Application of Hic1 inhibitors in the preparation of drugs for treating pulmonary hypertension

Abstract

The present invention relates to the field of biomedicine, and more particularly to the use of a Hic1 inhibitor in the preparation of a drug for treating pulmonary hypertension. The Hic1 inhibitor described herein is a molecule capable of degrading the mRNA of the Hic1 transcription factor. The Hic1 inhibitor obtained by the present invention can inhibit the expression of the Hic1 transcription factor, thereby achieving the therapeutic effect of pulmonary hypertension.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the use of a Hic1 inhibitor in preparing a drug for treating pulmonary hypertension. Background Art

[0002] Pulmonary hypertension (PAH) is a fatal disease characterized by a progressive increase in pulmonary vascular resistance, ultimately leading to right heart failure. Its pathological mechanisms involve pulmonary endothelial dysfunction, abnormal smooth muscle cell proliferation, and vascular remodeling, leading to vasoconstriction, thrombosis, and fibrosis. Common causes include idiopathic PAH, inherited gene mutations (such as BMPR2), connective tissue diseases (such as scleroderma), congenital heart disease, or drug / toxin-induced PAH. Clinical manifestations include dyspnea, fatigue, chest pain, and syncope, with signs of right heart failure (such as jugular venous distension and lower extremity edema) appearing in the late stages. Summary of the Invention

[0003] In order to solve the above technical problems, the present invention provides the use of a Hic1 inhibitor in the preparation of a drug for treating pulmonary hypertension. The Hic1 inhibitor of the present invention can inhibit the expression of the Hic1 transcription factor, thereby achieving the effect of treating pulmonary hypertension.

[0004] The present invention provides the use of the Hic1 inhibitor in the preparation of a drug for treating pulmonary hypertension, wherein the Hic1 inhibitor can inhibit the expression of the Hic1 transcription factor;

[0005] The Hic1 inhibitor includes a nucleic acid molecule capable of degrading the Hic1 transcription factor;

[0006] The Hic1 inhibitor includes at least one of antisense nucleic acid, siRNA, miRNA or shRNA; wherein,

[0007] The target sequence of the antisense nucleic acid, siRNA, miRNA or shRNA is located in any sequence on the mRNA of the Hic1 transcription factor.

[0008] Furthermore, the sequence of the siRNA includes a first sequence; wherein,

[0009] The sense strand of the first sequence includes the sequence shown in SEQ ID NO: 1, and the antisense strand of the first sequence includes the sequence shown in SEQ ID NO: 2.

[0010] Furthermore, the sequence of the siRNA includes a second sequence; wherein,

[0011] The sense strand of the second sequence includes the sequence shown in SEQ ID NO: 3, and the antisense strand of the second sequence includes the sequence shown in SEQ ID NO: 4.

[0012] Furthermore, the sequence of the siRNA includes a third sequence; wherein,

[0013] The sense strand of the third sequence includes the sequence shown in SEQ ID NO: 5, and the antisense strand of the third sequence includes the sequence shown in SEQ ID NO: 6.

[0014] Furthermore, the shRNA is constructed by siRNA targeting the mRNA of the Hic1 transcription factor;

[0015] The sequence of the siRNA includes a first sequence; wherein,

[0016] The sense strand of the first sequence includes the sequence shown in SEQ ID NO: 1, and the antisense strand of the first sequence includes the sequence shown in SEQ ID NO: 2;

[0017] Furthermore, the shRNA is constructed by siRNA targeting the mRNA of the Hic1 transcription factor; the sequence of the siRNA includes a second sequence; wherein,

[0018] The sense strand of the second sequence includes the sequence shown in SEQ ID NO: 3, and the antisense strand of the second sequence includes the sequence shown in SEQ ID NO: 4.

[0019] Furthermore, the shRNA is constructed by siRNA targeting the mRNA of the Hic1 transcription factor; the sequence of the siRNA includes a third sequence; wherein,

[0020] The sense strand of the third sequence includes the sequence shown in SEQ ID NO: 5, and the antisense strand of the third sequence includes the sequence shown in SEQ ID NO: 6.

[0021] As a feasible embodiment, the sequence of the siRNA includes:

[0022] Sense strand: having the sequence shown in SEQ ID NO: 1;

[0023] Antisense strand: has the sequence shown in SEQ ID NO: 2.

[0024] The present invention also provides a pharmaceutical composition for treating pulmonary hypertension, comprising: a Hic1 inhibitor, and a pharmaceutically acceptable carrier and excipients; wherein,

[0025] The definition of the Hic1 inhibitor is the same as the definition of the Hic1 inhibitor mentioned above.

[0026] Specifically, the Hic1 inhibitor refers to a nuclear molecule that can degrade the mRNA of the Hic1 transcription factor. The Hic1 inhibitor includes antisense nucleic acid, siRNA, miRNA or shRNA. The target sequence of the antisense nucleic acid, siRNA, miRNA or shRNA is located in any sequence on the mRNA of the Hic1 transcription factor.

[0027] Furthermore, the pharmaceutically acceptable carrier includes lipid nanoparticles or adeno-associated viruses.

[0028] As known feasible lipid nanoparticles, cationic liposomes LNP can be selected.

[0029] The present invention also discloses the use of a known Hic1 inhibitor in preparing a product having any of the following functions (1) to (4):

[0030] (1) Inhibit the expression of Hic1 transcription factor;

[0031] (2) Inhibit the transcription of Hic1 transcription factor;

[0032] (3) Alleviate pulmonary vascular inflammatory response;

[0033] (4) Pulmonary vascular remodeling;

[0034] The definition of the Hic1 inhibitor is the same as that of the aforementioned Hic1 inhibitor.

[0035] The embodiments of the present invention have the following technical effects:

[0036] 1. In the present invention, the Hic1 inhibitor targets the messenger RNA of the Hic1 transcription factor and degrades the mRNA to achieve gene silencing, thereby reducing the expression of the Hic1 transcription factor and ultimately achieving the purpose of treating pulmonary hypertension.

[0037] 2. The Si Hic1 / LNP of the present invention can alleviate the symptoms of rats with pulmonary hypertension and reduce inflammatory infiltration around the pulmonary vessels.

[0038] 3. During the course of pulmonary hypertension, the transcription factor Hic1 is recruited to be highly expressed in macrophages, regulating inflammation-related genes, promoting inflammatory infiltration of pulmonary vessels, and accelerating the course of pulmonary hypertension. Therefore, reducing the high expression of Hic1 can alleviate inflammation around pulmonary vessels and reduce pulmonary hypertension. Transcription factors lack a clear binding pocket and are traditionally considered "undruggable" for small molecules. The present invention utilizes the phagocytic function of recruited macrophages, uses LNP to carry siRNA, and reduces the expression of transcription factors in macrophages by recruiting macrophages to phagocytose Si Hic1 / LNP, thereby achieving the purpose of treating pulmonary hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0040] Figure 1 This is a schematic diagram of the Si-Hic1 / LNP connection provided by the present invention.

[0041] Figure 2 These are the particle size and zeta potential analysis results provided by the present invention, which represent LNP, Si-NC / LNP, and Si-Hic1 / LNP from left to right, respectively.

[0042] Figure 3 It is the expression level of Hic1 on macrophages provided by the present invention.

[0043] Figure 4 This is the expression of mRNA of Hic1 downstream inflammatory genes provided by the present invention.

[0044] Figure 5 This is a graph of mouse pulmonary hypertension data provided by the present invention.

[0045] Figure 6 This is the HE staining diagram provided by the present invention.

[0046] Figure 7 It is a Masson staining diagram provided by the present invention. DETAILED DESCRIPTION

[0047] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0048] In a first aspect, some embodiments of the present invention provide use of a Hic1 inhibitor in the preparation of a drug for treating pulmonary hypertension, wherein the Hic1 inhibitor refers to a nucleic acid molecule capable of degrading the mRNA of the Hic1 transcription factor.

[0049] In some embodiments, the Hic1 inhibitor comprises antisense nucleic acid, siRNA, miRNA or shRNA, wherein:

[0050] The target sequence of the antisense nucleic acid, siRNA, miRNA or shRNA is located in any sequence on the mRNA of the Hic1 transcription factor.

[0051] In some embodiments, the sequence of the siRNA may include a first sequence, a second sequence or a third sequence; the sense strand of the first sequence is SEQ ID NO: 1, and the antisense strand of the first sequence is SEQ ID NO: 2; the sense strand of the second sequence is SEQ ID NO: 3, and the antisense strand of the second sequence is SEQ ID NO: 4; the sense strand of the third sequence is SEQ ID NO: 5, and the antisense strand of the third sequence is SEQ ID NO: 6.

[0052] In some embodiments, the sequence of SEQ ID NO: 1 is GCUUUGGUGACAACCUGUATT.

[0053] In some embodiments, the sequence of SEQ ID NO: 2 is UACAGGUUGUCACCAAAGCTT.

[0054] In some embodiments, the sequence of SEQ ID NO: 3 is AGUUUGCUCAACAGCGCAATT.

[0055] In some embodiments, the sequence of SEQ ID NO: 4 is UUGCGCUGUUGAGCAAACUTT.

[0056] In some embodiments, the sequence of SEQ ID NO: 5 is CAAAUUCACUGCAGAGCUATT.

[0057] In some embodiments, the sequence of SEQ ID NO: 6 is UAGCUCUGCAGUGAAUUUGTT.

[0058] In some embodiments, the shRNA is constructed by siRNA targeting the mRNA of the Hic1 transcription factor, and the sequence of the siRNA includes a first sequence, a second sequence, or a third sequence; the sense chain of the first sequence is SEQ ID NO: 1, and the antisense chain of the first sequence is SEQ ID NO: 2; the sense chain of the second sequence is SEQ ID NO: 3, and the antisense chain of the second sequence is SEQ ID NO: 4; the sense chain of the third sequence is SEQ ID NO: 5, and the antisense chain of the third sequence is SEQ ID NO: 6.

[0059] In a second aspect, some embodiments of the present invention further provide a pharmaceutical composition for treating pulmonary hypertension, the pharmaceutical composition comprising: a Hic1 inhibitor, and a pharmaceutically acceptable carrier;

[0060] The Hic1 inhibitor refers to a molecule that can degrade the mRNA of the Hic1 transcription factor. The Hic1 inhibitor includes antisense nucleic acid, siRNA, miRNA or shRNA. The target sequence of the antisense nucleic acid, siRNA, miRNA or shRNA is located in any sequence on the mRNA of the Hic1 transcription factor.

[0061] In some embodiments, the pharmaceutically acceptable carrier comprises lipid nanoparticles or adeno-associated viruses.

[0062] Further verification is performed below in conjunction with specific embodiments.

[0063] Example 1 Construction of Si Hic1 / LNP

[0064] (1) Construction of siRNA:

[0065] Shanghai Hanheng Biotechnology synthesized small interfering siRNAs targeting the Hic1 transcription factor (SEQ ID NO: 1: GCUUUGGUGACAACCUGUATT, SEQ ID NO: 2: UACAGGUUGUCACCAAAGCTT, denoted as Si Hic1 molecules) and a comparative example Si-NC (the target site of the Si-NC is not on the Hic1 transcription factor mRNA, the positive strand is SEQ ID NO: 7, and the antisense strand is SEQ ID NO: 8. Sequence ID NO: 7 is UUCUCCGAACGUGUCACGUTT, and SEQ ID NO: 8 is ACGUGACACGUUCGGAGAATT). In sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 8, the TT sequence at the 3' end of the siRNAs helps Dicer more effectively cleave the siRNA, improving RNA efficiency.

[0066] (2) Construction of lipid nanoparticles:

[0067] DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium sulfate) (Shanghai Aiweituo Co., Ltd.) and cholesterol (Shanghai Aiweituo Co., Ltd.) were mixed in a 1:1 molar ratio, and the resulting powdered lipid mixture was dissolved in chloroform in a 50 mL round-bottom flask. The mixed solution was then rotated on a rotary evaporator (EYELA, Japan) at 30°C for 5 minutes to form a thin film. The flask containing the thin lipid film was dried under vacuum for 15 minutes. The film was then hydrated in 5% glucose to a final concentration of 20 mM DOTAP and 20 mM cholesterol. The hydrated lipid film was rotated in a 50°C water bath for 45 minutes, followed by rotation at 35°C for 10 minutes. The flask, covered with parfilm, was left at room temperature overnight. Low-frequency sonication was then applied at 50°C for 5 minutes, followed by heating at 50°C for 10 minutes. The mixture was sequentially extruded using a syringe through Whatman (Kent, UK) filters with filter sizes of 1 μm, 0.45 μm, 0.2 μm, and 0.1 μm to obtain cationic liposomes (LNPs).

[0068] (3) Connecting siRNA and lipid nanoparticles:

[0069] like Figure 1 According to the process shown, Si-Hic1 molecules, siRNA obtained from the comparative example, and LNPs were diluted in 5% glucose to prepare lipid nanoparticle-loaded siRNA. The Si-Hic1 solution and the comparative siRNA solution were added dropwise to the LNP solution at a mass ratio of 1:4. The mixture was incubated at room temperature for 30 minutes to prepare siRNA-loaded LNPs (experimental group: Si-Hic1 / LNP, comparative group: Si-NC / LNP).

[0070] The results of particle size and zeta potential analysis using Zetasizer NanoZS90 are as follows Figure 2 .

[0071] visible, Figure 2 The LNP carriers, Si-Hic1 / LNP and the comparative example Si-NC / LNP, were analyzed for particle size and zeta potential using a Zetasizer NanoZS90. After loading Si-Hic1 and Si-NC onto LNP, the zeta potential decreased from 43.7 mV to 27 mV (Si-Hic1 / LNP) and 29 mV (Si-NC / LNP), respectively, while the particle size increased from 157.3 nm to 179.3 nm and 173.4 nm, respectively. This indicates the successful formation of an electrostatic complex, indicating that the LNP and siRNA are electrostatically linked.

[0072] Example 2 Immunoblotting experiment

[0073] After incubating macrophages with Si Hic1 / LNP (100 nM, using 5% glucose as solvent) and comparative example Si NC / LNP (100 nM) for 48 hours, the expression level of Hic1 in macrophages was detected by immunoblotting. The results are shown in Figure 2. Figure 3 shown.

[0074] exist Figure 3 In the experiment, it was found that after 48 hours of incubation with Si Hic1 / LNP, the expression level of Hic1 on macrophages was effectively reduced. It can be seen that the drug combination with Hic1 inhibitor in the present invention can inhibit the expression of Hic1 transcription factor.

[0075] Example 3 qPCR detection of mRNA levels of inflammatory genes

[0076] After incubating macrophages with Si Hic1 / LNP (100 nM) for 48 h, the cells were collected for RNA extraction and RT-PCR detection of changes in the mRNA levels of Hic1 downstream inflammatory genes. The results are shown in Figure 2. Figure 4 shown.

[0077] exist Figure 4 In the case of Hic1 downstream inflammatory gene mRNA expression, high expression of inflammatory factors indicates an inflammatory response. Figure 4 The expression levels of inflammatory gene mRNA decreased, indicating that inflammatory factors decreased after Hic1 silencing, indicating a weakened inflammatory response. In summary, the Si Hic1 / LNP effectively inhibited the transcription of the Hic1 transcription factor, leading to gene silencing, reduced inflammation, and treatment of pulmonary hypertension.

[0078] Example 4 Animal Pulmonary Hypertension Model

[0079] Male Sprague-Dawley rats (weighing 200 g) were used for in vivo therapeutic experiments. A SuHx-induced rat pulmonary hypertension model was established. Rats were intraperitoneally injected with SU5416 (containing 0.5% (w / v) sodium carboxymethylcellulose, 0.9% (w / v) sodium chloride, 0.4% (v / v) polysorbate 80 and 0.9% (v / v) benzyl alcohol, with the remainder being deionized water) at an injection volume of 20 mg / kg (the injection volume was adjusted according to the weight of the rat). After injection, the rats were placed in a hypoxic environment (10% O2). After 3 weeks, the pulmonary hypertension of the rats was greater than 20 mm / hg, indicating that the model was successfully established. Subsequently, the animals were returned to a normoxic environment for 2 weeks, and the rats in the treatment group were given Si Hic1 / LNP (1 mg / kg; intravenous injection) every 3-4 days for a total of 5 treatments. The rats in the control group were injected with Si NC / LNP. The results are shown in Figure 2. Figure 5-Figure 7 shown.

[0080] like Figure 5As shown, the right ventricular systolic pressure of rats in the control group treated with Si NC / LNP was >20 mmHg, indicating severe pulmonary hypertension, demonstrating successful modeling. The right ventricular systolic pressure of rats in the treatment group treated with Si-Hic1 / LNP was >20 mmHg, indicating severe pulmonary hypertension, also demonstrating successful modeling. The right ventricular systolic pressure of rats in the treatment group was lower than that of rats in the control group, demonstrating that Si-Hic1 / LNP has a therapeutic effect on pulmonary hypertension.

[0081] The right ventricular systolic pressure of rats treated with Si-Hic1 / LNP decreased, indicating that Si-Hic1 / LNP has a therapeutic effect on pulmonary hypertension. Lungs were collected for histological analysis. Figure 6 As shown in Figure 3, HE staining showed that vascular remodeling decreased after Si-Hic1 / LNP treatment. Figure 7 As shown, Masson staining showed perivascular CD68 after Si-Hic1 / LNP treatment. + The decrease in cells (brown cells in the figure) indicates a decrease in macrophage infiltration, indicating that Si-Hic1 / LNP can alleviate pulmonary perivascular inflammation and vascular remodeling, thereby treating pulmonary hypertension.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention.

Claims

1. Use of a Hic1 inhibitor in the preparation of a drug for treating pulmonary hypertension, characterized in that: The Hic1 inhibitor can inhibit the expression of Hic1 transcription factor; The Hic1 inhibitor is a nucleic acid molecule capable of degrading the mRNA of the Hic1 transcription factor; The Hic1 inhibitor is an siRNA whose target sequence is located at any sequence on the mRNA of the Hic1 transcription factor, or an shRNA constructed by the siRNA targeting the mRNA of the Hic1 transcription factor; wherein, The sequence of the siRNA is the first sequence; wherein, The sense strand sequence of the first sequence is shown in SEQ ID NO: 1, and the antisense strand sequence of the first sequence is shown in SEQ ID NO:

2.

2. A pharmaceutical composition for treating pulmonary hypertension, characterized in that: The pharmaceutical composition comprises: a Hic1 inhibitor, and pharmaceutically acceptable excipients; wherein, The definition of the Hic1 inhibitor is the same as that of the Hic1 inhibitor in claim 1 .

3. The pharmaceutical composition according to claim 2, characterized in that The excipients include pharmaceutically acceptable carriers, fillers, binders, disintegrants, lubricants, preservatives, flavoring agents, colorants, thickeners, surfactants, coating agents or release-controlling agents.

4. The pharmaceutical composition according to claim 3, characterized in that The carriers include lipid nanoparticles and / or adeno-associated viruses.

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