Application of TLR1 receptor inhibitor in preparation of products for treating hypertension

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a TLR1 receptor inhibitor in preparation of products for treating hypertension. Existing medicines for clinical application of hypertension have different action mechanisms, and the research finds that sympathetic nervous excitation-induced renin synthesis and glucocorticoid-induced angiotensinogen synthesis are inhibited and the amount of angiotensin II is inhibited by inhibiting para-bulbar cells of the kidney and liver cell TLR1 receptors of the liver, so that the clinical application of hypertension is realized. The traditional Chinese medicine composition is a brand new mechanism for treating hypertension, and the mechanism and the method provide a new scheme for treatment of hypertension, especially treatment of nervous hypertension, and can also have a synergistic effect with existing treatment drugs. The invention finds that the TLR1 receptor is a new target for treating hypertension, development and application of hypertension treatment drugs mediated by the TLR1 receptor do not exist at present, and the invention provides sufficient theoretical basis and experimental basis for research and development of novel antihypertensive drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a TLR1 receptor inhibitor in the preparation of a product for treating hypertension. Background Art

[0002] Renin is a proteolytic enzyme synthesized and secreted by juxtaglomerular (JG) cells of the kidney, also known as angiotensinogenase. Juxtaglomerular cells are located at the tip of the afferent arteriole at the entrance of the glomerulus. Angiotensinogen (an α-globulin) is mainly synthesized and secreted by hepatocytes. The renin-angiotensin-aldosterone system (RAAS) is a hormone system and an important blood pressure regulation system. When blood pressure drops, the kidneys secrete renin, which catalyzes the hydrolysis of angiotensinogen to produce angiotensin I. Angiotensin I has basically no biological activity, but is formed by angiotensin converting enzyme cleaving the two amino acid residues at the C-terminus to form angiotensin II. Angiotensin II has a highly efficient vasoconstriction effect, thereby increasing blood pressure; angiotensin II can also stimulate the adrenal cortex to secrete aldosterone. Aldosterone can promote the reabsorption of water and sodium ions by the kidneys, thereby increasing body fluid volume and raising blood pressure. Therefore, renin and angiotensinogen can regulate the body's blood pressure through the renin-angiotensin-aldosterone system, and play an important role in the control of hypertension.

[0003] Currently, the drugs used in clinical practice to treat hypertension are mainly divided into seven categories:

[0004] 1. Calcium channel blockers (CCB): They mainly dilate blood vessels and lower blood pressure by blocking calcium ion channels on vascular smooth muscle cells.

[0005] 2. Angiotensin-converting enzyme inhibitor (ACEI): The mechanism of action is to inhibit angiotensin-converting enzyme, block the production of angiotensin II, and exert a antihypertensive effect.

[0006] 3. Angiotensin Receptor Blocker (ARB): The mechanism of action is to block angiotensin II type 1 receptor and exert a antihypertensive effect.

[0007] 4. Diuretics: They mainly lower blood pressure by promoting sodium excretion and reducing volume load.

[0008] 5. β-blockers: They mainly play a role in reducing blood pressure by inhibiting the over-activated sympathetic nerve activity, inhibiting myocardial contractility, and slowing down the heart rate. They can also form one of their blood pressure-lowering mechanisms by blocking the β1-receptors of juxtaglomerular cells to inhibit the release of renin.

[0009] 6. α-blockers: They selectively bind to α-adrenergic receptors, block the binding of corresponding neurotransmitters and drugs to α-receptors, and produce anti-adrenergic effects.

[0010] 7. Renin inhibitors: Their mechanism of action is to directly inhibit the activity of renin in the blood, and then reduce the production of angiotensin II, which can lower the blood pressure level of hypertensive patients. For example, aliskiren mainly binds to free renin in the blood to inhibit its activity.

[0011] TLR1 is one of Toll-like receptors (TLRs) 1-10, and there is no report on the correlation between the related TLR1 receptor and hypertension. Summary of the Invention

[0012] An object of the first aspect of the present invention is to provide the use of a TLR1 receptor inhibitor in the preparation of a product for treating hypertension.

[0013] An object of the second aspect of the present invention is to provide the use of TLR1 as a drug target in drugs for preventing and / or treating hypertension.

[0014] In order to achieve the above objects of the present invention, the technical solutions adopted by the present invention are as follows:

[0015] In the first aspect of the present invention, there is provided the use of a TLR1 receptor inhibitor in the preparation of a product for treating hypertension.

[0016] TLR1 is one of Toll-like receptors (TLRs).

[0017] In some embodiments of the present invention, the TLR1 receptor inhibitor includes at least one of the following:

[0018] (a1) A substance that inhibits the activity of TLR1 protein;

[0019] (a2) A substance that reduces the content of TLR1 protein;

[0020] (a3) A substance that knocks out the TLR1 gene;

[0021] (a4) A substance that inhibits the expression of the TLR1 gene.

[0022] In some embodiments of the present invention, the TLR1 receptor inhibitor includes nucleic acid molecules, protein molecules, and small molecule compounds.

[0023] In some embodiments of the present invention, the nucleic acid molecule is microRNA, siRNA, shRNA, dsRNA, sgRNA, and / or antisense oligonucleotide.

[0024] In some embodiments of the present invention, the protein molecule is a specific antibody against TLR2.

[0025] In some embodiments of the present invention, the small molecule compound is a small molecule that binds to TLR2.

[0026] In some embodiments of the present invention, the small molecule compound includes CU-CPT22, whose chemical name is hexyl 3,4,6-trihydroxy-2-methoxy-5-oxo-5H-benzocycloheptene-8-carboxylate, and the molecular formula is C 19 H 22 O 7 , and the structural formula is shown as follows:

[0027]

[0028] In some embodiments of the invention, the CU-CPT22 further includes a pharmaceutically acceptable salt and a pharmaceutically acceptable chemical modification.

[0029] In some embodiments of the invention, the pharmaceutically acceptable salt includes at least one of metal salt, ammonium salt, salt formed with inorganic acid, salt formed with organic base, salt formed with organic acid, salt formed with basic amino acid, and salt formed with acidic amino acid.

[0030] In some embodiments of the invention, the pharmaceutically acceptable modification includes at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation. For example, the lipophilicity can be enhanced by introducing fluorine atoms (fluoroethyl) to improve the blood-brain barrier permeability. The corresponding modification method can be selected according to the actual use of the drug.

[0031] In some embodiments of the invention, the product includes a drug.

[0032] In some embodiments of the invention, the drug includes a pharmaceutically acceptable excipient and / or any one or more other active ingredients.

[0033] In some embodiments of the invention, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickening agents, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, carriers.

[0034] The present invention provides a drug and a corresponding pharmaceutical dosage form for treating hypertension, with a TLR1 receptor inhibitor (especially CU-CPT22) as the active ingredient.

[0035] Among them, the drug exists in the form of an oral preparation, an injection or a topical administration preparation.

[0036] In particular, the oral preparation includes tablets, capsules, pills, powders, granules, syrups or solutions; the injection includes an injection dosage form or a freeze-dried powder for injection dosage form; the topical administration preparation includes creams, ointments, sprays, aerosols or patches.

[0037] Among them, the pharmaceutical preparation uses the TLR1 receptor inhibitor (especially CU-CPT22) as the effective active ingredient and includes other pharmaceutically acceptable carrier components.

[0038] The carriers in the drug include excipients such as starch, water, etc.; lubricants such as magnesium stearate, etc.; disintegrants such as microcrystalline cellulose, etc.; fillers such as lactose, etc.; binders such as pregelatinized starch, dextrin, etc.; sweeteners; antioxidants; preservatives; flavoring agents; fragrances, etc.

[0039] The carriers selected in the preparation of oral preparations can be conventional pharmaceutical excipients such as starch, dextrin or cyclodextrin and various chemically modified cyclodextrins, sucrose, stearates, etc. When preparing the freeze-dried powder for injection, it can be prepared by methods such as aseptic spray drying, low-temperature vacuum drying, freeze drying, etc. The later preparation processes and equipment of each preparation belong to the conventional technologies in the pharmaceutical field, and the present invention does not limit them.

[0040] The drug in the present invention exists in the form of tablets, capsules, pills, powders, granules, syrups, solutions, injections, sprays, aerosols, patches, gels, cataplasms, that is, the pharmaceutical preparation includes forms such as tablets, capsules, pills, powders, granules, syrups, solutions, injections, sprays, aerosols, patches, gels, cataplasms, etc., but is not limited to the above forms.

[0041] Further, for the convenience of drug administration, the active ingredient TLR1 receptor inhibitor (especially CU-CPT22) can be processed into a specific dosage form in combination with any one or several pharmaceutically acceptable excipients. These excipients can be diluents (such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, microcrystalline cellulose, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, light magnesium oxide, calcium carbonate, etc.), wetting agents (such as water, ethanol, etc.), binders (such as hydroxypropyl methylcellulose, povidone, starch paste, syrup, etc.), disintegrants (such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, cross-linked povidone, etc.), lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, colloidal silicon dioxide, etc.), coloring agents (such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide, etc.), coating materials (such as acrylic resin, hydroxypropyl methylcellulose, povidone, etc.), solvents (such as water for injection, ethanol, propylene glycol, glycerol, etc.), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid, sodium tartrate, etc.), antioxidants (such as sodium sulfite, sodium metabisulfite, sodium thiosulfate, etc.), bacteriostatic agents (such as phenol, benzyl alcohol, thimerosal, etc.), or can also be isotonic regulators (such as sodium chloride, glucose, etc.).

[0042] The above-mentioned pharmaceutically acceptable excipients are generally recognized for this purpose and serve as inactive ingredients of the medicament. A compilation of pharmaceutically acceptable excipients can be found in reference books such as Handbook of Pharmaceutical Excipients (2nd Edition, edited by A. Wade and P. J. Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994); and the list of names of pharmaceutical excipients in the Pharmacopoeia of the People's Republic of China, etc.

[0043] In some embodiments of the invention, the dosage form of the product includes a gastrointestinal dosage form or a parenteral dosage form.

[0044] In some embodiments of the invention, the gastrointestinal dosage form includes at least one of powders, tablets, granules, capsules, sustained-release preparations, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, chewable tablets.

[0045] In some embodiments of the invention, the parenteral dosage form includes at least one of injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[0046] In some embodiments of the invention, the subject of administration of the product is a mammal.

[0047] In some embodiments of the invention, the mammal includes humans.

[0048] The second aspect of the present invention provides the use of TLR1 as a drug target in the prevention and / or treatment of hypertensive drugs.

[0049] In some embodiments of the invention, the use is for non-diagnostic and non-therapeutic purposes.

[0050] The beneficial effects of the present invention are as follows:

[0051] The existing drugs for clinical application of hypertension have different mechanisms of action. This study found that by inhibiting the TLR1 receptors of juxtaglomerular cells in the kidney and hepatocytes in the liver, the synthesis of renin induced by sympathetic nerve excitation and the synthesis of angiotensinogen induced by glucocorticoid can be inhibited, and the amount of angiotensin II can be inhibited. This is a new mechanism for treating hypertension. This mechanism and method provide a new solution for the treatment of hypertension, especially for the treatment of neurogenic hypertension, and can also have a synergistic effect with existing therapeutic drugs.

[0052] The TLR1 receptor is a new target for the treatment of hypertension. At present, there is no development and application of antihypertensive drugs mediated by the TLR1 receptor, which provides a sufficient theoretical basis and experimental foundation for the research and development of new antihypertensive drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The present invention will be further described below in conjunction with the drawings and embodiments, wherein:

[0054] Figure 1 It is the result of the Western blot band of Renin protein in the renal cortex of mice in Example 4 of the present invention.

[0055] Figure 2 It is the result of the Western blot band of AGT protein in the liver of mice in Example 5 of the present invention.

[0056] Figure 3 It is the result of the Western blot band of Renin protein in juxtaglomerular cells in Example 7 of the present invention.

[0057] Figure 4 It is the result of the Western blot band of the expression of AGT protein in hepatocytes with high AGT induced by adrenaline by TLR1 inhibitor in Example 9 of the present invention.

[0058] Figure 5 It is the result of the Western blot band of the expression of AGT protein in hepatocytes with high AGT induced by cortisol by TLR1 inhibitor in Example 9 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0059] The concept of the present invention and the resulting technical effects will be clearly and completely described below in conjunction with embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0060] Example 1 Construction of a hypertensive mouse model

[0061] Currently, there are a variety of animal models of hypertension used in experimental research. The noise-induced hypertensive animal model is also a commonly used one. The main mechanism of noise-induced hypertension is that noise exposure stimulates the auditory organ to generate nerve impulses, which are transmitted to the sympathetic nerve through the central nervous system, resulting in sympathetic nerve excitation, causing changes in the functional state of the pituitary-adrenal axis, and then promoting the secretion of more catecholamine hormones (norepinephrine and epinephrine) by chromaffin cells of the adrenal medulla. Acting on the adrenergic receptors of blood vessels, the heart, and the kidneys, it leads to an increase in cardiac output, activation of the renin-angiotensin system (stimulating an increase in angiotensin II), and continuous tension and contraction of the smooth muscle of the blood vessel wall, resulting in an increase in blood pressure. In this study, a hypertensive mouse model was constructed through a noise exposure model, and the specific method is as follows:

[0062] All experimental animals were 7-week-old SPF-grade male C57BL / 6J mice, weighing approximately 14-16 g (Guangdong Provincial Medical Laboratory Animal Center). The experimental procedures were approved by the Animal Ethics Committee of Guangdong Pharmaceutical University (No.: GDPULACSPF2022124). The mice were housed at an environmental temperature of 20 ± 0.5 °C, a relative humidity of 55% ± 5%, a light / dark cycle of 12 h, and a background noise level of less than 40 dB SPL. Ten mice were exposed to a noise environment. The noise exposure was carried out using a 50 x 50 x 40 cm noise exposure chamber, and the noise was white noise at 103 dB SPL. The mice were exposed for 12 hours per day for a total of 7 days. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MBP, diastolic blood pressure + 1 / 3 pulse pressure difference) were measured before and after noise exposure in the mice using a Softron BP-2010A intelligent non-invasive mouse blood pressure monitor (Softron, Beijing, China). It was found that there were significant differences in systolic blood pressure between the mice before and after noise exposure (t = -12.55, P < 0.01), significant differences in diastolic blood pressure between the mice before and after exposure (t = -4.72, P < 0.01), and significant differences in mean arterial pressure between the mice before and after exposure (t = -6.74, P < 0.01). After noise exposure, the systolic blood pressure, diastolic blood pressure, and mean arterial pressure of the mice all increased significantly, as shown in Table 1.

[0063] Table 1 Changes in blood pressure of mice before and after noise exposure (mm / Hg, )

[0064]

[0065] The experimental results indicate that a hypertensive mouse model was successfully established by this noise exposure method.

[0066] Example 2 Experiment on the effect of TLR1 receptor inhibitor on blood pressure in hypertensive experimental mice

[0067] In this example, CU-CPT22 (3,4,6-trihydroxy-2-methoxy-5-oxo-5H-benzocycloheptene-8-carboxylic acid hexyl ester, molecular formula: C19H22O7) was selected from TLR1 receptor inhibitors for the experiment.

[0068] One day before the experiment, the blood pressure of the mice was measured as the baseline. Then the mice were randomly divided into 6 groups (12 mice in each group), namely the control group (Control), the noise exposure group (Noise), the noise exposure + low-concentration CU-CPT22 group (Noise + CU-CPTL), the noise exposure + medium-concentration CU-CPT22 group (Noise + CU-CPT22 M), the noise exposure + high-concentration CU-CPT22 group (Noise + CU-CPT22 H), and the CU-CPT22 group. The entire noise exposure period was 14 days. During the experiment, the mice were exposed to noise and intraperitoneally injected with drugs at a fixed time every day. After the blood pressure was measured on the 14th day, the animals were anesthetized and sacrificed for sampling. The TLR1 receptor inhibitor CU-CPT22 was divided into three groups with low concentration (0.5 mg / kg), medium concentration (2.5 mg / kg), and high concentration (5.0 mg / kg). The intraperitoneal injection volume was 0.2 ml / day, and the Control group was intraperitoneally injected with the same volume of normal saline.

[0069] The experimental results are shown in Table 2.

[0070] Table 2. Effects of TLR1 receptor inhibitor CU-CPT22 intervention on the blood pressure of mice

[0071]

[0072] Note: L in a represents low dose; M in b represents medium dose; H in c represents high dose.

[0073] By performing one-way analysis of variance on the blood pressure values of the 6 groups of mice respectively, it was found that there were significant differences in systolic blood pressure, diastolic blood pressure, and mean arterial pressure among the six groups of mice. After further multiple comparison analysis, it was found that the systolic blood pressure and mean arterial pressure of the Noise group and the Noise + CU-CPT22 L and M groups were significantly increased (P < 0.01, P < 0.01), and there was no difference in blood pressure between the Noise + CU-CPT22 H group and the CU-CPT22 group (P > 0.05). Compared with the Noise group, the systolic blood pressure and mean arterial pressure of the Noise + CU-CPT22 M and H groups were significantly decreased (P < 0.01, P < 0.01). There was no significant difference in diastolic blood pressure, systolic blood pressure, and mean arterial pressure between the CU-CPT22 group and the Control group (P > 0.05).

[0074] The research results show that simply inhibiting the TLR1 receptor has no effect on the blood pressure of normal mice; for mice with hypertension induced by noise exposure, inhibiting the TLR1 receptor has an obvious inhibitory effect on blood pressure, and this inhibitory effect becomes more obvious with the increase in the concentration of the inhibitor.

[0075] Example 3 Experiment on the effects of TLR1 receptor inhibitor on blood pressure-related factors in hypertensive mice

[0076] Since there is no relevant research report, the mechanism of the inhibitory effect of TLR1 receptor on blood pressure control in hypertensive mice is still unknown. In this example, the ELISA method was used to detect 7 hypertension-related factors or indicators in the serum of mice: angiotensin II (Ang II), renin, plasma renin activity (PRA), angiotensinogen (AGT), endothelin (ET), aldosterone (ALD), and atrial natriuretic peptide (ANP).

[0077] Specific method: 14 days after noise exposure, after the mice completed blood pressure detection, they were anesthetized with 0.2 ml / 20 g of 4% chloral hydrate. The orbital blood collection method was used to collect whole blood into a non-anticoagulant blood collection tube and placed at room temperature for 30 min. Centrifugation was carried out at 4°C and 1300 g for 10 min. The supernatant was transferred to a new centrifuge tube. The second centrifugation was carried out at 4°C and 16,000 g for 10 min. The detection steps were in accordance with the ELISA kit instructions.

[0078] The detection results are shown in Table 3.

[0079] Table 3. Effects of TLR1 receptor inhibitor on serum blood pressure active factors in mice (n = 12 / group)

[0080]

[0081] Note: ALD: aldosterone; ANP: atrial natriuretic peptide; AGT: angiotensinogen; Renin: renin; PRA: plasma renin activity; ET: endothelin; Ang II: angiotensin II.

[0082] The research results showed that there were significant differences in blood pressure-related active factors among the experimental groups of mice by analysis of variance (P<0.001); through inter-group comparison, it was found that after noise exposure, compared with the Control group, the levels of serum ALD (303.36 vs 362.85, P = 0.0049), ANP (4.06 vs 9.49, P = 0.0026), AGT (3056.10 vs 4024.89, P = 0.0071), Renin (64.47 vs 384.76, P = 0.0091), PRA (22.09 vs 35.42, P<0.001), and ET (57.49 vs 64.31, P<0.001) in the Noise group of mice increased significantly. Compared with the Noise group, the levels of ALD (362.85 vs 311.68, P = 0.0051), AGT (4024.89 vs 3394.85, P = 0.0018), Renin (384.76 vs 139.76, P = 0.00014), and PRA (35.42 vs 26.67, P = 0.0055) in the Noise+CU-CPT22 H group decreased significantly. Compared with the Noise+CU-CPT22 L group, the levels of ADL (366.06 vs 311.68, P<0.001), AGT (4485.61 vs 3394.85, P<0.001), and Renin (380.23 vs 139.76, P<0.001) in the Noise+CU-CPT22 H group decreased significantly.

[0083] This result indicates that after the occurrence of noise exposure-induced hypertension, the TLR1 receptor inhibitor mainly affects blood pressure changes by inhibiting the levels of serum aldosterone, angiotensinogen, renin, renin activity, and angiotensin II, suggesting that the TLR1 receptor inhibitor exerts its antihypertensive effect by inhibiting the activity of the renin-angiotensin-aldosterone system. It is speculated that the target of action may be in the juxtaglomerular cells that synthesize renin and hepatocytes that synthesize angiotensinogen, which requires further experimental verification.

[0084] Detection of Renin in the kidney tissue of experimental mice in Example 4

[0085] PCR detection of Renin mRNA expression level in kidney tissues: After the experimental mice were anesthetized and sacrificed, 1 g of kidney cortex tissue was taken from each mouse, ground with 200 μl of trizol, and then 800 μl of trizol was added; RNA was extracted by conventional methods. cDNA synthesis was carried out by reverse transcription of RNA according to the instructions of the cDNA Synthesis Kit. Fluorescent quantitative PCR primers were designed using Primer 3.0, and the primer sequences are shown in Table 4. The preparation of the reaction system and reaction parameters were referred to the instructions of the SYBR Premix Ex Taq II Kit, and the amplification curve and melting curve were carried out according to the operation method of the Real-Time PCR System: ① Preparation of the reaction system: 10.0 μl Premix Ex Taq TM , 0.8 μl of 10 μM upstream PCR primer, 0.8 μl of 10 μM downstream PCR primer, 2.0 μl of cDNA template, 6.4 μl of distilled water, a total of 20.0 μl, prepared on ice. ② Amplification program: Pre-denaturation at 95 °C for 10 min, cycle 44 times at 95 °C for 10 s, 60 °C for 22 s, 72 °C for 30 s, and obtain fluorescence signals at 72 °C; after the cycle ends, raise the temperature from 72 °C to 94 °C to obtain the melting curve. Data analysis was performed using the Manager Software 1.6 data analysis software supporting the Real Time PCR instrument, and the analysis method was the 2 -ΔΔCt -method. The results are shown in Table 5.

[0086] Western-Blot detection of Renin protein expression level in kidney tissues: After the experimental mice were anesthetized and sacrificed, 0.1 g of kidney cortex tissue was taken from each mouse, minced and ground with lysis buffer at low temperature, and the supernatant was collected as the total protein lysate. The protein concentration of each sample was determined by Coomassie Brilliant Blue and adjusted to the same level (4 - 8 μg / μl); after protein denaturation, SDS-PAGE electrophoresis was carried out at a constant voltage of 70 V × 20 min + 130 V × 30 min; transfer membrane at a constant current of 400 ma for 50 min, block at room temperature for 1 h; GAPDH: 1:1000; Renin: 1:3000, incubate the primary antibody overnight at 4 °C; secondary antibody (HRP-labeled goat anti-rabbit IgG: 1:1000). After exposure and development, the pictures were processed with the Image-J image processing software, and the gray values of the internal reference protein and the target protein bands were read. The internal reference was used as the base number, and the ratio of the gray value of other protein bands to it was the relative gray value. The relative expression of the target protein was evaluated by comparing the relative gray values of each group. The results are shown in Table 5 and Figure 1 .

[0087] Results: Compared with the Control group, the protein expression level of Renin in the Noise group was significantly increased, and the difference was statistically significant (P<0.05). Compared with the Noise group, the protein expression levels of Renin in the Noise+CU-CPT22 L, M, and H groups were all significantly decreased, and the differences were all statistically significant (P<0.05). The qRT-PCR experiment was used to detect the Renin mRNA expression level in the renal cortical tissues of mice in each group. Results: Compared with the Control group, the Renin mRNA expression levels in the Noise group, the Noise+CU+CPT22 L group, and the Noise+CU+CPT22 M group were all significantly increased (P<0.05). Compared with the Noise group, the Renin mRNA expression levels in the Noise+CU+CPT22M group and the Noise+CU+CPT22 H group were significantly decreased (P<0.05).

[0088] The experimental results suggest that noise exposure can significantly promote the synthesis of renin in the kidney, while the TLR1 receptor inhibitor can significantly inhibit the increase in the synthesis of renal renin caused by noise, but has no obvious effect on the synthesis of normal renal renin and renin activity. Combining with the inhibitory results of serum renin activity, it shows that the TLR1 receptor inhibitor controlling the synthesis of renal renin is an important factor in controlling noise-induced hypertension. However, further in vitro cell experiments are needed for verification.

[0089]

[0090] Example 5 Detection of angiotensinogen (AGT) in the liver of experimental mice

[0091] PCR was used to detect the AGT mRNA expression level in liver tissues: The method was referred to the method for detecting Renin in the kidney; the primer sequences are shown in Table 6. The results are shown in Table 7.

[0092] Western-Blot was used to detect the AGT protein expression level in liver tissues: The method was referred to the method for detecting Renin in the kidney; the results are shown in Table 7. Figure 2 .

[0093] Results showed that compared with the Control group, the AGT protein expression level in the Noise group was significantly increased, and the difference was statistically significant (P<0.05). Compared with the Noise group, the AGT protein expression levels in the Noise+CU-CPT L, M, and H groups were all significantly decreased, and the differences were statistically significant (P<0.05). RNA was extracted from the liver tissues of mice in each group for qRT-PCR experiments to detect the AGT mRNA expression level. The results showed that compared with the Control group, the AGT mRNA expression levels in the Noise group, the Noise+CU+CPT22 L group, and the Noise+CU+CPT22 M group were all significantly increased (P<0.05). Compared with the Noise group, the AGT mRNA expression levels in the Noise+CU+CPT22 M group and the Noise+CU+CPT22 H group were significantly decreased (P<0.05).

[0094] The experimental results suggest that noise exposure can significantly promote the synthesis of angiotensinogen in the liver, while the TLR1 receptor inhibitor can significantly inhibit the increase in the synthesis of angiotensinogen in the liver caused by noise, but has no obvious effect on the synthesis of normal liver angiotensinogen, indicating that the TLR1 receptor inhibitor controlling the synthesis of liver angiotensinogen is an important factor in controlling noise-induced hypertension. However, further in vitro cell experiments are needed for verification.

[0095]

[0096] Example 6 Construction of a pathological model of high renin-synthesizing cells in an in vitro juxtaglomerular cell line

[0097] The juxtaglomerular cell is an epithelial-like cell transformed from the smooth muscle cells of the afferent arteriole wall at the vascular pole of the renal corpuscle. Its main function is to synthesize and secrete renin. Since the juxtaglomerular cell has adrenergic receptors (β 2 type receptors) and is innervated by the sympathetic nerve, and the increase in blood pressure caused by sympathetic nerve excitation is an important mechanism for noise exposure-induced blood pressure increase, so constructing a pathological model of high renin-synthesizing juxtaglomerular cells by stimulating the synthesis of renin in cultured juxtaglomerular cells with epinephrine (E) can provide an effective research platform for the project research.

[0098] Mouse juxtaglomerular cell line (Shanghai Yaji Biotechnology Co., Ltd.), the culture conditions are 37°C, 5% CO 2Since renin is a secreted protein, the protein level in cells cannot represent the true gene expression level. Therefore, the qPCR method was used in the experiment to detect the renin mRNA level, which can more reasonably represent the true expression of this gene. To explore the optimal concentration and reaction time of adrenaline for promoting renin synthesis in juxtaglomerular cells and construct the most reasonable pathological model of juxtaglomerular cells with high renin synthesis, experiments with different concentration gradients of adrenaline and different stimulation times were selected. The results are shown in Table 8. The Renin mRNA expression level in juxtaglomerular cells has a dose-response relationship with adrenaline, and the synthesis peak occurs when stimulated with 1 μM adrenaline for 15 min. Therefore, the reaction conditions of 1 μM adrenaline concentration and 15 min intervention time are the best reaction conditions for constructing the pathological model of juxtaglomerular cells with high renin synthesis.

[0099] Table 8. Effects of different concentrations and different stimulation times of adrenaline on Renin synthesis in juxtaglomerular cells

[0100]

[0101] Example 7. Effects of TLR1 receptor inhibitor on renin synthesis in juxtaglomerular cells with high renin synthesis

[0102] Juxtaglomerular cells were seeded in 6-well plates. Adrenaline was diluted to 1 μM with culture medium, and the CU-CPT22 stock solution was diluted to three concentrations of 0.25 μM, 0.5 μM, and 1 μM with culture medium. The juxtaglomerular cells were digested and counted, and the cells were seeded at 2×10 6 / well on 6-well plates; the experimental groups were Control group, 1 μΜ adrenaline group, 1 μΜ adrenaline + 0.25 μΜ CU-CPT22 group, 1 μΜ adrenaline + 0.5 μΜ CU-CPT22 group, 1 μΜ adrenaline + 1 μΜ CU-CPT22 group, and the Control group was added with an equal amount of drug-free culture medium. Each group had 3 replicates, and the samples were collected after 15 min of culture at 37 °C and 5% CO 2 conditions to detect the Renin mRNA expression level and Renin protein expression level (PCR, Western-Blot). The results are shown in Table 9. Figure 3 .

[0103] Table 9. Effects of TLR1 receptor inhibitor on Renin mRNA and Renin expression levels in juxtaglomerular cells with high renin synthesis

[0104]

[0105]

[0106] The results showed that the expression level of Renin mRNA in juxtaglomerular cells was significantly increased under adrenaline stimulation (P<0.01). After treatment with CU-CPT22 at different concentrations, the expression level of Renin mRNA decreased with the increase of CU-CPT22 concentration, and the difference was statistically significant (F = 126.283, P<0.01). Renin protein was also significantly increased under adrenaline stimulation (P<0.01). After treatment with CU-CPT22 at different concentrations, the expression level of Renin mRNA decreased with the increase of CU-CPT22 concentration, and the difference was statistically significant (F = 55.572, P<0.01).

[0107] The experimental results suggested that juxtaglomerular cells could increase the synthesis and secretion of renin by activating β1 receptors under adrenaline stimulation, which was consistent with the theory that sympathetic nerve stimulation induced renin synthesis, indicating that the mechanism of noise-induced hypertension formation was consistent with the mechanism of sympathetic nerve excitation stimulating juxtaglomerular cells to synthesize and secrete a large amount of renin. The TLR1 receptor inhibitor could significantly inhibit the renin synthesis of juxtaglomerular cells with high renin synthesis, but had no obvious effect on the renin synthesis of normal juxtaglomerular cells, which was consistent with the experimental results of the TLR1 receptor inhibitor inhibiting blood pressure changes in mice with noise exposure-induced hypertension, further confirming that the TLR1 receptor inhibitor controlling the renin synthesis of juxtaglomerular cells in the kidney was an important factor in controlling noise-induced hypertension, and the TLR1 receptor was an important target for controlling the body's renin and renin activity.

[0108] Example 8 Construction of a pathological model of hepatocyte cell line with high angiotensinogen synthesis in vitro

[0109] Hepatocytes are the main secretory organs of angiotensinogen (AGT). AGT is a rate-limiting substrate of the Renin-Angiotensin System (RAS). It is catalyzed by renin to generate angiotensin I (ANG I), and ANG I is catalyzed by angiotensin-converting enzyme (ACE) to generate angiotensin II (ANG II), which can regulate blood pressure.

[0110] Mouse normal hepatocyte cell line (AML12) (Shanghai Jinyuan Biotechnology Co., Ltd.), the culture conditions were 37°C, 5% CO 2。Since angiotensinogen is a secreted protein, the intracellular protein level cannot represent the true gene expression level. Therefore, qPCR was used to detect the AGT mRNA level in hepatocytes, which can more reasonably represent the true expression of this gene. To explore the optimal stimulation concentration and time of adrenaline and cortisol (hydrocortisone) on AML12 cells, experiments with different concentration gradients and different stimulation times were conducted. The results are shown in Tables 10 and 11. There is a dose-response relationship between the AGT mRNA expression level of AML12 cells and adrenaline. The peak synthesis of AGT occurs at 60 min after stimulation with 1 μM adrenaline. Therefore, a pathological model of high angiotensinogen cells induced by adrenergic receptor stimulation was established under the conditions of 1 μM adrenaline concentration and 60 min intervention time. There is a dose-response relationship between the AGT mRNA expression level of AML12 cells and cortisol at 15 min and 60 min. The peak synthesis of AGT occurs at a cortisol concentration of 1.5 μM at 60 min of stimulation. Therefore, a pathological model of high angiotensinogen cells induced by cortisol receptor stimulation was established under the conditions of 1.5 μM cortisol concentration and 60 min intervention time.

[0111] Table 10. Effects of different concentrations and stimulation times of adrenaline on AGT synthesis in AML12 cells

[0112]

[0113] Table 11. Effects of different concentrations and stimulation times of cortisol on AGT synthesis in AML12 cells

[0114]

[0115] Example 9. Effects of TLR1 receptor inhibitor on angiotensinogen synthesis in the pathological model of high angiotensinogen hepatocytes

[0116] AML12 cells were seeded in 6-well plates. Adrenaline was diluted to 1 μM with medium, and the stock solution of CU-CPT22 was diluted to three concentrations of 0.25 μM, 0.5 μM, and 1 μM with medium. The AML12 cells were digested and counted, and the cells were seeded at 2×10 6 / well on 6-well plates. The experimental groups were Control group, 1 μΜ adrenaline group, 1 μΜ adrenaline + 0.25 μΜ CU-CPT22 group, 1 μΜ adrenaline + 0.5 μΜ CU-CPT22 group, and 1 μΜ adrenaline + 1 μΜ CU-CPT22 group. The Control group was added with an equal amount of medium without drugs. Each group had 3 replicate wells. At 37 °C and 5% CO 2Cultured under the conditions, samples were collected after 60 min to detect the expression levels of AGT mRNA and AGT protein (PCR, Western-Blot).

[0117] The results are shown in Table 12, Figure 4 .

[0118] Table 12. Effects of TLR1 inhibitor on AGT mRNA and AGT expression levels in adrenaline-induced high-AGT type hepatocytes

[0119]

[0120] The results showed that compared with Control, adrenaline stimulation could significantly increase the expression levels of AGT mRNA and AGT protein in AML12 cells (P<0.05), and CU-CPT22 at different concentrations could not inhibit the increased expression levels of AGT mRNA and AGT protein in AML12 cells (P>0.05). Combining previous studies and the animal model of this study, glucocorticoids can also stimulate hepatocytes to synthesize AGT. Considering the stimulating effect of cortisol (glucocorticoid) secreted by the adrenal cortex on hepatocytes, a cortisol stimulation experiment was continued to verify whether the TLR1 / 2 receptor inhibitor could inhibit the synthesis of AGT.

[0121] AML12 cells were seeded in 6-well plates. Cortisol was diluted to 1.5 μM with medium, and CU-CPT22 was diluted to three concentrations of 0.25 μM, 0.5 μM, and 1 μM with medium; AML12 cells were digested and counted, and the cells were seeded at 2×10 6 / well on 6-well plates; the experimental groups were Control group, 1.5 μΜ cortisol group, 1.5 μΜ cortisol + 0.25 μΜ CU-CPT22 group, 1.5 μΜ cortisol + 0.5 μΜ CU-CPT22 group, 1.5 μΜ cortisol + 1 μΜ CU-CPT22 group, and the Control group was added with an equal amount of medium without drugs. Each group had 3 replicate wells and was cultured under the conditions of 37 °C and 5% CO 2 Samples were collected after 60 min to detect the expression levels of AGT mRNA and AGT protein (PCR, Western-Blot). The results are shown in Table 13, Figure 5 .

[0122] Table 13. Effects of TLR1 inhibitor on AGT mRNA and AGT expression levels in cortisol-induced high-AGT type hepatocytes

[0123]

[0124] Results showed that compared with the Control, cortisol stimulation could significantly increase the expression levels of AGT mRNA and AGT protein in AML12 cells (P<0.01), and CU-CPT22 at different concentrations significantly inhibited the increased expression levels of AGT mRNA and AGT protein in AML12 cells (P<0.01).

[0125] The experimental results suggested that hepatocytes could increase the synthesis and secretion of angiotensinogen by activating β-type receptors under adrenaline stimulation, indicating that the mechanism of noise-induced hypertension formation was related to sympathetic nerve excitation stimulating hepatocytes to synthesize and secrete a large amount of angiotensinogen. However, the TLR1 receptor inhibitor could not control the synthesis of angiotensinogen caused by sympathetic nerve excitation, which was inconsistent with the animal experiment results. Further cortisol stimulation experiments proved that glucocorticoid (cortisol) secreted by the adrenal cortex was also one of the reasons for stimulating hepatocytes to synthesize and secrete a large amount of angiotensinogen under noise exposure, and the TLR1 receptor inhibitor could control the increase in angiotensinogen synthesis caused by cortisol stimulation. Therefore, it was inferred that sympathetic nerve excitation and increased adrenal glucocorticoid secretion were the main mechanisms for the increase in hepatocyte angiotensinogen synthesis caused by noise exposure and were also one of the important mechanisms for noise-induced hypertension. One of the main mechanisms for the TLR1 receptor inhibitor to control the increase in blood pressure in mice was to control the excessive synthesis of angiotensinogen in hepatocytes stimulated by glucocorticoids.

[0126] Thus, it can be seen that the TLR1 receptor has important significance in the mechanism of controlling noise-induced neurogenic hypertension, and the juxtaglomerular cells of the kidney and hepatocytes of the liver are the target cells for the TLR1 receptor to play its role. Controlling the synthesis and secretion of renin and angiotensinogen by inhibiting the formation of the TLR1 / 2 receptor heterodimer to achieve the mechanism of inhibiting the level of angiotensin II and inhibiting the activity of the renin-angiotensin-aldosterone system to control the occurrence of hypertension has good clinical application value, providing a sufficient theoretical basis and experimental foundation for the research and development of new antihypertensive drugs.

Claims

1. Application of TLR1 receptor inhibitors in the preparation of products for the treatment of hypertension.

2. The use according to claim 1, characterized in that: The TLR1 receptor inhibitor includes at least one of the following: (a1) Substances that inhibit TLR1 protein activity; (a2) substances that reduce the content of TLR1 protein; (a3) substances for knocking out the TLR1 gene; (a4) A substance that inhibits TLR1 gene expression.

3. The use according to claim 2, characterized in that: The TLR1 receptor inhibitors include nucleic acid molecules, protein molecules, and small molecule compounds; Preferably, the nucleic acid molecule is microRNA, siRNA, shRNA, dsRNA, sgRNA and / or antisense oligonucleotide; Preferably, the protein molecule is a specific antibody for TLR2; Preferably, the small molecule compound is a small molecule compound that binds to TLR2.

4. The use according to claim 3, characterized in that: The small molecule compound includes CU-CPT22.

5. The use according to claim 1, characterized in that: The products include pharmaceuticals.

6. The use according to claim 5, characterized in that: The drug includes pharmaceutically acceptable excipients, and / or any one or more other active ingredients.

7. The use according to claim 5, characterized in that: The pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

8. The use according to claim 7, characterized in that: The dosage form of the product includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration; Preferably, the dosage form for administration via the gastrointestinal tract includes at least one of powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet; Preferably, the non-intestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

9. The use according to claim 8, characterized in that: The product is administered to a mammal; Preferably, the mammal comprises a human.

10. Application of TLR1 as a drug target in the prevention and / or treatment of hypertension; The use is for non-diagnostic, non-therapeutic purposes.

Images