Application of SRSF1 in preparation of medicine for preventing and / or treating ejection fraction reserved heart failure

Abstract

The invention provides application of SRSF1 in preparation of a medicine for preventing and / or treating ejection fraction reserved heart failure, and belongs to the technical field of biological medicine. The invention determines that the SRSF1 can be used as a key target for preparation of heart failure related medicines for preventing and / or treating ejection fraction retention. Animal model construction and cellular level experiments prove that SRSF1 participates in regulation of the pathological process of the disease. Experiments of constructing a model in an SRSF1 endothelial cell specific knockout mouse show that by inhibiting SRSF1 expression, symptoms such as diastolic dysfunction and hypertension can be improved, pulmonary edema can be relieved, and myocardial fibrosis and reconstitution can be inhibited. The abnormal glucose and lipid metabolism can be improved, and the endothelial-mesenchymal transition process can be inhibited. The invention opens up a new path for new drug screening, provides a new target and method for treatment of ejection fraction reserved heart failure, and has important medical significance and value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application 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 various cardiovascular diseases, and its incidence and mortality rates are rising year by year. According to epidemiological data, heart failure with preserved ejection fraction (HFpEF) accounts for 50% of the total number of heart failures, and has become the most common type of heart failure. It is particularly noteworthy that with the continuous extension of human life expectancy, as well as the increasing incidence and normalization of obesity and diabetes, it is expected that the proportion of heart failure with preserved ejection fraction will 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 brings an extremely heavy burden to the health and life of patients.

[0003] In clinical practice, heart failure with preserved ejection fraction is defined as a disease characterized by: when ventricular systolic function is within the normal range, the relaxation characteristics and compliance of the ventricles are reduced, resulting in a decrease in ventricular filling volume and an increase in filling pressure, which ultimately leads to clinical syndrome manifestations of pulmonary and systemic congestion. The pathophysiological mechanisms of heart failure with preserved ejection fraction are complex and diverse. At present, there are five main mechanisms that have been deeply explored, namely 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, influence each other, and promote each other, greatly increasing the complexity of disease diagnosis and the difficulty of treatment. For this reason, in the current clinical field, it has been difficult to develop specific drugs specifically for heart failure with preserved ejection fraction, and effective treatment methods are extremely rare. Therefore, it is extremely important to deeply explore the molecular mechanisms of heart failure with preserved ejection fraction and to find 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 splicing functions, SRSF1 can also regulate processes such as mRNA transcription, stability, and protein translation. Studies have shown that under overexpression, SRSF1 can promote oncogene 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 a SRSF1 inhibitor in the preparation of a drug for preventing and / or treating heart failure with preserved ejection fraction, wherein the SRSF1 inhibitor includes a SRSF1 gene inhibitor or a 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 in 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, the drug comprising 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 is 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 giving full play to the positive role of alleviating the signs of heart failure. In addition, 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 (HUVEC), which further proves that inhibiting SRSF1 can effectively delay the development 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 sets 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 medical research and clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The physiological and biochemical conditions of animals after the heart failure model with preserved ejection fraction was established; 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 long-axis strain rate GLS of Chow and HFpEF mice; H: Maximum running distance of Chow and HFpEF mice in exercise endurance experiment; 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 cell 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 mice 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 Chow or HFD+L-NAME endo- / - Left ventricular longitudinal strain rate in mice GLS; I: WT and Srsf1 fed Chow or HFD+L-NAME endo- / - Maximum running distance of mice; J: WT and Srsf1 mice fed Chow or HFD+L-NAME endo- / - Systolic blood pressure of mice; K: WT and Srsf1 fed Chow or HFD+L-NAME 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 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 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: lectin staining (scale bar, 20 μm); E: WT and Srsf1 fed Chow or HFD+L-NAME endo- / - Masson staining of mouse heart tissue to evaluate myocardial fibrosis statistics; F: WT and Srsf1 fed Chow or HFD+L-NAME endo- / - Sirius red staining of mouse heart tissue to evaluate the statistical data of myocardial fibrosis; G: WT and Srsf1 fed Chow or HFD+L-NAME endo- / - Statistical data of the cross-sectional area of ​​cardiomyocytes stained with glutathione in mouse heart tissue; H: WT and Srsf1 fed with Chow or HFD+L-NAME endo- / - Plasma triglyceride levels in mice; I: WT and Srsf1 mice fed Chow or HFD+L-NAME endo- / - Plasma cholesterol levels in mice; J: WT and Srsf1 mice 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 is 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: Glycolytic 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 a drug for preventing and / or treating heart failure with preserved ejection fraction.

[0034] In the present invention, the SRSF1 includes SRSF1 protein or 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 SRSF1 protein or SRSF1 encoding gene.

[0035] In the present invention, C57BL / 6 mouse model, 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 particular restrictions on the method for constructing the C57BL / 6 mouse model. A high-fat diet (HFD) and L-NAME (nitric oxide synthase inhibitor N ω-nitro-L-arginine methyl ester) combined with induction to construct a HFpEF heart failure model with preserved ejection fraction. There is no special restriction on the model construction method of HUVEC cells. The high-sugar and high-fat cell model constructed by the combined stimulation of high glucose (HG) and palmitic acid (PA) 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 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 met the signs of heart failure with preserved ejection fraction. By jointly detecting the heart tissue and HUVEC cell model tissue of the animal model, it was found that SRSF1 was in a high expression state in the heart tissue and HUVEC cells, which revealed that SRSF1 was 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 state of pulmonary edema. 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] In the present invention, the use of SRSF1 inhibitors in the preparation of drugs for preventing and / or treating heart failure with preserved ejection fraction is provided. 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 its gene-derived products: siRNA, shRNA and sgRNA targeting the gene SRSF1. As an 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 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 lentivirus 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 to the SRSF1 protein. As an optional embodiment, the SRSF1 protein inhibitor can be Labetalol or Betaxolol, but is not limited to these. 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, the drug comprising the above-mentioned SRSF1 inhibitor and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes a buffer, an excipient, a stabilizer or a preservative, such as starch, lactose, magnesium stearate, water, saline, buffer, glycerol, ethanol, liposomes, lipids, proteins, protein-antibody conjugates, peptide substances, cellulose, nanogels or combinations thereof, but not limited to the above. The choice of the carrier should match the drug dosage form.

[0038] In the present application, the heart failure with preserved ejection fraction is heart failure with left ventricular ejection fraction LVEF ≥ 50%. The heart failure with preserved ejection fraction includes five types: HFpEF-1, HFpEF-2, HFpEF-3, HFpEF-4, and HFpEF-5. The HFpEF-1 is HFpEF associated with vascular diseases, 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 diseases, 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 diseases, 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 state, such as anemia, liver disease, hyperthyroidism, arteriovenous fistula, etc.; 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, which may be tablets, injections, inhalants, granules, pills, capsules, etc. Various dosage forms in the present invention also include pharmaceutically acceptable excipients, including one or more of diluents, colorants, sweeteners, coating agents, adhesives, absorbents, disintegrants, dispersants, wetting agents, cosolvents, buffers, and surfactants. The present invention does not have a particular limitation on the mode of administration, and the mode of oral, intravenous, parenteral, intramuscular, subcutaneous, intraperitoneal, intranasal, rectal administration, or topical administration may be conventionally selected according to the dosage form of the drug and actual needs.

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

[0041] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[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 in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0044] Example 1

[0045] Establishment of heart failure model with preserved ejection fraction: Ten 8-week-old C57BL / 6 male mice were selected and fed with a HFD diet containing 60% fat and drinking water containing 0.5 g / L L-NAME (pH adjusted to 7.4) for 8 weeks as the HFpEF group (model group). Ten C57BL / 6 male mice fed with a regular diet were used as the Chow group (control group).

[0046] The results are as follows Figure 1 As shown in the figure, after 8 weeks of feeding, the cardiac function of mice was detected by ultrasound. The results showed that compared with the Chow group, there was no significant change in the left ventricular ejection fraction and left ventricular shortening fraction (FS%, Fraction shortening) of the HFpEF group mice, indicating that the cardiac contractile function of the mice was not damaged. Under the conditions of Color Doppler-Mode and Pulse Wave, the blood flow velocity at the maximum opening of the mitral valve was detected, and the E wave and A wave were measured. It can be seen that the E / A ratio was significantly increased in the HFpEF group mice. Under the conditions of Tissue Doppler, the activity velocity of the mitral septum valve tissue e' was detected, and the statistical E / e' ratio showed that it was significantly increased in the HFpEF group mice. The left ventricular long axis image was obtained under B-mode, and the left ventricular strain rate was analyzed by speckle tracking Strain. It can be seen that the left ventricular strain rate of the HFpEF group mice was significantly reduced at the 8th week. The above results indicate that the cardiac diastolic function of HFpEF mice is impaired and obvious diastolic dysfunction occurs. Further testing of their exercise endurance by running distance showed that the running distance of HFpEF mice was significantly lower than that of Chow mice, indicating that the exercise endurance of HFpEF mice was impaired. In addition, by testing the blood pressure levels of the two groups of mice, the data showed that the systolic and diastolic blood pressure of HFpEF mice were significantly higher than those of Chow mice, indicating that HFpEF mice had signs of hypertension. The above confirmed that the mouse model of heart failure with preserved ejection fraction was successfully constructed.

[0047] Example 2

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

[0049] Eight 8-week-old C57BL / 6 male mice were selected and fed with a HFD diet containing 60% fat and drinking water containing 0.5 g / L L-NAME (pH adjusted to 7.4) for 8 weeks as the HFpEF group (model group). Eight C57BL / 6 male mice fed with a regular diet were used as the Chow group (control group). The expression level of SRSF1 in mice in the HFpEF group and the Chow group was detected.

[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 3C, the expression of SRSF1 in endothelial cells of the heart tissue of mice in the HFpEF group 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 glucose 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 in HUVEC cells were found to be significantly increased, 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., and the hybrid mice were identified by mouse tail gene PCR, confirming that the endothelial cell-specific promoter-driven SRSF1 knockout mice, namely Srsf1 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 HFD diet containing 60% fat and drinking water containing 0.5 g / L L-NAME (pH adjusted to 7.4) for 8 weeks as the HFpEF group. Among them, C57BL / 6 male mice were designated as the HFpEF+WT group (wide type) 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 as the Chow group, and compared with the above two groups, respectively, denoted as the WT+Chow group, 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 the HFpEF group 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 mice, 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 impaired diastolic function 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 -K. In summary, endothelial cell-specific knockout of SRSF1 can significantly improve diastolic dysfunction and hypertension signs in mice with heart failure with preserved ejection fraction, indicating that inhibiting the expression of SRSF1 can improve diastolic dysfunction and lower blood pressure.

[0060] Example 5

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

[0062] The lung tissue and heart tissue of each group of mice in Example 4 were respectively taken for detection, and the degree of pulmonary edema was evaluated by measuring and calculating the lung wet / dry weight ratio. The left ventricular myocardial tissue was taken for Masson and Sirius red staining (Sirius red stain), and the degree of myocardial fibrosis in each group was statistically calculated; wheat germagglutinin staining (WGA, wheat germagglutinin) was performed at the same time, and the cross-sectional area of ​​myocardial cells in each group was statistically calculated to evaluate the degree of myocardial hypertrophy. Metabolic function was also evaluated, and the plasma triglyceride (TG), cholesterol (TC) and blood glucose levels of mice were detected.

[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, showing more severe pulmonary edema, and endothelial cell-specific knockout of SRSF1 could significantly improve this situation. endo- / - Under high-fat + L-NAME feeding conditions, mice showed lower heart weight / tibialength (HW / TL) and heart weight / body weight (HW / BW), suggesting that SRSF1 gene knockout improved myocardial hypertrophy and alleviated signs of heart failure. Endothelial cell-specific knockout of SRSF1 significantly improved 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 (glucose tolerance test, GTT) experiment was used to detect their glucose tolerance. The data showed that the HFpEF group mice showed obvious abnormal glucose tolerance compared with the Chow group, while Srsf1 endo- / - This abnormality in mice was alleviated, and abnormal glucose metabolism was improved. In summary, specific knockout of SRSF1 in endothelial cells can significantly improve the signs of HFpEF heart failure and abnormal glucose and lipid metabolism, indicating that inhibiting the expression of SRSF1 can improve pulmonary edema, myocardial fibrosis, myocardial hypertrophy and muscle remodeling, and can significantly alleviate abnormal glucose and lipid metabolism.

[0064] Example 6

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

[0066] In order to verify the effect of endothelial cell-specific knockout of SRSF1 on HFpEF, the mechanism was further explored in endothelial cells, and HUVECs with SRSF1 knockdown and high expression were constructed, respectively.

[0067] 1. Knockdown of SRSF1

[0068] Firstly, two small interfering RNAs were designed for the coding region of SRSF1 using RNAi Designer 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). The double-stranded RNA-negative control of non-specific sequence (called Scrambled) was used as a control, and the cells were transfected with Lipofectamine RNAiMax for 72 hours.

[0069] The results are as follows Figure 5 As shown in the figure, after HUVEC was transfected with two different SRSF1 siRNAs, the protein level of SRSF1 was significantly reduced. After knocking down SRSF1 in HUVEC cells using SRSF1 siRNA, the mRNA levels of glycolysis pathway-related genes GLUT1, PFKFB3, and PKM2 were significantly reduced, and the protein levels of the above genes were also significantly lower than those in the control group. The supernatant culture medium of HUVEC with SRSF1 knocked down was collected to detect the glucose and lactate contents. The results showed that the glucose content in the supernatant of SRSF1 knocked down was significantly increased, and the lactate content was decreased, indicating that knocking down SRSF1 inhibited the glycolysis level of HUVEC. 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, and it was found that after knocking down SRSF1 in HUVEC, the protein expression of mesenchymal cell markers α-SMA and N-cadherin could be 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 adenovirus Ad-SRSF1-HA with an HA tag, which was constructed by Beijing Biochuang Biotechnology Co., Ltd. β-gal and SRSF1 adenovirus (Ad-SRSF1-HA) were thawed on ice, HUVEC cells were taken out, the original culture medium was sucked out with a suction pump, serum-free ECM culture medium was added, the virus was diluted and added to the culture dish or well plate, mixed, and placed in a CO2 incubator for 48 hours, and then the expression of SRSF1 and related proteins was detected.

[0072] The results are as follows Figure 6 As shown in the figure, after successfully overexpressing SRSF1 in HUVEC cells, the mRNA levels of glycolysis pathway-related genes GLUT1, PFKFB3 and PKM2 were significantly increased, and the protein levels of the above genes were also significantly higher than those in the control group. Seahorse was used to evaluate the glycolysis level of cells, and the results showed that overexpression of SRSF1 could significantly increase the glycolysis level of HUVEC. The supernatant culture medium of HUVEC overexpressing SRSF1 was collected to detect the glucose and lactate contents. The results showed that the glucose content in the supernatant overexpressing SRSF1 was significantly reduced, and the lactate content was increased, indicating that overexpression of SRSF1 promoted the glycolysis level of HUVEC. At the same time, it was found that overexpression of SRSF1 in HUVEC cells could significantly promote the protein expression of mesenchymal cell markers α-SMA and N-cadherin, and inhibit the protein expression of endothelial cell marker E-cadherin, indicating that overexpression of SRSF1 promoted the endothelial-mesenchymal transition process of HUVEC.

[0073] Conclusion: Endothelial-mesenchymal transition (Endo MT) is a potential disease-causing pathological biological process. Activating the mechanism of endothelial-mesenchymal transition will make mesenchymal characteristics prominent, fibroblasts overproliferate, further aggravate the ischemia and hypoxia of lung and heart tissues in the body, accelerate the fibrosis process, and aggravate the pathological process of heart failure with preserved ejection fraction. Therefore, inhibiting the expression of SRSF1 can inhibit the endothelial-mesenchymal transition process of HUVEC, thereby reducing cardiac fibrosis, so as to achieve the purpose 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of SRSF1 as a target in the preparation of drugs for the prevention and / or treatment of heart failure with preserved ejection fraction.

2. The use according to claim 1, characterized in that: The SRSF1 includes SRSF1 protein or SRSF1 encoding gene.

3. The use according to claim 1, characterized in that: The drug inhibits the expression of SRSF1 protein or SRSF1 encoding gene.

4. The use according to claim 3, characterized in that: The drug is any one or more of the following: 1) The drug is a drug for improving diastolic dysfunction; 2) The drug is a drug for lowering blood pressure; 3) The drug is a drug for alleviating pulmonary edema; 4) The drug is a drug for improving myocardial fibrosis; 5) The drug is a drug for alleviating myocardial hypertrophy and muscle remodeling; 6) The drug is a drug for improving abnormal lipid metabolism; 7) The drug is a drug for improving abnormal glucose metabolism; 8) The drug is a drug that inhibits the endothelial-mesenchymal transition process of HUVEC.

5. Use of a SRSF1 inhibitor in the preparation of a drug for preventing and / or treating heart failure with preserved ejection fraction, characterized in that: The SRSF1 inhibitor includes a SRSF1 gene inhibitor or a SRSF1 protein inhibitor.

6. The use according to claim 5, characterized in that: 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.

7. The use according to claim 6, characterized in that: The nucleotide sequences of the siRNA targeting the SRSF1 gene are shown in SEQ ID NO: 1 and SEQ ID NO:

2.

8. The use according to claim 5, characterized in that: 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.

9. A drug capable of preventing and / or treating heart failure with preserved ejection fraction, characterized in that: The medicament comprises the SRSF1 inhibitor according to claim 5 and a pharmaceutically acceptable carrier.

10. The drug according to claim 9, characterized in that The heart failure with preserved ejection fraction includes HFpEF-1, HFpEF-2, HFpEF-3, HFpEF-4, and HFpEF-5.

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