Application of active ingredient protopine of Dong medicine bletilla striata in anti-atherosclerosis medicine

Abstract

The invention relates to application of active ingredient protopine of Dong medicine bletilla striata in anti-atherosclerosis medicines. The invention relates to an application of protopine, which is an active ingredient from Dong medicine bletilla striata, in preparation of an anti-atherosclerosis medicine. The active ingredient can effectively inhibit formation of atherosclerotic plaques. The invention relates to a Dong medicine cynoglossum piloselloides active ingredient protopine which can effectively inhibit adhesion of mononuclear cells induced by APJ endogenous ligand Apelin-13 to vascular endothelium so as to inhibit atherosclerotic plaque formation. The active ingredient protopine of the Dong medicine cynoglossum piloselloides is used as a novel candidate medicine for resisting atherosclerosis in fundamental research of atherosclerosis diseases, and has potential application value in prevention and treatment of atherosclerosis.

Description

Technical Field

[0001] The invention belongs to the field of pharmacology research, and specifically relates to a new medical use of protopine, an active ingredient of the Dong medicine Sanguisorba officinalis, in an anti-atherosclerotic drug. Background Art

[0002] Atherosclerosis is the most common vascular disease, which is mainly caused by the adhesion of monocytes to the vascular endothelium, the formation of lipid deposition plaques, the accumulation of complex carbohydrates, the proliferation and calcification of smooth muscle tissue in the middle layer of the blood vessels, resulting in reduced elasticity of arterial vessels, narrowing of the lumen, occlusion, and ultimately hypoxic necrosis of tissues and organs. The adhesion of monocytes to the vascular endothelium is an important initiating link in the development of atherosclerosis. During the development of atherosclerosis, monocytes are actively recruited and adhere to the atherosclerotic lesions and promote the development of atherosclerotic plaques by maintaining a chronic inflammatory response. In addition, the adhered monocytes can also cross the endothelial cell gap under the drive of monocyte chemoattractant protein-1, enter the subendothelium, swim to the lesion site, differentiate into macrophages, participate in the formation of foam cells, and finally develop into typical atherosclerotic plaques. Previous studies have found that the seven-transmembrane G protein receptor APJ, which has a certain homology with the angiotensin type I receptor, and its endogenous ligand Apelin-13 can promote the adhesion of monocytes to the vascular endothelium and thus promote the development of atherosclerosis. Therefore, accelerating the development of drugs targeting blockers or agonists of APJ receptors is of great significance for the treatment of atherosclerotic diseases.

[0003] Modern research shows that the main active ingredients of Sanguisorba officinalis are alkaloids. So far, protopine, chelerythrine, sanguinarine, allocyptopine, etc. have been isolated from Sanguisorba officinalis. Among them, protopine has been reported to have multiple pharmacological effects such as anti-inflammatory, antioxidant and cell apoptosis. However, there is no report on whether it participates in the prevention of monocyte adhesion to vascular endothelial cells and the treatment of atherosclerotic diseases.

[0004] This patent mainly discusses the functional research of protopine, the active ingredient of the Dong medicine blood water grass, and the adhesion of monocytes to the vascular endothelium and the formation of atherosclerotic plaques. After repeated experiments, the inventor found that protopine, the active ingredient of the Dong medicine blood water grass, can effectively inhibit the adhesion of monocytes to the vascular endothelium induced by APJ endogenous ligand Apelin-13, thereby inhibiting the formation of atherosclerotic plaques. Summary of the invention

[0005] The purpose of the present invention is to provide a new medical use of protopine, an active ingredient of the Dong medicine Blood Water Grass, as an inhibitor of monocyte adhesion to the vascular endothelium induced by APJ endogenous ligand Apelin-13, and used together with other blockers or agonist drugs targeting APJ receptors to play a role in resisting monocyte adhesion to the vascular endothelium and treating atherosclerotic diseases. Therefore, protopine, an active ingredient of the Dong medicine Blood Water Grass, is expected to become a new candidate drug for the prevention and treatment of atherosclerosis.

[0006] The technical solution of the present invention:

[0007] Application of protopine, the active ingredient of the Dong medicine Sanshuicao, in the preparation of anti-atherosclerotic drugs.

[0008] Furthermore, the anti-atherosclerosis is achieved by inhibiting the adhesion of monocytes to the vascular endothelium induced by the APJ endogenous ligand Apelin-13, thereby inhibiting the formation of atherosclerotic plaques.

[0009] Furthermore, the dosage forms of the drug include powders, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.

[0010] The invention relates to protopine, an active ingredient of the Dong medicine blood water grass, which is used for preventing and improving the formation of atherosclerotic plaques, or the drug has good application prospects in treating atherosclerotic diseases caused by the adhesion of monocytes to vascular endothelium.

[0011] Beneficial technical effects of the present invention

[0012] The present invention first discovered that the active ingredient of the Dong medicine blood water grass protopine has an ideal effect of resisting the formation of atherosclerotic plaques, and verified its ideal anti-atherosclerotic effect from multiple directions. The active ingredient of the Dong medicine blood water grass protopine significantly downregulates the expression of vascular endothelial cell adhesion molecules ICAM-1 and VCAM-1, inhibits the adhesion of monocytes to the vascular endothelium, and alleviates the formation of atherosclerotic plaques, and can be used as a new candidate drug for the treatment of atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Protopine, the active ingredient of the Dong medicine Xueshuicao, inhibited the expression of vascular endothelial cell adhesion molecules induced by apelin-13. Protopine inhibited the expression of vascular endothelial cell adhesion molecules VCAM-1 and ICAM-1 promoted by Apelin-13 (n=3**P<0.01and***P<0.001vs Control; #P<0.05vs Apelin-13 group);

[0014] Figure 2Protopine, the active ingredient of the Dong medicine Xueshuicao, inhibits apelin-13-induced monocyte adhesion to the vascular endothelium;

[0015] Figure 3 Protopine, the active ingredient of the Dong medicine Sanshuicao, inhibits apelin-13-induced atherosclerotic plaque formation (n=5*P<0.05vs Control). DETAILED DESCRIPTION

[0016] The present invention firstly finds that the active ingredient protopine of the Dong medicine blood water grass has a new pharmacological function of anti-atherosclerosis through a series of in vitro cell experiments and in vivo animal experiments. Adhesion of monocytes to vascular endothelium mediated by adhesion molecules is the initiation link of atherosclerosis. Therefore, in the in vitro experiment, the present invention first verifies the influence of protopine, the active ingredient of the Dong medicine blood water grass, on the expression of adhesion molecules of vascular endothelial cells: first, the vascular endothelial cells are cultured to a suitable density and divided into different treatment groups, including a control group (without adding any drug), an apelin-13 group (only adding Apelin-13), a protopine group (only adding protopine) and an apelin-13+protopine group. By treating vascular endothelial cells with protopine in advance, and then adding apelin-13 to stimulate, and then using Western Blot protein immunoblotting experiments to detect the expression of vascular endothelial cell adhesion molecules ICAM-1 and VCAM-1, the effect of protopine on the expression of adhesion molecules induced by apelin-13 was observed to determine the effect of protopine on the adhesion properties of vascular endothelial cells in vitro. The results showed that protopine, the active ingredient of the Dong medicine blood water grass, has a new effect of downregulating the expression of vascular endothelial cell adhesion molecules ICAM-1 and VCAM-1. Then, a cell adhesion experiment was used to verify the effect of protopine, an active ingredient of the Dong medicine blood water grass, on the adhesion of monocytes to vascular endothelial cells in vitro: vascular endothelial cells were cultured in the same way, and after grouping and treatment, monocytes were labeled with Calcein AM, and the labeled monocytes were added to the treated vascular endothelial cell culture system. The adhesion rate of monocytes to the vascular endothelium was observed by fluorescence microscopy, and the inhibitory effect of protopine on apelin-13-induced monocyte adhesion to the vascular endothelium was evaluated, verifying the anti-adhesion effect of protopine in vitro from another perspective. The results showed that protopine, an active ingredient of the Dong medicine blood water grass, has a new effect of inhibiting the adhesion of monocytes to the vascular endothelium. The above in vitro cell experimental results show that protopine, an active ingredient of the Dong medicine blood water grass, has a new effect of downregulating the expression of vascular endothelial cell adhesion molecules ICAM-1 and VCAM-1 and inhibiting the adhesion of monocytes to the vascular endothelium.

[0017] Secondly, the present invention uses in vivo animal experiments to further clarify the effect of protopine, an active ingredient of the Dong medicine Xueshuicao, on the formation of atherosclerotic plaques: ApoE - / -Mice were fed with high-fat diet for 12 weeks to establish an atherosclerosis model. During the high-fat feeding process, different treatment groups were set up at the same time, and the active ingredients of the Dong medicine blood water grass, protopine (10 mg / kg), apelin-13 (5 mg / kg), and both were injected intraperitoneally for 12 weeks. The formation of atherosclerotic plaques in the aorta as a whole was observed by main body oil red O staining, and the area and severity of atherosclerotic plaques in mice in different treatment groups were compared. The inhibitory effect of protopine on the formation of atherosclerotic plaques induced by Apelin-13 was analyzed, thereby verifying the anti-atherosclerotic effect of protopine in vivo. The results showed that protoopine, the active ingredient of the Dong medicine Blood Water Grass, can effectively alleviate the formation of atherosclerotic plaques; therefore, combined with in vitro cell experiments and in vivo animal experiments, it was confirmed that protoopine, the active ingredient of the Dong medicine Blood Water Grass, can inhibit the adhesion of monocytes to the vascular endothelium by downregulating the expression of vascular endothelial cell adhesion molecules ICAM-1 and VCAM-1, thereby effectively alleviating the formation of atherosclerotic plaques, and it is expected to become a new candidate drug for the prevention and treatment of atherosclerosis.

[0018] The technical solution of the present invention is further described below in conjunction with the embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the protection scope of the present invention. The process equipment or devices not specifically noted in the following embodiments are all conventional equipment or devices in the art. If not specifically specified, the raw materials used in the embodiments of the present invention can be obtained commercially; if not specifically specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0019] In this experiment, the dosage of protopine, the active ingredient of the Dong medicine Xueshuicao, was 1 μmol / L in in vitro experiments and 10 mg / kg in in vivo animal experiments.

[0020] Example 1

[0021] Determination of the effect of protopine, an active ingredient of the Dong medicine Xueshuicao, on the expression of adhesion molecules in vascular endothelial cells

[0022] Western Blot protein immunoblotting was used to detect the expression of vascular endothelial cell adhesion molecules ICAM-1 and VCAM-1, and to determine the effect of protopine, the active ingredient of the Dong medicine blood water grass, on inhibiting the expression of vascular endothelial cell adhesion molecules induced by Apelin-13. The specific steps are as follows:

[0023] Vascular endothelial cells were cultured, 80%, and pretreated with protopine (1mmol / L), an active ingredient of the Dong medicine blood water grass, for 30 minutes, and then apelin-13 (1μmol / L) was added for 24 hours. Western Blot protein immunoblotting experiments were used to detect the effect of protopine, an active ingredient of the Dong medicine blood water grass, on the expression of endothelial cell adhesion molecules ICAM-1 and VCAM-1. Figure 1 The results showed that compared with the control group, apelin-13 significantly promoted the protein expression of vascular endothelial adhesion molecules ICAM-1 and VCAM-1, while the active ingredient of the Dong medicine Sanshuicao, protoopine, could significantly inhibit the expression of vascular endothelial adhesion molecules ICAM-1 and VCAM-1 induced by apelin-13. The above results indicate that the active ingredient of the Dong medicine Sanshuicao, protoopine, has a new effect of downregulating the expression of vascular endothelial adhesion molecules ICAM-1 and VCAM-1.

[0024] Example 2

[0025] Determination of the effect of protopine, an active ingredient of the Dong medicine Xueshuicao, on the adhesion of monocytes to the vascular endothelium

[0026] Calcein AM was used to label monocytes, and the adhesion rate of monocytes to the vascular endothelium was observed under a fluorescence microscope to determine the effect of protopine, the active ingredient of the Dong medicine Xueshuicao, on inhibiting the adhesion of monocytes to the vascular endothelium induced by Apelin-13. The specific steps are as follows:

[0027] Endothelial cells were cultured, and when the cells grew to 40-50%, protopine (1 mmol / L), an active ingredient of the Dong medicine blood water grass, was added for pretreatment for 30 minutes, and then apelin-13 (1 μmol / L) was added for 24 hours. Monocytes were labeled with Calcein AM and incubated for 1 hour. The effect of protopine, an active ingredient of the Dong medicine blood water grass, on the adhesion of monocytes to the vascular endothelium was observed under a fluorescence microscope. Figure 2 The results showed that compared with the group treated with apelin-13 alone, the apelin-13-induced monocyte adhesion to the vascular endothelium was significantly reduced after pretreatment with the active ingredient of the Dong medicine Sanguisorba officinalis, protoopine. The above results indicate that the active ingredient of the Dong medicine Sanguisorba officinalis, protoopine, has a new effect of inhibiting the adhesion of monocytes to the vascular endothelium.

[0028] Example 3

[0029] Measuring the effect of protopine, the active ingredient of the Dong medicine Xueshuicao, on the formation of atherosclerotic plaques

[0030] ApoE- / - male mice (6-8 weeks) were fed a high-fat diet for 12 weeks to establish an atherosclerosis model, and the effect of protopine, an active ingredient of the Dong medicine Sanshuicao, on inhibiting the formation of atherosclerotic plaques induced by Apelin-13 was determined.

[0031] High-fat fed ApoE- / - mice were intraperitoneally injected with protopine (10 mg / kg), the active ingredient of the Dong medicine Xueshuicao, and Apelin-13 (5 mg / kg) for 12 consecutive weeks. The overall atherosclerotic plaques in the aorta were observed using Oil Red O staining. Figure 3 The results showed that apelin-13 (5 mg / kg) promoted the formation of atherosclerotic plaques in ApoE- / - mice fed with high-fat diet, while protoopine (10 mg / kg), the active ingredient of the Dong medicine Sanshuicao, inhibited the formation of atherosclerotic plaques in ApoE- / - mice fed with high-fat diet induced by apelin-13 (5 mg / kg). The above results indicate that protoopine, the active ingredient of the Dong medicine Sanshuicao, has a new effect in alleviating atherosclerosis.

[0032] In summary, the Dong medicine blood water grass active ingredient protopine of the present invention has an ideal new effect of resisting monocyte adhesion to vascular endothelium and atherosclerotic plaque formation. Therefore, the Dong medicine blood water grass active ingredient protopine will be effectively used for the treatment of monocyte adhesion to vascular endothelium and atherosclerotic diseases.

Claims

1. A use of protopine in the preparation of an anti-atherosclerotic drug, characterized in that: The original opium alkaloid is taken from the active ingredients of the Dong medicine Xueshuicao.

2. The use of protopine according to claim 1 in the preparation of an anti-atherosclerotic drug, characterized in that: The anti-atherosclerosis method inhibits the adhesion of monocytes to vascular endothelium induced by APJ endogenous ligand Apelin-13, thereby inhibiting the formation of atherosclerotic plaques.

3. The use according to claim 1, characterized in that: The dosage forms of the drug include powders, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.

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