Application of alpha-ketoglutaric acid in preparation of hypertension angiogenesis promoting medicine

Abstract

The invention discloses an application of alpha-ketoglutaric acid in preparation of a medicine for promoting hypertension angiogenesis, belongs to the technical field of biological medicines, and finds that the alpha-ketoglutaric acid has the effects of promoting hypertension angiogenesis and improving microcirculation injury for the first time, and provides a new thought for diagnosis and treatment of sparse microcirculation. Alpha-ketoglutaric acid has good drug safety and a strong angiogenesis promoting effect, and can be used as a candidate drug for promoting angiogenesis. And the DM-alpha KG is alpha-ketoglutaric acid which can be absorbed by cells, can be dissolved in DMSO, and is good in stability, low in cytotoxicity and good in drug safety. Results show that the alpha-ketoglutaric acid has relatively good drug safety and relatively strong angiogenesis promoting effect, and can be used as a candidate drug for improving sparse blood vessels.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of α-ketoglutaric acid in the preparation of drugs for promoting angiogenesis in hypertension. Background Art

[0002] Cardiovascular diseases are the number one killer seriously threatening the health of residents in China, and hypertension is the most important risk factor. According to the latest data statistics, the number of hypertension patients in China is as high as 245 million, bringing a great economic burden to the whole society and families. Therefore, it is urgent to explore the pathogenesis of hypertension and find effective prevention and treatment strategies for hypertension.

[0003] The two major factors affecting blood pressure are volume and resistance, and the increase in peripheral vascular resistance is an important link in the occurrence and development of essential hypertension. Microcirculation, a microvascular network with a diameter of 20-200 microns, accounts for 90% of the total number of blood vessels in the body and is the most critical part determining peripheral resistance. Microcirculatory rarefaction is an important pathophysiological basis for hypertension-related target organ damage and cardiovascular diseases. Le Nobel et al. found that before the blood pressure rises in spontaneously hypertensive rats, structural rarefaction has already occurred in capillaries and arterioles; eNOS-deficient mice simultaneously develop hypertension and microvascular rarefaction changes, and the rarefaction phenomenon can be avoided by using vasodilators before the blood pressure rises. And endothelial cell angiogenesis is the key link to maintain microcirculatory homeostasis and repair microcirculatory damage in hypertension. Promoting endothelial cell angiogenesis in hypertension is an important treatment method to improve microcirculatory rarefaction. However, the diagnosis and treatment methods for improving microcirculatory rarefaction in hypertension are very limited. Therefore, it is of great clinical significance to deeply explore the pathogenesis of microcirculatory rarefaction in hypertension and find highly effective drugs to prevent or delay the occurrence and development of microcirculatory rarefaction in hypertension.

[0004] α-Ketoglutaric acid (αKG) is a metabolic intermediate of the tricarboxylic acid cycle, widely involved in processes such as biosynthesis, energy metabolism, and cellular homeostasis, thereby prolonging lifespan and improving healthspan. αKG helps the oxidation of nutrients such as amino acids, glucose, and fatty acids, and can provide energy for the cell metabolism process. In addition, it also has various physiological functions such as regulating metabolism, promoting autophagy, alleviating inflammation, delaying aging, and prolonging lifespan. Related studies have found that the circulating concentration of αKG decreases with aging; the calcium salt of αKG can prolong lifespan by reducing the levels of systemic inflammatory cytokines and inducing the anti-inflammatory cytokine IL-10; in addition, αKG also has potential benefits in promoting muscle growth, wound healing, cellular energy status, immunity, and faster recovery after surgery. However, whether αKG has the effect of promoting angiogenesis in hypertension is not clear; therefore, it is of great significance to deeply explore the regulatory effect of αKG on microcirculatory rarefaction in hypertension. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the present invention discovers that α-ketoglutaric acid has the effect of promoting angiogenesis in hypertension and improving microcirculation injury, providing a new idea for the diagnosis and treatment of sparse microcirculation.

[0006] The technical solution of the present invention is as follows:

[0007] In the first aspect of the present invention, there is provided the use of α-ketoglutaric acid in the preparation of a drug for preventing or delaying sparse microcirculation.

[0008] In some embodiments of the present invention, the sparse microcirculation is vascular calcification accompanied by or caused by hypertension.

[0009] In the second aspect of the present invention, there is provided the use of α-ketoglutaric acid or its cell-absorbable dimethyl α-ketoglutaric acid in the preparation of a drug for promoting angiogenesis.

[0010] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipients include at least one of diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, solubilizers, solubilizing agents, preservatives, pH regulators, osmotic pressure regulators, surfactants, coating materials, antioxidants, bacteriostatic agents or buffers.

[0011] In some embodiments of the present invention, the dosage form of the drug includes at least one of suspensions, granules, capsules, powders, tablets, emulsions, solutions, dripping pills, injections, oral preparations, suppositories, enemas, aerosols, patches or drops.

[0012] In some embodiments of the present invention, the administration route of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, aerosol administration or transdermal administration.

[0013] In the third aspect of the present invention, there is provided a drug for preventing and / or treating sparse microcirculation, the drug comprising α-ketoglutaric acid (αKG) or its pharmaceutically acceptable salt, and the αKG is a compound represented by formula (I):

[0014]

[0015] In the fourth aspect of the present invention, there is provided a drug for promoting angiogenesis, the drug comprising α-ketoglutaric acid or cell-absorbable dimethyl α-ketoglutaric acid (DM-αKG). It also includes its pharmaceutically acceptable salt, and DM-αKG is a compound represented by formula (Ⅱ):

[0016]

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] The present invention for the first time discovers that α-ketoglutaric acid has the effect of promoting angiogenesis in hypertension, providing a new idea for the treatment of vascular rarefaction. Moreover, α-ketoglutaric acid has good drug safety and strong angiogenesis-promoting effects, and can be used as a candidate drug for improving vascular rarefaction. And as a known compound, α-ketoglutaric acid has good water solubility, good stability, low cytotoxicity, and good drug safety. Brief Description of the Drawings

[0019] Figure 1 Dimethyl α-ketoglutaric acid promotes angiogenesis of endothelial cells. Figure 1 A shows that DM-αKG improves the reduction of scratch function of vascular endothelial cells caused by angiotensin II; Figure 1 B shows the statistical analysis results of the scratch assay of vascular endothelial cells induced by angiotensin II with DM-αKG; Figure 1 C shows that DM-αKG improves the reduction of migration function of vascular endothelial cells caused by angiotensin II; Figure 1 D shows the statistical analysis results of the migration assay of vascular endothelial cells induced by angiotensin II with DM-αKG; Figure 1 E shows that DM-αKG improves the reduction of adhesion function of vascular endothelial cells caused by angiotensin II; Figure 1 F shows the statistical analysis results of the adhesion assay of vascular endothelial cells induced by angiotensin II with DM-αKG; Figure 1 G shows that DM-αKG improves the reduction of tube formation function of vascular endothelial cells caused by angiotensin II; Figure 1 H shows the statistical analysis results of the tube formation assay of vascular endothelial cells induced by angiotensin II with DM-αKG (*P<0.05, **P<0.01, ***P<0.001).

[0020] Figure 2 Effects of dimethyl α-ketoglutaric acid on eNOS / p-eNOS and NO concentration in endothelial cells. Figure 2 A shows that eNOS / p-eNOS in vascular endothelial cells increases the reduction of eNOS / p-eNOS expression level in vascular endothelial cells caused by angiotensin II; Figure 2 B shows the statistical analysis results of the eNOS / p-eNOS expression level in vascular endothelial cells induced by angiotensin II with DM-αKG; Figure 2 C shows the effect of DM-αKG on the NO concentration in vascular endothelial cells induced by angiotensin II (*P<0.05, **P<0.01, ***P<0.001).

[0021] Figure 3 α-Ketoglutaric acid promotes angiogenesis in hypertensive mice. Figure 3A shows the experimental results of αKG improving angiogenesis in Matrigel of hypertensive mice; Figure 3 B shows the statistical analysis results of CD31 immunohistochemistry of Matrigel in each group of mice; Figure 3 C shows the statistical analysis results of CD31 immunofluorescence of Matrigel in each group of mice (*P<0.05, **P<0.01, ***P<0.001).

[0022] Figure 4 It is that α-ketoglutaric acid improves the sparse microcirculation of hypertensive mice. Figure 4 A shows the experimental results of αKG improving the retinal vascular density of hypertensive mice; Figure 4 B shows the statistical analysis results of the retinal vascular density of each group of mice (*P<0.05, **P<0.01, ***P<0.001).

[0023] Figure 5 It is that α-ketoglutaric acid reduces the expression of H3K27 me3. Figure 5 A shows the effects of DM-αKG on the expression of H3K27 me1, H3K27 me2, and H3K27 me3 in endothelial cells; Figure 5 B shows the statistical analysis results of the effects of DM-αKG on the expression of H3K27 me1, H3K27 me2, and H3K27 me3 in endothelial cells (*P<0.05, **P<0.01, ***P<0.001).

[0024] Figure 6 It is that α-ketoglutaric acid promotes the expression of angiogenesis-related genes. Figure 6 A shows the effects of DM-αKG on the expression of Angiopoietin-1 gene in endothelial cells; Figure 6 B shows the effects of DM-αKG on the expression of PIGF gene in endothelial cells; Figure 6 C shows the effects of DM-αKG on the expression of IGFBP-1 gene in endothelial cells; Figure 6 D shows the effects of DM-αKG on the expression of PDGF-AA gene in endothelial cells (*P<0.05, **P<0.01, ***P<0.001). Detailed implementation manners

[0025] The following further elaborates the content of the present invention in combination with specific examples.

[0026] Reagents, drugs, and cells:

[0027] Human umbilical vein endothelial cells (HUVECs) were all purchased from Cyagen Biosciences (Shanghai) Inc.; angiotensin II was purchased from Sigma Guangzhou Zhenhao Trading Co., Ltd.; α-ketoglutaric acid / dimethyl α-ketoglutaric acid was purchased from Shanghai Macklin Biochemical Co., Ltd.; castor oil polyoxyethylene ether and anhydrous glucose were purchased from Shanghai Macklin Biochemical Co., Ltd.; calcium content kit was purchased from Nanjing Jiancheng Bioengineering Institute; vitamin D3 was purchased from Sangon Biotech (Shanghai) Co., Ltd.; alizarin red dye was purchased from Regene Guangzhou Pengchen Biotechnology Co., Ltd.

[0028] Example 1

[0029] Dimethyl α-ketoglutaric acid promotes the angiogenesis function of HUVECs

[0030] In this example, angiotensin II was used to induce the impairment of the angiogenesis ability of HUVECs under hypertension, and dimethyl α-ketoglutaric acid was given for intervention for 2 days, followed by scratch, migration, adhesion, tube formation and other experiments to evaluate the promoting effect of dimethyl α-ketoglutaric acid on the angiogenesis ability of endothelial cells.

[0031] The specific operations include:

[0032] HUVECs of passages 6 - 12 were seeded at 1x10 5 / well in a 12-well plate, and 2 ml of ECM endothelial cell medium was added to each well. On the first day after plating, 1 mM angiotensin II was given to induce hypertension, and at the same time, 2 μM dimethyl α-ketoglutaric acid was given for intervention. On the third day, the cells were digested and centrifuged for migration, adhesion, tube formation and other experiments.

[0033] To further clarify the role of dimethyl α-ketoglutaric acid in promoting angiogenesis, angiotensin II was used to induce hypertension in HUVECs, and after 2 days of intervention with dimethyl α-ketoglutaric acid, the eNOS / p-eNOS content and NO concentration were detected.

[0034] The specific operations include:

[0035] HUVECs of passages 6 - 12 were seeded at 1x10 5 / well in a 12-well plate, and 2 ml of ECM endothelial cell medium was added to each well. On the first day after plating, 1 mM angiotensin II was given to induce hypertension, and at the same time, 2 μM dimethyl α-ketoglutaric acid was given for intervention. On the third day, the intracellular eNOS / p-eNOS content was measured and the NO concentration in the medium was measured.

[0036] As Figure 1As shown, angiotensin II treatment inhibited the scratch, migration, adhesion, and tube formation functions of endothelial cells, while supplementation with dimethyl α-ketoglutarate in endothelial cells significantly improved the scratch, migration, adhesion, and tube formation functions of endothelial cells ( Figure 1 ). The above experimental results suggest that angiotensin II treatment can significantly inhibit the neovascularization ability of endothelial cells, and supplementation with dimethyl α-ketoglutarate can improve the neovascularization ability of endothelial cells.

[0037] As Figure 2 shown, the content of eNOS / p-eNOS and the concentration of NO in endothelial cells were significantly decreased after angiotensin II treatment, while supplementation with dimethyl α-ketoglutarate could promote the expression of eNOS / p-eNOS and the concentration of NO in endothelial cells, which also proved that dimethyl α-ketoglutarate has the effect of promoting neovascularization.

[0038] Example 2

[0039] α-Ketoglutarate improves neovascularization disorder and microcirculation injury in hypertensive mice

[0040] In this example, angiotensin II was used to induce hypertension in mice, and after 1 month of intervention with α-ketoglutarate, a subcutaneous Matrigel neovascularization experiment was performed on the mice to evaluate the promoting effect of α-ketoglutarate on the neovascularization ability of mouse endothelial cells.

[0041] The specific operation was as follows:

[0042] C57BL / 6 male mice at 8 weeks of age with similar body weights (20 g - 22 g) were randomly divided into a blank group (n = 10), an α-ketoglutarate group (n = 10), a hypertension group (n = 10), and a hypertension + α-ketoglutarate group (n = 10). Mice in the hypertension group were continuously infused with angiotensin II (0.8 mg / kg / day) by subcutaneous injection osmotic pump for 28 days; mice in the α-ketoglutarate group were fed with a 2% α-ketoglutarate aqueous solution daily; the hypertension + α-ketoglutarate group was fed with a 2% α-ketoglutarate aqueous solution starting from Day 1 on the basis of continuous infusion of angiotensin II (0.8 mg / kg / day) by subcutaneous injection osmotic pump. Matrigel was implanted on the backs of the mice in each group after 14 days, and the Matrigel was removed after 28 days for CD31 immunohistochemistry and immunofluorescence staining.

[0043] As Figure 3As shown, in the angiotensin II-induced hypertensive mouse model, the number of neovascularizations in Matrigel was significantly reduced. CD31 immunohistochemistry and immunofluorescence staining also confirmed that the ability of neovascularization in hypertensive mice was weakened. After feeding α-ketoglutaric acid to hypertensive mice, the results of CD31 immunohistochemistry and immunofluorescence staining of Matrigel showed that α-ketoglutaric acid could significantly increase the vascular density in Matrigel, suggesting that α-ketoglutaric acid could significantly improve the neovascularization ability of hypertensive mice.

[0044] 2. To further verify the effect of α-ketoglutaric acid in improving the sparse microcirculation in hypertensive mice, angiotensin II was used to induce hypertension in mice, and after 1 month of α-ketoglutaric acid intervention, the retinal microvascular density of mice was detected to evaluate the improvement effect of α-ketoglutaric acid on the microcirculation density of mice.

[0045] The specific operation is as follows:

[0046] C57BL / 6 male mice at 8 weeks of age with similar body weights (20 g - 22 g) were randomly divided into a blank group (n = 10), an α-ketoglutaric acid group (n = 10), a hypertension group (n = 10), and a hypertension + α-ketoglutaric acid group (n = 10). Mice in the hypertension group were continuously infused with angiotensin II (0.8 mg / kg / day) by subcutaneous injection osmotic pump for 28 days; mice in the α-ketoglutaric acid group were fed with a 2% α-ketoglutaric acid aqueous solution daily; mice in the hypertension + α-ketoglutaric acid group were fed with a 2% α-ketoglutaric acid aqueous solution starting from Day 1 on the basis of continuous infusion of angiotensin II (0.8 mg / kg / day) by subcutaneous injection osmotic pump. After 28 days, the mice were sacrificed, the eyeball retinas of the mice were isolated, and IB4 immunofluorescence staining was performed.

[0047] As Figure 4 shown, in the angiotensin II-induced hypertensive mouse model, the retinal density of the eyeball was significantly reduced. After feeding α-ketoglutaric acid to hypertensive mice, the retinal density of the eyeball in hypertensive mice could be improved, suggesting that α-ketoglutaric acid could significantly improve the sparse microcirculation in hypertensive mice.

[0048] Example 3

[0049] α-Ketoglutaric acid reduces the expression of H3K27 me3 in endothelial cells and promotes the expression of angiogenesis-related genes

[0050] In this example, angiotensin II was used to induce the impairment of the ability of endothelial cell neovascularization under hypertension in HUVECs, and after 2 days of intervention with dimethyl α-ketoglutaric acid, the histone expression and the expression of angiogenesis-related genes in endothelial cells were detected.

[0051] To evaluate the molecular mechanism of α-ketoglutaric acid promoting angiogenesis in endothelial cells:

[0052] 1) HUVECs at passages 6 - 12 were seeded at a density of 1x10 5 / well in a 12 - well plate, and 2 ml of ECM endothelial cell medium was added to each well. On the first day after seeding, 1 mM angiotensin II was given to induce hypertension, and at the same time, 2 μM dimethyl-α-ketoglutarate was used for intervention. On the third day, the gene expression levels of H3K27 me1, H3K27me2, and H3K27 me3 in endothelial cells were detected, as Figure 5 shown.

[0053] 2) HUVECs at passages 6 - 12 were seeded at a density of 1x10 5 / well in a 12 - well plate, and 2 ml of ECM endothelial cell medium was added to each well. On the first day after seeding, 1 mM angiotensin II was given to induce hypertension, and at the same time, 2 μM dimethyl-α-ketoglutarate was used for intervention. On the third day, a CHIP experiment was performed to verify the effect of α-ketoglutarate on the expression of angiogenesis-related genes, as Figure 6 shown.

[0054] The above experiments confirmed that after stimulation with angiotensin II, the expression of H3K27 me3 in endothelial cells increased, and the expression of angiogenesis-related genes (Angiopoietin-1, PIGF, IGFBP-1, PDGF-AA) was inhibited. However, after supplementing α-ketoglutarate, the expression of H3K27 me3 was significantly reduced, the expression of Angiopoietin-1, PIGF, IGFBP-1, and PDGF-AA genes was promoted, and angiogenesis was improved, as Figure 5-6 shown.

[0055] Based on the above results, it can be seen that α-ketoglutarate / dimethyl-α-ketoglutarate can effectively promote the angiogenesis ability of endothelial cells, improve microcirculation rarefaction, and can be used as a drug for promoting angiogenesis.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that modifications or equivalent replacements of the technical solutions of the present invention do not depart from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of α-ketoglutaric acid in the preparation of drugs for preventing or delaying microcirculatory rarefaction.

2. The use according to claim 1, characterized in that: The microcirculation rarefaction is vascular calcification associated with or caused by hypertension.

3. Use of α-ketoglutarate or its dimethyl α-ketoglutarate that can be absorbed by cells in the preparation of drugs that promote angiogenesis.

4. The use according to claim 1 or 3, characterized in that: The drug also includes pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that: The pharmaceutically acceptable excipients include: at least one of a diluent, a binder, a wetting agent, a lubricant, a disintegrant, a solvent, an emulsifier, a cosolvent, a solubilizer, a preservative, a pH regulator, an osmotic pressure regulator, a surfactant, a coating material, an antioxidant, an antibacterial agent or a buffer.

6. The use according to claim 5, characterized in that: The dosage form of the drug includes at least one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, oral preparations, suppositories, enemas, aerosols, patches or drops.

7. The use according to claim 5, characterized in that: The administration route of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, atomization administration or transdermal administration.

8. A drug for preventing and / or treating microcirculatory sparseness, characterized in that: The medicament comprises alpha-ketoglutarate.

9. A drug for promoting angiogenesis, characterized in that: The drug comprises alpha-ketoglutarate or dimethyl alpha-ketoglutarate that can be taken up by cells.

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