Use of orm2 protein in the preparation of a medicament for preventing and / or treating heart failure with preserved ejection fraction

Abstract

The application relates to the technical field of biological medicine, and discloses application of ORM2 protein in preparation of a medicine for preventing and / or treating heart failure with preserved ejection fraction. The present application is directed to the problems that existing researches on heart failure with preserved ejection fraction (HFpEF) only focus on local heart pathology, the core molecule of liver-heart axis cross-organ regulation is not clear, and there is a lack of precise treatment targets for the core mechanism of HFpEF in clinic, and existing intervention means is non-specific, and for the first time, application of ORM2 protein from the liver in improving cardiac metabolic disorder of HFpEF is proposed. It is found through experiments that the ORM2 protein can effectively improve the diastolic function of the heart of HFpEF, reduce myocardial fibrosis and lipotoxic injury, and relieve cardiac metabolic disorder, can fill the research gap of the cross-organ regulation mechanism of HFpEF, provide a new precise treatment target for the disease, greatly improve the specificity and effectiveness of intervention, and have significant clinical transformation and drug research and development value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of ORM2 protein in the preparation of drugs for the prevention and / or treatment of heart failure with preserved ejection fraction. Background Technology

[0002] Heart failure with preserved ejection fraction (HFpEF) is a clinically prevalent subtype of heart failure. Myocardial metabolic disorders and energy metabolism disorders are the core pathological mechanisms driving its development. Imbalance in myocardial glucose and lipid metabolism and abnormal mitochondrial function can directly lead to myocardial diastolic dysfunction and impaired cardiac function, which are important inducing factors for the progression of HFpEF.

[0003] As a core metabolic regulatory organ, the liver can remotely regulate myocardial metabolism and cardiac physiological function through the hepatocardiac axis by secreting circulating proteins. The role of liver-derived secretory factors in regulating cardiac metabolic homeostasis is receiving increasing attention. However, there is currently a lack of endogenous liver-derived functional protein targets that can be used to improve cardiac metabolism in heart-free liver-associated ejection fraction (HFpEF), and a shortage of novel biotherapy candidates, which limits the research and translation of targeted biotherapy technologies for HFpEF.

[0004] Orosomucoid 2 (ORM2) is a circulating protein mainly synthesized and secreted by the liver, participating in the regulation of metabolic and physiological homeostasis. However, current research on ORM2 function mainly focuses on systemic inflammatory responses, disorders of glucose and lipid metabolism, and local pathological changes in the liver. Research on the interaction between the liver and heart has largely focused on classical hormones, inflammatory factors, and small metabolic molecules. No studies have reported the regulatory role of liver-derived ORM2 on cardiac metabolism in heart-free peritoneal ejection syndrome (HFpEF), nor have any related technologies and applications of ORM2 for improving myocardial metabolic abnormalities and protecting cardiac function in HFpEF been disclosed.

[0005] Therefore, existing technologies still need improvement and development. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide the application of ORM2 protein in the preparation of drugs for the prevention and / or treatment of heart failure with preserved ejection fraction, aiming to provide a new strategy for the treatment of heart failure with preserved ejection fraction.

[0007] The technical solution of the present invention is as follows: Firstly, it provides the application of ORM2 protein in the preparation of drugs for the prevention and / or treatment of heart failure with preserved ejection fraction.

[0008] In a preferred embodiment, the amino acid sequence of the ORM2 protein has at least 80%, 85%, 90%, or 95% identity with SEQ ID NO.7 or SEQ ID NO.8.

[0009] In a preferred embodiment, the amino acid sequence of the ORM2 protein is shown in SEQ ID NO.7 or SEQ ID NO.8.

[0010] In a preferred embodiment, the drug for preventing and / or treating heart failure with preserved ejection fraction is used to improve cardiac diastolic function, reduce myocardial fibrosis and lipotoxic damage, and alleviate cardiac metabolic disorders in heart failure with preserved ejection fraction.

[0011] In a second aspect, a medicament is provided for the prevention and / or treatment of heart failure with preserved ejection fraction, the medicament comprising one or more of: ORM2 protein, mRNA expressing ORM2 protein, transcriptional agonist of ORM2 protein, and secretion promoter of ORM2 protein.

[0012] In a preferred embodiment, the amino acid sequence of the ORM2 protein has at least 80%, 85%, 90%, or 95% identity with SEQ ID NO.7 or SEQ ID NO.8.

[0013] In a preferred embodiment, the amino acid sequence of the ORM2 protein is shown in SEQ ID NO.7 or SEQ ID NO.8.

[0014] In a preferred embodiment, the drug further includes: a pharmaceutically acceptable carrier and / or excipients.

[0015] In a preferred embodiment, the pharmaceutically acceptable carrier and / or excipients include one or more of the following: binders, buffers, antioxidants, solubilizers, thickeners, lubricants, disintegrants, diluents, stabilizers, preservatives, colorants, flavorings, solubilizers, emulsifiers, isotonic agents, fillers, surfactants, and adsorbents.

[0016] In a preferred embodiment, the dosage form of the drug is selected from one or more of the following: injection, tablet, liposome, nanoparticle, and exosome.

[0017] Beneficial Effects: Current treatments for heart-related paroxysmal ejection fraction (HFpEF) focus on symptomatic interventions targeting local cardiac and systemic metabolic changes, without proposing a cross-organ intervention approach of "targeting the heart with liver-secreted proteins." Furthermore, no protective effect of ORM2 on the heart in HFpEF has been found. Additionally, there is a lack of biomarkers for specific diagnosis, disease stratification, and prognostic assessment of HFpEF, hindering precise diagnosis and treatment. Compared to existing technologies, the advantages of this invention specifically include: (1) Mechanism innovation: For the first time, it has been confirmed that liver-derived ORM2 protein can directly target the heart through the liver-heart axis, improve HFpEF cardiac metabolic disorders, and break through the limitations of existing research and intervention that only focus on local cardiac pathology.

[0018] (2) Target innovation: ORM2 was discovered for the first time as a novel therapeutic target for HFpEF. Existing technologies have not revealed the regulatory and repairing effects of ORM2 on cardiac function in this disease.

[0019] (3) Innovative therapeutic effect: ORM2 protein can specifically repair the core lesions of HFpEF (myocardial lipotoxicity and cardiac diastolic dysfunction), which is different from the shortcomings of existing drugs that only relieve symptoms and have no targeted repair effect.

[0020] (4) Clinical innovation: For the first time, a liver-heart axis homologous targeted treatment plan was provided for HFpEF heart with fatty liver disease associated with metabolic dysfunction, filling the clinical gap of no exclusive precision treatment for such patients. Attached Figure Description

[0021] Figure 1 This is a graph showing the validation results of reduced ORM2 protein secreted by the liver to the heart in HFpEF mice. A shows the top 20 items in the GO enrichment analysis of differentially expressed proteomic proteins between HFpEF and normal hearts; B shows the number of differentially expressed proteomic proteins between HFpEF and normal hearts; C shows liver-specific secreted proteins among the differentially expressed proteins between HFpEF and normal hearts; D shows a negative correlation between the heart weight / tibia length ratio and plasma ORM2 levels in HFpEF mice; E shows ORM2 protein expression in the heart and liver of the HFpEF model; F shows quantitative analysis of ORM2 protein expression in the heart of the HFpEF model; G shows quantitative analysis of ORM2 protein expression in the liver of the HFpEF model; H shows the expression of ORM2 and the cardiomyocyte marker cTNT in cardiac tissue detected by immunofluorescence staining; and I shows the relative expression level of ORM2 in cardiac tissue detected by immunofluorescence staining.

[0022] Figure 2This is a graph showing the correlation between metabolic phenotype and cardiac function changes after specific knockdown of ORM2 in the liver of mice. A shows the expression results of ORM2 and its subtype mRNA in the liver of mice after specific knockdown of ORM2 using AAV8 with the TBG promoter; B shows the differences in body weight among the groups of mice after specific knockdown of ORM2; C shows the glucose tolerance curves of the groups of mice after specific knockdown of ORM2; D shows the area under the curve of the glucose tolerance curves of the groups of mice after specific knockdown of ORM2; E shows the ratio of heart weight to tibia length in the groups of mice after specific knockdown of ORM2; F shows the echocardiography of the groups of mice after specific knockdown of ORM2; G shows the left ventricular ejection fraction in the groups of mice after specific knockdown of ORM2; H shows the E / A ratio in the groups of mice after specific knockdown of ORM2; I shows the E / e' ratio in the groups of mice after specific knockdown of ORM2; and J shows the isovolumetric relaxation time of the heart in the groups of mice after specific knockdown of ORM2.

[0023] Figure 3 This is a graph showing the correlation between metabolic phenotype and cardiac function changes in HFpEF mice after treatment with exogenous ORM2. A shows a schematic diagram of the administration method to the mice; B shows the expression of cardiac ORM2 in wild-type mice after administration of exogenous ORM2; C shows the quantitative analysis of cardiac ORM2 in wild-type mice after administration of exogenous ORM2; D shows the body weight of mice in each group after administration of exogenous ORM2; E shows the glucose tolerance curves of mice in each group after administration of exogenous ORM2; F shows the results of treatment with exogenous ORM2 in each group. The area under the glucose tolerance curve in mice; G shows the ratio of heart weight to tibia length in each group of mice after administration of exogenous ORM2; H shows the echocardiography of each group of mice after administration of exogenous ORM2; I shows the left ventricular ejection fraction in each group of mice after administration of exogenous ORM2; J shows the E / A ratio in each group of mice after administration of exogenous ORM2; K shows the E / e' ratio in each group of mice after administration of exogenous ORM2; L shows the isovolumetric relaxation time of the heart in each group of mice after administration of exogenous ORM2.

[0024] Figure 4 The figures show the results of cardiac transcriptome sequencing in HFpEF mice after treatment with exogenous ORM2; where A is a volcano plot of the cardiac transcriptome; B is a heatmap of the cardiac transcriptome; C is GO enrichment analysis of differentially expressed genes in the cardiac transcriptome; D is KEGG enrichment analysis of differentially expressed genes in the cardiac transcriptome; and E is a bar chart of differentially expressed genes related to metabolism in the cardiac transcriptome. Detailed Implementation This invention provides the application of ORM2 protein in the preparation of drugs for the prevention and / or treatment of heart failure with preserved ejection fraction. To make the objectives, technical solutions and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0025] Current clinical and basic research on heart-stopped free cardiac ejection syndrome (HFpEF) generally recognizes the central role of cardiac metabolic abnormalities in its progression. However, current HFpEF intervention programs mostly focus on symptomatic treatment, and basic research largely revolves around local lesions such as myocardial inflammation, fibrosis, and endothelial damage. These approaches fail to target and correct the core pathological problem of cardiac metabolic disorders, only alleviating clinical symptoms and failing to improve myocardial function at the metabolic level, thus making it difficult to effectively curb the progression of HFpEF.

[0026] As a core metabolic regulatory organ, the liver can remotely regulate myocardial metabolism and cardiac function through the hepatocardiac axis by secreting circulating proteins. However, current technologies neglect the remote cardiac metabolic regulation mediated by hepatic secreted proteins, and the key molecular mechanisms of hepatocardiac metabolic interaction are poorly understood. Therefore, in-depth exploration of the regulatory role of liver-derived ORM2 in HFpEF cardiac metabolism can provide new research directions for the mechanistic elucidation and targeted intervention of HFpEF.

[0027] Based on this, embodiments of the present invention provide the application of ORM2 protein in the preparation of drugs for the prevention and / or treatment of heart failure with preserved ejection fraction.

[0028] Specifically, this invention employs a complete logic of targeting the disease through proteomics sequencing → verifying that endogenous deficiency aggravates the condition → confirming that exogenous supplementation can treat the disease → clarifying that the core therapeutic effect is metabolic repair. It is the first to demonstrate that exogenous supplementation of liver-derived ORM2 protein can treat heart failure with preserved ejection fraction (HFpEF) by improving cardiac metabolic disorders, breaking through the limitations of existing liver-heart axis-free targeted therapy for HFpEF and providing a new precision treatment option for clinical practice.

[0029] In this embodiment of the invention, firstly, it was verified that the reduction of endogenous ORM2 exacerbates HFpEF, confirming that ORM2 deficiency is an important inducing factor for the progression of HFpEF, thus laying the necessity and theoretical basis for ORM2 as a therapeutic target; secondly, through exogenous supplementation of recombinant ORM2 protein, the cardiac function damage caused by HFpEF was rapidly reversed, and diastolic function was significantly improved, thereby effectively treating HFpEF; furthermore, by repairing cardiac metabolic disorders with exogenous ORM2, the core pathological problems of HFpEF were addressed from the root, reversing myocardial lipotoxicity and energy metabolism imbalance, achieving precise targeted therapy, rather than merely alleviating clinical symptoms.

[0030] Understandably, the aforementioned heart failure with preserved ejection fraction includes: heart failure with preserved ejection fraction complicated by fatty liver disease with metabolic dysfunction, and heart failure with preserved ejection fraction related to obesity.

[0031] In some embodiments, the amino acid sequence of the ORM2 protein has at least 80%, 85%, 90%, or 95% identity with SEQ ID NO.7 or SEQ ID NO.8.

[0032] In some embodiments, the amino acid sequence of the ORM2 protein is shown in SEQ ID NO.7 or SEQ ID NO.8.

[0033] SEQ ID NO.7 (mouse-derived ORM2 protein) is as follows: MGWSCIILFLVATATGVHSQNPEHVNITIGDPITNETLSWLSDKWFFIGAAVLNPDYRQEIQKTQMVFFNLTPNLINDTMELREYHTIDDHCVYNSTHLGIQRE NGTLSKYVGGVKIFADLIVLKMHGAFMLAFDLKDEKKRGLSLNAKRPDITPELREVFQKAVTHVGMDESEIIFVDWKKDRCSQQEKQQLELEKETKKDPEEGQA.

[0034] SEQ ID NO.8 (human ORM2 protein) is as follows: MALSWVLTVLSLLPLLEAQIPLCANLVPVPITNATLDRITGKWFYIASAFRNEEYNKSVQEIQATFFYFTPNKTEDTIFLREYQTRQNQCFYNSSYLNVQR ENGTVSRYEGGREHVAHLLFLRDTKTLMFGSYLDDEKNWGLSFYADKPETTKEQLGEFYEALDCLCIPRSDVMYTDWKKDKCEPLEKQHEKERKQEEGES.

[0035] Specifically, this invention has verified in specific embodiments that the murine ORM2 protein in mice improves diastolic function in patients with heart failure with preserved ejection fraction, reduces myocardial fibrosis and lipotoxic damage, and alleviates cardiac metabolic disorders. It is understood that the human ORM2 protein should also possess the same or similar functions in humans.

[0036] In some embodiments, the drugs for preventing and / or treating heart failure with preserved ejection fraction are used to improve cardiac diastolic function in heart failure with preserved ejection fraction, reduce myocardial fibrosis and lipotoxic damage, and alleviate cardiac metabolic disorders, among one or more of these.

[0037] In some embodiments, the ORM2 protein may be modified by glycosylation, fatty acid coupling, or site-specific mutation; the site-specific mutation preserves the core active domain while mutating non-critical amino acid sites. Specifically, appropriate modification of the ORM2 protein can alter its molecular structure or physicochemical properties, prolonging its half-life and improving its stability.

[0038] Based on the same inventive concept, the present invention provides a medicament for the prevention and / or treatment of heart failure with preserved ejection fraction, the medicament comprising one or more of the following: ORM2 protein, mRNA expressing ORM2 protein, transcriptional agonist of ORM2 protein, and secretion promoter of ORM2 protein.

[0039] In some more specific embodiments, the transcriptional agonist of the ORM2 protein is selected from DNA methyltransferase inhibitors or histone acetylation regulators. Specifically, DNA methyltransferase inhibitors or histone acetylation regulators can indirectly activate the transcriptional expression of hepatic ORM2 through epigenetic mechanisms.

[0040] In some embodiments, the amino acid sequence of the ORM2 protein has at least 80%, 85%, 90%, or 95% identity with SEQ ID NO.7 or SEQ ID NO.8.

[0041] In some embodiments, the amino acid sequence of the ORM2 protein is shown in SEQ ID NO.7 or SEQ ID NO.8.

[0042] In some embodiments, the drug further includes: a pharmaceutically acceptable carrier and / or excipients.

[0043] In some embodiments, the pharmaceutically acceptable carrier and / or excipient includes one or more of the following: binders, buffers, antioxidants, solubilizers, thickeners, lubricants, disintegrants, diluents, stabilizers, preservatives, colorants, flavorings, solubilizers, emulsifiers, isotonic agents, fillers, surfactants, and adsorbents.

[0044] In some embodiments, the drug further includes one or more of the following: sodium-glucose cotransporter 2 (SGLT2) inhibitor, angiotensin receptor neprilysin inhibitor (ARNI), and diuretics.

[0045] In some embodiments, the dosage form of the drug is selected from one or more of the following: injection, tablet, liposome, nanoparticle, and exosome.

[0046] In some embodiments, the drug is administered via intraperitoneal / intravenous injection, using ORM2 protein as the active ingredient, at a dose of 1-5 mg / kg / day for 2-4 weeks, to improve HFpEF cardiac metabolic disorders, myocardial fibrosis, and diastolic dysfunction.

[0047] The present invention will be further described below through specific embodiments.

[0048] In the examples, unless otherwise stated, all experimental reagents and materials used were commercially available products. Six-week-old SPF-grade male C57BL / 6J mice were purchased from Guangdong Yaokang Biotechnology Co., Ltd., and housed in an SPF-grade environment at 20–22°C and 50–60% humidity, with a 12-hour light / 12-hour dark cycle and free access to food and water. Normal diet (Jiangsu Xietong Pharmaceutical Biotechnology, catalog number XTI01ZJ): 14.9 kcal% fat, 62.4 kcal% carbohydrates, 22.7 kcal% protein; high-fat diet (Research Diet, USA, catalog number D12492): 60 kcal% fat, 20 kcal% carbohydrates, 20 kcal% protein; N-nitro-L-arginine methyl ester (L-NAME) was purchased from Sigma-Aldrich (catalog number N5751).

[0049] Animal experiments were conducted in accordance with ethical guidelines. Mice were euthanized at the end of the experiment, and tissue and blood samples were collected in a standardized manner.

[0050] All statistical analyses were performed using GraphPad Prism 9.0 software. All data are expressed as mean ± standard error. Normality was tested for all continuous data. Independent samples t-tests were used for two groups, and analysis of variance was used for three or more groups. The significance level was set at P < 0.05.

[0051] Example 1 This embodiment explores the association between the ORM2 protein and HFpEF, as detailed below: (1) Construction of HFpEF mouse model Mice were randomly divided into a blank control group and an HFpEF model group, with no statistically significant difference in initial body weight. The blank control group was fed a normal diet and sterile purified water for 8 weeks; the HFpEF model group was fed a high-fat diet with 0.75 g / L L-NAME added to their drinking water, which was changed every 3 days for 8 weeks. During the modeling period, the mice's mental state, activity, food and water intake were observed daily, and they were weighed at a fixed time each week. After 8 weeks of modeling, EF, FS, LVID, E / A, E / e', and heart rate were measured using a Vevo 1100 ultrasound system under isoflurane anesthesia. The metabolic characteristics confirmed the successful establishment of the HFpEF model.

[0052] (2) Target screening Eight weeks after mouse modeling, mice in both groups were fasted for 12 hours, euthanized, and their hearts and livers were rapidly collected. After enzymatic digestion, desalting, and chromatographic separation, the whole-protein profile of the heart was analyzed using LC-MS / MS. Key differentially expressed proteins were screened through differential protein analysis and bioinformatics enrichment. Plasma ORM2 levels were detected using an ORM2 ELISA kit (Feien Biotech, catalog number EH1928); ORM2 expression in the heart and liver was detected by Western blot (ORM2 antibody: Invitrogen, PA5-119322; internal control HSP90: Wuhan Sanying, 60318-1-Ig); and the distribution of ORM2 in the heart was observed by immunofluorescence staining (ORM2 antibody catalog number: PA5-119322; cTNT antibody for cardiomyocyte localization, catalog number: MA5-12960). Image J was used for quantitative analysis of Western blot and immunofluorescence staining.

[0053] (3) Verification of the necessity of low expression of endogenous ORM2 Liver-specific ORM2 low-expression mice were constructed using an AAV8 vector with a TBG promoter. Six-week-old SPF-grade male C57BL / 6J mice were randomly divided into an AAV8-ORM2-shRNA group and an AAV8-NC-shRNA control group, and were injected with 1×10⁻⁶ tgV via tail vein. 11 The virus was administered to each animal (vg / animal) in a routinely fed environment for 3 weeks. qPCR was used to detect the expression of ORM1, ORM2, and ORM3 in the liver, confirming specific downregulation of ORM2. The primer sequences are as follows: ORM1-FORWARD (SEQ ID NO.1): 5'-CGAGTACAGGCAGGCAATTCA-3'; ORM1-REVERSE (SEQ ID NO.2): 5'-ACCTATTGTTTGAGACTCCCGA-3'; ORM2-FORWARD (SEQ ID NO.3): 5'-CAACATCACCATAGGCGACC-3'; ORM2-REVERSE (SEQ ID NO.4): 5'-ATTTCCTGCCGGTAATCAGGG-3'; ORM3-FORWARD (SEQ ID NO.5): 5'-GAACTACACACGGGTTCTCATCAT-3'; ORM3-REVERSE (SEQ ID NO. 6): 5'-CATTTGCCAGATAGCCAGCTC-3'.

[0054] Both groups were induced with a high-fat diet combined with 0.75 g / L L-NAME in drinking water for 8 weeks to establish an HFpEF model with endogenous ORM2 deficiency. After modeling, echocardiography and body weight were measured. Mice were fasted for 12 hours before the glucose tolerance test, but allowed free access to water. The weight of the mice was recorded, and sterile glucose solution (20% concentration) was injected intraperitoneally at a dose of 2.0 mg / kg body weight. Approximately 5 μL of blood was collected from the tip of the mouse tail using a micro-scanning pen before injection (0 min) and at 15, 30, 60, 90, and 120 min after injection. Blood glucose levels were measured and recorded using a portable blood glucose meter. Glucose tolerance curves were plotted with time on the x-axis and blood glucose concentration on the y-axis, and the area under the curve (AUC) was calculated to assess overall glucose metabolism and insulin sensitivity in mice. After the test, heart samples were collected, and the heart weight / tibia length ratio was calculated to assess the degree of cardiac hypertrophy.

[0055] (4) Validation of the therapeutic effect of exogenous ORM2 HFpEF mice that successfully developed the model were randomly divided into a model control group and an ORM2 intervention group, with a normal blank control group and a normal ORM2 intervention group also included. The ORM2 intervention group and the normal ORM2 intervention group received intraperitoneal injections of recombinant ORM2 protein at 2 mg / kg daily, while the model control group and the normal blank control group received an equal volume of PBS. The intervention lasted for 21 consecutive days. During the intervention, the mice maintained their corresponding diet and L-NAME water intake. After the intervention, Western blot was used to detect cardiac ORM2 levels, and ultrasound was used to detect diastolic function indicators such as E / A, E / e', and IVRT. Body weight and glucose tolerance were also monitored.

[0056] The recombinant ORM2 protein was synthesized by adding a His tag to the mouse ORM2 protein to form the mouse Orm2-6*His recombinant protein. Expression was performed using a mammalian expression system (CHO cells). The optimized gene sequence was cloned into an expression vector, transfected into CHO cells, and secreted for expression. After collecting the supernatant, further purification was performed by Genscript Biotech Inc., and the purity and specificity were verified by SDS-PAGE and Western blot. The mouse Orm2-6*His recombinant protein was concentrated by ultrafiltration, freeze-dried, resuspended in 1×PBS buffer (pH 7.4), aliquoted, and stored at -80°C. The amino acid sequence of the mouse ORM2 protein is shown in SEQ ID NO.7, as follows: MGWSCIILFLVATATGVHSQNPEHVNITIGDPITNETLSWLSDKWFFIGAAVLNPDYRQEIQKTQMVFFNLTPNLINDTMELREYHTIDDHCVYNSTHLGIQRE NGTLSKYVGGVKIFADLIVLKMHGAFMLAFDLKDEKKRGLSLNAKRPDITPELREVFQKAVTHVGMDESEIIFVDWKKDRCSQQEKQQLELEKETKKDPEEGQA.

[0057] (5) Validation of exogenous ORM2 improving cardiometabolic disorders After the intervention, heart tissues from the ORM2 intervention group and the model control group were collected, washed with PBS, flash-frozen in liquid nitrogen, and stored at -80℃. Total RNA was extracted using the Trizol method, and after quality control library construction, it was subjected to Illumina high-throughput sequencing for differential gene analysis, GO and KEGG pathway enrichment, with a focus on analyzing the expression of genes related to lipid metabolism, energy metabolism, and glucose metabolism.

[0058] The results of the above experiment are as follows: (1) LC-MS / MS full-protein proteometry analysis showed that, compared with the control group, differentially expressed proteins in the heart tissue of HFpEF mice were significantly enriched in metabolic-related pathways, among which the level of ORM2 protein, a liver-specific protein, was significantly downregulated. ELISA analysis showed that the plasma ORM2 level in mice was significantly negatively correlated with the heart weight / tibia length ratio. Western blot and immunofluorescence results were consistent, showing that ORM2 expression was significantly reduced in both the liver and heart tissues of HFpEF mice, establishing a direct association between ORM2 and HFpEF. Figure 1 ).

[0059] Three weeks after AAV8 injection and routine feeding, the expression of hepatic ORM1, ORM2, and ORM3 genes was detected by qPCR, indicating liver-specific ORM2 knockout. Following liver ORM2 knockout, HFpEF mice showed significantly worsened cardiac diastolic function, markedly increased cardiac hypertrophy, significant weight gain, and abnormal glucose tolerance. These results confirm that a reduction in endogenous ORM2 can significantly aggravate cardiac dysfunction and metabolic abnormalities in HFpEF, and that ORM2 is a key essential molecule regulating the development and progression of HFpEF. Figure 2 ).

[0060] Exogenous recombinant ORM2 intervention significantly increased ORM2 protein levels in the cardiac tissue of HFpEF mice. Compared with the model control group, the core indices of cardiac diastolic function (E / A, E / e', IVRT) in the ORM2 intervention group were significantly optimized, and systemic metabolic disorders were significantly improved, confirming that ORM2 has a clear therapeutic effect on HFpEF. Figure 3 ).

[0061] Transcriptome sequencing combined with pathological examination confirmed that exogenous ORM2 supplementation significantly reversed cardiac metabolic disorders in HFpEF mice, effectively improving myocardial lipotoxicity accumulation, inflammatory metabolic imbalance, and energy metabolism disorders. GO and KEGG enrichment analysis clarified that repairing cardiac metabolic disorders is the core mechanism of ORM2 treatment for HFpEF. Figure 4 ).

[0062] In summary, this invention, through in vivo experiments, is the first to discover that ORM2 expression is reduced in the liver tissue of a HFpEF mouse model, confirming that ORM2 can serve as a therapeutic target for HFpEF. Injection of recombinant ORM2 protein effectively improves related symptoms of HFpEF. It is believed that with more thorough experimental research and large-scale randomized controlled trials, recombinant ORM2 protein has promising applications in the treatment of HFpEF.

[0063] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. Application of ORM2 protein in the preparation of drugs for the prevention and / or treatment of heart failure with preserved ejection fraction.

2. The application according to claim 1, characterized in that, The amino acid sequence of the ORM2 protein has at least 80%, 85%, 90%, or 95% identity with SEQ ID NO. 7 or SEQ ID NO.

8.

3. The application according to claim 1, characterized in that, The amino acid sequence of the ORM2 protein is shown in SEQ ID NO.7 or SEQ ID NO.

8.

4. The application according to claim 1, characterized in that, The medications for the prevention and / or treatment of heart failure with preserved ejection fraction are used to improve cardiac diastolic function in heart failure with preserved ejection fraction, reduce myocardial fibrosis and lipotoxic damage, and alleviate cardiac metabolic disorders, among one or more of these.

5. A medicament for the prevention and / or treatment of heart failure with preserved ejection fraction, characterized in that, The drug comprises one or more of the following: ORM2 protein, mRNA expressing ORM2 protein, transcription agonist of ORM2 protein, and secretion promoter of ORM2 protein.

6. The drug according to claim 5, characterized in that, The amino acid sequence of the ORM2 protein has at least 80%, 85%, 90%, or 95% identity with SEQ ID NO. 7 or SEQ ID NO.

8.

7. The drug according to claim 5, characterized in that, The amino acid sequence of the ORM2 protein is shown in SEQ ID NO.7 or SEQ ID NO.

8.

8. The drug according to claim 5, characterized in that, The drug also includes: pharmaceutically acceptable carriers and / or excipients.

9. The drug according to claim 5, characterized in that, The pharmaceutically acceptable carriers and / or excipients include one or more of the following: binders, buffers, antioxidants, solubilizers, thickeners, lubricants, disintegrants, diluents, stabilizers, preservatives, colorants, flavorings, solubilizers, emulsifiers, isotonic agents, fillers, surfactants, and adsorbents.

10. The medicament according to claim 5, characterized in that, The dosage form of the drug is selected from one or more of the following: injection, tablet, liposome, nanoparticle, and exosome.

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