Application of CircRAB3IP in the preparation of drugs for treating heart failure
By using CircRAB3IP to prepare drugs, it promotes lymphatic regeneration, solves the problems of cardiac remodeling and myocardial fibrosis caused by mechanical stress in hypertension, and achieves the improvement of cardiac dysfunction and the prognosis diagnosis and treatment of heart failure.
Patent Information
- Application Number
- CN202310556158.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-05-17
- Publication Date
- 2025-07-22
- Estimated Expiration
- 2043-05-17
AI Technical Summary
In the prior art, cardiac remodeling, myocardial fibrosis and cardiac dysfunction caused by mechanical stress of hypertension lack effective regulatory factors and treatment methods, cardiac lymphangiopathy leads to myocardial edema and heart failure, and there is a lack of effective prevention and treatment methods.
CircRAB3IP is used in the preparation of drugs, and provides prognosis diagnosis and treatment of heart failure by promoting lymphatic regeneration, improving cardiac dysfunction, slowing cardiac remodeling and myocardial fibrosis.
CircRAB3IP can effectively improve cardiac dysfunction caused by mechanical stress in hypertension, slow down cardiac remodeling and myocardial fibrosis, promote lymphatic vessel regeneration, reduce cardiac inflammation, and provide new prognostic diagnosis and treatment methods for heart failure.
Smart Images
Figure CN116531398B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the application of CircRAB3IP in the preparation of drugs for treating heart failure, and belongs to the field of biomedical technology. Background Art
[0002] Cardiovascular disease (CVD) has become the leading cause of death in China and even the world, and the prevalence rate is still on the rise. Continuous mechanical stress stimulation of hypertension can lead to structural and functional changes in myocardial or non-myocardial cells, causing adverse cardiac remodeling, which is often manifested as damage to the cardiac vasculature system, myocardial interstitial fibrosis, and cardiomyocyte apoptosis, etc., ultimately leading to heart failure, seriously threatening public life and health. Therefore, actively exploring the regulatory factors of cardiac remodeling caused by mechanical stress of hypertension and clarifying the regulatory mechanisms involved in adverse cardiac remodeling have certain clinical significance for delaying or even reversing cardiac remodeling and improving cardiac function. The heart has a wide lymphatic network, which is distributed in the endocardium, epicardium, cardiac muscle layer, and various atrioventricular valves and atrioventricular conduction systems, with the largest number distributed in the left and right ventricles. The cardiac lymphatic network absorbs excess extracellular fluid, solutes, and metabolic wastes, and returns them from the heart to the blood circulation system to ensure the homeostasis of tissue fluid. It can also transport immune cells, antigens, etc. to lymph nodes to participate in the regulation of immune responses, which is crucial for maintaining the normal physiological function of the heart. Hypertension and heart failure can both lead to cardiac lymphatic dysfunction, causing myocardial edema and aggravating myocardial interstitial fibrosis, etc.
[0003] Circular RNAs are a class of non-coding RNA molecules that do not have a 5'-terminal cap and a 3'-terminal poly(A) tail, and form a circular structure through exon and / or intron back-splicing and covalent bonding. More and more studies have confirmed that circular RNAs (circRNAs) participate in the life activities of cells or individuals through different molecular mechanisms such as inhibiting the expression of microRNAs (miRNAs), binding to RNA-binding proteins (RBPs), and translating small peptides. More and more evidence indicates that circRNAs can participate in the regulation of the occurrence and development of various cardiovascular diseases, but the research on circrRNAs in lymphangiogenesis is still in its infancy.
[0004] In the prior art, there is a circRNA, namely circRAB3IP, in human lymphatic endothelial cells (hLECs). In the circbase database, the ID of this circRNA is: hsa_circ_0099132, and its genomic location is: chr12:70149163-70150443. Specifically, as attachedFigure 1 as shown in Summary of the Invention
[0005] The object of the present invention is to solve the technical problem of the application of CircRAB3IP in the preparation of drugs for treating heart failure.
[0006] To achieve the object of the present invention, the present invention provides the application of CircRAB3IP in the preparation of drugs for preventing or treating heart failure.
[0007] The present invention provides the application of CircRAB3IP in the preparation of drugs for preventing or treating myocardial fibrosis.
[0008] The present invention provides the application of CircRAB3IP in the preparation of drugs for preventing or treating myocarditis.
[0009] The present invention provides the application of CircRAB3IP in the preparation of drugs for promoting lymphangiogenesis.
[0010] Preferably, the dosage forms of the above drugs include tablets, powders, granules, capsules, oral liquids, injections or sustained-release agents.
[0011] The present invention provides the application of CircRAB3IP in the preparation of a heart failure prognosis diagnostic kit.
[0012] The present invention provides a heart failure prognosis judgment system, which includes substances for detecting the expression of CircRAB3IP.
[0013] The present invention provides a heart failure treatment system, which includes a drug administration system; the drug administration system contains CircRAB3IP.
[0014] Compared with the prior art, the present invention has the following beneficial effects:
[0015] The present invention proves through in vivo and in vitro experiments that circRAB3IP can promote lymphangiogenesis, and can effectively improve heart dysfunction caused by hypertensive mechanical stress, slow down cardiac remodeling, myocardial fibrosis, and reduce cardiac inflammation. The present invention provides a new target and certain theoretical basis for the treatment of hypertensive cardiac remodeling. Description of the Drawings
[0016] Figure 1 It is a schematic diagram of the genomic location of the gene expressing circRAB3IP;
[0017] Figure 2 It is a figure of the experimental results for confirming the presence of circRAB3IP in lymphatic endothelial cells;
[0018] Figure 3Comparison chart of echocardiogram analysis between circRAB3IP overexpressing mice and control mice after transverse aortic constriction (TAC).
[0019] Figure 4 Graph showing the experimental results of reduced myocardial hypertrophy induced by persistent mechanical stress in circRAB3IP overexpressing mice;
[0020] Figure 5 Graph showing the experimental results of reduced cardiac fibrosis induced by persistent mechanical stress in circRAB3IP overexpressing mice;
[0021] Figure 6 Graph showing the experimental results that overexpression of circRAB3IP can increase cardiac lymphangiogenesis and reduce inflammation in mice induced by persistent mechanical stress.
[0022] Figure 7 Graph showing the experimental results that overexpression of circRAB3IP promotes the proliferation, migration and tube formation of lymphatic endothelial cells. Detailed implementation methods
[0023] To make the present invention more obvious and understandable, preferred embodiments are hereby described in detail in conjunction with the accompanying drawings as follows:
[0024] The present invention provides the use of CircRAB3IP in the preparation of drugs for preventing or treating heart failure.
[0025] The present invention provides the use of CircRAB3IP in the preparation of drugs for preventing or treating myocardial fibrosis.
[0026] The present invention provides the use of CircRAB3IP in the preparation of drugs for preventing or treating myocarditis.
[0027] The present invention provides the use of CircRAB3IP in the preparation of drugs for promoting lymphangiogenesis.
[0028] The dosage forms of the above drugs include tablets, powders, granules, capsules, oral liquids, injections or sustained-release agents.
[0029] The present invention provides the use of CircRAB3IP in the preparation of a kit for diagnosing the prognosis of heart failure.
[0030] The present invention provides a heart failure prognosis judgment system, which includes substances for detecting the expression of CircRAB3IP.
[0031] The present invention provides a heart failure treatment system, which includes a drug administration system; the drug administration system contains CircRAB3IP.
[0032] Examples
[0033] In the following examples, the mice were provided by Jiangsu GICC Biopharmatech Co., Ltd., and the pK25ssAAV-circRAB3IP AAV9 virus and the pAV-CMV-GFP AAV9 control virus were purchased from Shandong Vigene Biosciences Co., Ltd. The remaining conventional reagents were all commercially available.
[0034] Example 1
[0035] Determination of circRAB3IP in lymphatic endothelial cells:
[0036] Extract the genomic DNA (gDNA) and total RNA (RNA reverse transcribed into cDNA) of hLEC. The results of the PCR experiment showed that using divergent primers, the circRAB3IP gene could be amplified from the cDNA of hLEC, but not from gDNA, while the corresponding linear transcript RAB3IP mRNA could be amplified by convergent primers. The product amplified by the divergent primers in the PCR experiment was subjected to Sanger sequencing, and the results confirmed the presence of the correct back-splicing site in the primer-amplified PCR product (as shown in Figure 2 Figure A). The results of the RNase R digestion experiment showed that after treatment with RNase R, the linear transcript of RAB3IP was digested and degraded, while circRAB3IP was resistant to RNase R treatment (as shown in Figure 2 Figure B). This experiment confirmed the actual existence of circRAB3IP in hLEC.
[0037] Example 2
[0038] Overexpression of circRAB3IP improves cardiac dysfunction in mice caused by persistent mechanical stress:
[0039] This experiment compared the cardiac function of mice overexpressing circRAB3IP with that of control mice under mechanical stress stimulation. Figure 3 It is a comparison chart of echocardiogram analysis of mice overexpressing circRAB3IP and control mice after transverse aortic constriction (TAC). Mice in the control group (GFP group) and mice in the circRAB3IP overexpression group (circRAB3IP group) underwent TAC or Sham surgery for 6 weeks, and cardiac ultrasound was detected 6 weeks after the operation. The results of echocardiography showed that compared with the GFP-Sham group, the left EF% and FS% of GFP-TAC mice were significantly reduced 6 weeks after TAC; (EF is the left ventricular ejection fraction; FS is the left ventricular shortening fraction); compared with circRAB3IP-Sham mice, the left ventricular ejection fraction and shortening fraction of circRAB3IP-TAC mice still decreased, but there was no statistical difference; the EF% and FS% of circRAB3IP-TAC mice were significantly increased compared with GFP-TAC mice (as shown in Figure 3Figures A - C).
[0040] Example 3
[0041] Overexpression of circRAB3IP in mice alleviates persistent mechanical stress - induced myocardial hypertrophy in mice:
[0042] This experiment compared the myocardial cell size and the expression of genes related to myocardial hypertrophy between circRAB3IP - overexpressing mice and control mice under mechanical stress stimulation.
[0043] Figure 4 H&E staining was performed on the heart tissues of GFP - Sham, GFP - TAC, circRAB3IP - Sham, and circRAB3IP - TAC mice ( Figure 4 Figure A in Figure 4 Figure B in Figure 4 Figure A in Figure 4 Figure B in Figure 4 Figures C and D in Figure 4 Figures E and F in
[0044] Example 4
[0045] Overexpression of circRAB3IP reduces persistent mechanical stress - induced cardiac fibrosis in mice:
[0046] This experiment compared the degree of myocardial fibrosis between circRAB3IP - overexpressing mice and control mice after TAC.
[0047] Figure 5 Masson staining of mouse heart tissues and statistical chart of fibrotic area, and qRT - PCR analysis of myocardial fibrosis markers. Compared with GFP - TAC group mice, the fibrotic area in circRAB3IP - TAC group mice decreased significantly ( Figure 5 Figures A and B in Figure 5 Figures C - E in
[0048] Example 5
[0049] Overexpression of circRAB3IP increases cardiac lymphangiogenesis and reduces inflammation in persistent mechanical stress - induced mice:
[0050] This experiment was to compare the number of cardiac lymphatic vessels and cardiac inflammation in mice overexpressing circRAB3IP and the control group after TAC surgery.
[0051] Figure 6 For immunofluorescence staining related to lymphatic vessels, ELISA analysis, qRT-PCR analysis, Western Blot analysis of mouse heart tissue, and qRT-PCR analysis of inflammation-related molecules in mouse heart tissue. Co-staining of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and CD31 in heart tissue, fluorescence staining of vascular endothelial growth factor receptor-3 (VEGFR3), and statistical results showed that: the number of lymphatic vessels positive for LYVE-1 and VEGFR3 in circRAB3IP-TAC mice was significantly increased compared with GFP-TAC mice ( Figure 6 in Figures A-C). The content of serum VEGF-C in circRAB3IP-TAC mice was significantly increased compared with GFP-TAC ( Figure 6 in Figure D). The results of qPCR analysis showed that the mRNA level of lymphangiogenesis markers in the circRAB3IP-TAC group was higher than that in the GFP-TAC group ( Figure 6 in Figures E-G). The WB experiment and statistical results also showed that the protein expression levels of lymphangiogenesis markers (VEGFR3 and VEGF-C) in the circRAB3IP-TAC group were higher than those in the GFP-TAC group ( Figure 6 in Figure H). qPCR analysis of the mRNA level of inflammatory factors showed that the mRNA level of inflammatory factors in the circRAB3IP-TAC group was lower than that in the GFP-TAC group ( Figure 6 in Figures I-K).
[0052] Example 6
[0053] Overexpression of circRAB3IP enhances the proliferation, migration and lymphatic vessel formation ability of lymphatic endothelial cells:
[0054] This experiment was to verify the effect of circRAB3IP on the proliferation, migration and lymphatic vessel formation ability of lymphatic endothelial cells at the cellular level. Through lentivirus infection and puromycin screening, a stable overexpression hLEC strain of circRAB3IP, circRAB3IP, and a stable knockdown cell strain of circRAB3IP, sh-circRAB3IP, were constructed.
[0055] Figure 7Changes in the proliferation, migration, and tube formation abilities of lymphatic endothelial cells after overexpressing circRAB3IP and knocking down circRAB3IP. The CCK8 assay showed that the number of cells in the circRAB3IP stable transfection strain was higher than that in the Vector cells at the 12-hour and 24-hour time points, and the number of cells in sh-circRAB3IP was slightly lower than that in the control shNC cells at 12 hours and 24 hours ( Figure 7 Figure A in Figure 7 ). The lymphatic vessel tube formation assay showed that the circRAB3IP cells formed tubes first ( Figure 7 Figure B in
[0056] ). The cell scratch assay and the scratch closure area statistical chart at 12 hours, 24 hours, and 48 hours showed that the scratch closure area of circRAB3IP cells was smaller than that of Vector cells at each time point, and the scratch closure area of circRAB3IP cells was significantly reduced compared with Vector cells at 48 hours ( Figure 7 Figure C and D in
[0056] ). The above in vivo and in vitro experiments demonstrated that circRAB3IP can promote lymphangiogenesis and effectively improve cardiac dysfunction caused by hypertensive mechanical stress, slow down cardiac remodeling, myocardial fibrosis, and reduce cardiac inflammation.
[0057] As described above, only the preferred embodiments of the present invention are provided, and there is no limitation to the present invention in any form or substance. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the premise of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. Those skilled in the art, without departing from the spirit and scope of the present invention, may make some changes, modifications, and equivalent variations by using the technical content disclosed above, which are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications, and variations made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.
Claims
1. Use of CircRAB3IP in the preparation of a drug for preventing or treating heart failure.
2. Use of CircRAB3IP in the preparation of a drug for preventing or treating myocardial fibrosis.
3. Use of CircRAB3IP in the preparation of a drug for preventing or treating myocarditis induced by persistent mechanical stress.
4. Use of CircRAB3IP in the preparation of a drug for improving cardiac dysfunction caused by mechanical stress in hypertension.
5. The application according to any one of claims 1-4, characterized in that, The dosage forms of the drug include tablets, powders, granules, capsules, oral liquids, injections or sustained-release agents.