CN111870639A

CN111870639A - Application of Tibetan medicine scindapsus aureus in preparing medicine for treating atherosclerosis

Info

Publication number: CN111870639A
Application number: CN202010727676.6A
Authority: CN
Inventors: 余军; 裘梁; 钟国跃; 唐乐; 秦嫚嫚
Original assignee: Jiangxi University of Traditional Chinese Medicine
Current assignee: Jiangxi University of Traditional Chinese Medicine
Priority date: 2020-08-11
Filing date: 2020-08-11
Publication date: 2020-11-03

Abstract

The invention relates to an application of Tibetan medicine scindapsus aureus in preparing a medicine for treating atherosclerosis, belonging to the technical field of traditional Chinese medicines; the invention shows that: on the animal level, the green bonnie flower alcohol extract can obviously reduce the atherosclerotic plaque area of mice, and the specific mechanism is shown as follows: on one hand, the cholesterol absorption is inhibited by reducing the SR-BI expression level, and on the other hand, the cholesterol metabolism is promoted to generate bile acid by increasing the CYP7A1 gene expression; at the cellular level, the green bonnie flower alcohol extract can obviously reduce the cholesterol uptake of cells, and reduce the expression of a macrophage marker CD36 to reduce the cholesterol uptake of macrophages, thereby reducing macrophage foaming. The invention provides scientific data and concept basis for the scindapsus aureus in clinical treatment of atherosclerosis, and provides data support for a new method and action targets for preventing and treating cardiovascular diseases more clinically.

Description

Application of Tibetan medicine scindapsus aureus in preparing medicine for treating atherosclerosis

Technical Field

The invention relates to the technical field of traditional Chinese medicines, in particular to application of Tibetan medicine scindapsus aureus in preparing a medicine for treating atherosclerosis.

Background

With the development of modern science and technology and the acceleration of pace of working and living, chronic diseases (such as cardiovascular diseases and diabetes) become a new focus of human health attention, wherein the cardiovascular diseases are the first killers which harm human health, and the mortality rate of the cardiovascular diseases is higher than that of diseases such as tumors and diabetes. According to WHO statistics, the cardiovascular disease will affect 2360 million people worldwide by 2030. According to the '2018 Chinese cardiovascular disease report', 2 deaths from cardiovascular diseases in every 5 deaths in China are shown, the morbidity of the deaths gradually rises, and the development trend of the deaths is low.

Atherosclerosis (AS) is the major pathological basis for the induction of cardiovascular disease and is a major cause of death in patients with cardiovascular disease. The atherosclerosis plaque is a progressive pathological process which is mainly characterized in that the structure or the function of vascular endothelial cells is damaged, the permeability of cell membranes is changed, and dyslipidemia is deposited under the intima, and is accompanied by inflammatory cell infiltration, mesoderm smooth muscle cell migration and proliferation, foam cell formation and extracellular matrix synthesis increase, and finally the atherosclerosis plaque is formed.

At present, the main method for treating atherosclerosis is to reduce high cholesterol level in serum, and clinically mainly relies on statins to prevent and treat AS, however, the method can only reduce the death rate of cardiovascular diseases by 30%, a large number of patients are still insensitive to statins, and atherosclerosis still deteriorates further and finally dies from cardiovascular diseases such AS myocardial infarction or apoplexy. Therefore, the search for new action targets and methods for effectively inhibiting the development of atherosclerosis has important scientific significance for reducing the morbidity and mortality of cardiovascular diseases.

The Tibetan medicine scindapsus aureus is a dry bud of oriental paperbush belonging to edgeworthia of the Thymelaeaceae, is mainly distributed in the Qinghai-Tibet and the colder regions of Dian with the elevation of more than three kilometers, is a unique ethnic medicine in the Tibet region, and is called as one of the 'eight treasures of the Qinghai-Tibet'. According to records in Tibetan medicine health preserving diagram, the scindapsus aureus has good auxiliary curative effect on cardiovascular diseases such as coronary heart disease. In folk, green bonnie flowers are often soaked in water for drinking to prevent coronary heart disease, hypertension, various vasculitis and other diseases. In recent years, researchers at home and abroad gradually increase the research heat of scindapsus aureus, but mostly stay in the analysis of crude extracts and extracts of scindapsus aureus, the scindapsus aureus mainly contains various compounds such as flavonoid glycoside substances, coumarin, total polyphenol, polysaccharide, volatile oil and the like, the medicine effect and pharmacological research mainly focuses on the aspects of reducing blood sugar, reducing blood fat, enhancing immunity, resisting oxidation and the like, and the scindapsus aureus is lack of comprehensive and deep research on cardiovascular diseases such as coronary heart disease and the like. Atherosclerosis is a main inducing factor and pathological basis of cardiovascular diseases such as coronary heart disease, and at present, no study report of using green bonnie flower to inhibit atherosclerosis exists.

In conclusion, it is necessary to research the application of the Tibetan medicine scindapsus aureus in preparing the medicine for treating atherosclerosis.

Disclosure of Invention

The invention aims to overcome the defects that the existing medicines for treating atherosclerosis have large toxic and side effects, cannot be tolerated by patients and influence the clinical treatment effect, provides a new application of scindapsus aureus in pharmacy, and particularly relates to the application of scindapsus aureus in preparing medicines for treating atherosclerosis.

The purpose of the invention can be realized by the following technical scheme:

the invention adopts high fat and high cholesterol to feed ApoE-/-mice to establish an atherosclerosis model, and the result shows that: the green bonnie flower can reduce the area of atherosclerotic plaques and has a good effect of preventing and treating atherosclerosis of mice; the extract of green bonnie flower can reduce atherosclerotic plaque by inhibiting cholesterol absorption, reducing macrophage foaming, and promoting cholesterol metabolism.

The invention has the beneficial effects that:

1. the in-vivo and in-vitro experiments prove that the green bonnie flower alcohol extract can obviously reduce the plaque area of apoE-/-mice fed with high fat and high cholesterol, increase the collagen content in the plaque, and further possibly promote the plaque stability, and the research result shows that the green bonnie flower intervenes in the direction of atherosclerosis, so that the application of the green bonnie flower in the preparation of the anti-atherosclerosis medicine is disclosed for the first time;

2. the invention provides an economical, effective, safe and reliable medicament without toxic side effect and adverse reaction for the clinical treatment of atherosclerosis;

3. the raw medicinal materials are common traditional Chinese medicinal materials, the sources are wide, and the raw material cost is low.

Drawings

In order to facilitate understanding for those skilled in the art, the present invention will be further described with reference to the accompanying drawings.

FIG. 1 is an analysis of atherosclerotic lesions of the full length aorta with an alcohol extract of green bonnie flowers; wherein, FIG. 1A is aortic oil Red O staining; fig. 1B shows the ratio of plaque area to total area of full-length aorta (left), full-length aortic plaque area (right).

FIG. 2 is a graph showing the effect of an alcohol extract of scindapsus aureus on the area of aortic root plaques; wherein, fig. 2A shows hematoxylin-eosin staining (HE) of frozen section of aortic root (left), Oil red O staining (Oil red O) (middle), Masson trichrome staining (right); FIG. 2B is a graph showing the statistical results of hematoxylin-eosin (HE) staining (left), Oil red O (center), and Masson's trichrome staining (right) of frozen sections of aortic roots.

FIG. 3 is a graph showing the effect of an alcohol extract of scindapsus aureus on the expression levels of genes involved in cholesterol transport and metabolism; FIG. 3A is a graph showing the expression levels of relevant genes involved in cholesterol metabolism balance in the liver; FIG. 3B is the expression level of the relevant genes involved in cholesterol transport in the small intestine.

FIG. 4 shows the cell viability assay of the scindapsus aureus extract on Bone Marrow Derived Macrophages (BMDM).

FIG. 5 is a graph of the effect of an alcohol extract of scindapsus aureus on foaming of BMDM induced by ox-LDL; wherein, FIG. 5A is foam cell oil red O staining; FIG. 5B is a statistical chart of the results of oil red O staining.

FIG. 6 is a graph of the effect of an alcohol extract of scindapsus aureus on macrophage foaming induced by lipoproteins.

FIG. 7 is a graph of the effect of an alcohol extract of scindapsus aureus on cholesterol absorption; wherein FIG. 7A is a peak diagram of fluorescent cells of each group; panel B shows the proportion of positive cells (top) and the average fluorescence intensity value (bottom).

FIG. 8 is a graph of the effect of an alcohol extract of scindapsus aureus on cholesterol binding.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 preparation of alcohol extract of Gloiopeltis Furcata

Weighing 500g of dried buds of scindapsus aureus, and equally putting the buds into two flasks of 5000 ml; 4000ml of 60% ethanol were added to each flask. Heating the flask by using a temperature-controlled electric heating jacket, and collecting 8000ml of extracting solution 2 hours after the liquid medicine boils; adding 4000ml of 60% ethanol into each flask again, and continuously decocting for 2 hours; collecting 16000ml ethanol extractive solution for two times, concentrating by rotary drying, and evaporating to dryness at 80 deg.C in water bath; weighing, and storing in a refrigerator at 4 deg.C. The dosage (g/d) commonly used by human/the weight (60kg) x 9.1 of a normal human is equal to the dosage (g/d.bw) of a mouse, 9g of traditional Chinese medicine materials is generally the maximum safe dosage, 2g (crude drug)/kg (weight) is converted to be a middle-stomach-perfused dosage group of the mouse, three low-middle-high (1g/kg, 2g/kg and 4g/kg) stomach-perfused dosage groups are set by diluting the medicine by 2 times, and the ointment is dissolved by using sterilized pure water and stored in a refrigerator at 4 ℃.

Example 2 analysis of atherosclerotic lesions of the full-Length aorta by Epilobium aureum alcohol extract

Male ApoE-/-mice (provided by Nanjing university-Nanjing biomedical research institute) were randomly divided into 4 groups from 9 weeks of age, namely a high fat high cholesterol diet group (HFD), a high fat high cholesterol + low dosage scindapsus aureus group (HFD +1g/kg EG), a high fat high cholesterol + medium dosage scindapsus aureus group (HFD +2g/kg EG), and a high fat high cholesterol + high dosage scindapsus aureus group (HFD +4g/kg EG), and the mice were given fat energy accounting for 42% of the total energy of HFD per group. The green bonnie flower administration group drenches the green bonnie flower ethanol extract with different concentrations every day, the drenching dosage is 10ml of liquid medicine/kg of mouse weight, and the drenching lasts for 16 weeks.

After 16 weeks, fasting for 12 hours, anesthetizing the mice, opening the abdominal cavity and the thoracic cavity of the mice, cutting the diaphragm muscle, taking 20ml of precooled Phosphate Buffer (PBS) by a 20ml syringe, pushing the 20ml of PBS into the left ventricle of the apex of the heart, changing the color of the muscle and the viscera of the mice from red to white, changing the effluent liquid to be clear, and stopping filling the PBS; 20ml of pre-cooled 4% paraformaldehyde solution (4% PFA) is injected into the same position of the cardiac apex at a constant speed. After perfusion is finished, the lung, the stomach and the small intestine are simply separated, and the heart position is exposed; after the full-length aorta is carefully and rapidly separated by using a micro-forceps under a body type microscope, the iliac arteries at two sides are cut off by using a micro-scissors; mouse blood vessels were fixed in 4% PFA for at least 24 hours before oil red O staining; rinsing with PBS for 15min to remove the fixative; the 60% isopropanol solution balances the blood vessel twice, each time for 5 min; dyeing the filtered 0.3% oil red O dyeing solution for 15min at dark room temperature; the 60% isopropanol solution differentiated for several seconds and stopped staining, the blood vessels changed from red to pink; cleaning redundant dye with pure water; and (4) continuously removing redundant adipose tissues under a body type microscope, cutting off blood vessels by using a pair of microscopical scissors, flattening the blood vessels on the transparent glue, sticking the blood vessels on the glass slide, and taking a picture.

The results are shown in fig. 1, and compared with the HFD model group, the aortic plaques of the mice in the low, medium and high dose groups of scindapsus aureus are significantly less, and the statistical results show significant differences (fig. 1A and 1B), but no significant difference exists among the low, medium and high dose groups.

Example 3 Effect of the alcohol extract of Gloiopeltis scintillans on the plaque area of aortic root

The heart of the mouse is transversely cut from the top to the middle position of the apex of the heart, and the upper half part of the heart is fixed on 4% PFA24 hours; flushing for 4 hours by running water; dehydrating 30% sucrose solution in a refrigerator at 4 deg.C overnight; frozen section embedding medium (OCT) embedded tissue; freezing with liquid nitrogen and then carrying out frozen slicing; the top of the heart of the mouse faces outwards, the thickness of the slice is 10 mu m, the circular blood vessel at the middle position of the slice to be sliced is changed into an irregular oval shape, and when two protruding valves exist, the slice is continuously collected by using an adhesive glass slide until no valve appears at the root of the aorta.

The sections were stained with hematoxylin and eosin, the procedure was as follows: washing off the OCT embedding agent by using a PBS solution; soaking in pure water for 5 min; staining cell nuclei for 3min with Harris hematoxylin; washing off excessive dye liquor with running water; differentiating with 1% hydrochloric acid alcohol and stopping staining; bluing with 60 deg.C warm water; staining cytoplasm for 1min with eosin staining solution, and washing off excessive staining solution with running water; dehydrating and sealing neutral gum; the photographs were observed under a microscope and analyzed using image analysis software.

The sections were stained with oil red O, the procedure was as follows: washing off the OCT embedding medium by using the PBS solution; soaking in pure water for 5 min; 60% isopropanol solution for 1 min; the oil red O dye solution is used for 15min at room temperature in a dark place; washing off the excess oil red O dye solution on the glass slide by using a 60% isopropanol solution; washing off isopropanol by pure water; staining cell nucleus for 1min with Harris hematoxylin staining solution; washing off the excess dye liquor by running water; differentiating with 1% hydrochloric acid alcohol and stopping staining; heating glycerol gelatin, and sealing; the photographs were observed under a microscope and analyzed using image analysis software.

Staining the section with collagen fiber, which comprises the following steps: washing off the OCT embedding medium by using the PBS solution; staining Bouin's solution for 1h at 37 ℃; washing the slices with running water; harris hematoxylin staining for 2 min; washing the redundant dye liquor; 1% hydrochloric acid alcohol differentiation; dyeing the ponceau acid fuchsin re-reddish liquid for 1 min; 1% glacial acetic acid water solution for 1 min; 1% aqueous molybdic acid solution for 5 min; dyeing the aniline blue solution for 2 min; soaking and washing with 1% glacial acetic acid water solution for 1 min; dehydrating and sealing neutral gum; the photographs were observed under a microscope and analyzed using image analysis software.

As a result, as shown in fig. 2, the lipid area of the scindapsus aureus-administered group was significantly reduced compared to the HFD model group.

Example 4 influence of the alcohol extract of Gloiopeltis scindaphnoides in the expression level of genes involved in cholesterol transport and metabolism

Grinding frozen liver or small intestine into powder, adding 50mg of powder into 1ml of TRIZOL reagent, and mixing; extracting total RNA, and synthesizing cDNA through reverse transcription; using this cDNA strand as a template, real time PCR analysis was performed on genes in the liver such as Abcg 5/8, Sr-b1, Cyp7a1 and genes in the small intestine such as Abcg 5/8 and Npc1l 1.

The results are shown in figure 3, scindapsus aureus did not alter the expression level of Abcg 5/8 gene compared to HFD group; the medium and high dose of scindapsus aureus can obviously inhibit the expression level of Sr-b1 gene; the expression level of Cyp7a1 gene is obviously increased by the aid of the low-dose scindapsus aureus, and the results show that the scindapsus aureus cannot promote cholesterol outflow but can inhibit cholesterol absorption and increase cholesterol metabolism to generate cholesterol juice; compared with the HFD group, the scindapsus aureus does not change the expression level of Abcg 5/8 gene of intestinal cells, but the scindapsus aureus with medium and high dose obviously inhibits the expression of Npc1l1, which indicates that the scindapsus aureus inhibits the absorption of intestinal cholesterol.

Example 5 cell viability assay of Epilobium aureus Total extract on BMDM

Dissolving the total extract of the scindapsus aureus in a 1640(B) culture medium, wherein the concentration is 1mg/ml, and filtering and sterilizing; laying 4 multiplied by 10^4 BMDM cells in each hole of a 96-hole plate, and culturing the BMDM cells in a bone marrow growth culture medium for 5 days; preparing medicated culture medium with different concentrations of 0 μ g/ml, 0.1 μ g/ml, 1 μ g/ml, 10 μ g/ml, 100 μ g/ml and 200 μ g/ml; discarding the culture medium, washing twice with PBS, and adding the culture medium containing the medicine; culturing for 24 h; preparing MTT solution with 1640(B) culture medium to a final concentration of 1 mg/ml; after 24h, cell administration was terminated, the medium was discarded, the cells were washed twice with 1 × PBS, 200 μ l of MTT solution was added to each well, and incubation was performed for 4 hours; removing MTT solution, adding 100 μ l DMSO into each well, and incubating at 37 deg.C for 10 min; the absorbance (OD 490nm) was measured.

As shown in FIG. 4, the concentrations of the Usnea barbata administered were 0.1. mu.g/ml, 1. mu.g/ml, 10. mu.g/ml, 100. mu.g/ml, and 200. mu.g/ml, which were not cytotoxic to BMDM cells.

Example 6 Effect of Usnea on foaming of BMDM induced by ox-LDL

The mouse bone marrow derived monocyte is taken and induced into macrophage, and then is induced into foam cell by oxidized low density lipoprotein, and the influence of the green bonnie flower on the foam of the macrophage is observed. The specific implementation is as follows: paving cell slide in advance on a 24-pore plate, paving 10 multiplied by 10^4 BMDM cells in each pore, and culturing for 5 days; setting a group: blank group (Vehicle), lipoprotein group (ox-LDL), lipoprotein with different concentrations and scindapsus aureus group (ox-LDL +100ng EG, ox-LDL +1 mug EG, ox-LDL +10 mug gEG, ox-LDL +100 mug EG), the final concentration of lipoprotein is 80 mg/ml; after 24h, foam cell oil red O staining was performed as in example 2.

As shown in FIG. 5, 1. mu.g/ml EG significantly reduced macrophage foaming after 24h compared to the ox-LDL group.

Example 7 Effect of Usnea on Cholesterol absorption

12 well plates were plated with 4X 10^5 BMDM cells, set up groups: blank group (Vehicle), lipoprotein group (Dil-oxLDL), lipoprotein + scin + scindapsus aureus group (Dil-oxLDL + EG), the lipoprotein is marked by Dil fluorescence, the final concentration is 10 mug/ml, and the scindapsus aureus concentration is 1 mug/ml; setting 3 time points of 2h, 4h and 6 h; after the culture is finished, removing the culture medium, washing with acidic PBS for 4 times, and each time for 5 min; digesting with pancreatin for 5min, removing digestive juice, and adding culture medium to blow off cells; centrifuging at 1000rpm for 3 min; discarding the supernatant, and resuspending the cells with PBS; transferring the cell to a flow-type sample loading tube, regulating current and voltage by taking a blank group as a reference after a flow cytometer is started, setting enclosed cells as negative cell groups, expressing no fluorescence, taking the cell groups as a gate, taking the cell groups outside the gate as positive cell groups, carrying a fluorescence label, and counting the number of the positive cells and the average fluorescence intensity after the sample loading is finished.

As shown in FIG. 6, the administration of Usnea barbata reduced lipid absorption and decreased foaming of macrophages.

Example 8 Effect of Usnea on Cholesterol binding

Laying 4X 10^5 BMDMs on a 12-hole plate, and setting the group as follows: blank group (Vehicle), lipoprotein group (Dil-oxLDL), lipoprotein + scin + scindapsus aureus group (Dil-oxLDL + EG), the lipoprotein is marked by Dil fluorescence, the final concentration is 10 mug/ml, and the scindapsus aureus concentration is 1 mug/ml; setting 4 time points, 30min, 60min, 90min and 120 min; after the culture is finished, removing the culture medium, washing with acidic PBS for 4 times, and each time for 5 min; digesting with pancreatin for 5min, removing digestive juice, and adding culture medium to blow off cells; centrifuging at 1000rpm for 3 min; discarding the supernatant, and resuspending the cells with PBS; transferring the cells into a flow-type sample loading tube, regulating current and voltage by taking a blank group as a reference after a flow cytometer is started, setting the enclosed cells as negative cell groups, expressing no fluorescence, taking the negative cell groups as a gate, taking the extra-gate cell groups as positive cell groups with fluorescence labels, and counting the number of the positive cells and the average fluorescence intensity after the sample loading is finished.

As a result, as shown in FIG. 7, the combination of the oxidized low density lipoprotein and the cells was not affected by the Usnea barbata.

Example 9 Effect of Usnea on the expression level of genes involved in cholesterol transport in foam cells

The 12-well plate was plated with cells in advance, 4X 10^5 BMDM cells per well, set the group: blank group (Vehicle), lipoprotein group (ox-LDL), lipoprotein + scin + scindapsus aureus group (ox-LDL +1 mug EG), final concentration of lipoprotein 40mg/ml, drug stimulation for 6 h; the culture medium is discarded, and PBS is used for washing for 2 times; placing a 12-hole plate on ice, and adding 1ml of Trizol lysate into each hole; transferring the liquid in the pore plate to a 1.5ml centrifuge tube, and standing for 5min on ice; adding 0.2ml of chloroform into each hole, shaking and uniformly mixing for 30 seconds, and standing for 10min on ice; centrifuging at 12000rpm for 10min at 4 deg.C; taking 0.4ml of supernatant to a new centrifuge tube; adding equal volume of isopropanol, and standing at room temperature for 10min under reciprocal of top and bottom; centrifuging at 12000rpm for 10min at 4 deg.C; discarding the supernatant, adding 1ml of 75% ethanol to wash the RNA precipitate; centrifuging at 12000rpm for 5min at 4 deg.C; discarding the supernatant, absorbing residual liquid, and drying; adding 20 μ L DEPC water, and performing metal bath at 60 deg.C for 5 min; taking 1 mu g of total RNA to carry out reverse transcription to form cDNA, taking the cDNA as a template to carry out the next fluorescent quantitative PCR, and detecting the expression levels of the related genes of Actin, CD36, SR-B1, ABCA1 and ABCG 1.

As shown in FIG. 8, the gene expression of CD36 and SR-B1 after the treatment of scindapsus aureus showed a tendency to decrease compared to the model group (ox-LDL), but there was no difference in the gene expression of ABCA1 and ABCG 1. We speculate that the extract of Gloiopeltis aureus probably does not play a major role in cholesterol excretion.

The working principle of the invention is as follows: .

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. Application of Tibetan medicine scindapsus aureus in preparing target organ protecting medicine is provided.

2. Application of Tibetan medicine scindapsus aureus in preparing medicine for treating atherosclerosis is provided.

3. The use according to claim 2, wherein said use is for reducing the formation of atherosclerotic plaque areas.

4. The use according to claim 3, characterized in that it is intended to influence cholesterol transport and metabolism, on the one hand by inhibiting cholesterol absorption by lowering the SR-BI expression level and on the other hand by increasing the Cyp7a1 gene expression level to promote cholesterol metabolism to bile acids.

5. The use according to claim 3, wherein the use is for reducing macrophage foaming induced by oxidized low density lipoprotein and reducing cholesterol uptake by macrophages; but does not affect the binding of cholesterol to cell membranes.

6. The use according to claim 3, wherein said use is capable of reducing macrophage foaming by reducing macrophage uptake of cholesterol by reducing the expression of macrophage marker CD 36.

7. The medicine for treating atherosclerosis is characterized by comprising Tibetan medicine scindapsus aureus.