Application of dracocephalum moldavica total flavonoids in preparation of medicine for preventing and / or treating heart failure with preserved ejection fraction

Abstract

The invention provides application of dracocephalum moldavica total flavonoids in preparation of a medicine for preventing and / or treating heart failure with preserved ejection fraction, and belongs to the technical field of biological medicine. After a heart failure model mouse with reserved ejection fraction is constructed and dracocephalum moldavica general flavone is administrated, research finds that compared with an HFpEF model group mouse, the myocardial cell damage degree of the dracocephalum moldavica general flavone group mouse is obviously reduced, the myocardial hypertrophy is obviously reduced, and the myocardial hypertrophy is obviously reduced. The total flavonoids of dracocephalum moldavica can significantly reduce pathological cardiac hypertrophy and myocardial fibrosis of HFpEF model mice, improve cardiac diastolic function, and reduce the degree of liver cell injury and lipid metabolism imbalance, thereby preventing and / or treating heart failure with ejection fraction retention.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of total flavonoids of Dracocephalum moldavica L. in preparation of a medicine for preventing and / or treating heart failure with preserved ejection fraction. BACKGROUND

[0002] Heart failure is a group of complex clinical syndromes caused by abnormal changes in cardiac structure and / or function, which leads to ventricular systolic and / or diastolic dysfunction. According to the different left ventricular ejection fraction (LVEF) and changes after treatment, heart failure is divided into four categories, which are heart failure with reduced ejection fraction (HFrEF), heart failure with improved ejection fraction (HFimpEF), heart failure with mildly reduced ejection fraction (HFmrEF) and heart failure with preserved ejection fraction (HFpEF). HFpEF is a new public health problem. So far, although the existing treatment methods have improved some specific phenotypes within the overall range of HFpEF, there is no treatment method that can convincingly reduce the mortality and morbidity of HFpEF patients. Therefore, further clinical and basic research is needed to explore effective drugs for treating HFpEF.

[0003] Total Flavonoids of Dracocephalum moldavica L. Dracocephalum moldavica L. Through in vivo and in vitro experiments, it is proved that total flavonoids of Dracocephalum moldavica L. can regulate myocardial cell mitochondrial function, such as increasing mitochondrial membrane potential and reducing the release of reactive oxygen species (ROS), thereby reducing myocardial ischemia-reperfusion injury, and showing good cardiac protection. Total flavonoids of Dracocephalum moldavica L. have multiple pharmacological effects such as anti-inflammatory, antioxidant, anti-atherosclerosis, blood pressure reduction and blood lipid reduction. However, the mechanism of total flavonoids of Dracocephalum moldavica L. in treating heart failure with preserved ejection fraction has not been reported in detail. SUMMARY

[0004] In view of this, the purpose of the present application is to provide application of total flavonoids of Dracocephalum moldavica L. in preparation of a medicine for preventing and / or treating heart failure with preserved ejection fraction.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides application of total flavonoids of Dracocephalum moldavica L. in preparation of a medicine for preventing and / or treating heart failure with preserved ejection fraction.

[0006] Preferably, the heart failure with preserved ejection fraction is N-nitro-L-arginine methyl ester and high-fat diet induced heart failure with preserved ejection fraction.

[0007] Preferably, the total flavonoids of Dracocephalum moldavica L. have the effects of preventing and / or treating myocardial cell injury.

[0008] Preferably, the total flavones of Dracocephalum moldavica have the effect of preventing and / or treating myocardial fibrosis.

[0009] Preferably, the total flavones of Dracocephalum moldavica have the effect of preventing and / or treating myocardial hypertrophy.

[0010] Preferably, the total flavones of Dracocephalum moldavica have the effect of preventing and / or treating abnormal liver function indicators.

[0011] Preferably, the total flavones of Dracocephalum moldavica have the effect of preventing and / or treating lipid metabolism imbalance.

[0012] Preferably, the total flavones of Dracocephalum moldavica have the effect of preventing and / or treating diastolic heart failure.

[0013] Preferably, the medicine further comprises a pharmaceutically acceptable excipient.

[0014] Preferably, the total flavones of Dracocephalum moldavica account for 10% to 90% of the mass percentage of the medicine.

[0015] Compared with the prior art, the present application has the following beneficial effects: The present application provides an application of total flavones of Dracocephalum moldavica in preparing a medicine for preventing and / or treating heart failure. After constructing a heart failure model mouse with preserved ejection fraction, and giving TFDM, it is found that, compared with the HFpEF model mouse, the myocardial cell damage degree of the mouse in the total flavones of Dracocephalum moldavica group is obviously reduced, the myocardial hypertrophy is obviously reduced, and the E / e' ratio is obviously reduced. The total flavones of Dracocephalum moldavica can obviously reduce the pathological myocardial hypertrophy of the HFpEF model mouse, myocardial fibrosis, improve diastolic heart failure, and reduce the liver cell damage degree and lipid metabolism imbalance, so as to prevent and / or treat heart failure with preserved ejection fraction. It is found in the present application that TFDM inhibits the NF-κB inflammatory pathway through the Zbtb16 target point, reduces downstream inflammatory factors such as IL-6 and TNF-α, and thus achieves the purposes of treating HFpEF and inhibiting inflammation caused by HFpEF. The present application provides an innovative drug for HFpEF treatment. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 A, an experimental flowchart, a diagram showing the complete experimental design of mouse grouping, model construction, TFDM intervention and index detection; B, the final body weight of mice in different groups at the end of treatment; C, the heart weight of mice in different groups; D, the heart weight / tibia length ratio (HW / TL) of mice in different groups; E, the serum lactate dehydrogenase (LDH) level of mice in different groups; the data is represented by Mean±SEM; Figure 2A, Representative echocardiography images: M-mode echocardiography in the first row, mitral valve pulsed-wave Doppler flow in the second row, and mitral annulus tissue Doppler in the third row; B, Left ventricular ejection fraction (LVEF) in different groups; C, Left ventricular fractional shortening (LVFS) in different groups; D, E / e' ratio in different groups; E, Interventricular septal thickness at diastole (IVSd) in different groups; F, Interventricular septal thickness at systole (IVSs) in different groups; G, Left ventricular posterior wall thickness at diastole (LVPWd) in different groups; H, Left ventricular posterior wall thickness at systole (LVPWs) in different groups; I, Left ventricular mass in different groups; Figure 3 A, Serum aspartate aminotransferase (AST) levels in different groups; B, Serum alanine aminotransferase (ALT) levels in different groups; C, Serum triglyceride (TG) levels in different groups; D, Serum low-density lipoprotein cholesterol (LDL-C) levels in different groups; E, Serum high-density lipoprotein cholesterol (HDL-C) levels in different groups; F, Liver tissue staining images in different groups: H&E staining in the upper panel and Oil Red O (ORO) staining in the lower panel; G, ORO staining positive area statistics in different groups; H, Liver triglyceride (TG) levels in different groups; I, Liver total cholesterol (TC) levels in different groups; Figure 4 A, Representative staining images of heart tissue in different groups: from top to bottom, H&E staining, Masson's trichrome staining, and wheat germ agglutinin (WGA) fluorescent staining; B, Masson's trichrome staining fibrosis area statistics in different groups; C, WGA staining myocardial cell cross-sectional area statistics in different groups; Figure 5 A, Drug and disease target Venn diagram; B, Analysis of important potential targets; C, GO function enrichment analysis results; D, KEGG pathway enrichment analysis results; Figure 6 A, Volcano plot of differentially expressed genes; B, Heat map of core differentially expressed genes; C, mRNA levels of key target Zbtb16; D, KEGG pathway enrichment analysis results; E, GO function enrichment analysis results; Figure 7For in vivo validation of the TFDM pathway, the following data are presented: A) Western blot results of each protein in different groups; B) Zbtb16 protein expression results in different groups; C) Comparison of p-NF-κB / NF-κB in different groups; D) IL-6 protein expression results in different groups; E) TNF-α protein expression results in different groups; F) IL-6 mRNA expression level in different groups; G) TNF-α mRNA expression level in different groups; H) BNP mRNA expression level in different groups. Detailed Implementation

[0017] This invention provides the application of total flavonoids from *Cymbidium goeringii* in the preparation of drugs for the prevention and / or treatment of heart failure with preserved ejection fraction.

[0018] In this invention, the heart failure with preserved ejection fraction is a heart failure with preserved ejection fraction induced by N-nitro-L-arginine methyl ester (L-Name) and a high-fat diet. As one embodiment, the method for constructing the heart failure with preserved ejection fraction includes the following steps: feeding animals with drinking water containing L-Name and a high-fat diet for 10 consecutive weeks. The final concentration of L-Name in the drinking water containing L-Name is 0.5 g / L; the high-fat diet is a high-fat diet purchased from Research Diets, catalog number D12492. The animals are preferably mice or rats, such as C57BL / 6N mice. Figures 1-4 The results showed that, compared with the control group, the mice in the heart failure model group with preserved ejection fraction had significantly increased body weight, heart weight, HW / TL ratio, and serum LDH, while the left ventricular ejection fraction remained unchanged. The E / e' ratio was significantly increased. The diastolic interventricular septal thickness, systolic interventricular septal thickness, diastolic left ventricular posterior wall thickness, systolic left ventricular posterior wall thickness, and left ventricular mass were all significantly increased compared with the control group. The AST and ALT levels, serum TG levels, LDL-C, HDL-C, ORO area, liver TG level, liver TC level, myocardial fibrosis area, and cardiomyocyte cross-sectional area were all significantly increased compared with the control group. Therefore, the present invention successfully constructed a heart failure model animal with preserved ejection fraction.

[0019] In this invention, it was found that, compared with the control group, the total flavonoids of *Cinnamomum camphora* group mice showed significantly reduced myocardial hypertrophy and serum LDH levels, improved myocardial cell damage, and significantly reduced the E / e' ratio. TFDM can significantly alleviate pathological myocardial hypertrophy in HFpEF model mice, improve myocardial fibrosis, hepatocyte damage, and lipid metabolism imbalance, and improve diastolic dysfunction in HFpEF model mice. Therefore, this invention can effectively prevent and / or treat heart failure with preserved ejection fraction.

[0020] In this invention, the drug further includes pharmaceutically acceptable excipients, which include one or more of solvents, binders, diluents, disintegrants, antioxidants, dispersants, wetting agents, solubilizers, buffers, and surfactants. The preferred dosage forms of the drug include suspensions, tablets, pills, solutions, injections, granules, capsules, or sprays. The drug of this invention can be administered orally, rectally, intraperitoneally, subcutaneously, intramuscularly, intravenously, or nasally. This invention does not specifically limit the source of total flavonoids from *Illicium verum*; products known in the art or commercially available products are acceptable.

[0021] In this invention, the total flavonoids of *Heliotropium indicum* account for 10% to 90% of the mass percentage of the drug. In this invention, the total flavonoids of *Heliotropium indicum* can be used as the sole active ingredient to treat heart failure with preserved ejection fraction, or diseases caused by heart failure with preserved ejection fraction, or can be used in combination with other active ingredients to treat heart failure with preserved ejection fraction, or diseases caused by heart failure with preserved ejection fraction.

[0022] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] In the following examples, statistical analysis was performed using one-way ANOVA combined with Tukey's multiple comparison test; p <0.05、 p <0.01、 p <0.001 indicates a comparison between the model and the blank or between the drug-treated group and the model group.

[0025] Example 1 1. Materials and Reagents In this embodiment, C57BL / 6N wild-type mice were purchased from Beijing Spaford, and the sources of the reagents are shown in Table 1 below.

[0026] Table 1 Reagent Information

[0027] In addition to the reagents listed in Table 1, all other materials and reagents used in this embodiment are commercially available products.

[0028] 2. Animal Experimentation Guidelines In this embodiment, all animal studies were conducted under the guidance of the Experimental Animal Center of the Ethics Review Committee of Shihezi University. All mice were housed in the same environment, and were randomly grouped during the experiment. Echocardiographic analysis was performed by independent researchers unaware of the research objectives.

[0029] 3. Induction of a heart failure model with preserved ejection fraction C57BL / 6N mice were fed drinking water with a final concentration of 0.5 g / L L-Name adjusted to pH 7.4 for 10 consecutive weeks, and were also fed a high-fat diet (purchased from Research Diets, catalog number D12492), i.e., high-fat diet + L-Name mice. Control mice were fed standard food and normal drinking water without L-Name. After completing the 10-week dietary regimen, cardiac function, blood pressure, and blood glucose were measured. Mice with similar baseline characteristics were then randomly divided into three groups (n=8 per group): the control group (Ctr) consisted of mice that had completed the 10-week diet and were fed a standard diet and normal drinking water; the HFpEF model group (Mod) consisted of mice that had completed the 10-week diet on a lipid-based + L-Name diet and were fed HFD and 0.5 g / L L-Name, and were administered the same amount of solvent (the solvent for dissolving total flavonoids of *Cymbidium faberi*) as the HFpEF + TFDM group by gavage for 4 weeks; and the HFpEF + TFDM treatment group (TFDM) consisted of mice that had completed the 10-week diet on a lipid-based + L-Name diet and were fed HFD and 0.5 g / L L-Name, and were administered 90 mg / kg / day of total flavonoids of *Cymbidium faberi* by gavage for 4 weeks. Body weight and food intake were monitored weekly.

[0030] 4. The therapeutic effect of total flavonoids from *Cymbidium faberi* on HFpEF The following measurements were taken after completing 4 weeks of treatment with total flavonoids from Qinglan: 4.1 Routine Ultrasonic Testing Mice were anesthetized with oxygen and isoflurane (1–2%). Left ventricular ejection fraction and other systolic function indices were obtained by short-axis M-mode scanning of the mid-ventricular segment. Diastolic function was measured at the mitral valve level using pulsed wave and tissue Doppler. Collected parameters included: LVFS, IVSd, IVSs, LVPWd, LVPWs, LVFS, LVEF, LV Mass, and E / e'.

[0031] 4.2 Pathological tissue monitoring Mouse heart tissue was collected: one part was fixed overnight with 4% paraformaldehyde, then dehydrated in a gradient manner, cleared, embedded in paraffin, and cut into 5μm thick cross-sectional sections; the other part was made into frozen sections for preservation.

[0032] Paraffin sections were stained according to the manufacturer's instructions using an HE staining kit (Servicebio, catalog number G1003) and a Masson trichrome staining kit (Servicebio, catalog number G1006). The stained sections were then imaged using a Pannoramic MIDI digital scanner (3DHISTECH, Budapest, Hungary). The Masson-positive blue areas were quantitatively analyzed using ImageJ software to calculate myocardial collagen content.

[0033] 4.3 Biochemical Detection Mice were fasted for 6 hours (with free access to water) and then euthanized by cervical dislocation. Blood was collected from the orbital sinus into anticoagulant-free centrifuge tubes, allowed to stand at room temperature to clot, and then centrifuged at 3500 rpm for 15 minutes at 4°C. The supernatant serum (avoiding red blood cell contamination) was collected and transferred to enzyme-free EP tubes, stored at -80°C (avoiding repeated freeze-thaw cycles). Using Nanjing Jiancheng reagent kits, following the kit instructions, the serum levels of AST (C010-2-1), ALT (C009-2-1), TG (A110-1-1), TC (A111-1-1), LDL-C (A113-1-1), and HDL-C (A112-1-1) were measured.

[0034] 4.4 Real-time quantitative reverse transcription PCR detection method First, high-quality total RNA was obtained from mouse left ventricular tissue using a full-gold assay kit, ensuring the purity and integrity of the RNA to avoid interference with subsequent experimental results. Then, using the extracted RNA as a template, the easily degradable RNA was reverse transcribed into stable complementary DNA (cDNA) under the catalysis of reverse transcriptase. Next, a PCR reaction system was prepared and placed in a real-time quantitative PCR instrument. Cyclic amplification was performed according to a preset denaturation, annealing, and extension program, and the changes in fluorescence signals generated during amplification were monitored by the instrument. Finally, the Ct value method (cyclic threshold method) was used for data processing. By comparing the Ct values ​​of the target gene and the internal reference gene, and combining with appropriate calculation methods, the relative expression level of the target gene was obtained, thereby achieving precise quantitative analysis of the target gene expression level in mouse left ventricular tissue.

[0035] 4.5 Western blot detection The left ventricle of a mouse was placed in an EP tube, and pre-cooled lysis buffer (RIPA lysis buffer: PMSF: phosphatase inhibitor volume ratio of 100:1:1) was added. Lysis was carried out at 4°C for 30 minutes. The sample was then centrifuged at 22500×g for 15 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA protein quantification kit (Beyotime, Shanghai, China). The protein samples were then subjected to heat denaturation. After separation by SDS-PAGE electrophoresis, the protein was transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk powder for 2 hours, followed by incubation with primary antibody at 4°C overnight, and then co-incubated with secondary antibody for 1 hour. Images were acquired using ECL chemiluminescence reagent and a Tianneng 5200 multicolor fully automated chemiluminescence imaging system, and the grayscale values ​​of the protein bands were calculated using ImageJ software.

[0036] 5. Experimental Results and Discussion After a 10-week modeling period, the experimental mice underwent a 4-week TFDM gavage intervention. For details of the administration regimen, please refer to [link to relevant documentation]. Figure 1 A in the middle.

[0037] 5.1 Effects of TFDM intervention on improving general parameters in HFpEF mice First, after 14 weeks of feeding mice with a high-fat diet (HFD), there were significant differences in body weight between the groups. The body weight of the model group was significantly higher than that of the blank control group. Although the body weight of the TFDM-treated group tended to decrease compared with the model group, this difference did not reach a statistically significant level. Figure 1 In contrast, cardiac-related indicators showed a clear intervention effect. The heart weight and cardiac organ index (calculated as the ratio of heart weight to tibia length, i.e., HW / TL) of mice in the model group were significantly higher than those in the blank control group, while both of these indicators in the TFDM-treated group were significantly lower than those in the model group. Figure 1 The results (including CD) suggest that TFDM can effectively alleviate myocardial hypertrophy in HFpEF model mice. The LDH levels measured in this experiment showed abnormally elevated serum LDH in the model group mice, which was significantly reduced after TFDM treatment. This indicates that the HFpEF model constructed with HFD+L-NAME caused significant cell damage in mice, and that TFDM can effectively improve the above abnormalities. Figure 1 (E).

[0038] 5.2 TFDM can alleviate left ventricular diastolic dysfunction in HFpEF model mice. Echocardiography is a common method for assessing cardiac function, and it can simultaneously detect systolic and diastolic function: when atrial pressure increases, the peak velocity of mitral valve blood flow in early diastole (E wave) increases; while tissue Doppler imaging can directly capture the motion of the mitral valve annulus, and its peak velocity of myocardial motion in early diastole (e' wave) will be further weakened in pathological conditions. Therefore, when diastolic dysfunction occurs, the E / e' ratio (the ratio of E wave to e' wave) will be significantly increased.

[0039] After 14 weeks of animal experiments, echocardiography was performed on the mice. Figure 2 (A) The results showed that the left ventricular ejection fraction (LVEF) of mice in each group was... Figure 2 (B) and left ventricular fractional shortening (LVFS) Figure 2 In the middle group (C), there was no significant difference between groups, but the E / e' ratio in the HFpEF model group was significantly higher than that in the control group. After TFDM intervention, the further increase in the E / e' ratio in mice was effectively inhibited. Figure 2 (Middle D), suggesting that TFDM can improve diastolic dysfunction in HFpEF model mice; in addition, left ventricular hypertrophy is the core pathological feature of HFpEF, as shown by echocardiography ( Figure 2 The results (A) showed that the HFpEF model group mice had significantly increased IVSd, IVSs, LVPWd, LVPWs and left ventricular mass compared with the control group, and TFDM could reverse the above pathological changes. Figure 2 The results (EI) indicate that this dose of TFDM can significantly reduce pathological myocardial hypertrophy in HFpEF model mice, thereby alleviating left ventricular diastolic dysfunction.

[0040] 5.3 TFDM can alleviate liver pathological damage and abnormal serum markers in HFpEF model mice. Serum marker tests showed that the HFpEF model group mice had significant liver function abnormalities and lipid metabolism disorders, with liver function-related ALT and AST levels significantly higher than those in the control group. Figure 3 (A and B) indicates significant hepatocyte damage in the HFpEF model group mice. After TFDM intervention, the abnormal elevation of these two liver function indicators was significantly suppressed, indicating that TFDM can alleviate the degree of hepatocyte damage in HFpEF model mice. Among lipid-related serum indicators, the serum TG and TC levels in the model group mice were significantly higher than those in the control group (…). Figure 3 TFDM intervention significantly reduced serum TG levels. Furthermore, serum HDL-C and LDL-C levels in the model group mice were significantly increased (in C). Figure 3TFDM intervention significantly reduced both D and E levels, indicating that it can regulate systemic lipid metabolism disorders in HFpEF model mice. Liver histopathology and lipid index detection further corroborated the hepatoprotective effect of TFDM. Liver HE staining results showed that the number of vacuolar degeneration of hepatocytes in the HFpEF model group mice was significantly increased, indicating aggravated hepatocyte structural damage. Figure 3 In the model group, Oil Red O staining revealed a significantly increased area of ​​lipid droplet deposition in the liver (reflecting abnormal hepatic lipid accumulation). Figure 3 In the HFpEF model group, after TFDM intervention, the number of hepatocyte vacuolar degenerations decreased, the lipid droplet deposition area was reduced, and liver pathological damage was effectively improved. Furthermore, the levels of TG and TC in the liver tissue of the HFpEF model group mice were significantly increased. Figure 3 After TFDM intervention, both hepatic lipid levels (H and I) significantly decreased, and liver lipid content recovered to levels close to the control group, indicating that TFDM can directly improve lipid accumulation in the liver tissue of HFpEF model mice. These results suggest that HFpEF model mice exhibit liver pathological damage (hepatocyte ischemic injury, structural destruction, and lipid accumulation) and systemic lipid metabolism imbalance related to myocardial injury. TFDM can effectively alleviate these pathological abnormalities by reducing hepatocyte damage and bidirectionally regulating liver and serum lipid metabolism, providing experimental evidence for its protective effect on liver function and improvement of metabolic disorders in HFpEF model mice. This result demonstrates that TFDM intervention can significantly improve abnormal liver function indicators and lipid metabolism imbalance in HFpEF model mice.

[0041] 5.4 TFDM can alleviate cardiac pathological damage in HFpEF model mice. Previous studies have confirmed that HFpEF model mice exhibit typical pathological features such as altered cardiomyocyte morphology, abnormal volume, and aggravated myocardial fibrosis. Therefore, this study systematically evaluated the intervention effect of TFDM on myocardial pathological damage by performing HE staining, Masson's trichrome staining, and WGA fluorescence staining on myocardial tissue from HFpEF model mice. HE staining results showed that the cardiomyocytes in the HFpEF model group were disordered, significantly enlarged, and had blurred cell boundaries, indicating significant damage to the cardiomyocyte structure. Figure 4 (A) After TFDM intervention, the neatness of cardiomyocyte arrangement was significantly improved, the clarity of cell boundaries was effectively restored, and the cell volume was significantly smaller than that of the model group and closer to the level of the normal control group. Figure 4 (A) indicates that TFDM can effectively improve the pathological morphological abnormalities of cardiomyocytes in HFpEF model mice.

[0042] Masson's trichrome staining results further showed that the area of ​​myocardial fibrosis in the HFpEF model group was significantly increased compared with the control group, indicating a significant aggravation of myocardial fibrosis; after TFDM intervention, the pathological changes in the area of ​​myocardial fibrosis were significantly restored. Figure 4 (A, B) suggests that TFDM can further reduce myocardial pathological damage by inhibiting the process of myocardial fibrosis in HFpEF model mice.

[0043] WGA fluorescence staining results confirmed that the cross-sectional area of ​​cardiomyocytes in the HFpEF model group mice was significantly increased, indicating cardiomyocyte hypertrophy; after TFDM intervention, the cross-sectional area of ​​cardiomyocytes was significantly reduced and returned to near normal levels. Figure 4 (A, C) indicates that TFDM can participate in the regulation of myocardial pathological state in HFpEF model mice by inhibiting cardiomyocyte hypertrophy.

[0044] Therefore, it can be inferred that TFDM can improve the pathological morphology of cells by correcting disordered arrangement of cardiomyocytes and reducing the abnormally enlarged cell volume, inhibit the process of myocardial fibrosis by reducing collagen deposition, and alleviate cell hypertrophy by reducing the cross-sectional area of ​​cardiomyocytes, thereby alleviating myocardial pathological damage in HFpEF model mice.

[0045] 5.5 Mechanism of action of TFDM in treating HFpEF based on systems pharmacology prediction To explore the potential therapeutic targets and pathways of TFDM in treating HFpEF, this study employed systems pharmacology to analyze and infer its possible therapeutic mechanism. First, relevant literature and public target databases were systematically searched to collect targets related to the active ingredients of TFDM and disease-related targets of HFpEF. Then, online bioinformatics analysis tools were used to map these two types of targets, identifying overlapping targets. A total of 247 TFDM-HFpEF overlapping targets were obtained. Figure 5 (A)

[0046] The core interaction network of the aforementioned intersection targets was constructed using Cytoscape 3.9.1 software. The results showed that the network contained 245 nodes (representing target points) and 7226 edges (representing the interaction relationships between target points). Based on the "Connectivity (Degree)" value in network topology analysis, core target points were progressively screened: first, nodes with a Degree ≥ 90 were screened, resulting in 57 potential core target points; then, the screening threshold was further increased to a Degree ≥ 152, ultimately identifying 10 key core target points, including interleukin-6 (IL-6). Figure 5 (B) These targets may be the core effector targets for TFDM to exert its anti-HFpEF effect.

[0047] 247 TFDM-HFpEF intersection targets were imported into the DAVID database for GO functional annotation and KEGG pathway enrichment analysis. GO functional annotation results showed 1069 biological process (BP) entries, 237 cellular component (CC) entries, and 137 molecular function (MF) entries. The top 10 entries were selected by count for visualization analysis. Figure 5 The C pathway enrichment analysis revealed that BP was primarily enriched in dynamic biological processes such as gene expression regulation, cell signal transduction, apoptosis and inflammatory response, and cell proliferation and migration, suggesting that TFDM may participate in the pathological improvement of HFpEF by regulating these biological processes. KEGG pathway enrichment analysis showed that 44 pathways with statistically significant differences were identified. p <0.05); Select the top 20 pathways sorted by Count for visualization (if the relevant attached figures are not labeled with specific serial numbers, they can be referred to as "corresponding attached figures"). Figure 5 (Middle D). Enrichment results suggest that these pathways are mainly concentrated in the fields of immune regulation and inflammation-related signaling pathways, indicating that TFDM may exert its therapeutic effect on HFpEF by regulating inflammation and immune-related pathways.

[0048] 5.6 Investigating the Mechanism of Action of TFDM Against HFpEF Using Transcriptomics and Molecular Biology Methods To further explore and validate the potential targets and signaling pathways of TFDM in treating HFpEF, this study performed transcriptomic analysis on the heart tissues of HFpEF model mice and TFDM intervention group mice to elucidate the molecular mechanism by which TFDM regulates HFpEF. The specific results are as follows: Transcriptome sequencing results were compared between the HFpEF model group and the TFDM intervention group, and volcano plot analysis was used to screen differentially expressed genes (DEGs). The results showed a total of 463 DEGs in both groups, of which 219 were upregulated and 244 were downregulated. Zbtb16 was a representative gene among the upregulated DEGs, suggesting that these DEGs may be potential targets for TFDM intervention in HFpEF. Figure 6 (A). Hierarchical clustering heatmaps further visualized the differential expression patterns of all DEGs in the two groups, and the results showed that key genes such as Zbtb16 had significant expression differences between the model group and the TFDM intervention group. Figure 6 (B), and qRT-PCR results showed that the expression trend of the Zbtb16 gene was consistent with the overall transcriptome differences (B). Figure 6 The presence of C further suggests that it is a core candidate target for TFDM to regulate HFpEF.

[0049] To clarify the biological functions and signaling pathways involved in DEGs, GO functional annotation and KEGG pathway enrichment analysis were performed on the screened DEGs. GO functional annotation results showed that DEGs were mainly enriched in biological processes (BP) such as signaling pathways related to inflammatory responses and immune responses, suggesting that TFDM may improve the pathological state of HFpEF by regulating inflammation-related biological processes. Figure 6 The KEGG pathway enrichment analysis further confirmed that DEGs were significantly enriched in inflammation-related pathways (D); Figure 6 The presence of E in the middle indicates that inflammation-related pathways are key pathways for TFDM to exert its anti-HFpEF effect.

[0050] 5.7 In vivo validation of the TFDM pathway Proteins were extracted from the heart tissues of Ctr mice, HFpEF model mice, and TFDM intervention group mice, and analyzed by Western blotting. The mRNA expression levels of IL-6, TNF-α, and B-type brain natriuretic peptide (BNP) were detected by qRT-PCR.

[0051] Figure 7 The results showed that, compared with the HFpEF model mouse group, TFDM treatment in HFpEF mice significantly increased Zbtb16 expression and significantly decreased the p-NF-κB / NF-κB ratio, IL-6 protein expression, and TNF-α protein expression (see [link to study]). Figure 7 (A~E in the text), and the mRNA expression levels of IL-6 and TNF-α were significantly decreased (see A~E in the text). Figure 7 (F~G in the text). Simultaneously, compared with the HFpEF model mouse group, the total flavonoids of *Cymbidium faberi* significantly reduced the mRNA expression level of BNP in HFpEF mice after treatment (see...). Figure 7 The H in the middle improved the pathological condition of HFpEF. Figure 7 The results showed that TFDM inhibited the NF-κB inflammatory pathway through the Zbtb16 target, reducing downstream inflammatory factors such as IL-6 and TNF-α, thereby achieving the purpose of treating HFpEF and inhibiting the inflammation caused by HFpEF.

[0052] TFDM, as a natural drug extract, has significantly improved diastolic function, reduced myocardial remodeling and inflammatory damage in HFpEF mice, providing an innovative drug for the treatment of HFpEF.

[0053] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of total flavonoids from *Cymbidium goeringii* 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 heart failure with preserved ejection fraction is heart failure with preserved ejection fraction induced by N-nitro-L-arginine methyl ester and a high-fat diet.

3. The application according to claim 1, characterized in that, The total flavonoids from the fragrant orchid have the ability to prevent and / or treat myocardial cell damage.

4. The application according to claim 1, characterized in that, The total flavonoids from the fragrant orchid have the effect of preventing and / or treating myocardial fibrosis.

5. The application according to claim 1, characterized in that, The total flavonoids from the fragrant orchid have the effect of preventing and / or treating myocardial hypertrophy.

6. The application according to claim 1, characterized in that, The total flavonoids from *Cymbidium goeringii* have the effect of preventing and / or treating abnormal liver function indicators.

7. The application according to claim 1, characterized in that, The total flavonoids from *Cymbidium goeringii* have the effect of preventing and / or treating lipid metabolism imbalance.

8. The application according to claim 1, characterized in that, The total flavonoids from *Cymbidium goeringii* have the effect of preventing and / or treating diastolic heart failure.

9. The application according to any one of claims 1 to 8, characterized in that, The drug also includes pharmaceutically acceptable excipients.

10. The application according to claim 9, characterized in that, The total flavonoids of *Cymbidium ensifolium* account for 10% to 90% of the mass of the drug.

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