Use of MrgD as a target in the preparation of drugs for treating and / or preventing hypertension

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to the application of MrgD as a target in the preparation of drugs for treating and / or preventing hypertension. The present invention verified the role of MrgD as an inhibitory target in a hypertensive mouse model through three aspects: gene knockout, small molecule inhibitors, and interfering RNA, all of which showed good antihypertensive effects, providing more possibilities for the application of the MrgD target in the prevention and treatment of hypertension diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the use of MrgD as a target in the preparation of drugs for treating and / or preventing hypertension. Background Art

[0002] Hypertension is a common and frequently-occurring disease in clinic and an important independent risk factor for cardiovascular and cerebrovascular events. The incidence of hypertension increases year by year, and its harm is mainly target organ damage, including cardiovascular remodeling, stroke, renal failure, eye diseases, etc. The regulation of hypertension mainly includes hormonal regulation and neural regulation. The former is mainly regulated by the renin-angiotensin-aldosterone system (RAAS). Clinically recommended direct renin inhibitors, angiotensin-converting enzyme inhibitors, and angiotensin receptor antagonists can all be used as first-line treatment options for hypertension. They can form "upstream, midstream, and downstream" multi-target interventions and constitute an effective inhibition of the entire RAAS process, forming "upstream, midstream, and downstream" multi-target interventions and constituting an effective inhibition of the entire RAAS process. Among them, angiotensin receptors are an important part of the renin-angiotensin system and are G protein-coupled receptors with angiotensin as a ligand.

[0003] G protein-coupled receptors are a group of membrane surface glycoproteins coupled with GTP-binding proteins, which are composed of transmembrane subunits formed by 7 polypeptide chains, forming a spatial conformation of three extracellular loops and three intracellular loops. It is the largest class of receptors discovered so far and is widely distributed in various organs and tissues. Main receptors on blood vessels such as angiotensin II receptors, adrenergic receptors, cholinergic receptors, prostatic receptors, 5-hydroxytryptamine receptors, etc. all belong to G protein-coupled receptors, and they play a leading role in blood pressure regulation. In patients with hypertension, the discovered anti-G protein-coupled receptor autoantibodies mainly include: angiotensin II type I receptor autoantibodies (anti-AT1 receptor antibodies), α1 adrenergic receptor autoantibodies (anti-α1 receptor antibodies), β1 adrenergic receptor autoantibodies (anti-β1 receptor antibodies), and M2 cholinergic receptor autoantibodies (anti-M2 receptor antibodies). Studies have found that these G protein-coupled receptor autoantibodies can mimic normal physiological signals (such as angiotensin II, adrenaline), activate the corresponding G protein-coupled receptors, and play roles similar to those of angiotensin II, adrenaline, etc.

[0004] Valsartan is an angiotensin II type I (AT1) antagonist, which can selectively block the binding of Ang II to the AT1 receptor, thereby inhibiting vasoconstriction and the release of aldosterone, producing a blood pressure lowering effect; it is used to treat mild to moderate essential hypertension; the exact blood pressure lowering effect can be achieved within 2 weeks of medication, and the maximum efficacy can be achieved after 4 weeks.

[0005] Research findings indicate that Mas-related G protein-coupled receptor D (MrgD) is a major receptor regulated by the renin-angiotensin system and plays an important role in the regulation of the renin-angiotensin system. However, there is currently little research on the MrgD target. D-Pro 7 -Ang-(1–7) is an inhibitor of MrgD for the receptor.

[0006] Chinese Patent with Publication No. CN112409473B discloses an MrgD immunogenic antigen fragment, an MrgD antibody, and their preparation methods and applications. This antibody can bind to rat Mas-related MrgD and has good specificity. Combined with immunofluorescence, it can quickly detect changes in the expression level of MrgD under cardiac pathophysiological conditions, providing a reliable basis for the occurrence and development process of the pathology.

[0007] Chinese Patent with Publication No. CN112168970A discloses a G protein-coupled receptor MrgD, which can be used in the prevention and treatment of myocardial hypertrophy and fibrosis diseases. Silencing or inhibiting the expression of MrgD can significantly reduce the degree of myocardial hypertrophy and fibrosis.

[0008] Chinese Patent with Publication No. CN115645408A provides a series of MrgD receptor inhibitors and drugs for improving myocardial hypertrophy and cardiac fibrosis by targeting MrgD, which can be used for the treatment of myocardial hypertrophy or cardiac fibrosis.

[0009] In the prior art, antibodies of G protein-coupled receptor MrgD, their preparation methods, and their applications in the prevention and treatment of myocardial hypertrophy and fibrosis diseases are disclosed. However, there is currently no research proving their application in the prevention and treatment of hypertension. Summary of the Invention

[0010] To solve the above problems, the present invention discovers that the technical effect of reducing hypertension can be achieved by silencing / inhibiting the MrgD receptor, and verification tests are conducted. It provides more possibilities for the application of the MrgD target in the prevention and treatment of hypertension diseases. The present invention specifically provides the application of MrgD as a target in the preparation of drugs for treating and / or preventing hypertension.

[0011] On the one hand, the present invention provides the application of MrgD as a target in the preparation of drugs for treating and / or preventing hypertension.

[0012] Specifically, the drugs targeting MrgD are administered to play a role in treating and / or preventing hypertension.

[0013] More specifically, the drugs that inhibit MrgD are administered to play a role in treating and / or preventing hypertension.

[0014] Specifically, the drug includes, but is not limited to, any one or more of MrgD antagonists, MrgD interfering RNAs, and MrgD gene knockout drugs.

[0015] On the other hand, the present invention provides the use of MrgD inhibitors in the preparation of drugs for treating and / or preventing hypertension.

[0016] Specifically, the drug includes, but is not limited to, any one or more of MrgD antagonists, MrgD interfering RNAs, and MrgD gene knockout drugs.

[0017] Preferably, the MrgD interfering RNA achieves MrgD inhibition by constructing an interfering virus.

[0018] Preferably, the MrgD interfering RNA includes any one or more of SEQ ID NO: 1-3.

[0019] More preferably, the MrgD interfering RNA includes SEQ ID NO: 1.

[0020] Preferably, the preparation method of the interfering virus includes: inserting the MrgD interfering RNA into an adenovirus vector.

[0021] More preferably, the preparation method includes: co-transfecting cells with the adenovirus vector and an auxiliary packaging plasmid.

[0022] Even more preferably, the adenovirus vector in the preparation method includes, but is not limited to, GV119; the cells include, but are not limited to, HEK293 cells.

[0023] In some embodiments, the preparation method includes: inserting the MrgD interfering RNA into an adenovirus vector, co-transfecting cells with the adenovirus vector and an auxiliary packaging plasmid; the adenovirus vector includes GV119; the cells include HEK293 cells.

[0024] Specifically, the MrgD antagonist includes, but is not limited to, D-Pro 7 -Ang-(1–7).

[0025] Specifically, the hypertension includes essential hypertension or secondary hypertension.

[0026] Preferably, the treatment and / or prevention of hypertension includes reducing systolic blood pressure, diastolic blood pressure, and / or mean arterial pressure.

[0027] On yet another aspect, a drug for treating and / or preventing hypertension.

[0028] Specifically, the drug includes any one or more of the interfering RNAs shown in SEQ ID NO: 1-3 or the interfering virus constructed by SEQ ID NO: 1-3.

[0029] Specifically, the drug includes, but is not limited to, injectable drugs or oral drugs, preferably injectable drugs.

[0030] Specifically, the drug also includes pharmaceutical excipients.

[0031] In some embodiments, the drug includes any one of SEQ ID NO: 1-3.

[0032] In some embodiments, the drug includes any one of the interfering viruses constructed by SEQ ID NO: 1-3.

[0033] Advantages of the present invention:

[0034] The present invention provides the application of MrgD as a target in the preparation of drugs for treating and / or preventing hypertension. The present invention verified the role of MrgD as an inhibitory target in a hypertensive mouse model through three aspects: gene knockout, small molecule inhibitors, and interfering RNAs, all showing good antihypertensive effects, providing more possibilities for the application of the MrgD target in the prevention and treatment of hypertension. Description of the drawings

[0035] Figure 1 Shows the experimental results of Ang II-induced hypertension in MrgD knockout mice.

[0036] Figure 2 Shows the experimental results of blood pressure reduction by the antagonist of MrgD.

[0037] Figure 3 Shows the plasmid map of the adenovirus carrying the target gene.

[0038] Figure 4 Shows the experimental results of blood pressure reduction by the MrgD interfering virus. Specific embodiments

[0039] The following combines specific embodiments to further elaborate on the present invention. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments, unless otherwise specified, and the experimental methods without specific conditions noted in the embodiments are usually carried out under conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0040] Example 1 Experiment on the improvement of Ang II-induced hypertension by MrgD knockout

[0041] Experimental procedures: MrgD knockout (MrgD KO) or wild-type (MrgD WT) mice (Cyagen Biosciences; CKOCMP-211578-Mrgprd-B6J-VA) were selected, with 6 mice in each group. A micro-osmotic pump (Alzet; 2004) was implanted subcutaneously to perfuse angiotensin II (Ang II) (Sigma; A9525; 1.44 mg / kg / day) to induce hypertension. A non-invasive tail artery blood pressure monitor (Kent; Coda4) was used to monitor the systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure of the two groups of mice via the tail artery every week.

[0042] Experimental results: The results are as Figure 1 shown. In the figure, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The results showed that compared with the Ang II + MrgD WT group, the systolic blood pressure, diastolic blood pressure, and mean arterial pressure in the Ang II + MrgD KO group were significantly decreased, indicating that MrgD gene knockout significantly improved Ang II-induced hypertension.

[0043] Example 2: Antihypertensive experiment of MrgD antagonist

[0044] Experimental procedures: Spontaneously hypertensive rats (SHR) (Beijing Vital River Laboratory Animal Technology Co., Ltd.; 121) were selected, with 6 rats in each group. In the experimental group, a sustained-release pump (Alzet; 2006) was implanted subcutaneously to slowly release the MrgD antagonist D-Pro 7 -Ang-(1–7) (Phoenix Pharmaceuticals; 002-15), and in the control group, normal saline (Saline) was slowly released. The systolic blood pressure, diastolic blood pressure, and mean arterial pressure were monitored via the tail artery every week.

[0045] Experimental results: The results are as Figure 2 shown. In the figure, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The results showed that compared with the Saline group, the blood pressure in the D-Pro 7 -Ang-(1–7) group was significantly decreased, indicating that blocking MrgD significantly improved hypertension.

[0046] Example 3: Antihypertensive experiment of MrgD interfering virus

[0047] (1) Preparation of MrgD interfering virus:

[0048] ① Design of interfering sequence

[0049] The MrgD Accession ID is NM_001001506. Three interfering sequences were designed as follows:

[0050] No. 1 (the sequence is SEQ ID NO:1) TGATTTGGCTACTGAGTTT;

[0051] No. 2 (the sequence is SEQ ID NO:2) TCAACTGGTTCTTACTCTA;

[0052] No. 3 (the sequence is SEQ ID NO:3) TCTTCATCCGCATGCGAAA.

[0053] ② Preparation of interfering virus

[0054] The preparation of the interfering virus was entrusted to GeneChem (Shanghai, China).

[0055] Taking the No. 1 interfering sequence as an example, the target gene fragment was obtained, and the adenovirus shuttle plasmid was constructed using GV119. The adenovirus shuttle plasmid carrying the target gene (such as Figure 3 ) was co-transfected with the helper packaging plasmid carrying most of the adenovirus genome (E1 / E3 deleted) into HEK293 cells (ATCC, cat#CRL-1573). Using the Cre-loxP (or FLP / frt) recombinase cleavage system, a non-replicating recombinant adenovirus carrying the foreign gene can be generated. The quality of the adenovirus was detected by the end-point dilution method, and the titers are shown in Table 1.

[0056] Using the end-point dilution method, the virus titer = 10 (x+0.8) (PFU / mL)*:

[0057] X —— the sum of the CPE positive rates at 10 -1 to 10 -13 successive dilution degrees;

[0058] * Conditions for using the formula:

[0059] A The negative control has no CPE and growth inhibition phenomenon;

[0060] B All wells with the virus crude extract at the minimum dilution concentration have CPE.

[0061] Table 1 Titers of each interfering virus

[0062]

[0063]

[0064] ③ Detection of virus interference efficiency

[0065] Detection process:

[0066] Primary rat vascular smooth muscle cells were cultured, and the preparation steps were as follows: Under sterile conditions, anesthetized SD rats (male, 8 weeks old, Vital River) were placed in the supine position and their chests were opened. The thoracic aorta was removed and transferred to a 10 cm cell culture dish (Corning, 430167) containing pre-cooled (4 °C) DMEM (Gibco, 11965092) with 0.5% double antibiotics (Gibco, 15140122). Immediately afterwards, it was successively immersed in 10 cm dishes containing DMEM with different concentrations of double antibiotics (5%; 2%; 1%; 0.5%), and the adipose tissue around the artery was carefully and gently removed. Subsequently, the artery was longitudinally cut and placed in another 10 cm cell culture dish containing pre-cooled DMEM. Then, the intima was gently scraped with a pair of curved ophthalmic forceps to remove endothelial cells. The artery was cut into small square pieces with a side length of about 1 mm and transferred to the wall of a T25 cell culture flask (Corning, 430168). A small amount of DMEM containing 20% fetal bovine serum (FBS; Gibco, 10099141C) was added to the bottom. The T25 cell culture flask was placed upright in the cell culture incubator for about 4 hours to allow the small tissue pieces to adhere to the wall of the flask. The flask was carefully laid down so that the culture medium submerged the artery pieces while the pieces did not fall off the wall of the flask. The tissue pieces were incubated in the cell culture incubator, and the medium was changed for the first time after 3 days. Thereafter, the medium was changed every 3 days until the bottom of the flask was covered with smooth muscle cells, and then digestion and subculture were carried out.

[0067] Three viruses numbered 1, 2, and 3 were respectively applied. After 24 hours, mRNA was extracted from each group and the infection efficiency was verified by PCR.

[0068] After verification, the result showed that No. 1 decreased the MrgD mRNA level most significantly; indicating that the interference efficiency of No. 1 was the best, and No. 1 was selected for subsequent experiments.

[0069] (2) MrgD interference virus blood pressure lowering experiment

[0070] Spontaneously hypertensive rats (SHRs) were selected, 6 rats in each group. Adenovirus-mediated MrgD interference virus (MrgD shRNA) was injected into the tail vein, and the injection dose was 10 9 PFU / rat; virus GFP shRNA (GeneChem) was used as a control, and systolic blood pressure, diastolic blood pressure, and mean arterial pressure were monitored via the tail artery weekly.

[0071] Experimental results: As Figure 4As shown in the figure, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The results showed that compared with the SHR-GFP shRNA group, the systolic blood pressure, diastolic blood pressure, and mean arterial pressure in the SHR-MrgD shRNA group were significantly decreased, indicating that interfering with the MrgD gene could significantly improve hypertension.

Claims

1. Use of MrgD inhibitor in the preparation of a medicament for treating hypertension, characterized in that, The drug described is MrgD interfering RNA, and the sequence of the MrgD interfering RNA is as shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that The MrgD interfering RNA inhibits MrgD by constructing interfering viruses.

3. The application according to claim 2, characterized in that, The method for constructing the interfering virus includes inserting the MrgD interfering RNA into an adenovirus vector.

4. The application according to any one of claims 1 to 3, characterized in that The hypertension described includes essential hypertension or secondary hypertension.

5. The application according to claim 4, wherein The treatment of hypertension includes reducing systolic blood pressure, diastolic blood pressure, and / or mean arterial pressure.

6. A drug for treating hypertension, characterized in that, It includes any one or more of the interfering RNA shown in SEQ ID NO:1 or the interfering virus constructed by SEQ ID NO:1.

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