CN110893187A

CN110893187A - Application of tanshinone IIA sodium sulfonate in preparation of medicine for treating acute or chronic diseases accompanied with increase of homocysteine in blood

Info

Publication number: CN110893187A
Application number: CN201811065431.0A
Authority: CN
Inventors: 唐靖一; 陈昕琳; 周忠焱; 马子霖; 赵外荣; 肖颖; 施雯婷
Original assignee: Longhua Hospital Affiliated to Shanghai University of TCM
Current assignee: Longhua Hospital Affiliated to Shanghai University of TCM
Priority date: 2018-09-13
Filing date: 2018-09-13
Publication date: 2020-03-20

Abstract

The invention relates to application of tanshinone IIA sodium sulfonate in preparation of drugs for treating acute or chronic diseases accompanied with increase of homocysteine in blood independently or as an effective component. The invention uses vascular endothelial cells, myocardial cells, nerve cells and isolated blood vessel models to simulate the damage effect of homocysteine on the cardiovascular and cerebrovascular systems, finds that the tanshinone IIA sodium sulfonate has the protection and/or treatment effect on the cardiovascular and cerebrovascular damage caused by homocysteine, and fills the blank of protection and/or treatment medicines for resisting the cardiovascular and cerebrovascular damage caused by homocysteine. The medicine provided by the invention is suitable for diseases causing abnormal increase of homocysteine in blood, can play a role in protecting and/or treating cardiovascular and cerebrovascular injuries caused by resisting homocysteine only by reaching effective concentration in blood, and provides a better choice for treating cardiovascular and cerebrovascular injuries caused by diseases causing the increase of homocysteine in the blood.

Description

Application of tanshinone IIA sodium sulfonate in preparation of medicine for treating acute or chronic diseases accompanied with increase of homocysteine in blood

Technical Field

The invention belongs to the field of pharmacy, and relates to application of tanshinone IIA sodium sulfonate in preparation of medicines for preventing and/or treating cardiovascular and cerebrovascular injury related diseases caused by hyperhomocysteine

Background

Homocysteine is an independent risk factor of cardiovascular and cerebrovascular diseases such as coronary heart disease, atherosclerosis and the like, and the pathogenesis of homocysteine comprises the influence on the function of vascular endothelium, the promotion of smooth muscle proliferation, the promotion of thrombosis, cardiovascular toxicity and the like. Some nephropathy, liver disease, metabolic disease, nervous system disease, cancer, some medicine intake and other clinical manifestations are abnormal elevation of homocysteine in blood. At present, the medicines for clinically preventing and treating the homocysteine are mainly compound preparations containing B vitamins and folic acid for interfering in a methyl transferring way or a sulfur transferring way of the homocysteine or simultaneously interfering in the two ways, but the improvement effect of the medicines on the incidence rate and the death rate of cardiovascular and cerebrovascular diseases such as atherosclerosis and the like is supported by lack of authoritative evidence. Therefore, the development of a protective agent for resisting the cardiovascular damage caused by the hyperhomocysteine has important significance for treating related diseases, and has wide application prospect.

The tanshinone IIA sodium sulfonate is a water-soluble derivative of tanshinone IIA in traditional Chinese medicine salvia miltiorrhiza bunge, greatly enhances water solubility by introducing sodium sulfonate groups, is clinically prepared into an injection, overcomes the problems of poor water solubility, low oral bioavailability and difficulty in drug formation of the tanshinone IIA, and has the CAS No. as follows: 696659-80-9, the structural formula is:

the tanshinone IIA sodium sulfonate injection is widely applied to treatment of diseases such as clinical coronary heart disease, angina pectoris, ventricular premature beat and the like, modern pharmacology shows that the tanshinone IIA sodium sulfonate injection has pharmacological activities such as oxidation resistance, inflammation resistance and the like, and has a definite cardiovascular protection effect, but the tanshinone IIA sodium sulfonate injection has no report on resisting cardiovascular damage caused by hyperhomocysteine.

Disclosure of Invention

In view of the above-mentioned deficiencies of the prior art, according to the embodiments of the present invention, it is desirable to provide an application of tanshinone IIA sodium sulfonate in preparing a medicine for treating and preventing cardiovascular and cerebrovascular injury related diseases accompanied by cysteine increase.

In order to complete the invention, the inventor adopts a plurality of models to simulate the cardiovascular and cerebrovascular injury effect of the homocysteine, and researches the efficacy of the tanshinone IIA sodium sulfonate for treating and preventing the cardiovascular and cerebrovascular injury caused by the homocysteine.

In the invention, the tanshinone IIA sodium sulfonate is used alone or as an effective component in preparing the medicine for treating acute or chronic diseases accompanied with the increase of homocysteine in blood.

According to the embodiment of the present invention, the aforementioned acute and/or chronic diseases accompanied with elevated homocysteine in blood include, but are not limited to, (1) cardiovascular and cerebrovascular diseases such as hypertension, coronary heart disease, heart failure, arrhythmia, atherosclerosis, stroke, etc., (2) nervous system diseases such as cognitive disorder, senile dementia, migraine, etc., (3) renal system diseases such as acute and chronic renal failure, etc., (4) metabolic diseases such as diabetes, gout, osteoporosis, etc., (5) mental diseases such as schizophrenia, etc., (6) eye diseases such as cataract, glaucoma, etc., (7) cancer (8) congenital diseases (9) elevated homocysteine in blood caused by drugs, etc.

According to an embodiment of the present invention, the dosage form of the tanshinone IIA sodium sulfonate of the present invention is a compound preparation using tanshinone IIA sodium sulfonate as a raw material preparation or using tanshinone IIA sodium sulfonate as a component, and includes, but is not limited to, an injection, a tablet, a capsule, a liposome, a granule, an oral liquid, a pill, and the like.

The homocysteine obviously causes the toxicity of vascular endothelial cells and myocardial cells, the migration inhibition of the endothelial cells and the dysfunction of vasoconstriction and relaxation, and shows that the homocysteine has obvious toxicity to the cardiovascular system. Research data of the invention show that tanshinone IIA sodium sulfonate can effectively inhibit cardiovascular damage caused by homocysteine, and can play a role in preventing and treating cardiovascular damage for various diseases which show that homocysteine in blood is increased. In addition, the tanshinone IIA sodium sulfonate is a clinically used patent medicine, and has a huge market application prospect for expanding the clinical indications.

Drawings

Figure 1 toxic effect of homocysteine (Hcy) on endothelial cells (P <0.001 compared to control).

Figure 2 tanshinone IIA sodium sulfonate (STS) caused a protective effect on homocysteine (Hcy) toxicity of vascular endothelial cells (# P <0.001 compared to control, P <0.01 compared to model, P < 0.001).

FIG. 3 shows the protective effect of tanshinone IIA sodium sulfonate (STS) on homocysteine (Hcy) induced inhibition of vascular endothelial cell migration (P # 0.001, # P <0.05 compared to control group; P # 0.05, P # 0.01 compared to model group).

Figure 4 toxic effect of homocysteine (Hcy) on cardiomyocytes (P <0.05, P <0.001 compared to control group).

Figure 5 protective effect of tanshinone IIA sodium sulfonate (STS) on homocysteine (Hcy) induced toxicity of cardiomyocytes (# P <0.001 compared to control, P <0.01 compared to model, P < 0.001).

Figure 6 effect of homocysteine (Hcy) on the impaired thoracic aortic systolic function in rats (P <0.001 compared to control).

FIG. 7 shows the protective effect of tanshinone IIA sodium sulfonate (STS) on homocysteine (Hcy) on the systolic function impairment of rat thoracic aorta (ratio # # # P <0.001 to control group, and ratio # # P <0.001 to model group).

Figure 8 toxic effect of homocysteine (Hcy) on nerve cells (P <0.001 compared to control).

Figure 9 protective effect of tanshinone IIA sodium sulfonate (STS) on homocysteine (Hcy) induced toxicity of nerve cells (# P <0.001 compared to control, P <0.01 compared to model, P < 0.001).

Detailed Description

The invention is further illustrated with reference to the following figures and specific examples. These examples are to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever. After reading the description of the invention, one skilled in the art can make various changes and modifications to the invention, and such equivalent changes and modifications also fall into the scope of the invention defined by the claims.

Example 1 (protective action of sodium tanshinone IIA sulfonate on endothelial cell damage caused by homocysteine in vitro)

The main reagents of the experiment are as follows: homocysteine was purchased from sigma, having a product number and specification of: H4628-5G; MTT is purchased from sigma, Inc., and the goods number and specification are: M2128-5G; tanshinone IIA sodium sulfonate is purchased from Dowmaste company, and the product number and specification are as follows: a0058-20 mg. HUVEC endothelial cells were purchased from ATCC, USA, and the catalog numbers are: CRL-1730;

1.1 toxic Effect of homocysteine on vascular endothelial cells

Human umbilical vein endothelial cells were treated according to 1X10⁴one/mL of the cells were seeded in 96-well plates at a density of 100. mu.l per well in CO₂Incubator (37 ℃, 5% CO)₂) And culturing for 24 h. According to the blank control group and the homocysteine different dose groups (1, 2, 4 and 8mM), the medicine is respectively added for 24h, and the cell activity is detected (MTT method). The results show that homocysteine significantly affects the cellular activity of vascular endothelial cells (see fig. 1).

1.2 protective action of tanshinone IIA sodium sulfonate on toxicity of blood vessel endothelial cell caused by homocysteine

Human umbilical vein endothelial cells were treated according to 1X10⁴one/mL of the cells were seeded in 96-well plates at a density of 100. mu.l per well in CO₂Incubator (37 ℃, 5% CO)₂) And culturing for 24 h. According to a blank control group, a model group (homocysteine 8mM) and different doses of tanshinone IIA sodium sulfonate intervention groups (25, 50 and 100 mu M), respectively adding medicines for 24h, and detecting the cell activity (MTT method). The results show that tanshinone IIA sodium sulfonate significantly protects homocysteine-induced vascular endothelial cytotoxicity (as shown in FIG. 2).

1.2 protective action of tanshinone IIA sodium sulfonate on inhibition of migration of vascular endothelial cells caused by homocysteine

Human umbilical vein endothelial cells were treated according to 10X10⁴one/mL of the cells were seeded in 12-well plates, 1mL per well, in CO₂Incubator (37 ℃, 5% CO)₂) And culturing for 24 h. Crosses were drawn in the well plate with a 1ml tip and photographed. According to a blank control group, a model group (homocysteine 4mM) and different doses of tanshinone IIA sodium sulfonate intervention groups (25, 50 and 100 mu M), the medicines are respectively added for 12h and 24h, photographing observation is carried out, and the migration distance is measured by using Image J. The results show that tanshinone IIA sodium sulfonate significantly protects homocysteine to cause the inhibition of vascular endothelial cell migration (as shown in figure 3).

Example 2 (protective action of tanshinone IIA sodium sulfonate on myocardial cell injury caused by homocysteine in vitro)

The main reagents of the experiment are as follows: homocysteine was purchased from sigma, having a product number and specification of: H4628-5G; MTT is purchased from sigma, Inc., and the goods number and specification are: M2128-5G; tanshinone IIA sodium sulfonate is purchased from Dowmaste company, and the product number and specification are as follows: a0058-20 mg. H9c2 cardiomyocytes were purchased from shanghai academy of sciences cell bank under the accession number: GNR 5;

2.1 toxic Effect of homocysteine on myocardial cells

Human cardiac myocytes were plated at 0.8X10⁴one/mL of the cells were seeded in 96-well plates at a density of 100. mu.l per well in CO₂Incubator (37 ℃, 5% CO)₂) And culturing for 24 h. According to the blank control group and the homocysteine different dose groups (0.5, 1, 2 and 4mM), the cell activity is detected by adding medicines for 24h respectively (MTT method). The results show that homocysteine significantly affected the cellular activity of cardiomyocytes (see figure 4).

2.2 protective action of tanshinone IIA sodium sulfonate on toxicity of cardiac muscle cells caused by homocysteine

Human cardiac myocytes were plated at 0.8X10⁴one/mL of the cells were seeded in 96-well plates at a density of 100. mu.l per well in CO₂Incubator (37 ℃, 5% CO)₂) And culturing for 24 h. According to a blank control group, a model group (homocysteine 4mM) and different doses of tanshinone IIA sodium sulfonate intervention groups (10, 30 and 100 mu M), respectively adding medicines for 24h, and detecting the cell activity (MTT method). The results show that tanshinone IIA sodium sulfonate significantly protects homocysteine-induced vascular endothelial cytotoxicity (see FIG. 5).

Example 3 protective action of tanshinone IIA sodium sulfonate on in vitro homocysteine induced impairment of thoracic aorta contractile function in rats)

The main reagents of the experiment are as follows: homocysteine was purchased from sigma, having a product number and specification of: H4628-5G; tanshinone IIA sodium sulfonate is purchased from Dowmaste company, and the product number and specification are as follows: a0058-20 mg. Wistar rats were provided by the SPF-scale animal testing center, affiliated longhua hospital, university of medicine, shanghai.

3.1 Damage Effect of homocysteine on thoracic aorta contractile function in rats

After the thoracic aortic vascular rings were isolated from the rats, they were cultured in the tai-lian tissue perfusion system and their changes in systolic relaxation tension were recorded. The culture conditions were: krebs physiological equilibrium liquid, 37 ℃, 5% of carbon dioxide and 95% of oxygen. After the vessels were equilibrated for 1h under basal tension, the vessels were contracted with 60mM KCl, then re-equilibrated with Krebs physiological equilibrating solution, and repeated three times. The maximal vasodilation was greater than 80% with 1 μ M phenylephrine (Phe) pre-constriction and cumulative concentrations of acetylcholine (Ach) from 1nM to 3 μ M vasodilation. According to the blank control group, the homocysteine different dose groups (4, 8mM) are respectively dosed for 30min, the blood vessels are pre-contracted by 1 mu M phenylephrine (Phe), and the blood vessels are dilated by cumulative concentration of acetylcholine (Ach) between 1nM and 3 mu M. The results show that tanshinone IIA sodium sulfonate improves hyperhomocysteine-induced arterial vasodilation disorder of rats (as shown in figure 6).

3.2 protective action of sodium tanshinone IIA sulfonate on damage of homocysteine to thoracic aorta systole function of rat

After the thoracic aortic vascular rings were isolated from the rats, they were cultured in the tai-lian tissue perfusion system and their changes in systolic relaxation tension were recorded. The culture conditions were: krebs physiological equilibrium liquid, 37 ℃, 5% of carbon dioxide and 95% of oxygen. After the vessels were equilibrated for 1h under basal tension, the vessels were contracted with 60mM KCl, then re-equilibrated with Krebs physiological equilibrating solution, and repeated three times. The maximal vasodilation was greater than 80% with 1 μ M phenylephrine (Phe) pre-constriction and cumulative concentrations of acetylcholine (Ach) from 1nM to 3 μ M vasodilation. The blood vessels were pre-contracted with 1 μ M phenylephrine (Phe) and vasodilated with cumulative concentration acetylcholine (Ach) of 1nM-3 μ M, each for 30min, according to the blank control group, model group (homocysteine 4mM), tanshinone IIA sodium sulfonate intervention group (50 μ M). The results show that tanshinone IIA sodium sulfonate improves hyperhomocysteine-induced arterial vasodilation disorder in rats (as shown in figure 7).

Example 4 protective action of tanshinone IIA sodium sulfonate on in vitro homocysteine-induced nerve cell injury

The main reagents of the experiment are as follows: homocysteine was purchased from sigma, having a product number and specification of: H4628-5G; MTT is purchased from sigma, Inc., and the goods number and specification are: M2128-5G; tanshinone IIA sodium sulfonate is purchased from Dowmaste company, and the product number and specification are as follows: a0058-20 mg. SH-SY5Y human neural cells were purchased from American ATCC cell bank under the accession number: CRL-2266;

4.1 toxic Effect of homocysteine on nerve cells

Human nerve cells were treated as 1X10⁴one/mL of the cells were seeded in 96-well plates at a density of 100. mu.l per well in CO₂Incubator (37 ℃, 5% CO)₂) And culturing for 24 h. According to the blank control group and the homocysteine different dose groups (1, 2, 4 and 8mM), the medicine is respectively added for 24h, and the cell activity is detected (MTT method). The results show that homocysteine significantly reduces the cell activity of nerve cells (as shown in fig. 8), and high concentration of Hcy has an injury effect on nerve cells.

4.2 protective action of tanshinone IIA sodium sulfonate on toxicity of nerve cells caused by homocysteine

Human nerve cells were treated as 1X10⁴Each/mL was inoculated into a 96-well plate at a density of 100. mu.l per well and incubated for 24h in a CO2 incubator (37 ℃, 5% CO 2). According to a blank control group, a model group (homocysteine 4mM) and different doses of tanshinone IIA sodium sulfonate intervention groups (25, 50 and 100 mu M), respectively adding medicines for 24h, and detecting the cell activity (MTT method). The results show that tanshinone IIA sodium sulfonate significantly protected homocysteine-induced neurocytotoxicity (see FIG. 9).

Endothelial cell damage is a pathological process of occurrence and development of various cardiovascular and cerebrovascular diseases such as atherosclerosis, hypertension and the like, homocysteine influences the activity of endothelial cells to cause the endothelial cell damage, and experiments in examples 1-4 prove that tanshinone IIA sodium sulfonate has a protective effect on the endothelial cell damage caused by homocysteine.

The experiment of the example 1-4 proves that the tanshinone IIA sodium sulfonate has protective effect on the myocardial cell injury caused by homocysteine, further can conclude that the tanshinone IIA sodium sulfonate can be used for preventing and treating the cardiovascular system diseases accompanied with the increase of homocysteine,

the nerve cells are basic composition units of a nervous system, the homocysteine can cause the activity of the nerve cells to be reduced and even cause the damage of the nerve cells, a series of cerebrovascular system diseases accompanied with the increase of the homocysteine are caused, and the tanshinone IIA sodium sulfonate has a protective effect on the damage of the nerve cells caused by the homocysteine.

The basic pathogenesis of various system diseases relates to vascular lesions, endothelial cells and functions thereof are important factors influencing the structure and the function of blood vessels, and the vascular endothelial cells are direct action targets of the toxicity of homocysteine in blood. The experiments in examples 1-4 show that tanshinone IIA sodium sulfonate has a protective effect on endothelial cell damage caused by hyperhomocysteine, and further can conclude that tanshinone IIA sodium sulfonate has a preventive or therapeutic effect on systemic diseases caused by concomitant increase in homocysteine.

Claims

1. Application of tanshinone IIA sodium sulfonate in preparing medicine for treating acute or chronic diseases accompanied with increase of homocysteine in blood independently or as effective component is provided.

2. Use according to claim 1, wherein the acute and/or chronic disease accompanied by an increase in homocysteine in the blood is hypertension, coronary heart disease, heart failure, arrhythmia, atherosclerosis or stroke.

3. The use according to claim 1, wherein the acute and/or chronic disease accompanied by an increase in homocysteine in the blood is cognitive disorders, senile dementia or migraine.

4. The use according to claim 1, wherein the acute and/or chronic disease associated with elevated homocysteine in the blood is acute or chronic renal failure.

5. The use according to claim 1, wherein the acute and/or chronic disease accompanied by an increase in homocysteine in the blood is diabetes, gout or osteoporosis.

6. The use according to claim 1, wherein the acute and/or chronic disease accompanied by an increase in homocysteine in the blood is schizophrenia.

7. The use according to claim 1, wherein the acute and/or chronic disease associated with elevated homocysteine in the blood is cataract or glaucoma.

8. The use according to claim 1, wherein the acute and/or chronic disease accompanied by an increase in homocysteine in the blood is cancer or a congenital disease.

9. The use according to claim 1, wherein the acute and/or chronic disease associated with elevated homocysteine in blood is drug-induced elevated homocysteine in blood.

10. The use as claimed in claim 1, wherein the tanshinone IIA sodium sulfonate is in the form of an injection, tablet, capsule, liposome, granule, oral liquid or pill using tanshinone IIA sodium sulfonate as raw material or sodium tanshinone IIA sulfonate as component.