Application of SRSF1 in the preparation of drugs for preventing and / or treating heart failure with preserved ejection fraction

Abstract

The present invention provides the use of SRSF1 in the preparation of drugs for preventing and / or treating heart failure with preserved ejection fraction, and belongs to the field of biomedicine technology. The present invention determines that SRSF1 can be used as a key target for the preparation of drugs related to the prevention and / or treatment of heart failure with preserved ejection fraction. By constructing animal models and cell-level experiments, it was confirmed that SRSF1 is involved in regulating the pathological process of the disease. The model experiments constructed in SRSF1 endothelial cell-specific knockout mice showed that inhibiting SRSF1 expression can improve symptoms such as diastolic dysfunction and hypertension, reduce pulmonary edema, and inhibit myocardial fibrosis and remodeling. It can also improve abnormal glucose and lipid metabolism and inhibit the process of endothelial-mesenchymal transition. The present invention opens up a new path for new drug screening, provides new targets and methods for the treatment of heart failure with preserved ejection fraction, and has important medical significance and value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the use of SRSF1 in preparing a drug for preventing and / or treating heart failure with preserved ejection fraction. Background Art

[0002] Heart failure (HF) is the terminal stage of the development of many cardiovascular diseases, and its incidence and mortality rates are showing an upward trend year by year. According to epidemiological data, heart failure with preserved ejection fraction (HFpEF) accounts for 50% of the total number of heart failure cases, and has become the most common type of heart failure. It is particularly noteworthy that with the continuous extension of human life expectancy, and the increasing prevalence and normalization of obesity and diabetes, the proportion of heart failure with preserved ejection fraction is expected to continue to grow. It is worth noting that heart failure with preserved ejection fraction not only has a high incidence rate and a serious aging trend, but also has a high mortality rate. It is also very easy to cause various complications, which imposes an extremely heavy burden on the health and life of patients.

[0003] In clinical practice, heart failure with preserved ejection fraction is defined as a condition characterized by decreased ventricular relaxation and compliance while ventricular systolic function is within the normal range, resulting in reduced ventricular filling volume and increased filling pressures, ultimately leading to a clinical syndrome of pulmonary and systemic congestion. The pathophysiology of heart failure with preserved ejection fraction is complex and diverse. Five major mechanisms have been thoroughly investigated: diastolic dysfunction, inflammation and oxidative stress / endothelial dysfunction, chronotropic insufficiency and cardiac reserve dysfunction, pulmonary hypertension, and ventricular-arterial coupling abnormalities. These five mechanisms are intertwined, influencing, and mutually reinforcing, significantly increasing the complexity of disease diagnosis and the difficulty of treatment. Consequently, effective medications specifically targeting heart failure with preserved ejection fraction have been elusive, and effective treatments are extremely scarce. Therefore, it is extremely important to deeply explore the molecular mechanisms of heart failure with preserved ejection fraction and to fully search for new therapeutic targets. Moreover, time is of the essence and there is no time to lose. This plays a vital role in improving patients' survival, promoting the advancement of cardiovascular disease treatment technology, and reducing the medical burden.

[0004] SRSF1 (Serine / arginine splicing factor 1), also known as ASF / SF2, belongs to the SR protein family and is an important splicing regulator that is indispensable for mRNA splicing and alternative splicing. As a typical member of the highly conserved SR protein family, SRSF1 also plays a key role in maintaining genome stability, cell viability, and cell cycle progression. In addition to its splicing function, SRSF1 can also regulate processes such as mRNA transcription, stability, and protein translation. Studies have shown that when overexpressed, SRSF1 can promote oncogenic transformation of fibroblasts and epithelial cells by promoting proliferation and inhibiting apoptosis. However, there have been no reports on the role of SRSF1 in heart failure with preserved ejection fraction. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an application of SRSF1 as a target in the preparation of a drug for preventing and / or treating heart failure with preserved ejection fraction, which can significantly improve heart failure with preserved ejection fraction and its complications by inhibiting the expression of SRSF1 gene or protein.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides the use of SRSF1 as a target in the preparation of a drug for preventing and / or treating heart failure with preserved ejection fraction.

[0008] As one embodiment, the SRSF1 includes SRSF1 protein or SRSF1 encoding gene.

[0009] Preferably, the drug inhibits the expression of SRSF1 protein or SRSF1 encoding gene.

[0010] As an embodiment, the drug is any one or more of the following drugs:

[0011] 1) The drug is a drug for improving diastolic dysfunction;

[0012] 2) The drug is a drug for lowering blood pressure;

[0013] 3) The drug is a drug for alleviating pulmonary edema;

[0014] 4) The drug is a drug for improving myocardial fibrosis;

[0015] 5) The drug is a drug for alleviating myocardial hypertrophy and muscle remodeling;

[0016] 6) The drug is a drug for improving abnormal lipid metabolism;

[0017] 7) The drug is a drug for improving abnormal glucose metabolism;

[0018] 8) The drug is a drug that inhibits the endothelial-mesenchymal transition process of HUVEC.

[0019] The present invention also provides use of an SRSF1 inhibitor in preparing a drug for preventing and / or treating heart failure with preserved ejection fraction. The SRSF1 inhibitor includes an SRSF1 gene inhibitor or an SRSF1 protein inhibitor.

[0020] As an embodiment, the SRSF1 gene inhibitor includes at least one of the following RNA or its gene-derived products: siRNA, shRNA and sgRNA targeting the gene SRSF1.

[0021] As an embodiment, the nucleotide sequence of the siRNA targeting the gene SRSF1 is shown as SEQ ID NO: 1 and SEQ ID NO: 2.

[0022] Preferably, the SRSF1 protein inhibitor includes an antibody to the SRSF1 protein, a binding molecule to the SRSF1 protein, or a degrader to the SRSF1 protein.

[0023] The present invention also provides a drug capable of preventing and / or treating heart failure with preserved ejection fraction, wherein the drug comprises the aforementioned SRSF1 inhibitor and a pharmaceutically acceptable carrier.

[0024] In the present invention, the heart failure with preserved ejection fraction includes HFpEF-1, HFpEF-2, HFpEF-3, HFpEF-4, and HFpEF-5.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention determines that SRSF1 can be used as a key target for the preparation of drugs related to the prevention and / or treatment of heart failure with preserved ejection fraction. In the research process of the present invention, by constructing an animal model of heart failure with preserved ejection fraction and conducting in-depth experiments at the cellular level, it has been strongly confirmed that SRSF1 is deeply involved in and regulates the pathological development process of heart failure with preserved ejection fraction. A heart failure model with preserved ejection fraction was constructed in mice with SRSF1 endothelial cell-specific knockout for experiments. The results showed that inhibiting the expression of SRSF1 can effectively improve the diastolic dysfunction and hypertension symptoms induced by heart failure with preserved ejection fraction. At the same time, it can significantly reduce pulmonary edema, inhibit the process of myocardial fibrosis, and improve the myocardial remodeling caused by myocardial hypertrophy, thereby fully exerting the positive effect of alleviating the signs of heart failure. Furthermore, inhibiting the expression of SRSF1 also has the ability to improve the body's abnormal glucose and lipid metabolism, and can significantly inhibit the endothelial-mesenchymal transition process of human umbilical vein endothelial cells (HUVECs), which further proves that inhibiting SRSF1 can effectively delay the progression of heart failure with preserved ejection fraction, and also has certain positive significance and potential value in reducing the occurrence of complications related to the disease. The present invention uses SRSF1 as a target for the prevention and treatment of heart failure with preserved ejection fraction, which not only opens up a new path for screening new drugs of great value, but also provides new targets and methods for the treatment of heart failure with preserved ejection fraction, which has extremely critical significance and value in both medical research and clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The physiological and biochemical conditions of animals after the establishment of a heart failure model with preserved ejection fraction; Figure 1 A: Representative M-mode images of Chow and HFpEF mice; B: Left ventricular ejection fraction (LVEF) of Chow and HFpEF mice; C: Left ventricular fractional shortening (LVFS) of Chow and HFpEF mice; D: Representative Pulse-Wave images and Tissue Doppler images of Chow and HFpEF mice; E: E / A ratio of Chow and HFpEF mice; F: E / e' ratio of Chow and HFpEF mice; G: Left ventricular longitudinal strain rate (GLS) of Chow and HFpEF mice; H: Maximum running distance of Chow and HFpEF mice in exercise endurance test; I: Systolic blood pressure of Chow and HFpEF mice; J: Diastolic blood pressure of Chow and HFpEF mice; Chow was the control group and HFpEF was the model group.

[0028] Figure 2 The expression of SRSF1 at the animal and cellular levels; Figure 2 A: mRNA level of SRSF1 in the hearts of Chow and HFpEF mice; B: representative western blot and average data of SRSF1 in the hearts of Chow and HFpEF mice; C: representative immunofluorescence images of SRSF1 and CD31 in the hearts of Chow and HFpEF mice (scale bar, 20 μm); D: mRNA level of SRSF1 in HUVECs stimulated with high glucose for 24 hours; E: representative western blot and average data of SRSF1 in HUVECs stimulated with high glucose for 24 hours; F: mRNA level of SRSF1 in HUVECs stimulated with palmitic acid for 24 hours; G: representative western blot and average data of SRSF1 in HUVECs stimulated with palmitic acid for 24 hours; Chow is the control group and HFpEF is the model group.

[0029] Figure 3 The physiological and biochemical conditions of SRSF1 endothelial cell-specific knockout mice; Figure 3 Middle A: WT and Srsf1 endo- / - Representative images of mouse tail DNA genomic PCR identification; B: WT and Srsf1 fed Chow or HFD+L-NAME endo- / - Representative M-mode images of mice; C: WT and Srsf1 mice fed Chow or HFD+L-NAME endo- / - Left ventricular ejection fraction (LVEF) of mice; D: WT and Srsf1 fed Chow or HFD+L-NAME endo- / - Left ventricular fractional shortening (LVFS) of mice; E: WT and Srsf1 fed Chow or HFD+L-NAME endo- / - Representative Pulse-Wave images and Tissue Doppler images of mice; F: WT and Srsf1 mice fed Chow or HFD+L-NAME endo- / - E / A ratio of mice; G: WT and Srsf1 fed Chow or HFD+L-NAME endo- / - E / e' ratio of mice; H: WT and Srsf1 fed with Chow or HFD+L-NAME endo- / - Left ventricular longitudinal strain rate GLS; I: WT and Srsf1 fed with Chow or HFD+L-NAME endo- / - Maximum running distance of mice; J: WT and Srsf1 fed Chow or HFD+L-NAME endo- / - Systolic blood pressure of K:Chow or HFD+L-NAME-fed WT and Srsf1 mice endo- / - Diastolic blood pressure of mice; WT is wild type, Srsf1endo- / - The SRSF1 endothelial cell-specific knockout mice were used, Chow was used as the control group, and HFpEF was used as the model group.

[0030] Figure 4 These are indicators of lung, heart tissue, and glucose and lipid metabolism in SRSF1 endothelial cell-specific knockout mice; Figure 4 Middle A: WT and Srsf1 fed with Chow or HFD+L-NAME endo- / - Lung wet / dry weight ratio of mice; B: WT and Srsf1 fed Chow or HFD+L-NAME endo- / - Heart weight / tibia ratio (HW / TL) of mice; C: WT and Srsf1 mice fed Chow or HFD+L-NAME endo- / - Heart weight / body weight ratio (HW / BW) of mice; D: WT and Srsf1 mice fed with Chow or HFD+L-NAME endo- / - Characteristic pathological staining images of mouse heart tissue, top: Masson staining (scale bar, 50 μm); middle: Sirius red staining (scale bar, 50 μm); bottom: wheat agglutinin staining (scale bar, 20 μm); E: WT and Srsf1 fed Chow or HFD+L-NAME endo- / - Masson staining of mouse heart tissue to assess myocardial fibrosis statistics; F: WT and Srsf1 fed Chow or HFD+L-NAME endo- / - Sirius red staining of mouse heart tissue to evaluate myocardial fibrosis statistics; G: WT and Srsf1 fed Chow or HFD+L-NAME endo- / - Statistical data of cardiomyocyte cross-sectional area in mouse heart tissue stained with wheat agglutinin; H: WT and Srsf1 fed with Chow or HFD+L-NAME endo- / - Plasma triglyceride levels in mice; I: WT and Srsf1 fed Chow or HFD+L-NAME endo- / - Plasma cholesterol levels in mice; J: WT and Srsf1 fed Chow or HFD+L-NAME endo- / - Blood glucose levels (left) and area under the curve (right) of mice at different time points, WT is wild type, Srsf1 endo- / - The SRSF1 endothelial cell-specific knockout mice were used, Chow was used as the control group, and HFpEF was used as the model group.

[0031] Figure 5 To investigate the effect of knocking down SRSF1 on HUVEC; Figure 5A: Representative western blot and average data of SRSF1 in HUVECs treated with SRSF1-targeting siRNA; B: mRNA levels of GLUT1, PFKFB3 and PKM2 in HUVECs treated with SRSF1-targeting siRNA; C: Representative western blot and average data of GLUT1, PFKFB3 and PKM2 in HUVECs treated with SRSF1-targeting siRNA; D: Glucose content in the culture medium of HUVECs treated with SRSF1-targeting siRNA; E: Lactate content in the culture medium of HUVECs treated with SRSF1-targeting siRNA; F: Representative western blot and average data of α-SMA, N-cadherin and E-cadherin in HUVECs treated with SRSF1-targeting siRNA.

[0032] Figure 6 The effect of overexpression of SRSF1 on HUVEC; Figure 6 A: Representative western blot of SRSF1 in HUVECs infected with Ad-SRSF1 for 48 hours; B: mRNA levels of GLUT1, PFKFB3 and PKM2 in HUVECs infected with Ad-SRSF1; C: Representative western blot and average data of GLUT1, PFKFB3 and PKM2 in HUVECs infected with Ad-SRSF1; D: Representative graph of ECAR in HUVECs infected with Ad-SRSF1; E: Glycolysis level of HUVECs infected with Ad-SRSF1; F: Glycolytic reserve level of HUVECs infected with Ad-SRSF1; G: Glucose content in the culture medium of HUVECs infected with Ad-SRSF1; H: Lactate content in the culture medium of HUVECs infected with Ad-SRSF1; I: Representative western blot and average data of α-SMA, N-cadherin and E-cadherin in HUVECs infected with Ad-SRSF1. DETAILED DESCRIPTION

[0033] The present invention studies heart failure with preserved ejection fraction and discovers a new target SRSF1 for treating heart failure with preserved ejection fraction. Therefore, the present invention provides the use of SRSF1 as a target in the preparation of drugs for preventing and / or treating heart failure with preserved ejection fraction.

[0034] In the present invention, the SRSF1 includes the SRSF1 protein or the SRSF1 encoding gene. In the present invention, the human SRSF1 gene sequence number is Gene ID: 6426, and the mouse SRSF1 gene sequence number is Gene ID: 110809. In the present invention, the drug is a drug that inhibits the expression of the SRSF1 protein or the SRSF1 encoding gene.

[0035] In the present invention, C57BL / 6 mouse model and SRSF1 endothelial cell-specific knockout mouse (Srsf1 endo- / - ) and HUVEC cells (umbilical vein endothelial cells) were used as experimental subjects to explore the role of SRSF1 in heart failure with preserved ejection fraction. The present invention has no special restrictions on the method for constructing the C57BL / 6 mouse model. A high-fat diet (HFD) and L-NAME (nitric oxide synthase inhibitor N) well known in the art were used. ω-nitro-L-arginine methyl ester) to induce the construction of a HFpEF heart failure model with preserved ejection fraction. There is no special limitation on the method for constructing the HUVEC cell model. A high-sugar, high-fat cell model constructed by the combined stimulation of high glucose (HG) and palmitic acid (PA) well known in the art can be used. Through the heart failure model mice with preserved ejection fraction, it was found that the left ventricular strain rate of the induced mice was significantly reduced, that is, the diastolic function of the animal's heart was impaired, and obvious diastolic dysfunction occurred. At the same time, the animal's exercise tolerance decreased, proving that the model was successfully established and consistent with the signs of heart failure with preserved ejection fraction. By jointly detecting the heart tissue of the animal model and the HUVEC cell model tissue, it was found that SRSF1 was in a high expression state in the heart tissue and HUVEC cells, which revealed that SRSF1 is involved in regulating the pathological process of heart failure with preserved ejection fraction. Experiments on SRSF1 endothelial cell-specific knockout mice found that SRSF1 endothelial cell-specific knockout can significantly improve the diastolic dysfunction and hypertension signs of heart failure mice with preserved ejection fraction, and significantly improve the pulmonary edema state. At the same time, staining of myocardial tissue sections revealed that endothelial cell-specific knockout of SRSF1 can significantly improve myocardial remodeling of myocardial fibrosis and myocardial hypertrophy. It was further found that endothelial cell-specific knockout of SRSF1 also has a significant effect on metabolic regulation, and can significantly improve abnormal sugar and lipid metabolism. The present invention also interferes with the expression of SRSF1 in HUVEC cells and finds that knocking down SRSF1 can inhibit the endothelial-mesenchymal transition process of HUVEC, and overexpressing SRSF1 can promote the endothelial-mesenchymal transition process of HUVEC. In summary, endothelial cell-specific knockout and interference of SRSF1 can play a role in alleviating heart failure, thereby playing a role in treating heart failure with preserved ejection fraction. The findings of the present invention contribute to the current understanding of the pathogenesis of heart failure with preserved ejection fraction, and therapeutic intervention by inhibiting SRSF1 can provide a potential new treatment strategy for heart failure with preserved ejection fraction.

[0036] The present invention provides the use of an SRSF1 inhibitor in the preparation of a drug for preventing and / or treating heart failure with preserved ejection fraction. In the present invention, the SRSF1 inhibitor includes an SRSF1 gene inhibitor or an SRSF1 protein inhibitor. The SRSF1 gene inhibitor includes at least one of the following RNAs or gene-derived products thereof: siRNA, shRNA, and sgRNA targeting the gene SRSF1. In one embodiment, the nucleotide sequence of the siRNA targeting the gene SRSF1 is shown in SEQ ID NO: 1 and SEQ ID NO: 2. The SRSF1 gene inhibitor can also be a nucleic acid construct or a lentivirus. The nucleic acid construct is a gene fragment containing the gene encoding the above-mentioned nucleic acid molecule and can express the above-mentioned nucleic acid molecule. The lentivirus is formed by viral packaging of the above-mentioned nucleic acid construct with the assistance of a lentiviral packaging plasmid and a cell line. The SRSF1 protein inhibitor includes an antibody to the SRSF1 protein, a binding molecule to the SRSF1 protein, or a degrader of the SRSF1 protein. In an alternative embodiment, the SRSF1 protein inhibitor can be, but is not limited to, labetalol or betaxolol. Drugs such as labetalol or betaxolol bind to the SRSF1 protein, thereby affecting the function of the SRSF1 protein.

[0037] The present invention also provides a drug capable of preventing and / or treating heart failure with preserved ejection fraction, comprising the aforementioned SRSF1 inhibitor and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes a buffer, excipient, stabilizer, or preservative, such as, but not limited to, starch, lactose, magnesium stearate, water, saline, buffer, glycerol, ethanol, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be compatible with the dosage form.

[0038] In this application, the heart failure with preserved ejection fraction is heart failure with a left ventricular ejection fraction (LVEF) ≥ 50%, including five types: HFpEF-1, HFpEF-2, HFpEF-3, HFpEF-4, and HFpEF-5. The HFpEF-1 is HFpEF associated with vascular disease, which is associated with hypertension, coronary artery disease and coronary microvascular dysfunction; the HFpEF-2 is HFpEF associated with cardiomyopathy, which is HFpEF caused by hypertrophic cardiomyopathy, infiltrative cardiomyopathy such as cardiac amyloidosis and Fabry disease; the HFpEF-3 is HFpEF associated with right heart and pulmonary artery disease, that is, HFpEF caused by pulmonary hypertension with or without right ventricular dysfunction; the HFpEF-4 is HFpEF associated with valvular heart disease and arrhythmia, that is, HFpEF caused by valvular heart disease and atrial fibrillation; the HFpEF-5 is HFpEF associated with extracardiac disease, that is, HFpEF caused by extracardiac diseases, mainly including metabolic diseases such as diabetes, obesity or metabolic syndrome; diseases that often lead to high-output states, such as anemia, liver disease, hyperthyroidism, arteriovenous fistula, etc.; and other diseases, such as chronic kidney disease, tumor treatment, etc.

[0039] In the present invention, there is no particular limitation on the dosage form of the drug, and the drug may be in the form of tablets, injections, inhalants, granules, pills, and capsules. Each dosage form in the present invention may also contain pharmaceutically acceptable excipients, including one or more of diluents, colorants, sweeteners, coating agents, binders, absorbents, disintegrants, dispersants, wetting agents, solubilizers, buffers, and surfactants. The present invention does not particularly limit the route of administration, and the drug may be administered orally, intravenously, parenterally, intramuscularly, subcutaneously, intraperitoneally, intranasally, orally, or topically, depending on the dosage form and actual needs.

[0040] In the examples of the present invention, all data were statistically analyzed using GraphPad Prism 9.0 software, and the data were expressed as mean ± standard deviation. A significant difference was defined when P < 0.05.

[0041] In the following examples, unless otherwise specified, all methods are conventional.

[0042] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0043] The technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] To establish a heart failure model with preserved ejection fraction, ten eight-week-old C57BL / 6 male mice were fed a 60% fat HFD diet and 0.5 g / L L-NAME in drinking water (pH adjusted to 7.4) for eight weeks to serve as the HFpEF group (model group). Ten C57BL / 6 male mice fed a conventional diet served as the Chow group (control group).

[0046] The results are as follows Figure 1 As shown, after 8 weeks of feeding, cardiac function was assessed by ultrasound. Results showed no significant changes in left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (FS%) in the HFpEF group compared with the Chow group, suggesting intact cardiac systolic function. Blood flow velocity at the maximum opening of the mitral valve was measured using Color Doppler-Mode and Pulse Wave, with E wave and A wave measurements. The E / A ratio was significantly increased in the HFpEF group. Tissue Doppler measurements of the mitral septal tissue velocity, e', and the E / e' ratio were significantly increased in the HFpEF group. Left ventricular long-axis images were acquired using B-mode, and speckle tracking analysis of left ventricular strain rate revealed a significant decrease in the HFpEF group at 8 weeks. These results suggest that diastolic function in HFpEF mice is impaired, with significant diastolic dysfunction. Further testing of exercise endurance by running distance revealed that the HFpEF group ran significantly less distance than the Chow group, suggesting impaired exercise endurance in these mice. Furthermore, blood pressure measurements in both groups revealed that both systolic and diastolic blood pressure in the HFpEF group were significantly higher than those in the Chow group, suggesting signs of hypertension in these mice. This confirms the successful establishment of a mouse model of heart failure with preserved ejection fraction.

[0047] Example 2

[0048] Determination of SRSF1 expression level in mice with heart failure and preserved ejection fraction

[0049] Eight 8-week-old C57BL / 6 male mice were fed a 60% fat HFD diet and 0.5 g / L L-NAME in drinking water (pH adjusted to 7.4) for 8 weeks to serve as the HFpEF group (model group). Eight C57BL / 6 male mice fed a conventional diet served as the Chow group (control group). SRSF1 expression levels were measured in the HFpEF and Chow groups.

[0050] The results are as follows Figure 2 -A and Figure 2-B, compared with the Chow group, the mRNA and protein levels of SRSF1 in the HFpEF group were significantly increased. Immunofluorescence staining was performed using the heart tissues of the two groups of mice. Figure 2 As shown in Figure 3-C, the expression of SRSF1 in endothelial cells of the heart tissue of HFpEF mice was significantly increased, confirming that SRSF1 is involved in regulating the pathological process of HFpEF.

[0051] Example 3

[0052] Determination of SRSF1 expression level in high glucose and high fat HUVEC cell models

[0053] HUVEC cells were induced with DMEM medium containing 5.5 mmol / L glucose for 24 hours as the control group (NG), HUVEC cells were induced with DMEM medium containing 33 mmol / L glucose for 24 hours as the high glucose treatment group (HG), and HUVEC cells were induced with 0.2 mmol / L palmitic acid for 24 hours to construct a high lipid cell model.

[0054] After high sugar and high fat induction, the expression level of SRSF1 was detected. Figure 2 -D, Figure 2 -E, Figure 2 -F, Figure 2 As shown in Figure 5-G, the mRNA and protein levels of SRSF1 were found to be significantly increased in HUVEC cells, confirming that SRSF1 is involved in regulating the pathological process of heart failure with preserved ejection fraction.

[0055] Example 4

[0056] Validation of the potential role of SRSF1 in heart failure with preserved ejection fraction

[0057] SRSF1 endothelial cell-specific knockout mice (Srsf1 endo- / - ) construction: SRSF1 was provided by Professor Fu Xiangdong's team flox / flox The mice were hybridized with Tie2-Cre mice provided by Beijing Weishanglide Biotechnology Co., Ltd. to obtain hybrid mice. The SRSF1 knockout mice driven by the endothelial cell-specific promoter were confirmed by mouse tail gene PCR. endo- / - mouse.

[0058] Ten C57BL / 6 male mice aged 8 to 12 weeks and ten Srsf1 mice aged 8 to 12 weeks were selected. endo- / -Mice were fed a 60% fat HFD diet and 0.5 g / L L-NAME in drinking water (pH adjusted to 7.4) for 8 weeks and served as the HFpEF group. Among them, C57BL / 6 male mice were designated as the HFpEF+WT group (wide type, wild type group), and Srsf1 endo- / - Mice were designated as HFpEF+Srsf1 endo- / - Group 10 C57BL / 6 male mice aged 8 to 12 weeks and 10 Srsf1 mice aged 8 to 12 weeks were selected. endo- / - The mice were fed with a regular diet for 8 weeks and used as the Chow group to control the above two groups, which were respectively recorded as the WT+Chow group and the Srsf1 endo- / - +Chow group.

[0059] The results are as follows Figure 3 , cardiac function of mice was assessed by ultrasound. Figure 3 -B. Figure 3 -C and Figure 3 -D showed that the left ventricular ejection fraction and left ventricular contraction fraction of HFpEF mice were not significantly reduced compared with the Chow group, indicating that the mouse heart maintained normal contractile function. The values ​​of E, A, and e' were measured under Pulse Wave and Tissue Doppler conditions, and the results were obtained through data processing and analysis. In the HFpEF group, Srsf1 endo- / - The E / A and E / e' of mice were significantly lower than those of WT group. The statistical results of global longitudinal strain (GLS) of left ventricle suggested that Srsf1 endo- / - The left ventricular strain rate of mice was significantly higher than that of the WT group. This suggests that after SRSF1 gene knockout, the diastolic function impairment of HFpEF was improved. The results of the exercise endurance experiment showed that in mice fed with high fat + L-NAME, Srsf1 endo- / - The maximum running distance of the mice in the WT group was significantly higher than that in the SRSF1 group, which further indicated that SRSF1 gene knockout enhanced the exercise tolerance of HFpEF. Figure 3 In addition, SRSF1 gene knockout significantly reduced systolic and diastolic blood pressure levels in HFpEF mice, as shown in Figure 3 -J and Figure 3 In summary, endothelial cell-specific knockout of SRSF1 significantly improved diastolic dysfunction and hypertension in mice with heart failure and preserved ejection fraction, indicating that inhibiting SRSF1 expression can improve diastolic dysfunction and lower blood pressure.

[0060] Example 5

[0061] Verify the effect of SRSF1 on cardiopulmonary tissue and lipid metabolism in heart failure with preserved ejection fraction

[0062] Lung and heart tissues were taken from each group of mice in Example 4 for testing, and the degree of pulmonary edema was assessed by measuring and calculating the lung wet / dry weight ratio. Left ventricular myocardial tissue was stained with Masson and Sirius red, and the degree of myocardial fibrosis in each group was statistically calculated. Wheat germ agglutinin (WGA) staining was also performed, and the cross-sectional area of ​​myocardial cells in each group was statistically calculated to assess the degree of myocardial hypertrophy. Metabolic function was also assessed, and plasma triglyceride (TG), cholesterol (TC), and blood glucose levels in the mice were measured.

[0063] The results are as follows Figure 4 As shown in the results, the lung wet / dry weight ratio of the HFpEF group was significantly increased compared with the Chow group, indicating more severe pulmonary edema, while endothelial cell-specific knockout of SRSF1 significantly improved this situation. endo- / - Under high-fat + L-NAME feeding conditions, mice showed lower heart weight / tibia length (HW / TL, heart weight / tibialength) and heart weight / body weight (HW / BW, heart weight / body weight) ratios, suggesting that SRSF1 gene knockout improved myocardial hypertrophy and alleviated signs of heart failure. Endothelial cell-specific knockout of SRSF1 significantly improved myocardial remodeling with myocardial fibrosis and hypertrophy induced by HFD+L-NAME diet, as manifested by reduced collagen deposition and reduced cross-sectional area of ​​cardiomyocytes. Metabolic function was subsequently evaluated, and consistent with the above results, SRSF1 knockout could reverse the high triglyceride and high cholesterol levels induced by high fat + L-NAME and improve lipid metabolism. At the same time, the glucose tolerance test (GTT) was used to detect glucose tolerance. The data showed that the HFpEF group mice showed obvious abnormal glucose tolerance compared with the Chow group, and Srsf1 endo- / - These abnormalities in mice were alleviated, and abnormal glucose metabolism was improved. In summary, specific knockout of SRSF1 in endothelial cells significantly improved the signs of heart failure and abnormal glucose and lipid metabolism in HFpEF, indicating that inhibiting SRSF1 expression can improve pulmonary edema, myocardial fibrosis, myocardial hypertrophy, and muscle remodeling, and significantly alleviate abnormal glucose and lipid metabolism.

[0064] Example 6

[0065] Effects of SRSF1 knockdown and overexpression on endothelial cell metabolism and function

[0066] To verify the effect of endothelial cell-specific knockout of SRSF1 on HFpEF, we further explored the mechanism in endothelial cells and constructed HUVECs with SRSF1 knockdown and SRSF1 overexpression, respectively.

[0067] 1. Knockdown of SRSF1

[0068] First, two small interfering RNAs were designed using RNAi Designer targeting the coding region of SRSF1 to specifically knock down the expression level of SRSF1. The SRSF1 si1 sequence was AGACUGUGAUAUU GUGUAAAU (SEQ ID NO: 1), and the SRSF1 si2 sequence was ATGTATGTTATGCTGA TGTTTAC (SEQ ID NO: 2). A double-stranded RNA-negative control with a non-specific sequence (called Scrambled) was used as a control. Cells were transfected with Lipofectamine RNAiMax for 72 hours.

[0069] The results are as follows Figure 5 As shown in the figure, after HUVECs were transfected with two different SRSF1 siRNAs, SRSF1 protein levels were significantly reduced. After knocking down SRSF1 in HUVEC cells using SRSF1 siRNA, the mRNA levels of glycolysis-related genes GLUT1, PFKFB3, and PKM2 were significantly reduced, and the protein levels of these genes were also significantly lower than those in the control group. Supernatant culture medium from SRSF1-knockdown HUVECs was collected and assayed for glucose and lactate levels. The results showed that glucose content in the supernatant of SRSF1-knockdown cells was significantly increased, while lactate content was decreased, indicating that knocking down SRSF1 inhibited glycolysis in HUVECs. Abnormal glycolysis metabolism is an important factor causing endothelial-mesenchymal transition (Endo-MT). Therefore, the protein expression of endothelial-mesenchymal transition-related genes was detected. It was found that after knocking down SRSF1 in HUVEC, the protein expression of mesenchymal cell markers α-SMA and N-cadherin was significantly reduced, and the protein expression of endothelial cell marker E-cadherin was promoted, indicating that knocking down SRSF1 inhibited the endothelial-mesenchymal transition process of HUVEC.

[0070] 2. High expression of SRSF1

[0071] First, molecular biology methods were used to construct an HA-tagged adenovirus, Ad-SRSF1-HA, which was constructed by Beijing Biochuang Biotechnology Co., Ltd. The β-gal and SRSF1 adenovirus (Ad-SRSF1-HA) were thawed on ice. HUVEC cells were removed, and the original culture medium was aspirated using a suction pump. Serum-free ECM culture medium was added, and the virus was diluted and added to culture dishes or well plates. The cells were mixed and cultured in a CO2 incubator for 48 hours. The expression of SRSF1 and related proteins was then detected.

[0072] The results are as follows Figure 6 As shown, after successful overexpression of SRSF1 in HUVEC cells, mRNA levels of glycolysis-related genes GLUT1, PFKFB3, and PKM2 were significantly increased, while protein levels of these genes were also significantly higher than those in the control group. Cellular glycolysis was assessed using Seahorse, and the results showed that SRSF1 overexpression significantly increased glycolysis in HUVEC. Supernatant culture medium from HUVEC cells overexpressing SRSF1 was collected and assayed for glucose and lactate content. Results showed that glucose content was significantly decreased and lactate content was increased in the supernatant of cells overexpressing SRSF1, indicating that SRSF1 overexpression promoted glycolysis in HUVEC. Furthermore, SRSF1 overexpression in HUVEC cells significantly increased protein expression of mesenchymal cell markers α-SMA and N-cadherin, while inhibiting protein expression of the endothelial cell marker E-cadherin, suggesting that SRSF1 overexpression promotes the endothelial-mesenchymal transition (EMT) in HUVEC.

[0073] Conclusion: Endothelial-to-mesenchymal transition (EndoMT) is a potential pathological process. Activation of the EMT mechanism leads to prominent mesenchymal characteristics and excessive fibroblast proliferation, further aggravating ischemia and hypoxia in lung and cardiac tissues, accelerating fibrosis, and exacerbating the pathological process of heart failure with preserved ejection fraction. Therefore, inhibiting SRSF1 expression may inhibit the EMT process of HUVECs, thereby reducing cardiac fibrosis, thereby achieving the goal of treating heart failure with preserved ejection fraction and alleviating related symptoms.

[0074] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of an SRSF1 inhibitor in the preparation of a drug for preventing and / or treating heart failure with preserved ejection fraction, the drug comprising an siRNA targeting the gene SRSF1; the nucleotide sequence of the siRNA targeting the gene SRSF1 is shown in SEQ ID NO: 1 and SEQ ID NO:

2.

2. The use according to claim 1, characterized in that 1) The SRSF1 inhibitor prevents and / or treats heart failure with preserved ejection fraction by improving diastolic dysfunction; 2) The SRSF1 inhibitor prevents and / or treats heart failure with preserved ejection fraction by lowering blood pressure; 3) The SRSF1 inhibitor prevents and / or treats heart failure with preserved ejection fraction by alleviating pulmonary edema; 4) The SRSF1 inhibitor prevents and / or treats heart failure with preserved ejection fraction by improving myocardial fibrosis; 5) The SRSF1 inhibitor prevents and / or treats heart failure with preserved ejection fraction by alleviating myocardial hypertrophy and muscle remodeling; 6) The SRSF1 inhibitor prevents and / or treats heart failure with preserved ejection fraction by improving abnormal lipid metabolism; 7) The SRSF1 inhibitor prevents and / or treats heart failure with preserved ejection fraction by improving abnormal glucose metabolism; or 8) The SRSF1 inhibitor prevents and / or treats heart failure with preserved ejection fraction by inhibiting the endothelial-mesenchymal transition process of HUVECs.

3. A drug capable of preventing and / or treating heart failure with preserved ejection fraction, characterized in that: The drug comprises siRNA targeting gene SRSF1 and a pharmaceutically acceptable carrier; the siRNA targeting gene SRSF1 is shown as SEQ ID NO: 1 and SEQ ID NO:

2.

4. The drug according to claim 3, characterized in that The heart failure with preserved ejection fraction includes HFpEF-1, HFpEF-2, HFpEF-3, HFpEF-4, and HFpEF-5.

Images