIA: puerarin =1, and the reduction amplitude of the group is maximal.
2. The influence of tanshinone IIA and puerarin on ventricular structure and cardiac function:
as shown in FIGS. 1 c-1 g, 2a, 2h and 2i, in different periods after MI, M-mode ultrasonic evaluation shows that the ventricular cavity of the model group is progressively expanded compared with the ventricular cavity of the dummy operation group, the anterior wall of the left ventricle is obviously changed, the motion is weakened, and the ventricular wall is thinned. After administration, the ejection fraction and the short axis shortening rate of each group were significantly increased compared to the model group.
As shown in FIGS. 2 a-2 b, the left ventricular anterior wall end diastole (IVS; d) and end systole (IVS; s) thicknesses of the model groups were significantly reduced 28 days after myocardial infarction compared to the sham group. Tanshinone IIA: puerarin =1 group after treatment, the left anterior ventricular end diastolic thickness was significantly increased (p <0.05 vs) compared to the model group, while there was no significant difference between the other groups. Compared to the model group, the left ventricular anterior wall end-diastolic and end-systolic thicknesses were significantly increased (p < 0.05) for each of the remaining groups, except for the puerarin group (p < 0.05).
As shown in FIGS. 2 b-2 d, the inner diameters of 28-day end diastole (LVID; d) and end systole (LVID; s) of the model group were significantly increased (p <0.05vs control, p <0.01vs control), tanshinone IIA: puerarin =1 group 1, end diastole and end systole internal diameters decreased significantly (p <0.05vs model group, p < 0.05). p <0.01vs model. Compared with the model group, the wall thickness after the end-diastolic phase of the left ventricle (LVPW; d) of each group has no significant difference, but the ratio of tanshinone IIA to tanshinone IIA: puerarin =1 group can significantly increase left ventricular end-diastolic wall thickness. Contraction (LVPW; s) (p <0.05vs model).
As shown in FIGS. 2 e-2 f, the left ventricular end-systolic and end-diastolic volumes of the model groups increased significantly 28 days after myocardial infarction (p < 0.01). After administration, except for the positive drugs, the other groups were significantly down-regulated, tanshinone IIA: puerarin =1, group 1 showed the greatest reduction (p <0.05vs model, p <0.01vs model). Meanwhile, tanshinone IIA: puerarin =1 group of rats with a significant decrease in left ventricular mass 28 days after myocardial infarction (p < 0.01). The tanshinone IIA is suggested: puerarin =1 group can improve ventricular contraction and relaxation functions during myocardial infarction.
3. The influence of tanshinone IIA and puerarin on the hemodynamics of myocardial ischemia model:
as shown in fig. 3a, at 28 days, the blood flow in mice can be significantly reduced (p <0.01 compared to control group) because different drugs can increase blood flow.
As shown in fig. 3b, meanwhile, tanshinone iia: puerarin =1 group showed the most significant increase in blood flow (p <0.01vs model).
As shown in FIGS. 3 c-3 e, 28 days after myocardial infarction, the isovolumetric contraction phase (p < 0.01) of the model group was significantly increased (p < 0.01), and the relaxation phase (p < 0.01) was significantly decreased (p < 0.01). Tanshinone IIA: puerarin =1 group significantly shortened the systolic and diastolic intervals in mice (p <0.05,vs model), while increasing the ejection time in mice (p <0.01,vs model).
As shown in FIGS. 3 f-3 g, 28 days after myocardial infarction, the systolic pressure of the model group was significantly decreased (p < 0.01) and the differential pressure was significantly decreased (p < 0.01). After the tanshinone IIA and puerarin are treated for 28 days in a combined mode, the systolic pressure is obviously increased (p is less than 0.01) and the differential pressure is increased (p is less than 0.01) compared with a model group.
As shown in FIGS. 3 h-3 i, the maximal increase rate (+ dp/dt max) and maximal decrease rate (-dp/dtmax) were decreased at 3, 7, 14, 28 days after myocardial infarction in the model mice (p <0.01, p <0.01 in the control group). After the tanshinone IIA and the puerarin are administrated for 3, 7 and 14 days, the maximum rising rate and the maximum falling rate of the mouse are not obviously improved. And tanshinone IIA: puerarin =1 and 1. Tanshinone IIA: puerarin =1 group compared to the single dose puerarin group, the maximum rate of increase was significantly enhanced (p <0.05vs pue).
4. The influence of tanshinone IIA and puerarin on myocardial ischemia model M1/M2 type macrophage:
as shown in fig. 4 a-4 b, peripheral blood mononuclear cells infiltrated damaged myocardial tissue in the early stage of myocardial infarction. Compared with the control group, the mononuclear cell infiltration of the model group is obviously increased. The administration of metoprolol, puerarin, tanshinone IIA type: after puerarin =1 and 1. Meanwhile, tanshinone IIA: puerarin =1 group decreased most significantly, but not significantly differently.
As shown in fig. 4c to 4d, on day 7 after myocardial infarction, the mononuclear cell infiltration of the model group was significantly increased compared to the sham operation group, and only tanshinone iia: puerarin =1 group showed significant differences in the combination treatment group. The tanshinone IIA is prompted: puerarin =1 group can inhibit cd11b monocyte infiltration after myocardial infarction.
As shown in FIGS. 5a to 5c, 3 days after myocardial infarction, a large number of monocytes in the myocardial damaged tissue first differentiated into M1 macrophages. Model set F4/80 + Ly6C + Macrophages and CD11b + Ly6C + Monocytes increase significantly, thereby activating the inflammatory response. Tanshinone IIA: puerarin =1 group, which can significantly inhibit F4/80 + Ly6C + Macrophages and CD11b + Ly6C + Mononuclear cells, thereby effectively reducing the inflammation degree in the early stage of myocardial infarction.
As shown in FIGS. 5d to 5e, and, at the same time, F4/80 + Ly6C - Macrophages and CD11b + Ly6C - The regulation effect of the mononuclear cells in the early stage of myocardial infarction is not obvious. The tanshinone IIA is prompted: puerarin =1 group can effectively reduce early inflammatory cells after myocardial infarction, thereby delaying further deterioration of myocardial tissues.
As shown in FIGS. 5 f-5 h, monocytes in myocardial tissue differentiated into M2 macrophages at 7d after myocardial infarction, which play a protective and defensive role in suppressing inflammatory responses. Model group myocardial infarction F4/80 after 7d + Ly6C - The number of macrophages was increased compared to 3 d. Treatment groups F4/80 after administration + Ly6C - Macrophages are all presented withIncreasing trend, tanshinone iia: puerarin =1, the rise is more obvious in the group 1.
As shown in FIGS. 5i to 5j, on the other hand, it can suppress F4/80 + Ly6C + Macrophages and CD11b + Ly6C + Upregulation of monocytes. The tanshinone IIA is prompted: puerarin =1 group 1 can obviously activate F4/80 in the later period of myocardial infarction + Ly6C - The macrophage is up-regulated to play an anti-inflammatory role.
Inducible Nitric Oxide Synthase (iNOS) is a marker of mouse M1 macrophages, and it can promote inflammation. As shown in FIGS. 6a to 6c, immunohistochemistry results showed that the expression of iNOS positive cells in the model group was significantly increased at 3d in myocardial infarction 3d and 7 d. iNOS expression was gradually decreased in 7d of myocardial infarction in the model group. The expression of iNOS in each group of myocardial infarction at 3d is lower than that in the model group, and the expression of iNOS at 7d is obviously lower than that in the model group, particularly the expression of tanshinone IIA: puerarin =1 group. Indicating that the expression of M1 macrophages dominates in the early phase of inflammation. Tanshinone IIA: puerarin =1 group more inhibits the expression of M1-type macrophages in the early stage of inflammation, thereby inhibiting the occurrence of inflammation. Arginase 1 (Arg 1) is a marker of mouse M2 macrophages and has an anti-inflammatory effect. The immunohistochemical result shows that the expression of Arg1 positive cells in heart tissues of the model group is gradually increased from 3d to 7d, and the peak is reached in 7 d. Meanwhile, the expression of Arg1 macrophages in each group was higher than that in the model group.
5. The influence of tanshinone IIA and puerarin on myocardial ischemia model inflammatory factors is as follows:
as shown in FIGS. 7 a-7 b, there was no significant difference in serum IL-1. Beta. And TNF-. Alpha.levels 3 days after ischemia. However, significant differences were observed in the TNF-. Alpha.content between the model group and the tanshinone IIA single dose group and the tanshinone IIA and puerarin combination group.
As shown in FIGS. 7c to 7e, RT-PCR results showed that the expression of the myocardial tissue inflammatory cytokines IL-6, IL-1. Beta., IL-10 and iNOS in the post-myocardial infarction 3d model group was significantly higher than that in the sham operation group. Compared with the model group, the expressions of IL-6, IL-1 beta and iNOS in each group are all obviously reduced, and the tanshinone IIA: puerarin =1 group reduction was more significant.
As shown in fig. 7f, after administration, expression of IL-10 was increased, tanshinone iia: puerarin =1, the increase of group 1 is more significant.
These results suggest that after administration, proinflammatory inflammatory cytokines can be significantly inhibited and anti-inflammatory cytokines can be upregulated.
6. The influence of tanshinone IIA combined with puerarin on ventricular remodeling after myocardial ischemia:
as shown in FIG. 3j, 28 days after myocardial infarction of the mice, the hearts of the model groups were significantly dilated, and the myocardial mass index was significantly higher than that of the sham-operated group. After drug treatment, the myocardial mass was significantly reduced compared to the model group.
As shown in FIG. 4e, the ELISA results showed that the model group showed an increase in TGF-. Beta.release in the serum, and the treatment group showed a significant decrease in TGF-. Beta.release compared to the sham operation group, with statistical significance of the difference compared to the model group (P < 0.05). HE staining morphological observation shows that the degree of myocardial fibrosis of the 3 d-14 d model group after myocardial infarction is obviously higher than that of the administration group. Collagen fibers in the marginal area of myocardial infarction are mixed with myocardium and cells. However, in the model group, the myocardial infarction area is free of viable myocardial cells or only a few viable myocardial cells in 28 days, and severe fibrosis causes thinning of the wall of the chamber. Tanshinone IIA: puerarin =1 group compared with the model group, pathological changes were mild.
As shown in fig. 5k, masson staining showed that fibrosis in the marginal regions changed after myocardial infarction, collagen content was higher and residual myocardium was less in the model group, global fibrosis was observed, and the degree of fibrosis was lower in the treatment group compared to the model group. To some extent, tanshinone iia: puerarin =1, and the group 1 can obviously improve myocardial fibrosis. The degree of fibrosis was increased at 28 days as compared to 14, 7 and 3 days, while the residual myocardium was distributed in islands around the vessels in the administration group. In particular to tanshinone IIA: puerarin =1, has obvious inhibiting effect on myocardial fibrosis, and the distribution characteristics of cells around blood vessels can be seen in pathological images.
As shown in fig. 8a, tanshinone iia: puerarin =1 group can inhibit myocardial fibrosis, thereby resist ventricular remodeling and improve cardiac function. Masson stained myocardium panoramas showed significant fibrosis of the left ventricular walls 3d, 7d, 14d, 28d of the model groups, thinning of the ventricular walls, clear borders with normal myocardium, and only a small amount of myocardium was present. On day 28, the myocardial infarction area of each group was increased to a different extent than in the other 3 periods, and the wall thickness was reduced. However, the range of variation of the administration group was smaller than that of the model group.
As shown in FIG. 6d and FIG. 8b, sirius red staining showed severe fibrosis at the myocardial infarction margin of the model group 28 days after myocardial infarction, increased type I collagen, and significantly increased proportions of type I and type III collagen. The fibrosis degree of the metoprolol group is obviously reduced, the collagen I is obviously reduced, but the proportion change of the collagen I and the collagen III is not obvious. Tanshinone IIA: puerarin =1 and 1. Compared with a model group, the collagen I and III are obviously reduced, the collagen III is obviously increased, the proportion of the collagen I and III is obviously changed, and particularly, the tanshinone IIA: puerarin =1 group.
As shown in fig. 8c and 8d, the α -SMA immunohistochemical staining results showed that myocardial fibroblasts increased 28 days after myocardial infarction in the model group, which resulted in ventricular fibrosis and ventricular remodeling. The number of the fibroblasts of each component is obviously reduced after the medicine treatment. Quantitative analysis shows that compared with a normal group, the number of the fiber cells formed by the model is obviously increased, and the ratio of the metoprolol group to the tanshinone IIA is as follows: puerarin =1 component fiber cell number significantly decreased. These results suggest that tanshinone iia: puerarin =1 group can slow down proliferation of myocardial tissue fibroblasts after myocardial infarction and inhibit ventricular remodeling.
7. The combined application of tanshinone IIA and puerarin has effects on macrophage, myocardial cell and endothelial cell:
as shown in fig. 9 a-9 b, there was no significant difference in survival between the two groups of RAW264.7 and H9C2 cells at day 1. The culture was continued for 3 days and 5 days, and MTT method showed that the culture was carried out with tanshinone IIA: puerarin =1 the proliferation of the two cells treated above was significantly enhanced.
As shown in fig. 9 c-9 e, we also investigated whether different drug treatments could improve endothelial cell luminal formation in vitro by luminal formation experiments. When tanshinone iia puerarin =1 was used in the culture, the connection between endothelial cells increased and lumen formation occurred. Tanshinone IIA: puerarin =1 there were more nodules and grids in group 1 than in the other groups.
8. The combined application of tanshinone IIA and puerarin has the following effects on the lipopolysaccharide-stimulated RAW264.7 cell inflammation pathway key protein:
as shown in FIGS. 10 a-10 b, in the present invention, we also examined the expression of proteins involved in the inflammatory pathway. Lipopolysaccharide causes significant elevation of TLR4 protein. The tanshinone IIA or puerarin inhibits the expression of TLR4 protein, but the tanshinone IIA: puerarin =1 has more remarkable inhibiting effect.
As shown in fig. 10a and 10c, simultaneously, the tanshinone iia: puerarin =1, group 1C/EBP- β protein expression was up-regulated.
The results show that the combined application of tanshinone IIA and puerarin can regulate the inflammatory reaction of LPS stimulating RAW264.7 cells by influencing key proteins in inflammatory pathways.
Myocardial infarction is the most common cause of myocardial injury, leading to the loss of a large number of myocardial cells, and finally ischemic myocardial diseases and heart failure. Inflammation is the first manifestation after myocardial infarction, increased monocyte macrophage expression, increased inflammatory factor release, TGF-beta activation, and accelerated myocardial fibrosis. In addition, apoptosis of myocardial cells occurs with expansion of infarct size, further leading to myocardial fibrosis and poor ventricular dilation. The tanshinone IIA and the puerarin are combined to treat the myocardial infarction, the tanshinone IIA and the puerarin are found to have obvious curative effect on treating the myocardial infarction, play an important role in inhibiting ventricular remodeling at the later stage of the myocardial infarction by inhibiting inflammatory reaction at the early stage of the myocardial infarction, and can be combined to improve the cardiac function of a mouse after the myocardial infarction, improve the hemodynamics, reduce the number of myocardial cells and reduce the synthesis of collagen; through comprehensive evaluation of cardiac function, hemodynamics and inflammatory factors, the optimal ratio of tanshinone IIA to puerarin is determined to be 1.
Cardiac injury activates innate immunity and triggers an inflammatory response. Myocardial cell death leads to replacement of scar tissue and ventricular remodeling after myocardial infarction, further impairing cardiac function. Early cardiac wound healing is characterized by immune cells, particularly neutrophils and monocytes/megakaryocytesPhagocytic cells, which penetrate the myocardium congenital. Macrophages may not be apparent in a healthy heart, but after myocardial damage, macrophage numbers increase dramatically. After myocardial infarction, chemokines induce and recruit a large number of pro-inflammatory monocytes to differentiate into macrophages for phagocytosis. Wound clearance from dead cells and matrix debris triggers an anti-inflammatory cascade that inhibits leukocyte recruitment. Infarcted macrophages exhibit significant phenotypic and functional heterogeneity and may regulate cellular processes during cardiac repair. The non-infarcted myocardium has a lower number of monocytes/macrophages than the ischemic myocardium. The time course is consistent with the two monocyte phases described in infarction, but the kinetics are slower. The number of cells reached a peak at day 10 after myocardial infarction, 5 days later than in the ischemic area. Tissues obtained from clinical necropsy showed a large increase in macrophages in the distal myocardium of patients who died 5 to 14 days after the ischemic event occurred. The experiment firstly analyzes mononuclear macrophages and inflammatory factors in myocardial tissues through flow cytometry, immunohistochemistry and RT-PCR detection, and discovers that the mononuclear cells in the myocardial tissues are obviously increased after myocardial infarction. Tanshinone IIA and puerarin can reduce CD11b + Ly6C + 、F4/80 + Ly6C + And the number of iNOS positive (M1) monocytes/macrophages. Myocardial infarction for 7 days, F4/80 + Ly6C - And increased Arg 1-positive (M2) macrophages, while also decreasing Ly6C + CD11b + And F4/80 + Ly6C + Cell number, wherein tanshinone iia: puerarin =1 group was most significant. The tanshinone IIA and puerarin are combined to play a bidirectional inhibition role in MI-7d inflammation.
The invention shows that the combination of tanshinone IIA and puerarin (1). iNOS and IL-6 (mainly secreted by M1 macrophages) and increases the expression and secretion of the anti-inflammatory factor IL-10 (mainly secreted by M2 macrophages). The tanshinone IIA and the puerarin (1) are combined to be applied to the early stage of the myocardial infarction inflammatory reaction, so that the proportion of M1 macrophages can be reduced, and the proportion of M2 macrophages can be increased. In addition, TLR4 and C/EBP- β are key proteins that regulate inflammatory pathways, the former mediating pro-inflammatory responses and the latter playing a role in regulating the conversion of macrophages to the M2 phenotype, i.e. in regulating anti-inflammatory responses. TLR 4-mediated inflammatory pathways can promote the expression and secretion of various proinflammatory factors such as IL-1 beta, TNF-alpha and the like. In our study, TLR4 and C/EBP- β were quantified in RAW264.7 cells. The results show that the combined medicine can effectively inhibit the expression of TLR4 protein and increase the expression of C/EBP-beta protein, and explains the reason of inhibiting the expression of inflammatory markers by the medicine from a signal transduction mechanism. In addition, the anti-inflammatory action mechanism of the tanshinone IIA and the puerarin is preliminarily proved by detecting the expression of the tanshinone IIA and the puerarin.
Ventricular remodeling following myocardial ischemia is a clinically common ongoing pathophysiological process. Ventricular remodeling develops in proportion to the infarct size. The larger the infarct size, the greater the number of myocardial cell necrosis, the thinner the wall of the chamber, the bulging, and left ventricular remodeling. Myocardial fibrosis is an important pathological change for reasonable remodeling of heart diseases and has important significance in various diseases such as arrhythmia, heart failure, sudden cardiac death and the like. Therefore, inhibition of myocardial fibrosis is one of the important means for clinical treatment of heart disease. Transforming growth factor beta (TGF-. Beta.) has been widely accepted and recognized by medical workers as an important marker of ventricular remodeling. Some even argue that it is an index for evaluating ventricular remodeling. In the myocardial ischemia process, as the ischemia time is prolonged, the M2 macrophage releases TGF-beta in the myocardium and is highly expressed, so that fibroblast proliferation and collagen secretion are caused, and myocardial fibrosis and ventricular remodeling aggravation are accelerated. The experiment detects the content of TGF-beta in serum of a mouse in 28 days, the content of TGF-beta in a model group is obviously higher than that in a control group, and ventricular remodeling is shown. But there was a varying degree of decline in TGF- β levels following administration. The tanshinone IIA is most obviously matched with the doses of 1, 1 and 2 of puerarin in a weight ratio. The combined application of tanshinone IIA and puerarin can reduce the proportion of I, III type collagen in injured tissues and the number of myoblasts after myocardial infarction. Therefore, the combined application of tanshinone IIA and puerarin can inhibit ventricular remodeling in the later stage of myocardial infarction. These results suggest that tanshinone IIA in combination with puerarin may play a role in protecting the heart by inhibiting the number of macrophages in the early stage of inflammation. Thereby reducing the expression of TGF-beta and inhibiting myocardial fibrosis and ventricular remodeling in the later period of myocardial infarction.
Tanshinone IIA and puerarin alone have been studied. Taking tanshinone IIA as an example, more researches are focused on the anticancer effect, such as non-small cell lung cancer, osteosarcoma, colon cancer and breast cancer, wherein some of the tanshinone IIA plays the anticancer effect by regulating inflammatory response; in addition, tanshinone IIA affects diabetic peripheral neuropathic pain through regulation of endoplasmic reticulum stress pathway, spinal cord dorsal horn neural circuit; puerarin inhibiting hepatic fibrosis and central nervous system injury through inflammation is a hotspot of puerarin research in recent years.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
SEQUENCE LISTING
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<120> a traditional Chinese medicine composition based on combined use of tanshinone IIA and puerarin and application thereof
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Claims (1)

1. An application of a Chinese medicinal composition based on compatibility of tanshinone IIA and puerarin in preparing medicine for treating myocardial infarction is provided;
the traditional Chinese medicine composition is prepared from the following raw materials in parts by mass:
1 part of tanshinone IIA and 1-2 parts of puerarin.
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