Use of a competitive inhibitor of snat2 in the preparation of a medicament for the prevention and / or treatment of a hypertensive condition

Abstract

The application discloses application of a SNAT2 competitive inhibitor in preparation of a medicine for preventing and / or treating a hypertension disease. The SANT2 competitive inhibitor is alpha-amino isobutyric acid (MeAIB). The application verifies that the competitive inhibitor MeAIB of SNAT2 and knockout of the SNAT2 gene can prevent and treat hypertension, has important value in preventing and treating primary hypertension, and provides evidence that SNAT2 is a potential target for drug research and development and intervention means of primary hypertension.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of SNAT2 competitive inhibitors in the preparation of drugs for the prevention and / or treatment of essential hypertension. Background Technology

[0002] Primary hypertension is a cardiovascular syndrome characterized primarily by elevated systemic arterial blood pressure, commonly referred to simply as hypertension. Hypertension is a growing global public health problem, typically defined as a diastolic blood pressure above 90 mmHg and a systolic blood pressure above 140 mmHg. By 2015, 1.15 billion people worldwide suffered from hypertension. Hypertension poses serious risks, potentially leading to complications such as cerebral hemorrhage, cerebral infarction, retinal blindness, myocardial infarction, and kidney disease.

[0003] The heart, kidneys, and blood vessels are the main target organs of hypertension's pathophysiological effects. Early stages may show no obvious pathological changes, but long-term hypertension primarily causes left ventricular hypertrophy and dilation. Systemic small artery disease mainly results in an increased wall-to-lumen ratio and narrowed lumen diameter, leading to ischemia in vital target organs such as the heart, brain, and kidneys. Long-term hypertension and associated risk factors can promote the formation and development of atherosclerosis. Currently, endothelial dysfunction is considered the earliest and most significant vascular injury caused by hypertension.

[0004] Clinical evidence shows that a decrease in systolic blood pressure of 10-20 mmHg or a decrease in diastolic blood pressure of 5-6 mmHg can reduce the mortality rates of stroke, coronary heart disease, and cardiovascular and cerebrovascular diseases by 38%, 16%, and 20%, respectively, and reduce heart failure by more than 50% within 3-5 years. The ultimate goal of antihypertensive treatment is to reduce the incidence and mortality of cardiovascular, cerebrovascular, and cerebrovascular diseases, as well as the occurrence of renal complications in hypertensive patients. Currently, antihypertensive drugs can be classified into five major categories: diuretics, beta-blockers, calcium channel blockers (CCBs), angiotensin-converting enzyme inhibitors (ACEIs), and angiotensin II receptor blockers (ARBs). However, these drugs mainly alleviate symptoms and are not very helpful in improving the overall prognosis of the disease, nor are they very effective in significantly improving pathological changes such as vascular remodeling. Moreover, these drug treatments can cause adverse reactions such as fatigue, increased urine output, abnormal heart rate, cold extremities, facial flushing, dry cough, and angioedema. Therefore, the efficacy and safety of existing drugs are not ideal. Summary of the Invention

[0005] The purpose of this invention is to provide the use of SNAT2 competitive inhibitors in the preparation of medicaments for the prevention and / or treatment of essential hypertension.

[0006] Furthermore, the object of the present invention is to provide the use of substances having competitive inhibitory activity against SNAT2 in the preparation of medicaments for the prevention and / or treatment of essential hypertension.

[0007] The substance with competitive inhibitory activity against SNAT2 may specifically be α-aminoisobutyric acid (MeAIB).

[0008] More preferably, the drug is a drug having any of the following functions:

[0009] 1) Medications that lower basal blood pressure levels;

[0010] 2) Medications for the prevention and / or treatment of hypertension;

[0011] 3) Drugs that promote the production of the vasodilator NO.

[0012] Preferably, the drug uses a competitive inhibitor of SNAT2 or a substance with competitive inhibitory activity of SNAT2 as its active ingredient, and may also include pharmaceutically acceptable excipients.

[0013] Preferably, the drug is a systemic or local therapeutic agent or method that targets the SNAT2 gene and its products (mRNA and protein).

[0014] Preferably, the dosage form of the drug includes: powder, paste, granules, pills, tablets, capsules, granules, ointments, decoctions, sprays, or injections.

[0015] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without exceeding the concept and protection scope of this invention.

[0016] The present invention also provides a method for preparing a cell model for screening antihypertensive drugs, the method comprising the following steps:

[0017] 1) Obtaining vascular endothelial cells from animals,

[0018] 2) Treat the vascular endothelial cells with a substance that has the activity of inhibiting the SNAT2 gene and its products (mRNA and protein) or that can knock out the SNAT2 gene, thereby obtaining vascular endothelial cells with reduced expression levels of the SNAT2 gene and its products (mRNA and protein) or with the SNAT2 gene knocked out.

[0019] 3) The NO content in vascular endothelial cells with reduced expression levels of the SNAT2 gene and its products (mRNA and protein) obtained in step 2) or with the SNAT2 gene knocked out was used as an indicator of blood pressure level.

[0020] The animal may specifically be a wild-type mouse, more specifically a wild-type C57BL / 6 mouse;

[0021] The substance that inhibits the activity of the SNAT2 gene and its products (mRNA and protein) may specifically be MeAIB;

[0022] The NO content in vascular endothelial cells was detected using a total nitric oxide assay kit.

[0023] Cell models for screening antihypertensive drugs were obtained using the above preparation method.

[0024] A method for screening antihypertensive drugs using the above-mentioned cell model includes the following steps:

[0025] 1) Grouping: test drug group, positive control group and blank control group. The positive control group was treated with arginine and the blank control group was treated with an equal volume of PBS.

[0026] 2) Treat the cell models using the respective groups of drugs;

[0027] 3) Detect the NO content in the treated cells. If the NO content level obtained in the test drug treatment group is higher than or equal to the NO content obtained in the positive control group, and there is a statistically significant difference compared with the blank control group, then the test drug has antihypertensive activity. If the NO content level obtained in the test drug treatment group is lower than the NO content obtained in the positive control group, and there is no statistically significant difference compared with the blank control group, then the test drug has no antihypertensive activity.

[0028] This invention is the first to demonstrate that inhibition and knockout of SNAT2 can improve the signs and symptoms of essential hypertension, specifically by reducing basal blood pressure (basal vascular resistance) and resisting the increase in blood pressure caused by a high-salt diet (salt sensitivity). Therefore, the SNAT2 gene and protein can be used as potential targets for the development of drugs for essential hypertension, and related animal and cell models can be prepared for screening antihypertensive drugs.

[0029] This invention discovers that MeAIB, as a competitive inhibitor of SNAT2, has significant value in the prevention and treatment of essential hypertension.

[0030] Furthermore, our study found that SNAT2 is highly expressed in vascular endothelium. The competitive inhibitor MeAIB of SNAT2 significantly reduced blood pressure in wild-type mice, and the blood pressure of systemic knockout and endothelium-specific SNAT2 knockout mice was significantly lower than that of wild-type mice. This finding provides a theoretical and experimental basis for screening drugs for the prevention and / or treatment of essential hypertension by specifically inhibiting endothelial SNAT2. Attached Figure Description

[0031] Figure 1This is a schematic diagram showing the results of the SNAT2 competitive inhibitor (MeAIB) significantly reducing the baseline blood pressure level of wild-type mice in Example 1 of the present invention (7 wild-type mice, MeAIB was dissolved in water to a final concentration of 1 g / L, and the blood pressure of the mice decreased after drinking MeAIB water for 2 weeks. The results are expressed as mean ± standard error, * indicates p<0.05).

[0032] Figure 2 This is a schematic diagram (A) showing the SNAT2 systemic gene knockout mice obtained by deleting 10bp (GCGATTGTGG) in Exon4 using CRISPR / Cas9 technology to create a frameshift mutation in Example 2 of the present invention, and the DNA sequencing results (B).

[0033] Figure 3 This is a schematic diagram showing the results of the systemic gene knockout of SNAT2 in Example 2 of the present invention, which significantly reduced the baseline blood pressure level in mice (where: A. systolic blood pressure; B. diastolic blood pressure; C. mean arterial pressure. Approximately 30 mice per group, and the results are expressed as mean ± standard error, *** indicates p<0.001).

[0034] Figure 4 This is a schematic diagram (B) illustrating how, in Example 3 of the present invention, the ends of exons 5 and 10 of the SNAT2 (Slc38a2) gene were modified with flux using the homologous recombination principle in fertilized eggs (A), and the gene was identified by DNA gel electrophoresis.

[0035] Figure 5 This is a schematic diagram showing the results of endothelial-specific gene knockout of SNAT2 in Example 3 of the present invention significantly reducing the baseline blood pressure level in mice (where: A. systolic blood pressure; B. diastolic blood pressure; C. mean arterial pressure. Each group consisted of 13 mice, and the results are expressed as mean ± standard error, ** indicates p < 0.01, *** indicates p < 0.001).

[0036] Figure 6 This is a schematic diagram showing the effect of SNAT2 knockout significantly improving high blood pressure caused by a high-salt diet in Example 4 of the present invention (4-8 mice per group, results are expressed as mean ± standard error, *p<0.05, ** indicates p<0.01).

[0037] Figure 7 This is a schematic diagram showing the results of SNAT2 knockout significantly increasing the level of the vasodilatory substance NO in mouse serum in Example 5 of the present invention (6 mice in each group, serum NO content was measured. Results are expressed as mean ± standard error, * indicates p<0.05).

[0038] Figure 8In Example 6 of this invention, the SNAT2 inhibitor MeAIB dose-dependently increased the production of NO and the activity of endothelial NO synthase in human umbilical vein endothelial cells (HUVECs) (wherein: A: changes in cell morphology after dose-dependent MeAIB treatment of HUVECs were observed using optical microscopy; B: NO content in cell supernatant after dose-dependent MeAIB treatment of HUVECs was measured; C: endothelial NO synthase (eNOS) and its phosphorylation (p-eNOS) were detected by Western blotting after dose-dependent MeAIB treatment of HUVECs). Ser1177 The expression level of the protein was measured. The experiment was repeated three times, and the results are expressed as mean ± standard error. ** indicates p < 0.01, and *** indicates p < 0.001. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0041] The specific technical solution adopted is as follows:

[0042] High-salt diet-induced hypertension model: Mice were fed a high-salt diet containing 3.5% NaCl (this is the mass concentration, 100g of grain contains 3.5g NaCl) (Medicience Ltd). After 28 days, a mouse hypertension model was induced.

[0043] Blood pressure measurement in mice using the tail cuff method: Blood pressure was measured in mice using a non-invasive tail cuff device (BP-2010 series Softron blood pressure monitor). The sensor was placed over the mouse's tail, and blood flow signals were monitored simultaneously by inflating and deflating the tail artery to obtain the blood pressure value. This method is non-invasive and does not require surgery. Animals underwent two weeks of pre-training to fully adapt to the environment. Before recording, the mouse was allowed to rest for at least 10 minutes until it was comfortable and quiet in its cage.

[0044] Human umbilical vein endothelial cell (HUVECs) culture: Place the umbilical cord (within 24 hours of delivery) in sterile 1×Hepes buffer; gently wipe away blood and buffer solution from the umbilical cord with sterile gauze; dry the end of the umbilical cord and locate the umbilical vein; insert a metal needle into the umbilical vein and clamp it with hemostats; mount the metal needle in a 50mL syringe filled with 1×Hepes buffer, then repeatedly flush the umbilical vein with Hepes buffer to ensure thorough flushing; after flushing, insert the metal needle into the other end of the umbilical vein and secure it with hemostats; slowly inject trypsin into the umbilical cord; when the trypsin reaches the hemostat, seal the opening with a 1mL syringe and continue. Inject the remaining trypsin into the umbilical cord; place the umbilical cord into a sterilized cup containing approximately 20 mL of pre-warmed 1×Hepes buffer, and incubate the umbilical cord in a 37°C water bath for 10 min; then, carefully loosen the hemostat and flush the umbilical cord with a syringe containing 20 mL of buffer in a 50 mL centrifuge tube containing 5 mL of endothelial cell (EC) medium; spread the cell suspension into a T25 culture flask pre-coated with rat tail collagen, and incubate at 37°C in a 5% CO2 incubator (change the medium after 2 h); after approximately 3-6 days, when the cells have reached confluence, transfer them to a T75 culture flask; this is the first generation (P1). Use P3-P9 generation cells for cell experiments.

[0045] Determination of nitric oxide (NO) content: The total nitric oxide assay kit uses nitrate reductase to reduce nitrate to nitrite, and then uses the classic Griess reagent to detect nitrite, thereby determining the total nitric oxide. Nitric oxide itself is extremely unstable and is quickly metabolized into nitrate and nitrite in cells. By measuring the total amount of nitrate and nitrite using the above method, the total amount of nitric oxide can be calculated.

[0046] Molecular biology experiments: using techniques such as Western blotting for detection and analysis.

[0047] Example 1

[0048] This study found that the SNAT2 competitive inhibitor (MeAIB) can reduce blood pressure levels in wild-type mice at basal levels.

[0049] Primary hypertension is a cardiovascular syndrome characterized by elevated systemic arterial blood pressure, commonly referred to simply as hypertension. Hypertension is typically defined as a cardiovascular disease with a systolic blood pressure higher than 140 mmHg and / or a diastolic blood pressure higher than 90 mmHg. This study used wild-type C57BL / 6 mice (Liaoning Changsheng Biotechnology Co., Ltd.). Baseline blood pressure was measured using the tail-cuff method. The mice were then given MeAIB (1 g / L) in drinking water for two weeks, and their blood pressure (systolic blood pressure, SBP) was measured after drinking the water. The results showed that MeAIB reduced systolic blood pressure in the mice after drinking the water. Figure 1 ).

[0050] Example 2

[0051] This study investigated how knocking out the SNAT2 gene (SNAT2- / -) led to a decrease in blood pressure (systolic, diastolic, and mean arterial pressure) in mice at baseline.

[0052] SNAT2 systemic gene knockout mice were obtained by deleting a 10bp (GCGATTGTGG) mutation in Exon4 using CRISPR / Cas9 technology (see [link to CRISPR / Cas9]). Figure 2 A), and was identified by DNA sequencing (see A). Figure 2 B). SNAT2 knockout mice under the C57BL / 6 background exhibit a homozygous lethal phenotype. To obtain a sufficient number of SNAT2 WT and systemic SNAT2 knockout (KO) mice, we backcrossed heterozygous male SNAT2 mice under the C57BL / 6 background with female mice under the wild-type 129 background. Adult mice obtained after 5 generations of backcrossing were used for subsequent experiments. Compared to wild-type mice (SNAT2+ / +), SNAT2 knockout mice (SNAT2- / -) had significantly lower systolic blood pressure (SBP). Figure 3 A) Diastolic blood pressure (DBP, Figure 3 B) Mean arterial pressure (MBP) Figure 3 C) Lower than wild-type mice.

[0053] Example 3

[0054] This study investigated how specific knockout of SNAT2 vascular endothelium (EC-SNAT2cKO) led to a decrease in blood pressure (systolic, diastolic, and mean arterial pressure) in mice under basal conditions.

[0055] Using the principle of homologous recombination, flux modification was performed on both ends of exons 5 and 10 of the SNAT2 (Slc38a2) gene via homologous recombination in fertilized eggs, and the gene was identified by DNA gel electrophoresis (see [link to article]). Figure 4To further elucidate the role of vascular endothelial SNAT2 in blood pressure regulation, we crossed SNAT2 flox / flox mice with vascular endothelial-specific Cre mice (VE-Cadherin-Cre) (The Jackson Laboratory 017968) to obtain endothelial-specific SNAT2 knockout mice (EC-SNAT2-cKO). We then measured the blood pressure of the mice using the tail-cuff method. Compared with wild-type mice (SNAT2+ / +, WT), the systolic blood pressure (SBP) of vascular endothelial SNAT2 gene-specific knockout mice (EC-SNAT2 cKO) was significantly lower. Figure 5 A) Diastolic blood pressure (DBP, Figure 5 B) Mean arterial pressure (MBP) Figure 5 C) was significantly lower than that of wild-type mice.

[0056] Example 4

[0057] This study investigated how knocking out the SNAT2 gene resisted an increase in blood pressure (systolic blood pressure) in mice induced by a high-salt diet.

[0058] This study divided mice into wild-type (SNAT2+ / +) and SNAT2 knockout mice (SNAT2- / -). Blood pressure was measured at baseline using the tail-cuff method. Mice were then fed a high-salt (3.5% NaCl) diet for 4 weeks, with blood pressure measured weekly. The results showed that the high-salt diet increased systolic blood pressure (SBP) in SNAT2+ / + mice, while SNAT2- / - mice were resistant to the high-salt diet-induced increase in systolic blood pressure. Figure 6 ).

[0059] Example 5

[0060] This study investigated the increase in serum NO in mice caused by the knockout of the SNAT2 gene.

[0061] This study divided mice into wild-type (SNAT2+ / +) and SNAT2 gene knockout mice (SNAT2- / -). Blood was collected from the inner canthal vein of the mice, incubated at room temperature for 2 hours, and then centrifuged at 3000 rpm for 10 minutes. The supernatant was collected as serum. The total NO content in the serum was measured by Griesis assay. The results showed that the NO content in the serum of SNAT2- / - mice was increased ( Figure 7 ).

[0062] Example 6

[0063] This study investigated how MeAIB, a competitive inhibitor of SNAT2, can increase NO content in human umbilical vein endothelial (HUVEC) cells in a dose-dependent manner.

[0064] Cultured human umbilical vein endothelial cells (HUVECs) Figure 8A) When cell confluence reached 80%, MeAIB was administered in a dose-dependent manner (0, 5, 10, 20, 50, 100 mM) for 24 h. The NO content in the cell supernatant was detected by Griesis assay. The results showed that MeAIB-dependently increased the NO content in the cell supernatant. Figure 8 B), Western blot analysis was used to detect the expression levels of eNOS and p-eNOS (Ser1177) proteins in cells. The results showed that the expression level of p-eNOS (Ser1177) protein was increased. Figure 8 C) indicates an increase in eNOS activity associated with NO synthesis.

[0065] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of substances with competitive SNAT2 inhibitory activity in the preparation of drugs for the prevention and / or treatment of essential hypertension; The substance with competitive inhibitory activity against SNAT2 is α-aminoisobutyric acid.

2. The application according to claim 1, characterized in that: The drug is a drug that has any of the following functions: 1) Medications that lower baseline blood pressure levels; 2) Medications for the prevention and / or treatment of hypertension; 3) Drugs that promote the production of the vasodilator NO.

3. A method for preparing a cell model for screening antihypertensive drugs, comprising the following steps: 1) Obtaining vascular endothelial cells from animals; 2) Treat the vascular endothelial cells with a substance that has the activity of inhibiting the SNAT2 gene and its products (mRNA and protein) or that can knock out the SNAT2 gene, thereby obtaining vascular endothelial cells with reduced expression levels of the SNAT2 gene and its products (mRNA and protein) or with the SNAT2 gene knocked out. 3) The NO content in vascular endothelial cells with reduced expression levels of the SNAT2 gene and its products (mRNA and protein) obtained in step 2) or with the SNAT2 gene knocked out is used as an indicator of blood pressure level.

4. A cell model for screening antihypertensive drugs obtained by the preparation method described in claim 3.

5. A method for screening antihypertensive drugs using the cell model of claim 4, comprising the following steps: 1) Grouping: test drug group, positive control group and blank control group. The positive control group was treated with an equal amount of arginine, and the blank control group was treated with an equal volume of PBS. 2) Treat the cell models of each group with the drugs used in each group; 3) Detect the NO content of the treated cells. If the NO content level obtained in the test drug treatment group is higher than or equal to the NO content obtained in the positive control group, and there is a statistically significant difference compared with the blank control group, then the test drug has antihypertensive activity; if the NO content level obtained in the test drug treatment group is lower than the NO content obtained in the positive control group, and there is no statistically significant difference compared with the blank control group, then the test drug has no antihypertensive activity.

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