Use of a tlr1 receptor inhibitor in the manufacture of a medicament for treating hypertension

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of TLR1 receptor inhibitors in the preparation of products for treating hypertension. Existing drugs used clinically for hypertension have varying mechanisms of action. This study discovers a novel mechanism for treating hypertension: by inhibiting TLR1 receptors in juxtaglomerular cells of the kidney and hepatocytes of the liver, sympathetic nerve excitation-induced renin synthesis and glucocorticoid-induced angiotensinogen synthesis are suppressed, thereby inhibiting angiotensin II levels. This mechanism and method provide a new treatment option for hypertension, particularly neurogenic hypertension, and can also exhibit synergistic effects with existing therapeutic drugs. This invention identifies the TLR1 receptor as a novel target for treating hypertension. Currently, there are no developed and applied hypertension treatment drugs mediated by TLR1 receptors. This invention provides sufficient theoretical and experimental basis for the development of novel antihypertensive drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of TLR1 receptor inhibitors in the preparation of products for treating hypertension. Background Technology

[0002] Renin is a proteolytic enzyme, also known as angiotensinogenase, synthesized and secreted by juxtaglomerular (JG) cells in the kidney. These cells are located at the tips of the afferent arterioles at the glomerular inlet. Angiotensinogen (an α-globulin) is primarily synthesized and secreted by hepatocytes. The renin-angiotensin-aldosterone system (RAAS) is a hormonal system and an important blood pressure regulatory system. When blood pressure decreases, the kidneys secrete renin. Renin catalyzes the hydrolysis of angiotensinogen to produce angiotensin I. Angiotensin I has virtually no biological activity. Instead, it is cleaved by angiotensin-converting enzyme, which breaks down the two C-terminal amino acid residues to form angiotensin II. Angiotensin II has a highly effective vasoconstrictive effect, thereby increasing blood pressure. Angiotensin II also stimulates the adrenal cortex to secrete aldosterone. Aldosterone promotes the reabsorption of water and sodium ions by the kidneys, thereby increasing fluid volume and raising blood pressure. Therefore, renin and angiotensinogen can regulate blood pressure through the renin-angiotensin-aldosterone system, playing an important role in controlling hypertension.

[0003] Currently, the main types of hypertension medications used in clinical practice are divided into seven categories:

[0004] 1. Calcium Channel Blockers (CCBs): They mainly exert their effect of dilating blood vessels and lowering blood pressure by blocking calcium ion channels on vascular smooth muscle cells.

[0005] 2. Angiotensin-converting enzyme inhibitors (ACEIs): Their mechanism of action is to inhibit angiotensin-converting enzyme, blocking the production of angiotensin II, thereby exerting a hypotensive effect.

[0006] 3. Angiotensin receptor blockers (ARBs): Their mechanism of action is to block angiotensin II type 1 receptors to exert a blood pressure-lowering effect.

[0007] 4. Diuretics: They mainly exert their antihypertensive effect by promoting sodium excretion and reducing volume overload.

[0008] 5. Beta-blockers: They primarily exert their antihypertensive effect by inhibiting overactive sympathetic nerve activity, suppressing myocardial contractility, and slowing heart rate. They can also exert their antihypertensive mechanism by blocking β1-receptors in juxtaglomerular organelle cells, thus inhibiting renin release.

[0009] 6. Alpha receptor blockers: These selectively bind to alpha adrenergic receptors, blocking the binding of corresponding neurotransmitters and drugs to alpha receptors, thus producing an anti-adrenergic effect.

[0010] 7. Renin inhibitors: Their mechanism of action is to directly inhibit the activity of renin in the blood, thereby reducing the production of angiotensin II and lowering blood pressure levels in hypertensive patients. For example, aliskiren mainly binds to free renin in the blood and inhibits its activity.

[0011] TLR1 is one of the Toll-like receptors (TLRs) 1-10, and no studies have been reported on the correlation between TLR1 receptors and hypertension. Summary of the Invention

[0012] The first aspect of this invention aims to provide the application of TLR1 receptor inhibitors in the preparation of products for treating hypertension.

[0013] A second aspect of the present invention aims to provide the use of TLR1 as a drug target in drugs for the prevention and / or treatment of hypertension.

[0014] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0015] A first aspect of the present invention provides the use of a TLR1 receptor inhibitor in the preparation of a product for treating hypertension.

[0016] TLR1 is one of the 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) Substances that inhibit the activity of TLR1 protein;

[0019] (a2) Substances that reduce TLR1 protein levels;

[0020] (a3) Substances that knock out the TLR1 gene;

[0021] (a4) Substances that inhibit TLR1 gene expression.

[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 TLR2.

[0026] In some embodiments of the present invention, the small molecule compound includes CU-CPT22, whose chemical name is 3,4,6-trihydroxy-2-methoxy-5-oxo-5H-benzocycloheptene-8-carboxylic acid hexyl ester, and whose molecular formula is C 19 H 22 O7, the structural formula is as follows:

[0027]

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

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

[0030] In some embodiments of the invention, the pharmaceutically acceptable modifications include at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation. For example, lipophilicity can be enhanced and blood-brain barrier permeability improved by introducing a fluorine atom (fluoroethyl). The appropriate modification method can be selected based on 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 medicament includes pharmaceutically acceptable excipients 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 the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, release inhibitors, and carriers.

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

[0035] The drug is present in the form of an oral preparation, an injection, or a topical preparation.

[0036] In particular, the oral preparations include tablets, capsules, pills, powders, granules, syrups, or solutions; the injectable preparations include injection solutions or lyophilized powder for injection; and the topical preparations include creams, ointments, sprays, aerosols, or patches.

[0037] The pharmaceutical formulation uses a TLR1 receptor inhibitor (especially CU-CPT22) as the active ingredient and includes other pharmaceutically acceptable carrier components.

[0038] Carriers in pharmaceuticals include excipients such as starch and water; lubricants such as magnesium stearate; disintegrants such as microcrystalline cellulose; fillers such as lactose; binders such as pregelatinized starch and dextrin; sweeteners; antioxidants; preservatives; flavoring agents; and fragrances.

[0039] The carriers used in the preparation of oral formulations can be conventional pharmaceutical excipients such as starch, dextrin, cyclodextrin, various chemically modified cyclodextrins, sucrose, and stearates. Lyophilized powder injections can be prepared using methods such as aseptic spray drying, low-temperature vacuum drying, and freeze-drying. The subsequent preparation processes and equipment for each formulation are all conventional technologies in the pharmaceutical field, and this invention does not limit their application.

[0040] The drugs described in this invention exist in the form of tablets, capsules, pills, powders, granules, syrups, solutions, injections, sprays, aerosols, patches, gels, and poultices. That is, the drug preparations include, but are not limited to, the forms of tablets, capsules, pills, powders, granules, syrups, solutions, injections, sprays, aerosols, patches, gels, and poultices.

[0041] Furthermore, to facilitate medication, the active ingredient, a TLR1 receptor inhibitor (especially CU-CPT22), can be formulated into a specific dosage form with one or more pharmaceutically acceptable excipients. These excipients can be diluents (e.g., starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, and microcrystalline cellulose), absorbents (e.g., calcium sulfate, dicalcium phosphate, light magnesium oxide, and calcium carbonate), wetting agents (e.g., water and ethanol), binders (e.g., hydroxypropyl methylcellulose, povidone, starch paste, and syrup), disintegrants (e.g., dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, and crospovidone), and lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, and micronized powders). The following are examples of agents: silica gel, colorants (such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide), coating materials (such as acrylic resin, hydroxypropyl methylcellulose, and povidone), solvents (such as water for injection, ethanol, propylene glycol, and glycerin), acid-base adjusters (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid, and sodium tartrate), antioxidants (such as sodium sulfite, sodium metabisulfite, and sodium thiosulfate), antibacterial agents (such as phenol, benzyl alcohol, and thimerosal), and isotonic adjusters (such as sodium chloride and glucose).

[0042] The pharmaceutically acceptable excipients mentioned above are generally recognized for use in this purpose and as inactive ingredients in the pharmaceutical preparation. Compilations of pharmaceutically acceptable excipients can be found in reference books such as the *Handbook of Pharmaceutical Excipients* (2nd edition, edited by A. Wade and P.J. Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical 6Gess, London, 1994) and the *Pharmacopoeia of the People's Republic of China - List of Pharmaceutical Excipients*.

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

[0044] In some embodiments of the invention, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

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

[0046] In some embodiments of the invention, the product is applied to mammals.

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

[0048] A second aspect of the invention provides the use of TLR1 as a drug target in drugs for the prevention and / or treatment of hypertension.

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

[0050] The beneficial effects of this invention are:

[0051] The mechanisms of action of existing drugs used in clinical hypertension treatment vary. This study found that by inhibiting the TLR1 receptors in juxtaglomerular cells of the kidney and hepatocytes of the liver, the synthesis of renin induced by sympathetic nerve excitation and the synthesis of angiotensinogen induced by glucocorticoids are suppressed, thereby inhibiting the amount of angiotensin II. This is a novel mechanism for treating hypertension. This mechanism and method provide a new treatment option for hypertension, especially for neurogenic hypertension, and may also have a synergistic effect with existing therapeutic drugs.

[0052] The TLR1 receptor is a novel target for the treatment of hypertension. Currently, there are no drugs for the treatment of hypertension mediated by the TLR1 receptor, which provides sufficient theoretical basis and experimental foundation for the research and development of new antihypertensive drugs. Attached Figure Description

[0053] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0054] Figure 1 This is the result of the Renin protein blot in the mouse kidney cortex in Example 4 of the present invention.

[0055] Figure 2 This is the result of AGT protein blot in mouse liver in Example 5 of the present invention.

[0056] Figure 3 This is the result of Renin protein blotting in juxtaglomerular cells in Example 7 of the present invention.

[0057] Figure 4 This is the result of the TLR1 inhibitor's effect on adrenaline-induced AGT protein expression in high-AGT hepatocytes in Example 9 of the present invention.

[0058] Figure 5 This is the result of the TLR1 inhibitor blot on cortisol-induced AGT protein expression in high-AGT hepatocytes in Example 9 of the present invention. Detailed Implementation

[0059] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0060] Example 1: Construction of a Hypertensive Mouse Model

[0061] Currently, there are many types of animal models of hypertension used in experimental research, and noise-induced hypertension is one of the commonly used ones. The main mechanism of noise-induced hypertension is that noise exposure stimulates the auditory organs to generate nerve impulses, which are transmitted to the sympathetic nervous system through the central nervous system, leading to sympathetic nerve excitation. This causes changes in the functional state of the pituitary-adrenergic axis, which in turn promotes the secretion of more catecholamine hormones (norepinephrine and epinephrine) by the chromaffin cells of the adrenal medulla. These hormones act on adrenergic receptors in blood vessels, heart, and kidneys, resulting in increased cardiac output, activation of the renin-angiotensin system (stimulation of increased angiotensin II), and sustained tension and contraction of smooth muscle in the vascular wall, thus causing an increase in blood pressure. This study constructed a hypertensive mouse model using a noise exposure model, and the specific methods are as follows:

[0062] All experimental animals were 7-week-old SPF-grade male c57bl / 6j mice, weighing approximately 14-16g (Guangdong Provincial Medical Laboratory Animal Center). The experimental procedures were approved by the Animal Ethics Committee of Guangdong Pharmaceutical University (No.: gdpulacspf2022124). Mice were housed in an environment with a temperature of 20±0.5℃, relative humidity of 55%±5%, a light / dark cycle of 12h, and background noise less than 40dB SPL. Ten mice were exposed to a noisy environment using a 50x50x40cm noise exposure chamber with white noise at 103dB SPL, for 12 hours daily for 7 days. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MBP, diastolic blood pressure + 1 / 3 pulse pressure) were measured before and after noise exposure using a Softron BP-2010A intelligent non-invasive mouse blood pressure monitor (Softron, Beijing, China). The results showed that there were significant differences in systolic blood pressure between mice before and after noise exposure (t = -12.55, P < 0.01), diastolic blood pressure between mice before and after noise exposure (t = -4.72, P < 0.01), and mean arterial pressure between mice before and after noise exposure (t = -6.74, P < 0.01). After noise exposure, systolic blood pressure, diastolic blood pressure, and mean arterial pressure in mice all increased significantly (see Table 1).

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

[0064]

[0065] The experimental results demonstrate that a hypertensive mouse model was successfully constructed using this noise exposure method.

[0066] Example 2: Effect of TLR1 receptor inhibitors on blood pressure in hypertensive mice.

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

[0068] Blood pressure was measured in mice one day before the experiment to serve as a baseline. Mice were then randomly divided into six groups (n=12 per group): Control, Noise, Noise + CU-CPT22 (low concentration), Noise + CU-CPT22 (medium concentration), Noise + CU-CPT22 (high concentration), and CU-CPT22. The noise exposure period lasted 14 days. During the experiment, mice were exposed to noise and injected intraperitoneally with the drug daily. On day 14, after blood pressure measurements were completed, the animals were anesthetized and sacrificed for sampling. The TLR1 receptor inhibitor CU-CPT22 was administered at three concentrations: low (0.5 mg / kg), medium (2.5 mg / kg), and high (5.0 mg / kg), with an intraperitoneal injection volume of 0.2 ml / day. The Control group received an equal volume of physiological saline intraperitoneally.

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

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

[0071]

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

[0073] One-way ANOVA was performed on the blood pressure values ​​of the six groups of mice, revealing significant differences in systolic blood pressure, diastolic blood pressure, and mean arterial pressure among the six groups. Further multiple comparison analysis showed that the systolic blood pressure and mean arterial pressure were significantly increased in the Noise group and the Noise+CU-CPT22 L and M groups (P<0.01, P<0.01), while there was no difference in blood pressure between the Noise+CU-CPT22H group and the CU-CPT22 group (P>0.05). Compared with the Noise group, the systolic blood pressure and mean arterial pressure were significantly decreased in the Noise+CU-CPT22 M and H groups (P<0.01, P<0.01). There were no significant differences in diastolic blood pressure, systolic blood pressure, and mean arterial pressure between the CU-CPT22 group and the Contro group (P>0.05).

[0074] The results showed that simply inhibiting the TLR1 receptor had no effect on blood pressure in normal mice; however, in mice with noise-induced hypertension, inhibiting the TLR1 receptor had a significant inhibitory effect on blood pressure, and this inhibitory effect became more pronounced with increasing inhibitor concentration.

[0075] Example 3: Effects of TLR1 receptor inhibitors on blood pressure-related factors in hypertensive mice.

[0076] Since there are no relevant research reports, the mechanism by which inhibiting TLR1 receptors in hypertensive mice controls blood pressure is still unknown. In this example, the ELISA method was used to detect seven hypertension-related factors or indicators in mouse serum: angiotensin II (Ang II), renin, plasma renin activity (PRA), angiotensinogen (AGT), endothelin (ET), aldosterone (ALD), and atrial natriuretic peptide (ANP).

[0077] Specific methods: After 14 days of noise exposure, mice were anesthetized with 0.2 ml / 20 g of 4% chloral hydrate after blood pressure measurement. Whole blood was collected using the orbital blood sampling method into anticoagulant-free blood collection tubes and incubated at room temperature for 30 min. The tubes were then centrifuged at 1300 g for 10 min at 4°C. The supernatant was transferred to new centrifuge tubes. A second centrifugation was performed at 16,000 g for 10 min at 4°C. The assay procedure followed the ELISA kit instructions.

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

[0079] Table 3. Effects of TLR1 receptor inhibitors 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: renin activity; ET: endothelin; Ang II: angiotensin II.

[0082] The results showed that there were significant differences in blood pressure-related active factors among the experimental groups (P<0.001) according to the analysis of variance. Intergroup comparisons revealed that, compared with the Control group, the Noise group mice showed significantly higher 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) levels after noise exposure. Compared with the Noise group, the Noise+CU-CPT22 H group showed significantly lower 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). Compared with the Noise+CU-CPT22L group, the Noise+CU-CPT22 H group showed significantly lower 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).

[0083] These results indicate that after noise exposure-induced hypertension, TLR1 receptor inhibitors primarily affect blood pressure changes by inhibiting serum aldosterone, angiotensinogen, renin, renin activity, and angiotensin II levels. This suggests that TLR1 receptor inhibitors exert their antihypertensive effect by inhibiting the renin-angiotensin-aldosterone system. The target sites are speculated to be juxtaglomerular cells that synthesize renin and hepatocytes that synthesize angiotensinogen, requiring further experimental confirmation.

[0084] Example 4: Detection of renin in mouse kidney tissue

[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 RNA reverse transcription was carried out 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 carried out according 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 of 2× 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, cycles of 95 °C for 10 s, 60 °C for 22 s, and 72 °C for 30 s for 44 times, and fluorescence signals were obtained at 72 °C; after the cycle ended, the temperature was raised from 72 °C to 94 °C to obtain the melting curve. Data analysis was carried out using the Manager Software 1.6 data analysis software supporting the Real Time PCR instrument, and the analysis method used 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 at low temperature with lysis buffer, 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, constant voltage 70V×20min + 130V×30min; transferred at a constant current of 400 ma for 50 min, blocked at room temperature for 1 h; GAPDH: 1:1000; Renin: 1:3000, incubated with 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 (P<0.05). Compared with the Noise group, the protein expression level of Renin in the Noise+CU-CPT22 L, M, and H groups was significantly decreased (P<0.05). qRT-PCR was used to detect the Renin mRNA expression level in the renal cortex tissue of mice in each group. Results: Compared with the Control group, the Renin mRNA expression level in the Noise group, Noise+CU+CPT22 L group, and Noise+CU+CPT22 M group was significantly increased (P<0.05). Compared with the Noise group, the Renin mRNA expression level in the Noise+CU+CPT22M group and Noise+CU+CPT22 H group was significantly decreased (P<0.05).

[0088] The experimental results suggest that noise exposure significantly promotes renin synthesis in the kidneys, while TLR1 receptor inhibitors significantly inhibit the increase in renin synthesis induced by noise. However, they have no significant effect on the synthesis and activity of renin in normal kidneys. Combined with the results of inhibition of serum renin activity, this indicates that TLR1 receptor inhibitors are an important factor in controlling renin synthesis in the kidneys and thus controlling noise-induced hypertension. However, further in vitro cell experiments are needed to verify this.

[0089]

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

[0091] PCR detection of AGT mRNA expression levels in liver tissue: The method was the same as that used for Renin detection in kidney tissue; primer sequences are shown in Table 6. Results are shown in Table 7.

[0092] Western blotting was used to detect AGT protein expression levels in liver tissue: the method was the same as that used for renin detection in kidney tissue; the results are shown in Table 7. Figure 2 .

[0093] The results showed that, compared with the Control group, the AGT protein expression level in the Noise group was significantly increased (P<0.05). Compared with the Noise group, the AGT protein expression levels in the Noise+CU-CPT22L, Noise+CU-CPT22M, and Noise+CU-CPT22M groups were significantly decreased (P<0.05). RNA was extracted from the liver tissue of mice in each group and qRT-PCR was used 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, Noise+CU+CPT22L group, and Noise+CU+CPT22M group were significantly increased (P<0.05), while compared with the Noise group, the AGT mRNA expression levels in the Noise+CU+CPT22M and Noise+CU+CPT22H groups 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 TLR1 receptor inhibitors can significantly inhibit the increase in hepatic angiotensinogen synthesis caused by noise, but have no significant effect on the synthesis of angiotensinogen in normal liver. This indicates that TLR1 receptor inhibitors are an important factor in controlling hepatic angiotensinogen synthesis and noise-induced hypertension. However, further in vitro cell experiments are needed to verify this.

[0095]

[0096] Example 6: Construction of an in vitro juxtaglomerular cell line high-renin synthesis cell pathological model

[0097] Juxtaglomerular cells are epithelial-like cells transformed from smooth muscle cells in the walls of afferent arterioles near the vascular pole of the renal corpuscle. Their main function is to synthesize and secrete renin. Since juxtaglomerular cells possess adrenergic receptors (β2 receptors) and are innervated by the sympathetic nervous system, and sympathetic nerve excitation leading to increased blood pressure is an important mechanism by which noise exposure induces elevated blood pressure, stimulating cultured juxtaglomerular cells with epinephrine (E) to synthesize renin and constructing a high-renin-synthesizing juxtaglomerular cell pathological model can provide an effective research platform for this project.

[0098] The mouse juxtaglomerular cell line (Shanghai Yaji Biotechnology Co., Ltd.) was cultured at 37℃ with 5% CO2. Because renin is a secreted protein, its intracellular protein level cannot represent the true gene expression level. Therefore, qPCR was used to detect renin mRNA levels, which can more reasonably represent the true expression of this gene. To investigate the optimal concentration and reaction time for adrenaline to promote renin synthesis in juxtaglomerular cells and to construct the most reasonable high-renin-synthesis juxtaglomerular cell pathological model, experiments were conducted using different concentration gradients of adrenaline and different stimulation times. The results are shown in Table 8. The renin mRNA expression level in juxtaglomerular cells showed a dose-response relationship with adrenaline, with the synthesis peak at 1 μM adrenaline stimulation for 15 min. Therefore, the reaction conditions of 1 μM adrenaline concentration and 15 min intervention time are the best reaction conditions for constructing a high-renin-synthesis juxtaglomerular cell pathological model.

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

[0100]

[0101] Example 7: Effect of TLR1 receptor inhibitors on renin synthesis in high-renin-synthesizing juxtaglomerular cells

[0102] Juxtaglomerular cells were seeded in 6-well plates. Epinephrine was diluted to 1 μM with culture medium, and CU-CPT22 stock solution was diluted to three concentrations: 0.25 μM, 0.5 μM, and 1 μM. The juxtaglomerular cells were digested and counted, and then divided into groups of 2 × 10⁶ cells / well. 6 The renin mRNA and renin protein expression levels were seeded onto 6-well plates. Experimental groups included a Control group, a 1 μM epinephrine group, a 1 μM epinephrine + 0.25 μM CU-CPT22 group, a 1 μM epinephrine + 0.5 μM CU-CPT22 group, and a 1 μM epinephrine + 1 μM CU-CPT22 group. The Control group received an equal volume of drug-free culture medium. Each group was divided into three replicates. The plates were incubated at 37°C and 5% CO2. After 15 minutes, samples were collected to detect renin mRNA and renin protein expression levels (PCR, Western blotting). Results are shown in Table 9. Figure 3 .

[0103] Table 9. Effects of TLR1 receptor inhibitors on Renin mRNA and Renin expression levels in high-renin-synthesizing juxtaglomerular cells

[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 different concentrations of CU-CUT22, the expression level of Renin mRNA decreased with increasing 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 different concentrations of CU-CUT22, the expression level of Renin mRNA decreased with increasing CU-CPT22 concentration, and the difference was statistically significant (F=55.572, P<0.01).

[0107] The experimental results suggest that juxtaglomerular cells, under adrenaline stimulation, can increase renin synthesis and secretion by activating β1 receptors, consistent with the theory that sympathetic nerve stimulation induces renin synthesis. This indicates that the mechanism of noise-induced hypertension aligns with the mechanism of sympathetic nerve excitation stimulating juxtaglomerular cells to synthesize and secrete large amounts of renin. TLR1 receptor inhibitors significantly inhibited renin synthesis in high-renin-synthesizing juxtaglomerular cells, but had no significant effect on renin synthesis in normal juxtaglomerular cells. This is consistent with the experimental results of TLR1 receptor inhibitors inhibiting blood pressure changes in mice with noise-induced hypertension, further confirming that TLR1 receptor inhibitors controlling renin synthesis in renal juxtaglomerular cells are an important factor in controlling noise-induced hypertension, and that TLR1 receptors are important targets for controlling renin and its activity in the body.

[0108] Example 8: Construction of an in vitro hepatocyte cell line with high angiotensinogen synthesis-type cellular pathological model

[0109] Hepatocytes are the main secretory organs of angiotensinogen (AGT), which is a rate-limiting substrate of the renin-angiotensin system (RAS). AGT is catalyzed by reninase to produce angiotensin I (ANGI). ANGI is then catalyzed by angiotensin-converting enzyme (ACE) to produce angiotensin II (ANGII). ANGII can regulate blood pressure.

[0110] Normal mouse hepatocytes (AML12) (Shanghai Jinyuan Biotechnology Co., Ltd.) were cultured at 37℃ with 5% CO2. Because angiotensinogen is a secreted protein, intracellular protein levels cannot represent the true gene expression level. Therefore, qPCR was used to detect AGT mRNA levels in hepatocytes, which can more reasonably represent the true expression of this gene. To investigate the optimal stimulation concentration and time of adrenaline and cortisol (hydrocortisone) on AML12 cells, experiments were conducted with different concentration gradients and stimulation times. The results are shown in Tables 10 and 11. A dose-response relationship was observed between AGT mRNA expression in AML12 cells and adrenaline. The peak synthesis of AGT occurred at 1 μM adrenaline stimulation for 60 min. Therefore, a hyperangiotensinogen cell pathological model induced by adrenergic receptor stimulation was established using a concentration of 1 μM adrenaline and an intervention time of 60 min. The expression level of AGT mRNA in AML12 cells showed a dose-response relationship with cortisol at 15 min and 60 min. The peak of AGT synthesis occurred at a cortisol concentration of 1.5 μM at 60 min of stimulation. Therefore, the experiment used a cortisol concentration of 1.5 μM and an intervention time of 60 min to establish a cortisol receptor-induced hyperangiotensinogen cell pathological model.

[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 cortisol concentrations and stimulation times on AGT synthesis in AML12 cells

[0114]

[0115] Example 9: Effects of TLR1 receptor inhibitors on angiotensinogen synthesis in a hepatocyte pathological model with high angiotensinogen levels.

[0116] AML12 cells were seeded in 6-well plates. Epinephrine was diluted to 1 μM with culture medium, and CU-CPT22 stock solution was diluted to three concentrations: 0.25 μM, 0.5 μM, and 1 μM. AML12 cells were digested and counted, and cells were divided into groups of 2 × 10⁻⁶ cells per well. 6The plates were seeded in 6-well plates; the experimental groups were: Control group, 1 μM epinephrine group, 1 μM epinephrine + 0.25 μM CU-CPT22 group, 1 μM epinephrine + 0.5 μM CU-CPT22 group, and 1 μM epinephrine + 1 μM CU-CPT22 group. The Control group was supplemented with an equal volume of drug-free culture medium. Each group was divided into 3 replicates and cultured at 37℃ and 5% CO2. After 60 min, samples were collected to detect AGT mRNA expression level and AGT protein expression level (PCR, Western blotting). The results are shown in Table 12. Figure 4 .

[0117] Table 12. Effects of TLR1 inhibitors on AGT mRNA and AGT expression levels in adrenaline-induced high-AGT hepatocytes

[0118]

[0119] The results showed that, compared with the control group, adrenaline stimulation significantly increased the expression levels of AGT mRNA and AGT protein in AML12 cells (P<0.05), while different concentrations of CU-CPT22 did not inhibit the increased expression levels of AGT mRNA and AGT protein in AML12 cells (P>0.05). Based on previous studies and the animal model used in this study, glucocorticoids can also stimulate AGT synthesis in hepatocytes. Considering the stimulatory effect of cortisol (glucocorticoids) secreted by the adrenal cortex on hepatocytes, further cortisol stimulation experiments were conducted to verify whether TLR1 / 2 receptor inhibitors could inhibit AGT synthesis.

[0120] AML12 cells were seeded in 6-well plates. Cortisol was diluted to 1.5 μM with culture medium, and CU-CPT22 was diluted to three concentrations: 0.25 μM, 0.5 μM, and 1 μM with culture medium. AML12 cells were digested and counted, and the cells were divided into groups of 2 × 10⁻⁶ cells. 6 The plates were seeded onto 6-well plates. Experimental groups included: Control group, 1.5 μM cortisol group, 1.5 μM cortisol + 0.25 μM CU-CPT22 group, 1.5 μM cortisol + 0.5 μM CU-CPT22 group, and 1.5 μM cortisol + 1 μM CU-CPT22 group. The Control group received an equal volume of drug-free culture medium. Each group had 3 replicates. The plates were incubated at 37℃ and 5% CO2 for 60 min, and AGT mRNA and protein expression levels were detected using PCR and Western blotting. Results are shown in Table 13. Figure 5 .

[0121] Table 13. Effects of TLR1 inhibitors on AGT mRNA and AGT expression levels in cortisol-induced high-AGT hepatocytes

[0122]

[0123] The results showed that, compared with the control group, cortisol stimulation significantly increased the expression levels of AGT mRNA and AGT protein in AML12 cells (P<0.01), and different concentrations of CU-CPT22 significantly inhibited the increased expression levels of AGT mRNA and AGT protein in AML12 cells (P<0.01).

[0124] Experimental results suggest that hepatocytes, under adrenaline stimulation, can increase the synthesis and secretion of angiotensinogen by activating β-receptors, indicating that the mechanism of noise-induced hypertension is related to sympathetic nerve excitation stimulating hepatocytes to synthesize and secrete large amounts of angiotensinogen. However, TLR1 receptor inhibitors cannot control the synthesis of angiotensinogen induced by sympathetic nerve excitation, which is inconsistent with animal experimental results. Further cortisol stimulation experiments demonstrated that glucocorticoids (cortisol) secreted by the adrenal cortex are also one of the reasons for stimulating the synthesis and secretion of large amounts of angiotensinogen in hepatocytes under noise exposure. Moreover, TLR1 receptor inhibitors can control the increase in angiotensinogen synthesis induced by cortisol stimulation. Therefore, it is inferred that sympathetic nerve excitation and increased secretion of adrenal glucocorticoids are the main mechanisms by which noise exposure leads to increased angiotensinogen synthesis in hepatocytes, and are also one of the important mechanisms of noise-induced hypertension. The control of excessive angiotensinogen synthesis in hepatocytes stimulated by glucocorticoids by TLR1 receptor inhibitors is one of the main mechanisms by which they control the increase in blood pressure in mice.

[0125] Therefore, the TLR1 receptor plays a crucial role in controlling noise-induced neurogenic hypertension, with juxtaglomerular cells of the kidney and hepatocytes of the liver serving as target cells for its function. Controlling the synthesis and secretion of renin and angiotensinogen by inhibiting the formation of TLR1 / 2 receptor heterodimers, thereby suppressing angiotensin II levels and inhibiting the activity of the renin-angiotensin-aldosterone system, has significant clinical application value in controlling the pathogenesis of hypertension and provides a strong theoretical and experimental basis for the development of novel antihypertensive drugs.

Claims

1. Application of TLR1 receptor inhibitors in the preparation of drugs for treating hypertension; The TLR1 receptor inhibitor is CU-CPT22.

2. The application according to claim 1, characterized in that: The drug includes pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that: The dosage forms of the drug include those administered via the gastrointestinal tract or those administered outside the gastrointestinal tract.

4. The application according to claim 3, characterized in that: The gastrointestinal dosage forms include at least one of the following: powder, tablet, granule, capsule, sustained-release, solution, dry suspension, emulsion, suspension, syrup, and drops; The non-gastrointestinal dosage forms include at least one of the following: injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

5. The application according to claim 4, characterized in that: The drug is administered to mammals; The mammals mentioned include humans.

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