Application of reagent for inhibiting expression of LRP2 in preparation of medicine for treating myocardial fibrosis

Abstract

The invention relates to the field of biological medicine research, in particular to application of a reagent for inhibiting expression of LRP2 in preparation of a medicine for treating myocardial fibrosis. The effect and mechanism of the low-density lipoprotein receptor related protein LRP2 as a non-classical receptor in TGF-beta induced fibroblast activation and myocardial fibrosis are studied, and the result shows that in the myocardial fibrosis process, the expression quantity of the LRP2 protein is increased, and the expression quantity of the LRP2 protein is increased. In addition, an Ang II model mouse of which the LRP2 is knocked down through specific siRNA shows a lower cardiac fibrosis level, and myocardial cell hypertrophy is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to application of a reagent for inhibiting expression of low-density lipoprotein receptor-related protein LRP2 in preparation of a drug for treating myocardial fibrosis. BACKGROUND

[0002] Myocardial fibrosis is a necessary path for high blood pressure, ischemic heart disease and other heart diseases to develop into heart failure, and also runs through the whole process of heart injury. Myocardial fibrosis is a pathological change of imbalance between extracellular matrix (ECM) production and degradation, which leads to accumulation of scar tissue, decrease of myocardial elasticity, and affects cardiac output, and finally leads to heart failure. The formation factors of myocardial fibrosis are complex, and its pathogenesis is numerous, and there is a complex relationship among different mechanisms. At present, there is still lack of treatment method with significant effect on myocardial fibrosis. It is very important to actively study the pathogenic causes and pathological mechanisms, and find the treatment target for inhibiting myocardial fibrosis. At present, the treatment method for myocardial fibrosis in clinic is mainly drug treatment, including angiotensin converting enzyme inhibitors, beta receptor blockers, sodium-glucose co-transporter 2 inhibitors and the like; other treatment methods include targeted transportation of some biological materials, such as nanoparticle materials represented by liposomes, hydrogel materials represented by hyaluronic acid, and exosomes secreted by mesenchymal stem cells, cardiac progenitor cells or engineered cells. However, these methods cannot significantly and effectively control the progress of myocardial fibrosis.

[0003] Low-density lipoprotein receptor-related protein 2 (LRP2), also known as megalin, is an endocytosis receptor, which is a member of the low-density lipoprotein family. The members of this receptor family are cell surface receptors and widely participate in processes such as cholesterol metabolism, intracellular transport and cell signal transduction, and play an important role in cardiovascular diseases that cannot be ignored. However, there are still many research gaps between LRP2 protein and myocardial fibrosis. SUMMARY

[0004] The technical problem to be solved by the present application is to provide application of a reagent for inhibiting expression of LRP2 in preparation of a drug for treating myocardial fibrosis, aiming at the deficiencies of the prior art.

[0005] In order to solve the above technical problem, the present application discloses application of a reagent for inhibiting expression of LRP2 in preparation of a drug for treating myocardial fibrosis. The present application studies the role and mechanism of low-density lipoprotein receptor-related protein LRP2 as a non-classical receptor in myocardial fibrosis. The specific technical scheme is as follows: This invention provides the use of a reagent that inhibits the expression of low-density lipoprotein receptor-associated protein LRP2 in the preparation of drugs for the prevention and / or treatment of myocardial fibrosis.

[0006] Myocardial fibrosis is a key characteristic of myocardial remodeling. The reagent described in this invention for inhibiting the expression of low-density lipoprotein receptor-associated protein LRP2 can also be used to prepare drugs for the prevention and / or treatment of myocardial hypertrophy. Myocardial hypertrophy also falls under the category of myocardial remodeling.

[0007] The myocardial fibrosis mentioned herein is caused by any one of the following (1) to (3): (1) Myocardial fibrosis caused by myocardial ischemia; in some embodiments of the present invention, the myocardial ischemia includes myocardial infarction; (2) Myocardial fibrosis caused by angiotensin II; (3) TGF-β-induced myocardial fibrosis.

[0008] In cell experiments, TGF-β-induced activation and fibrosis of NIH / 3T3 cells promoted increased LRP2 expression. Knockdown of LRP2 inhibited TGF-β-induced activation and fibrosis of NIH / 3T3 cells and reduced the expression of fibroblast activation markers.

[0009] In animal experiments, LRP2 protein or its encoding gene was increased in the heart tissue of Ang II-induced model mice and myocardial infarction model mice, and myocardial fibrosis markers also increased synchronously. Ang II model mice with extensive LRP2 knockdown exhibited lower levels of cardiac fibrosis and improved cardiomyocyte hypertrophy.

[0010] The reagent for inhibiting the expression of low-density lipoprotein receptor-associated protein (LRP2) targets LRP2 to inhibit the transcription or translation of the LRP2 encoding gene, or to inhibit the production of LRP2. The inhibition of LRP2 expression described in this invention includes blocking or reducing LRP2 expression.

[0011] The reagent for inhibiting the expression of low-density lipoprotein receptor-associated protein LRP2 includes any one or more combinations of nucleic acid molecules, nucleic acid constructs, small molecule chemical drugs, or antibody drugs. Preferably, it is a nucleic acid molecule, including any one of siRNA, shRNA, or miRNA. Preferably, it is siRNA, which includes si-LRP2, the nucleotide sequence of which is shown in SEQ ID No. 1 and SEQ ID No. 2. si-LRP2 includes a sense strand and an antisense strand; the sequence shown in SEQ ID No. 1 is the sense strand, and the sequence shown in SEQ ID No. 2 is the antisense strand.

[0012] In this embodiment, the 3' end of the si-LRP2 is suspended by dTdT.

[0013] The reagents mentioned herein may be in the form of any one of injections, infusions, powders, capsules, or tablets.

[0014] Secondly, this invention provides a drug for the prevention and / or treatment of myocardial remodeling, comprising siRNA, the nucleotide sequence of which is shown in SEQ ID No. 1 and SEQ ID No. 2. The siRNA can inhibit the expression of low-density lipoprotein receptor-associated protein LRP2, thereby preventing and / or treating myocardial remodeling. The myocardial remodeling includes myocardial fibrosis and / or myocardial hypertrophy, both of which are important characteristics of myocardial remodeling. This invention has successfully verified the ameliorative effect of si-LRP2 on myocardial fibrosis and myocardial hypertrophy through both cell and animal experiments.

[0015] In this embodiment, the 3' end of the si-LRP2 is suspended by dTdT.

[0016] Beneficial effects: This invention provides the application of reagents that inhibit LRP2 expression in the preparation of drugs for the prevention and / or treatment of myocardial fibrosis, thereby providing a drug for the prevention and / or treatment of myocardial fibrosis. This invention has observed increased expression of LRP2 protein or its encoding gene in TGF-β-induced fibroblast activation and fibrosis, Ang II-induced myocardial fibrosis, and myocardial ischemia-induced myocardial fibrosis models. Further experiments revealed that Ang II-induced LRP2 knockdown mice exhibited lower levels of cardiac fibrosis and improved cardiomyocyte hypertrophy. Attached Figure Description

[0017] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0018] Figure 1 This is a Western blot diagram of LRP2 protein in the sham group and the myocardial infarction group (MI).

[0019] Figure 2 This is a Western blot diagram of LRP2 and fibrosis activation markers in mouse heart tissue after injection of Ang II.

[0020] Figure 3 Immunofluorescence staining of LRP2 in mouse heart tissue after Ang II injection is shown. A represents the control group, and B represents the Ang II injection group. In the figure, green represents the expression level of fibrosis activation marker α-SMA, yellow represents the expression level of LRP2, and blue represents DAPI.

[0021] Figure 4 Western blot diagram of LRP2 after stimulation of mouse embryonic fibroblasts with different concentrations of TGF-β.

[0022] Figure 5 Immunofluorescence staining of LRP2 in mouse embryonic fibroblasts after stimulation with different concentrations of TGF-β.

[0023] Figure 6 This is a Western blot diagram showing the LRP2 and fibrosis activation markers after treating mouse embryonic fibroblasts with si-LRP2.

[0024] Figure 7 Immunofluorescence staining of LRP2 and fibronectin proteins after treatment of mouse embryonic fibroblasts with si-LRP2.

[0025] Figure 8 The level of cardiac fibrosis in mice after treatment with si-LRP2.

[0026] Figure 9 To illustrate the myocardial hypertrophy in mice treated with si-LRP2. Detailed Implementation

[0027] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0028] In the following examples, the LRP2 protein has an NCBI reference sequence of NM_004525.3, and the LRP2 gene has a Gene ID of 4036.

[0029] Example 1 The myocardial infarction (MI) mouse model was constructed as follows: C57BL / 6 mice were acclimatized for one week, then anesthetized with pentobarbital and connected to a ventilator. The left anterior descending coronary artery was ligated at the opening of the fourth intercostal space, and the proximal end of the left anterior descending coronary artery was ligated with 7-0 sutures while preserving the pericardium. Paleness at the apex confirmed infarction.

[0030] The control group (Sham) underwent only open-chest surgery. Mice hearts were collected 28 days post-surgery for examination.

[0031] Eight-week-old male C57BL / 6 mice (18-25g) were used to establish a myocardial infarction (MI) model using the method described above. The control group (Sham group) underwent only open-chest surgery. Western blot analysis (Tubulin as internal control) revealed increased LRP2 protein expression in the heart tissue of MI mice compared to the Sham group.Figure 1 As shown.

[0032] Seven-week-old male C57BL / 6 mice (18-25g) were acclimatized for one week, and then subcutaneously implanted with a micro-osmotic pump containing angiotensin II (Ang II), an aortic and myocardial hypertrophy-inducing agent (Alzet Mini-Osmotic Pumps 2002, 1.44 mg / kg / day), for two weeks. The control group received the same dose of saline instead of Ang II. Western blot analysis (Tubulin as internal control) revealed increased LRP2 protein expression in the heart tissue of Ang II-injected mice compared to the control group. Simultaneously, the expression of fibrosis activation markers such as α-SMA, COL1A1, and COL3A1 was also increased (e.g., ...). Figure 2 (As shown). Immunofluorescence staining of heart sections from Ang II-injected mice and control groups revealed increased LRP2 expression and α-SMA expression in the hearts of Ang II-injected mice (as shown). Figure 3 (As shown).

[0033] In summary, this embodiment found that the expression of LRP2 gene or protein increased in the heart tissue of Ang II-induced model mice and myocardial infarction model mice, and the markers of myocardial fibrosis also increased synchronously.

[0034] Example 2 In this embodiment, mouse embryonic fibroblasts NIH / 3T3 were stimulated with different concentrations (0, 3, 6, and 9 ng / mL) of TGF-β. Specifically, NIH / 3T3 cells were cultured until the cell density reached approximately 50%, then different concentrations of TGF-β were added. After 24 hours of culture, cells were harvested for testing. The cell culture medium was DMEM (containing 4.5 g / L glucose and 10% v / v FBS). Western blot experiments were performed (with Tubulin as the internal control). Figure 4 (as shown) and immunofluorescence assay (as shown) Figure 5 As shown in the figure, it was found that the expression level of LRP2 increased with the increase of TGF-β concentration.

[0035] This example demonstrates that TGF-β-induced activation and fibrosis in NIH / 3T3 cells promote increased LRP2 expression.

[0036] Example 3 This embodiment utilizes si-LRP2 (si-LRP2 refers to a small interfering RNA that can interfere with the translation of LRP2, ultimately reducing the expression level of LRP2 protein). The specific steps are as follows: when NIH / 3T3 cells are cultured to a density of approximately 60%~70%, si-LRP2 (the final concentration of si-LRP2 added is 50 nM) and transfection reagent (Lipofectamine 2000) are added. After 6 hours of static transfection, the medium is changed to fresh DMEM medium (containing 4.5 g / L glucose and 10% v / v FBS). 24 hours later, TGF-β is added to a final concentration of 10 ng / mL. 24 hours after adding TGF-β, the cells are harvested to obtain the si-LRP2+TGF-β group.

[0037] The treatment method is the same as above, except that si-LRP2 is replaced with si-NC as the control group, resulting in the si-NC+TGF-β group.

[0038] Similarly, the treatment method is the same as described above, except that after adding si-LRP2 for 24 hours, TGF-β was replaced with an equal dose of PBS to obtain the si-LRP2 group; while after adding si-NC for 24 hours, TGF-β was replaced with an equal dose of PBS to obtain the si-NC group.

[0039] This embodiment reduces LRP2 expression levels in NIH / 3T3 cells by transfecting si-LRP2, then activates the transfected NIH / 3T3 cells with TGF-β, and sets up a corresponding control group. Western blot experiments (with Tubulin as the internal control) were performed. Figure 6 As shown in the figure, compared with cells not treated with si-LRP2, cells treated with si-LRP2 significantly knocked down the expression level of LRP2, while the expression levels of fibrosis markers and myocardial hypertrophy markers (COL1A1, COL3A1 and MYH7) were also significantly reduced, indicating that fibrosis in NIH / 3T3 cells was inhibited.

[0040] In addition, fibroblasts activated by TGF-β significantly upregulate fibronectin, and the detection of fibronectin protein can reflect fibroblast activation to some extent, as shown in immunofluorescence experiments (e.g., Figure 7 As shown in the figure, fibrosis in NIH / 3T3 cells was inhibited.

[0041] The specific sequence of si-LRP2 used in this embodiment is as follows: Chain of Justice: 5'-GGATCGATGCAGAGAAACA-3' (SEQ ID No. 1); Antisense chain: 5'-TGTTTCTCTGCATCGATCC-3' (SEQ ID No. 2); The above-mentioned si-LRP2 is modified by adding dTdT to the 3' end to enhance the suppression efficiency.

[0042] The si-NC used in this embodiment is an siRNA sequence unrelated to the research subjects, serving as a negative control group. The specific sequence is as follows: The specific sequence of the si-NC used in this embodiment is as follows: Chain of Justice: 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID No. 3); Antonym: 5'-ACGTGACACGTTCGGAGAA-3' (SEQ ID No. 4).

[0043] This embodiment demonstrates that knocking down LRP2 inhibits TGF-β-induced activation and fibrosis in NIH / 3T3 cells, and reduces the expression of fibroblast activation markers.

[0044] Example 4 The si-LPR2 described in Example 3 was directly injected via the tail vein into 8-week-old male C57BL / 6 mice. si-LPR2 was dissolved in physiological saline, and the injection dose was 8 mg / kg to broadly inhibit LRP2 expression levels in mice. After injection, mice were subcutaneously implanted with an Ang II micro-osmotic pump (Alzet Mini-Osmotic Pumps Model 2002, 1.44 mg / kg / day) for 2 weeks. The control group received only an equivalent dose of si-NC and / or an equivalent dose of physiological saline (saline) to Ang II. The specific groupings were as follows: Saline+si-NC group: Injected with the same dose of si-NC and normal saline; Saline+si-LRP2 group: injected with the same dose of si-LRP2 and normal saline; Ang II + si-NC group: Injected with equal doses of si-NC and Ang II; Ang II + si-LRP2 group: Injected with the same dose of si-LRP2 and Ang II; Masson staining was used to detect the level of fibrosis in the mouse heart, and the results are as follows: Figure 8 As shown, inhibiting LRP2 expression reduced the level of cardiac fibrosis in Ang II mice.

[0045] In this embodiment, WGA staining was used to detect myocardial hypertrophy in mice, and the results are as follows:Figure 9 As shown, Figure 9 The study revealed the morphology of cardiomyocytes in different groups of mice. Cardiomyocytes in the Saline+si-NC group and the Saline+si-LRP2 group showed normal morphology. Cardiomyocytes in the AngII+si-NC group were twisted, elongated and enlarged. Cardiomyocytes in the AngII+si-LRP2 group showed reduced hypertrophy. Inhibition of LRP2 expression also reduced cardiomyocyte hypertrophy in AngII mice.

[0046] Therefore, Ang II model mice with extensive LRP2 knockdown exhibited lower levels of cardiac fibrosis and improved cardiomyocyte hypertrophy.

[0047] In summary, LRP2, as a non-canonical receptor, induces the activation of cardiac fibroblasts and promotes myocardial fibrosis, while inhibiting LRP2 improves this phenomenon, providing a new target for the treatment of myocardial fibrosis.

[0048] This invention provides a method and approach for using a reagent that inhibits LRP2 expression in the preparation of drugs for treating myocardial fibrosis. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. The application of reagents that inhibit the expression of low-density lipoprotein receptor-associated protein LRP2 in the preparation of drugs for the prevention and / or treatment of myocardial fibrosis.

2. The application according to claim 1, characterized in that, The aforementioned myocardial fibrosis is caused by any one of the following (1) to (3): (1) Myocardial fibrosis caused by myocardial ischemia; (2) Myocardial fibrosis caused by angiotensin II; (3) TGF-β-induced myocardial fibrosis.

3. The application according to claim 1, characterized in that, The reagent for inhibiting the expression of low-density lipoprotein receptor-associated protein LRP2 is a reagent for inhibiting the transcription or translation of the LRP2 encoding gene, or a reagent for inhibiting the production of LRP2 protein.

4. The application according to claim 1, characterized in that, The reagents used to inhibit the expression of low-density lipoprotein receptor-associated protein LRP2 include any one or more combinations of nucleic acid molecules, nucleic acid constructs, small molecule chemical drugs, or antibody drugs.

5. The application according to claim 4, characterized in that, The nucleic acid molecules mentioned include any one of siRNA, shRNA, or miRNA.

6. The application according to claim 5, characterized in that, The siRNA is si-LRP2, and its nucleotide sequence is shown in SEQ ID No. 1 and SEQ ID No.

2.

7. The application according to claim 6, characterized in that, The 3' end of the si-LRP2 is suspended by dTdT.

8. The application according to claim 1, characterized in that, The reagent may be in the form of any one of injection, infusion, powder, capsule or tablet.

9. A medicament for the prevention and / or treatment of myocardial remodeling, comprising siRNA, the nucleotide sequence of which is shown in SEQ ID No. 1 and SEQ ID No. 2, wherein the siRNA can inhibit the expression of low-density lipoprotein receptor-associated protein LRP2 to prevent and / or treat myocardial remodeling.

10. The medicament according to claim 9, characterized in that, The siRNA has dTdT hanging from its 3' end.

Images