Mbl2 inhibitors and their use in the treatment of heart failure with preserved ejection fraction

Abstract

The application belongs to the field of biological medicine and disease diagnosis, and particularly relates to an MBL2 inhibitor and application thereof in treating heart failure with preserved ejection fraction. The application provides an MBL2 inhibitor, which is an MBL2 silencing preparation, the MBL2 silencing preparation is siRNA and / or shRNA, the siRNA comprises three pairs of siRNA double strands targeting the Mbl2 gene, the nucleotide sequences of the sense strand and the antisense strand are respectively shown as SEQ ID NO:1-6, the three pairs of siRNA double strands are mixed in a 1:1:1 molar ratio, the nucleotide sequence of the shRNA is shown as SEQ ID NO:9, and the application of the MBL2 inhibitor in preparing a drug for treating and / or preventing heart failure. The application explicitly indicates that MBL2 can be used as a potential diagnostic marker and a treatment target of heart failure with preserved ejection fraction, and provides a new thought and experimental basis for the clinical diagnosis and treatment of HFpEF.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and disease diagnosis, specifically involving MBL2 inhibitors and their application in the treatment of heart failure with preserved ejection fraction, as well as diagnostic methods for heart failure with preserved ejection fraction. Background Technology

[0002] Heart failure (HF) is a serious and life-threatening clinical syndrome with a high global prevalence and rising mortality rate. Heart failure with preserved ejection fraction (HFpEF) is one type of HF, characterized by myocardial stiffness and impaired diastolic function, but with an unchanged ejection fraction (EF). However, understanding its etiology and treatment remains a significant challenge. Current treatments for HFpEF primarily focus on symptom control and lifestyle modifications, such as using diuretics to reduce edema and ACE inhibitors or ARBs to lower blood pressure. While these treatments can alleviate symptoms to some extent, they cannot fundamentally improve the pathophysiological processes of HFpEF and have limited impact on long-term prognosis. The pathophysiological mechanisms of HFpEF are complex, involving multiple factors including metabolic abnormalities, inflammatory responses, and neuroendocrine activation. Currently, there is a lack of fundamental treatment strategies targeting the pathogenesis of HFpEF. For example, HFpEF patients often have comorbidities such as metabolic syndrome, obesity, and hypertension, but current treatments are ineffective in addressing these comorbidities.

[0003] In recent years, studies have shown that the liver plays a crucial role in the development and progression of heart failure. The liver interacts with the heart in areas such as repair after cardiac injury, metabolic regulation, and inflammatory responses. Liver dysfunction can affect cardiac function, and vice versa; a complex pathophysiological relationship exists between the two. However, current research still requires further investigation into the specific molecular mechanisms and interactions between HFpEF and the liver. Our previous research indicated that the liver-secreting factor F11 regulates cardiac function. Against this backdrop, this invention focuses on a liver-specific secretory protein, mannose-binding lectin 2 (MBL2), to investigate its role in heart failure with preserved ejection fraction (HFpEF). We found that MBL2 is upregulated in pathological myocardial hypertrophy, and overexpression of MBL2 promotes myocardial hypertrophy and the progression of heart failure in mice; knockdown of hepatic MBL2 significantly improved the progression of heart failure.

[0004] Existing technologies have included studies on MBL2’s involvement in infection immunity, inflammatory response, and fibrosis in some organs. However, no patents or literature disclose that MBL2 is involved in the development of HFpEF as a liver-secreting factor. Furthermore, no studies disclose that MBL2 regulates cardiac diastolic dysfunction, myocardial hypertrophy, and fibrosis through the PKA-STAT3 pathway. Also, no studies disclose that MBL2 is used as a diagnostic biomarker or therapeutic target for HFpEF.

[0005] The present invention is applicable to the early screening, disease assessment and targeted treatment of heart failure with preserved ejection fraction (HFpEF); the applicable subjects include HFpEF patients with obesity, metabolic syndrome and hypertension and HFpEF animal models, and it is especially suitable for people with abnormal liver metabolism, decreased cardiac diastolic function but relatively preserved left ventricular ejection fraction. Summary of the Invention

[0006] The technical problem this invention aims to solve is that current treatments for heart failure with heart failure at the pathogenesis stage (HFpEF) are limited, mainly focusing on symptom control and lifestyle modifications, lacking fundamental treatment strategies targeting its pathogenesis. Traditional heart failure treatments primarily focus on interventions within the heart itself, neglecting the crucial role of other organs such as the liver in the progression of heart failure. Furthermore, existing research has not fully elucidated the molecular mechanisms of HFpEF, particularly the molecular communication network between the liver and heart, and there is a lack of sensitive and specific diagnostic biomarkers and targeted therapeutic targets.

[0007] The purpose of this invention is to provide an MBL2 inhibitor and its application in the treatment of heart failure with preserved ejection fraction.

[0008] In a first aspect, the present invention provides the application of a reagent for detecting MBL2 expression levels in the preparation of a diagnostic kit for heart failure.

[0009] Preferably, the reagent for detecting MBL2 expression level is a primer for detecting MBL2 or an anti-MBL2 antibody.

[0010] Secondly, the present invention provides the use of an MBL2 inhibitor in the preparation of a drug for the treatment and / or prevention of heart failure.

[0011] Preferably, the MBL2 inhibitor includes a small molecule inhibitor of MBL2, an anti-MBL2 monoclonal antibody, and an MBL2 silencing agent.

[0012] Preferably, the small molecule inhibitor of MBL2 is cysteine ​​(2-Aminoethanethiol) and / or taurine.

[0013] Preferably, the MBL2 silencing agent is siRNA and / or shRNA.

[0014] Preferably, the siRNA is delivered via lipid nanoparticles (LNPs).

[0015] Preferably, the shRNA is mediated by adeno-associated virus AAV8.

[0016] Preferably, the siRNA comprises three pairs of siRNA duplexes targeting the Mbl2 gene, the nucleotide sequences of the sense and antisense strands being as shown in SEQ ID NO:1-6, respectively, and they are mixed in a 1:1:1 molar ratio.

[0017] Preferably, the nucleotide sequence of the shRNA is as shown in SEQ ID NO: 9, and its sequence is: GGAAACAGAGTGCGCTATAT. Preferably, the heart failure is heart failure with reduced ejection fraction (HFrEF), heart failure with mildly reduced ejection fraction (HFmrEF), or heart failure with preserved ejection fraction (HFpEF).

[0018] Thirdly, the present invention provides an MBL2 inhibitor, wherein the MBL2 inhibitor is an MBL2 silencing agent, the MBL2 silencing agent is siRNA and / or shRNA, the siRNA comprises 3 pairs of siRNA double strands targeting the Mbl2 gene, the nucleotide sequences of the sense strand and the antisense strand are shown in SEQ ID NO:1-6 respectively, and they are mixed in a 1:1:1 molar ratio, and the nucleotide sequence of the shRNA is shown in SEQ ID NO:9.

[0019] Preferably, the siRNA is LNPs-siRNA and the shRNA is AAV8-shRNA.

[0020] Fourthly, the present invention provides a pharmaceutical composition comprising the MBL2 inhibitor described herein and a pharmaceutically acceptable carrier or excipient.

[0021] Beneficial effects This invention, through a systematic study of the role of MBL2 in HFpEF, enriches our understanding of the pathogenesis of HFpEF, particularly the understanding of the molecular communication network between the liver and heart, and provides a new direction for further in-depth research.

[0022] Clinical application prospects: Diagnostic aspects: Based on the fact that MBL2 is significantly elevated in the plasma of HFpEF patients and is correlated with the severity of the disease, an MBL2 detection kit (such as an ELISA kit) can be developed for early screening, disease assessment and prognosis of HFpEF, to solve the problem of insufficient specificity of existing HFpEF diagnostic biomarkers.

[0023] At the treatment level: Based on the feasibility of MBL2 as a therapeutic target for HFpEF, targeted interventions can be developed, including ① small molecule inhibitors targeting MBL2 (inhibiting MBL2 protein activity); ② anti-MBL2 monoclonal antibodies (neutralizing MBL2 protein in peripheral blood); ③ liver-specific MBL2 silencing agents (such as LNPs-siMBL2, AAV8-shMBL2) for etiological treatment of HFpEF, improving patient prognosis, reducing mortality and hospitalization rates, and overcoming the limitation of existing treatments that can only relieve symptoms.

[0024] The following will further explain the concept, specific structure, and technical effects of the present invention in order to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0025] Figure 1 This is the result of screening MBL2 as a key factor mediating liver-heart interaction; Figure 1 In this context, A represents human genetic data based on BXD recombinant inbred mice (BXD for short) and the UK Biobank. Figure 1 B in the text refers to liver-secreting proteins that can affect heart genes, selected from the BXD database. Figure 1 In this context, C represents the mRNA expression level of the Mbl2 gene in various tissues of adult male C57BL / 6J mice. Figure 1 In this context, D stands for Phenotypic Genome-Wide Association Analysis (PheWAS). Figure 1 E in the figure represents the expression level of MBL2 protein in the plasma of normal controls and patients with heart failure with preserved ejection fraction (HFpEF). Figure 1 F in the figure represents the analysis of ejection fraction, plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) level, plasma MBL2 protein level, and the correlation between plasma NT-proBNP and MBL2 protein levels in normal controls and HFpEF patients. Figure 1 G in the analysis represents the correlation between Mbl2 gene expression and cardiac-related phenotypes in the BXD mouse strain. The indicators included left ventricular diameter (LVID), left ventricular mass (LV Mass), left ventricular volume (LV Volume), and cardiac output (CO). Figure 1 H in the figure represents the expression level of MBL2 protein in the plasma of normal mice and HFpEF mice.

[0026] Figure 2 This is a result of MBL2 exacerbating diastolic dysfunction in the HFpEF mouse model; Figure 2 In this context, A represents the construction of the HFpEF mouse model; Figure 2 B in the figure represents the expression level of the Mbl2 gene in mice overexpressing GFP and MBL2. Figure 2In this context, C represents the expression level of MBL2 protein in mouse plasma as detected by Western blot. Figure 2 In this context, D represents the left ventricular mass, left ventricular mass / volume ratio, E / e' ratio, and left ventricular ejection fraction in mice overexpressing GFP and MBL2 in the normal feeding group and the HFpEF modeling group. Figure 2 In this context, E represents a representative image obtained through echocardiography. Figure 2 F in the figure represents the running distance of GFP and MBL2 overexpressing mice in the normal feeding group and the HFpEF modeling group. Figure 2 In this context, G represents the heart weight / body weight ratio, heart weight / tibia length ratio, and lung wet weight / dry weight ratio in GFP and MBL2 overexpressing mice. Figure 2 The H in the image represents a representative image of immunohistochemical staining of heart tissue from mice overexpressing GFP and MBL2, including HE staining, Masson's trichrome staining, and WGA staining. Figure 2 In this context, I represents the expression level of HFpEF-related genes in the heart tissue of GFP and MBL2 overexpressing mice. Figure 2 J in the figure represents the immunofluorescence image of GFP and MBL2 overexpressing mouse heart tissue and the statistical results of the number of positive cells.

[0027] Figure 3 This is because knocking out the Mbl2 gene can alleviate the development and progression of HFpEF in mice; Figure 3 In this context, A represents the Mbl2 gene knockout strategy. Figure 3 B in the figure represents the expression level of the Mbl2 gene in the livers of wild-type (WT), MBL2 heterozygous knockout (Mbl2-Het), and MBL2 homozygous knockout (Mbl2-Homo) mice. Figure 3 C in the figure represents the expression level of MBL2 protein in mice with different genotypes. Figure 3 D in the figure represents the construction of HFpEF mouse models from 8-week-old Mbl2-WT, Mbl2-het, and Mbl2-homo mice; Figure 3 E in the figure represents the ratio of heart weight to tibia length and the ratio of wet lung weight to dry weight in each group of mice. Figure 3 F in the figure represents the running distance of each group of mice. Figure 3 In this context, G represents the E / A ratio, E / e' ratio, left ventricular mass, left ventricular mass / volume ratio, left ventricular mass index, and left ventricular ejection fraction of the heart in each group of mice. Figure 3 H in the image represents a representative image obtained from echocardiography. Figure 3 In the image, I represents a representative image of immunohistochemical staining of mouse heart tissue, including HE staining, Masson trichrome staining, and WGA staining, as well as statistical results of cardiomyocyte cross-sectional area and fibrosis area. Figure 3J in the figure represents the expression level of genes related to HFpEF in cardiac tissue.

[0028] Figure 4 This is a result of MBL2 regulating diastolic dysfunction and cardiac hypertrophy through the PKA-STAT3 pathway; Figure 4 In this study, A represents the extraction of total RNA from mouse heart tissue, followed by screening and identification of differentially expressed genes in two groups using bulk RNA-seq technology; B represents the KEGG pathway enrichment analysis of the differentially expressed genes; and C represents the detection of the relative expression levels of p-PKA, total PKA, p-STAT3, and total STAT3 in the heart tissues of GFP control group and MBL2 overexpressing mice using Western blot. Figure 4 In the D–I section, 8-week-old male C57BL / 6J mice were injected with either AAV8-TBG-GFP or AAV8-TBG-MBL2 recombinant adeno-associated virus, and intraperitoneally injected twice weekly with H89 (10 mg / kg) to inhibit p-PKA, while simultaneously receiving a high-fat diet combined with L-NAME intervention for 7 weeks. D represents an overview of the H89 intervention experimental design; E represents ultrasound-detected cardiac parameters in mice, including left ventricular mass, E / A ratio, E / e' ratio, and left ventricular ejection fraction; F represents representative echocardiographic images of mice; G represents the results of the mouse running endurance test; H represents the heart weight / tibia length ratio and lung wet weight / dry weight ratio; and I represents the expression levels of heart failure-related genes. Figure 4 In the study, J–O consisted of 8-week-old male C57BL / 6J mice injected with either AAV8-TBG-GFP or AAV8-TBG-MBL2, and administered twice weekly intraperitoneal injections of Static (12 mg / kg) to inhibit p-STAT3. Simultaneously, they received a high-fat diet combined with L-NAME intervention for 7 weeks. J represents an overview of the Static intervention experimental design; K represents echocardiographic data of mouse cardiac parameters, including left ventricular mass, E / A ratio, E / e' ratio, and left ventricular ejection fraction (LVEF); L represents representative echocardiographic images of mice in the GFP+Static and MBL2+Static groups; M represents the results of mouse running distance measurements; N represents the heart weight / tibia length ratio and lung wet weight / dry weight ratio measurements; and O represents the expression levels of heart failure-related genes.

[0029] Figure 5 This is a result that inhibiting MBL2 can alleviate diastolic dysfunction and delay the progression of HFpEF; Figure 5 A in the diagram represents the experimental procedure for delivering siRNA to target and silence MBL2 in mouse liver using lipid nanoparticles (LNPs). Figure 5In this context, B represents the expression level and quantitative statistical results of MBL2 protein in the plasma of mice in the siControl and siMbl2 groups detected by Western blot. Figure 5 In the figure, C represents the cardiac function of mice in the siControl and siMbl2 groups at weeks 0, 5, and 10, as measured by dynamic ultrasound in small animals. This includes left ventricular mass, E / A ratio, E / e' ratio, multiplicative concentricity index, and left ventricular ejection fraction. Figure 5 D in the image represents a representative echocardiogram of the siControl and siMbl2 mice at week 10 of a high-fat diet. Figure 5 E in the figure represents the immunohistochemical staining images of the heart tissue of mice in the siControl and siMbl2 groups, including HE staining, Masson trichrome staining and WGA staining, as well as the statistical results of fibrosis area and cardiomyocyte cross-sectional area. Figure 5 In this context, F represents the treatment intervention in HFpEF model mice after 5 weeks of modeling. Figure 5 In this context, G represents the expression level of the Mbl2 gene in the heart tissue of mice in the shGFP and shMbl2 groups. Figure 5 In this context, H represents the running distance of mice in the Chow, shGFP, and shMbl2 groups; Figure 5 In this context, I represents the expression levels of heart failure and inflammation-related genes in the cardiac tissues of mice in the Chow, HFpEF+shGFP, and HFpEF+shMbl2 groups detected by qPCR. Figure 5 J in the table represents the cardiac function of mice in the Chow, HFpEF+shGFP, and HFpEF+shMbl2 groups at weeks 0, 5, and 10, including left ventricular mass, E / A ratio, E / e' ratio, left ventricular concentric hypertrophy index, and left ventricular ejection fraction. Figure 5 K in the image represents a representative echocardiographic image of mice in the Chow, HFpEF+shGFP, and HFpEF+shMbl2 groups at week 10.

[0030] Figure 6 This is the result of candidate drug screening based on the MBL2 target and the evaluation of its therapeutic effect on HFpEF mice; Figure 6 In this context, A represents the selected candidate compound; Figure 6 B in the text represents the therapeutic intervention for HFpEF mice; Figure 6 CD represents the E / A ratio, E / e' ratio, left ventricular mass, and left ventricular ejection fraction in mice treated with cysteine ​​and taurine, respectively. Figure 6 In this context, E represents the expression level of the cardiac Nppb gene detected by qPCR. Figure 6 In this context, FH represents the heart weight / body weight ratio, lung wet weight / dry weight ratio, and running distance. Figure 6 In this context, I represents mechanistic analysis. Detailed Implementation

[0031] The present invention will be described in detail below with reference to specific embodiments, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0032] Example 1: Construction of a liver-specific mouse model regulated by the MBL2 gene 1. Selection and Principles of Gene Editing Technology This invention employs multiple gene regulation techniques to construct HFpEF mouse models with different MBL2 gene expression levels, including gene overexpression, transient knockdown, stable knockdown, and gene knockout systems, to verify the regulatory role of the MBL2 gene in heart failure with preserved ejection fraction (HFpEF) from multiple dimensions.

[0033] Among them, the liver-specific overexpression system uses adeno-associated virus type 8 (AAV8) vector for in vivo gene delivery. The AAV8 vector has outstanding advantages such as strong hepatocyte targeting, high transfection efficiency, low immunogenicity, and long-lasting stable expression in vivo, which can efficiently target mouse liver tissue and achieve long-term expression of the target gene.

[0034] The transient knockdown system uses liver-targeting lipid nanoparticles (LNPs) to package siRNA, achieving rapid and transient silencing of the MBL2 gene in hepatocytes through the passive liver-targeting properties of LNPs. The stable knockdown system utilizes lentivirus-mediated shRNA to continuously interfere with the target gene. The gene knockout system uses Mbl2 gene-specific knockout mice to verify its function at the level of complete gene deletion.

[0035] 2. Construction of a multimodal regulatory system for the MBL2 gene 2.1 Construction of a liver-specific MBL2 gene overexpression system This invention constructs a mouse model of liver-specific overexpression of the MBL2 gene using a liver-specific AAV8 vector system. First, the complete coding sequence of the mouse Mbl2 gene is amplified by PCR and directionally cloned downstream of an AAV8 vector plasmid containing a liver-specific thyroid-binding globulin (TBG) promoter. The TBG promoter drives specific high expression of the MBL2 gene in liver cells. Simultaneously, an empty vector AAV8-GFP carrying a GFP fluorescent reporter gene is constructed as a negative control.

[0036] Recombinant AAV8-MBL2 plasmid and AAV8-GFP control plasmid were co-transfected into HEK293T cells with packaging plasmid and helper plasmid, respectively, to produce recombinant virus. After collecting the viral supernatant, high-purity AAV8 virus stock solution was obtained through concentration and purification, and the viral titer was calibrated to 1×10⁻⁶. 12 vg / mL. This titer is the standard effective intervention dose for liver-targeted AAV vectors. Preliminary experiments have verified that it can stably achieve highly efficient and specific expression of the target gene in mouse liver, and no obvious toxic side effects have been observed.

[0037] AAV8-MBL2 or AAV8-GFP viral solutions were delivered to experimental animals via tail vein injection. After viral particles targeted and infected hepatocytes, they mediated sustained and efficient expression of the exogenous MBL2 gene in the liver, thus obtaining a liver-specific MBL2 overexpression mouse model. This model was used to evaluate the effects of MBL2 gene overexpression on cardiac structure and function in HFpEF mice and to assess its potential application value in the prevention and treatment of HFpEF.

[0038] 2.2 Preparation and Construction of MBL2 Gene Transient Knockdown System (siRNA) This system uses lipid nanoparticles (LNPs) to encapsulate siRNA to achieve phased knockdown of the MBL2 gene in mouse liver. Since free siRNA lacks tissue targeting and is easily degraded by nucleases, this invention utilizes the passive targeting properties of LNPs in the liver to achieve protective delivery of siRNA and efficient hepatocyte enrichment, while setting up an irrelevant sequence siControl-LNP as a negative control.

[0039] Given that siRNA does not integrate into the genome and its interference is transient and reversible, this experiment initiated a 5-week intervention at week 5 of HFpEF modeling. Two tail vein siRNA injections were administered at weeks 5 and 7, respectively, to achieve sustained low MBL2 expression throughout the intervention period through transient knockdown. This transient knockdown maintained the sustained low expression of the MBL2 gene throughout the entire modeling period.

[0040] Three siRNA sequences targeting the mouse Mbl2 gene (Mbl2-mouse-86, Mbl2-mouse-242, and Mbl2-mouse-536) were selected for the experiment and mixed in a 1:1:1 molar ratio to prepare a siMbl2 mixed reagent, in order to improve interference efficiency and stability. The specific preparation process of siRNA-LNP is as follows: Ionized lipids DLin-MC3-DMA, distearate phosphatidylcholine (DSPC), cholesterol, and DMG-PEG2000 were mixed in a molar ratio of 50:10:38.5:1.5 and dissolved in anhydrous ethanol to prepare the lipid phase. The siMbl2 mixed sequence or control siRNA was dissolved in 10 mM, pH 3.0 citrate buffer to prepare the aqueous phase. Using a microfluidic mixer (iNano L+), with an ethanol-to-aqueous phase flow rate ratio of 1:3, rapid mixing resulted in the self-assembly of siRNA-loaded lipid nanoparticles (siRNA-LNPs). The suspension was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed against 1×PBS for 2 hours to remove ethanol and replace the buffer. After dialysis, the solution was filtered through a 0.22 μm sterile filter to obtain purified siRNA-LNPs. The siRNA encapsulation efficiency was measured using the Quant-iT RiboGreen kit. Once qualified, the solution was stored at 4°C protected from light for later use. Effective transient knockdown of the MBL2 gene in mouse hepatocytes was achieved through multiple tail vein injections.

[0041] Table 1 siRNA sequences

[0042] 2.3 Preparation and Construction of MBL2 Gene Stable Knockdown System (shRNA) This experiment employed lentivirus-mediated shRNA interference technology to construct a stable MBL2 gene knockdown system, using shGFP as a negative control. First, a specific shRNA sequence and a disordered control shRNA sequence were designed based on the mouse Mbl2 gene sequence. The nucleotide sequence of the specific shRNA is shown in SEQ ID NO: 9, and its sequence is: GGAAACAGAGTGCGCTATAT. The target shRNA fragment was cloned downstream of the lentiviral vector promoter to construct the recombinant lentiviral shMbl2 interference plasmid and the shGFP control plasmid.

[0043] The recombinant plasmid was co-transfected with lentiviral packaging plasmid and helper plasmid into HEK293T cells. After culturing for 48–72 hours, the viral supernatant was collected, and high-titer lentiviral particles were obtained by ultracentrifugation and filtration purification. After determining the viral titer, the particles were aliquoted and stored at -80°C. Long-term stable knockdown of the MBL2 gene in mouse liver was achieved through in vivo delivery methods such as tail vein injection.

[0044] 2.4 MBL2 gene knockout system This study used commercially available Mbl2 gene knockout mice to construct a gene deletion animal model. This strain targeted exons 4–6 of the transcript Mbl2-201 (ENSMUST00000025797.6) for fragment knockout. Mbl2 heterozygous knockout (Mbl2-Het) and homozygous knockout (Mbl2-Homo) mice were obtained through breeding, with wild-type mice of the same genetic background (Mbl2-WT) serving as a blank control. The function of MBL2 was verified at the level of complete gene deletion. This, along with overexpression and knockdown systems, constituted a complete control system of positive, negative, and gradient expression, further enhancing the reliability of the experimental conclusions.

[0045] 3. Grouping and treatment of animal models The experimental animals were C57BL / 6J male mice, which were randomly divided into the following groups: (1) AAV8-GFP control group + normal diet group; (2) AAV8-MBL2 overexpression + normal diet group; (3) AAV8-GFP control + HFpEF (l-NAME + HFD) group; (4) AAV8-MBL2 overexpression + HFpEF group; (5) Mbl2 wild type (Mbl2-WT) + HFpEF group; (6) Mbl2 heterozygous knockout (Mbl2-Het) + HFpEF group; (7) Mbl2 homozygous knockout (Mbl2-Homo) + HFpEF group; (8) HFpEF+siControl group; (9) HFpEF+siMbl2 group; (10) HFpEF+shGFP group; (11) HFpEF+shMbl2 group; (12) HFpEF+PBS group; (13) HFpEF+Taurine group; (14) HFpEF+2-Aminoethanethiol group.

[0046] All mice were raised in the same environment with free access to food and water.

[0047] Cardiac function was assessed by echocardiography at the beginning and end of the experiment. Mouse weight was measured at specific time points (e.g., 0, 3 weeks, 5 weeks, 7 weeks).

[0048] Model features Cardiac function: HFpEF characteristics are present, characterized by an E / e' ratio and a significantly increased LV mass, while LVEF remains unchanged.

[0049] Pathological changes: Cardiac cell hypertrophy, myocardial interstitial fibrosis, and left ventricular remodeling occur.

[0050] Example 2: Cardiac Function Testing and Assessment 1. Echocardiography: Echocardiography is a non-invasive, real-time, and radiation-free examination method widely used in the diagnosis and evaluation of heart diseases. It provides detailed information on cardiac structure and function, including chamber size, ventricular systolic function, and valvular function. In HFpEF studies, echocardiography is particularly suitable for assessing diastolic function; by measuring indicators such as the E / A ratio and E / e' ratio, it can accurately determine whether diastolic function is impaired. High-resolution echocardiography (such as the VisualSonics Vevo F2 system) was used to perform the examination in mice under mild anesthesia, and the measured indicators included: Left ventricular ejection fraction (LVEF): Reflects cardiac systolic function and is calculated by measuring the volume change of the left ventricle during systole and diastole. The formula is LVEF = (end-diastolic volume - end-systolic volume) / end-diastolic volume × 100%.

[0051] Left ventricular fractional shortening (LVFS): This is also an indicator for assessing cardiac systolic function. LVFS = (end-diastolic diameter - end-systolic diameter) / end-diastolic diameter × 100%.

[0052] The mitral valve flow spectrum parameters E / A ratio and E / e' ratio, as mentioned above, are used to assess cardiac diastolic function. A decreased E / A ratio or an increased E / e' ratio suggests impaired cardiac diastolic function.

[0053] Other indicators, such as left ventricular end-diastolic dimension (LVEDD) and left ventricular end-systolic dimension (LVESD), are used to comprehensively assess changes in cardiac structure and function.

[0054] Echocardiography was performed at different time points during the experiment (such as before treatment, 7 weeks after treatment, etc.) to dynamically observe changes in cardiac function.

[0055] 2. Histopathological examination of cardiac tissue. At the end of the experiment, mice were euthanized and heart tissue was quickly removed, fixed in 4% paraformaldehyde, routinely embedded in paraffin, and sectioned (approximately 4-6 μm thick). The following staining and analysis were performed: Hematoxylin-Eosin (HE) staining: to observe the morphology, size, arrangement, and nucleus morphology of cardiomyocytes, and to assess pathological changes such as cardiomyocyte hypertrophy, necrosis, or inflammatory cell infiltration.

[0056] Masson's Trichrome staining: used to detect the degree of myocardial fibrosis. Collagen fibers are stained blue, and myocardial cells are stained red. The proportion of collagen fiber area to the total area of ​​myocardial tissue is calculated by image analysis software to quantitatively assess the degree of myocardial fibrosis.

[0057] Wheat lectin (WGA) staining: used to label cardiomyocyte membranes, measure the cross-sectional area of ​​cardiomyocytes using image analysis software, and quantitatively assess the degree of cardiomyocyte hypertrophy.

[0058] Ly6G, CD3, and F4 / 80 staining were used to detect the infiltration of neutrophils (Ly6G labeling), T lymphocytes (CD3 labeling), and macrophages (F4 / 80 labeling) in myocardial tissue, respectively.

[0059] Example 3: Molecular biological detection 1. RNA extraction and real-time quantitative PCR (PCR) Mouse heart tissue was collected, and total RNA was extracted using Trizol reagent (a commonly used RNA extraction reagent that can effectively lyse cells and separate RNA). cDNA (complementary DNA) was then synthesized by reverse transcription.

[0060] Specific primers were designed for the MBL2 gene and genes related to cardiac function, inflammation, and fibrosis (such as myocardial contraction-related genes, inflammatory factor genes, and fibrosis-related genes), as shown in Table 2.

[0061] Table 2 Primer Sequences

[0062] PCR amplification was performed using a real-time quantitative PCR instrument to detect the mRNA expression levels of these genes in the heart tissues of mice in different groups. By comparing the differences in gene expression among different groups, the regulatory role of MBL2 on the expression of heart-related genes was analyzed, revealing the molecular mechanism by which MBL2 affects the pathophysiological processes of heart disease at the gene transcription level.

[0063] 2. RNA-seq analysis To gain a more comprehensive understanding of the role of MBL2 in HFpEF, we also performed batch RNA sequencing (RNA-seq) on mouse heart tissue. RNA-seq is a high-throughput sequencing technology that allows us to identify differentially expressed genes in MBL2 overexpression and reveal the biological pathways and processes involved by these genes through bioinformatics analysis (such as functional enrichment analysis). This method can complement the results of real-time quantitative PCR, providing deeper insights into the molecular mechanisms.

[0064] 3. Protein extraction and Western blot Total protein was extracted from mouse heart tissue. Cells were lysed using RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors to prevent protein degradation and dephosphorylation), and the supernatant was collected after centrifugation to obtain total protein.

[0065] Protein concentration was determined using the BCA protein quantification kit. Equal amounts of protein samples were subjected to SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, separating proteins according to their molecular weight) and then transferred to an NC membrane (nitrocellulose membrane).

[0066] NC membranes were incubated overnight at 4°C with specific primary antibodies (such as anti-MBL2 antibody or signaling pathway-related antibodies), followed by incubation at room temperature with corresponding secondary antibodies (horseradish peroxidase-labeled). Protein bands were detected by chemiluminescence immunoassay, and the grayscale values ​​of the protein bands were analyzed using image analysis software such as ImageJ. The expression levels of related proteins in the heart tissues of different groups of mice were quantitatively compared, further verifying the effect of MBL2 on the expression of cardiac function-related proteins at the protein level.

[0067] Data Analysis and Results Interpretation 1. Data collection and organization We systematically collected data from echocardiography, histopathological examination, molecular biological testing, and cell experiments. This included cardiac function indicators (such as LVEF, LVFS, E / A ratio, E / e' value, etc.), quantitative histological data (such as the proportion of myocardial fibrosis area, immunohistochemical positive cell count, etc.), gene and protein expression level data (Ct value of real-time quantitative PCR, gray value of protein bands in Western blotting, etc.), and data from cell immunofluorescence and gene expression in cell experiments. To ensure the accuracy and completeness of the data, each data point was meticulously recorded and labeled, indicating its experimental group, detection time point, and other information.

[0068] 2. Selection and Application of Statistical Analysis Methods In data analysis, appropriate statistical methods are selected based on different data types and experimental designs.

[0069] Comparing two sets of data: If the data are normally distributed and have homogeneous variance, use the independent samples Student's t test; if they are not normally distributed, use the Mann-Whitney U test.

[0070] For comparisons of three or more groups of data: First, perform tests for normality and homogeneity of variance. If the data are normally distributed and have homogeneity of variance, use one-way ANOVA, combined with Tukey's multiple comparison test for pairwise comparisons between groups; if the data are not normally distributed or have unequal variances, use the nonparametric Kruskal-Wallis test, and then use Dunn's multiple comparison test for comparisons between groups.

[0071] Correlation analysis: The correlation between two variables is analyzed using Pearson correlation analysis (if the data follows a normal distribution) or Spearman correlation analysis (if the data does not follow a normal distribution).

[0072] All statistical analyses were performed using GraphPad Prism software, with P < 0.05 used as the criterion for statistical significance.

[0073] 3. Interpretation of Results and Discussion of Mechanisms Based on data analysis, this study aims to explain the impact of MBL2 on the pathophysiological processes of heart failure with cardiac embolism (HFpEF). For example, if significant deterioration of cardiac function (such as increased E / A and E / e') is observed in the AAV8-MBL2 + HFpEF group, accompanied by pathological changes such as increased expression of myocardial inflammatory factors, it indicates that MBL2 further regulates cardiac dysfunction and increases pathological damage in HFpEF. Combining molecular biological detection results, the study explores the mechanism of action of MBL2 from the perspective of cell signaling pathways. Through in-depth interpretation of the results and exploration of the mechanisms, the study clarifies the importance of MBL2 in HFpEF and its potential therapeutic application value.

[0074] This study reveals for the first time that MBL2 is a novel mediator of liver-heart interaction, involved in the occurrence and development of heart failure with preserved ejection fraction (HFpEF). Therefore, MBL2 may serve as a potential diagnostic biomarker and therapeutic target for HFpEF.

[0075] There is a close regulatory relationship between the liver and the heart. Heart failure is often accompanied by abnormal liver function, suggesting that liver-secreted proteins may be involved in the regulation of cardiac function. To identify key factors mediating the liver-heart interaction, this invention uses BXD mouse population genetic data and bioinformatics analysis to screen liver-secreted proteins associated with cardiac gene expression, ultimately determining... Mbl2 The gene is a candidate gene mediating the liver-heart interaction. Figure 1 A in the text). MBL2 was identified as a liver-secreting protein that can affect cardiac genes in the BXD mouse database. A total of 2890 cardiac genes are associated with Mbl2. Figure 1 (B in the text). The results of detecting the mRNA expression level of the Mbl2 gene in various tissues of adult male C57BL / 6J mice clarified that Mbl2 is mainly expressed in the liver. Figure 1 (C in the middle).

[0076] To clarify the correlation between MBL2 and HFpEF, this study examined the expression levels of MBL2 protein in the plasma of normal controls and HFpEF patients. Figure 1 (D in the text); Simultaneously, the ejection fraction, plasma NT-proBNP levels, and the correlation between plasma NT-proBNP and MBL2 protein levels were analyzed in both groups. Figure 1 The results showed that the level of MBL2 protein in the plasma of HFpEF patients was significantly elevated and positively correlated with the level of NT-proBNP, a marker of HFpEF severity, suggesting that MBL2 may serve as a potential marker protein for HFpEF. Furthermore, the expression levels of MBL2 protein in the plasma of normal mice and HFpEF mice were measured. Figure 1 The results (F in the text) were consistent with those of HFpEF patients, showing significantly elevated plasma MBL2 protein levels. Analysis of the association between MBL2 gene expression and cardiac-related phenotypes (including left ventricular diameter LVID, left ventricular mass LV Mass, left ventricular volume LV Volume, and cardiac output CO) in the BXD mouse strain revealed that Mbl2 gene expression in the liver was significantly correlated with cardiac phenotype in the BXD mouse database. Figure 1 (G in the text). In summary, these results indicate that MBL2 is not only upregulated in HFpEF patients and mice, but its level is also closely related to disease severity and cardiac function indicators, supporting the value of MBL2 as a diagnostic biomarker and potential intervention target for HFpEF.

[0077] To investigate the functional role of MBL2 in HFpEF, this invention constructed an HFpEF mouse model overexpressing MBL2. The specific method is as follows: Male C57BL / 6J mice were injected with adeno-associated virus type 8 (AAV8-GFP) carrying green fluorescent protein (GFP) and adeno-associated virus type 8 (AAV8-MBL2) carrying the MBL2 gene, respectively. Subsequently, they were fed a high-fat diet (HFD) combined with NG-nitro-L-arginine methyl ester (L-NAME) for 7 weeks, successfully establishing the HFpEF mouse model. Figure 2 (A) The results showed that the expression levels of Mbl2 gene and plasma MBL2 protein in MBL2-overexpressing mice were significantly higher than those in the GFP control group. Figure 2 (B in 2, C in 2). Ultrasound examination of small animals showed that, compared with the GFP control group, MBL2 overexpression increased the E / e' ratio and left ventricular mass, while the left ventricular ejection fraction remained unchanged, significantly exacerbating diastolic dysfunction in HFpEF mice. Figure 2 D in D and E in E in E). MBL2 overexpression group mice had significantly shorter running distances, indicating a decline in their motor ability. Figure 2 The heart weight / body weight ratio, heart weight / tibia length ratio, and lung wet weight / dry weight ratio were all significantly increased, indicating that MBL2 overexpression can aggravate cardiac hypertrophy and pulmonary edema. Figure 2 (G in the text). Histopathological analysis of cardiac tissue showed that the cross-sectional area of ​​cardiomyocytes was significantly increased and the degree of myocardial fibrosis was significantly aggravated in MBL2-overexpressing mice. Figure 2 In H), the expression levels of hypertrophic and inflammatory genes in cardiac tissue were significantly increased ( Figure 2 I). Immunohistochemical staining results showed that immune cell infiltration was significantly increased in the heart tissue of MBL2-overexpressing mice. Figure 2 The above results indicate that MBL2 overexpression can promote cardiac hypertrophy, diastolic dysfunction, and myocardial inflammatory infiltration in HFpEF mice, thereby accelerating the progression of HFpEF disease.

[0078] To further verify the role of MBL2 in HFpEF, this invention constructed an Mbl2 gene knockout mouse model ( Figure 3 (A) The analysis showed that the expression levels of the Mbl2 gene in the liver and the MBL2 protein in vivo decreased in a gradient manner in wild-type (Mbl2-WT), MBL2 heterozygous knockout (Mbl2-Het), and MBL2 homozygous knockout (Mbl2-Homo) mice. Figure 3 (B in 3, C in 3). Eight-week-old mice of three genotypes were fed HFD combined with L-NAME for 7 weeks to construct an HFpEF model ( Figure 3(D in the text). Compared with WT mice, Mbl2 gene knockout significantly reduced the heart weight / tibia length ratio and lung wet weight / dry weight ratio in mice, alleviating cardiac hypertrophy and pulmonary edema. Figure 3 E); significantly increased running distance and improved motor ability in mice (in the context of E); Figure 3 (F in the text). Small animal ultrasound examination showed that Mbl2 gene knockout significantly improved diastolic function in mice, reduced the E / e' ratio, and regulated left ventricular mass and left ventricular mass / volume ratio. Figure 3 (G in G, H in 3). Pathological analysis of cardiac tissue showed that Mbl2 gene knockout significantly reduced the cross-sectional area of ​​cardiomyocytes and alleviated the degree of myocardial fibrosis. Figure 3 (I) Meanwhile, the expression levels of inflammation and hypertrophy-related genes decreased ( Figure 3 (J in the text). The above results confirm that Mbl2 gene knockout can effectively alleviate the occurrence and development of HFpEF in mice.

[0079] To elucidate the molecular mechanism by which MBL2 regulates HFpEF, this invention performed bulk RNA-seq on the heart tissues of mice in the HFpEF model-treated GFP control group and the MBL2 overexpression group. Transcriptome sequencing identified 324 differentially expressed genes (P < 0.05, Fold Change > 1.2 or < 0.8), of which 130 genes were downregulated and 194 genes were upregulated. Figure 4 A). KEGG pathway enrichment analysis showed that differentially expressed genes were mainly enriched in the PKA signaling pathway (A). Figure 4 (B in the original text). Western blot analysis confirmed that MBL2 overexpression significantly increased the relative expression levels of phosphorylated PKA (p-PKA) and phosphorylated STAT3 (p-STAT3) in mouse heart tissue, while the expression levels of total PKA and total STAT3 showed no significant change. Figure 4 (C in the text). This indicates that MBL2 can activate the PKA-STAT3 signaling pathway in cardiac tissue.

[0080] To verify the role of the PKA-STAT3 pathway in the regulation of HFpEF by MBL2, this invention used the PKA inhibitor H89 and the STAT3 inhibitor Static for intervention experiments. Eight-week-old male C57BL / 6J mice were injected with AAV8-TBG-GFP or AAV8-TBG-MBL2, followed by intraperitoneal injections of H89 (10 mg / kg) twice weekly, and simultaneously treated with HFD combined with L-NAME for 7 weeks. Figure 4 (D in the text). Ultrasound examination showed no significant differences in cardiac diastolic function, E / e' ratio, and left ventricular mass between the H89+GFP group and the H89+MBL group. Figure 4(E in 5, F in 5). Meanwhile, the running distance and athletic ability of the two groups of mice ( Figure 4 The values ​​in G (heart weight / tibia length ratio and lung wet weight / dry weight ratio) reflect the degree of cardiac hypertrophy and pulmonary edema. Figure 4 H in the heart failure), and the expression levels of heart failure-related genes (H in the heart failure). Figure 4 There were no significant differences in I) among the groups. Furthermore, 8-week-old male C57BL / 6J mice, after injection of AAV8-TBG-GFP or AAV8-TBG-MBL2, were given intraperitoneal injections of Static (12 mg / kg) twice weekly. Figure 4 The results showed that static intervention significantly improved cardiac hypertrophy remodeling, exercise capacity, and expression of heart failure-related genes in MBL2-overexpressing mice. Figure 4 In summary, MBL2 regulates diastolic dysfunction and cardiac hypertrophy in HFpEF mice by activating the PKA-STAT3 signaling pathway.

[0081] To verify the feasibility of MBL2 as a therapeutic target for HFpEF, this invention employs two methods to inhibit MBL2 expression. Method 1: Lipid nanoparticles (LNPs) are used to deliver siRNA, targeting and silencing MBL2 in mouse liver. The LNPs consist of Dlin-MC3-DMA, DSPC, DMG-PEG, and cholesterol, and siMbl2 is encapsulated using microfluidic mixing technology. HFpEF mice 5 weeks after modeling were injected via the tail vein with LNPs, and observation continued for another 5 weeks. Figure 5 (A) Western blot analysis showed that the expression level of MBL2 protein in the plasma of mice in the siMbl2 group was significantly lower than that in the siControl group (A). Figure 5 (B in the text). Small animal ultrasound examination showed that, compared with the siControl group, the cardiac function of mice in the siMbl2 group was significantly improved at week 10: left ventricular mass decreased, E / e' ratio decreased, and left ventricular concentric hypertrophy indices improved ( ). Figure 5 (C in 6, D in 6). Pathological analysis of cardiac tissue showed that the cross-sectional area of ​​cardiomyocytes was reduced and the degree of myocardial fibrosis was alleviated in the siMbl2 group mice. Figure 5 Method 2: Using adeno-associated virus-mediated RNA interference technology. HFpEF mice 5 weeks after modeling were injected intravenously with AAV8-shGFP or AAV8-shMbl2 for a 5-week treatment intervention. Figure 5 The F in the data shows that the expression level of the Mbl2 gene in the heart tissue of mice in the shMbl2 group was significantly lower than that in the shGFP group. Figure 5 (G in the text). Motor ability testing showed that the running distance of mice in the shMbl2 group was significantly longer than that in the shGFP group, and close to that in the normal-fed group (Chow group). Figure 5The H in the text is missing. qPCR detection showed that, compared with the HFpEF+shGFP group, the expression levels of heart failure and inflammation-related genes in the cardiac tissue of mice in the HFpEF+shMbl2 group were significantly downregulated. Figure 5 (I) Dynamic ultrasound examination in small animals showed that shMbl2 intervention significantly improved cardiac function in HFpEF mice, reduced the E / e' ratio, decreased left ventricular mass, and reduced indicators of concentric hypertrophy (I). Figure 5 (J in J, K in 6). The above results confirm that inhibiting MBL2 can effectively alleviate diastolic dysfunction in HFpEF mice and delay disease progression.

[0082] To screen potential drugs targeting MBL2, this invention performs molecular docking of the MBL2 protein with an FDA drug library. Based on binding energy and potential binding modes, cysteine ​​(2-Aminoethanethiol) and taurine were selected as candidate compounds. Figure 6 (A) To verify the therapeutic effect of the above compounds, HFpEF mice 5 weeks after modeling were treated with cysteine ​​(20 mg / kg) or taurine (400 mg / kg) three times a week for 5 weeks. Figure 6 (B in the text). Ultrasound examination of mice showed that, compared with the control group, the E / e' and E / A ratios were significantly lower in the cysteine ​​treatment group and the taurine treatment group, and the left ventricular mass was decreased (B in the text). Figure 6 CD in the heart). qPCR detection showed a significant decrease in cardiac Nppb gene expression (CD). Figure 6 (E in the text). Simultaneously, the heart weight / body weight ratio and lung wet weight / dry weight ratio decreased, and exercise capacity also improved. Figure 6 (FH in the text). The above results indicate that both cysteine ​​and taurine can effectively improve diastolic dysfunction in HFpEF mice. Combined with molecular docking results, this suggests that the above drugs may exert cardioprotective effects by targeting MBL2, further validating the feasibility of MBL2 as a therapeutic target for HFpEF.

[0083] In summary, this invention is the first to clearly demonstrate that MBL2, as a novel mediator of liver-heart interaction, plays a pathogenic role in the development and progression of HFpEF. Specifically, MBL2 expression is significantly elevated in HFpEF mice and patients; overexpression of MBL2 can activate the PKA-STAT3 signaling pathway in cardiomyocytes, promoting cardiomyocyte hypertrophy, myocardial inflammation and fibrosis, and exacerbating diastolic dysfunction; while inhibiting MBL2 expression can effectively improve cardiac function and exercise capacity in HFpEF mice and alleviate myocardial damage. Figure 6(I) Meanwhile, validation using human plasma samples showed that MBL2 protein levels were positively correlated with NT-proBNP, a marker of HFpEF severity. Therefore, MBL2 can serve as a potential diagnostic marker and therapeutic target for heart failure with preserved ejection fraction, providing new insights and experimental evidence for the clinical diagnosis and treatment of HFpEF.

[0084] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. The application of a reagent for detecting MBL2 expression levels in the preparation of a diagnostic kit for heart failure.

2. The application according to claim 1, characterized in that, The reagents used to detect MBL2 expression levels are primers for detecting MBL2 or anti-MBL2 antibodies.

3. The use of an MBL2 inhibitor in the preparation of drugs for the treatment and / or prevention of heart failure.

4. The application according to claim 3, characterized in that, The MBL2 inhibitors include small molecule inhibitors of MBL2, anti-MBL2 monoclonal antibodies, and MBL2 silencing agents.

5. The application according to claim 4, characterized in that, The small molecule inhibitors of MBL2 are cysteine ​​(2-Aminoethanethiol) and / or taurine.

6. The application according to claim 4, characterized in that, The MBL2 silencing agent is siRNA and / or shRNA.

7. The application according to claim 6, characterized in that, The siRNA is delivered via lipid nanoparticles (LNPs), and the shRNA is mediated by adeno-associated virus AAV8.

8. The application according to claim 6, characterized in that, The siRNA comprises three pairs of double-stranded siRNAs targeting the Mbl2 gene, the nucleotide sequences of which are shown in SEQ ID NO:1-6, respectively, and are mixed in a 1:1:1 molar ratio. The nucleotide sequence of the shRNA is shown in SEQ ID NO:

9.

9. An MBL2 inhibitor, wherein the MBL2 inhibitor is an MBL2 silencing agent, the MBL2 silencing agent is siRNA and / or shRNA, wherein the siRNA comprises 3 pairs of siRNA double strands targeting the Mbl2 gene, the nucleotide sequences of the sense strand and the antisense strand are shown in SEQ ID NO:1-6 respectively, and they are mixed in a 1:1:1 molar ratio, and the nucleotide sequence of the shRNA is shown in SEQ ID NO:

9.

10. A pharmaceutical composition comprising the MBL2 inhibitor of claim 9 and a pharmaceutically acceptable carrier or excipient.

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