Application of fibroblast growth factor 9 in medicine for treating diabetic cardiomyopathy

Abstract

The invention discloses an application of a fibroblast growth factor 9 in a medicine for treating diabetic cardiomyopathy, and particularly relates to the application that the fibroblast growth factor 9 promotes expression of Nrf2 protein in a heart with the diabetic cardiomyopathy by promoting autophagy regulation of Keap1 so as to treat the diabetic cardiomyopathy. Compared with the prior art, the fibroblast growth factor 9 is applied to the medicine for treating the diabetic cardiomyopathy.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an application of fibroblast growth factor 9 in a drug for treating diabetic cardiomyopathy. Background Art

[0002] The global prevalence of diabetes, especially type 2 diabetes (T2D), has been rising at an alarming rate over the past few decades. By 2045, the number of people with diabetes worldwide is expected to reach approximately 693 million. Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality in patients with diabetes, leading to heart failure in up to 80% of diabetic patients. However, the precise mechanisms driving diabetic cardiomyopathy and subsequent cardiovascular complications remain inadequately understood.

[0003] Fibroblast growth factor (FGF) is a large family of growth factors. FGFs mediate a variety of biological and pathophysiological processes, including angiogenesis, wound healing, embryonic development, and metabolic regulation, using paracrine or endocrine signals. Fibroblast growth factor 9 (FGF9) is a member of this family that selectively binds to fibroblast growth factor receptor 3 (FGFR3) and plays a key role in the development of organs such as the pancreas, lung, osteogenesis, and chondrogenesis. FGF9 is also involved in adipogenesis and systemic energy metabolism by regulating uncoupling protein 1 (UCP1).

[0004] Although several studies have explored the role of FGF9 in non-cardiac diseases, such as embryonic development and metabolic disorders, these studies have focused on its potential role in cardiovascular diseases. For example, FGF9 has been shown to regulate myocardial differentiation in adult mice and reduce mortality after myocardial infarction; in addition, FGF9 expression has been shown to be reduced in diabetic nephropathy and associated with atrial fibrillation-related stroke. However, the role of FGF9 in the pathophysiology of diabetic cardiomyopathy remains poorly understood. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an application of fibroblast growth factor 9 in a drug for treating diabetic cardiomyopathy in view of the above technical status.

[0006] The technical solution adopted by the present invention to solve the above technical problem is: application of fibroblast growth factor 9 in drugs for treating diabetic cardiomyopathy.

[0007] Preferably, the drug promotes the expression of Nrf2 protein.

[0008] Preferably, the drug promotes Keap1 autophagy. Selective autophagy adaptor protein 1 (sequestosome1, SQSTM1 / p62) and nuclear factor erythroid-derived 2-like 2 (NF-E2-related factor 2, Nrf2) competitively interact with Kelch-like ECH-associated protein 1 (Kelch-like ECH-associated protein 1, Keap1), and the binding of Keap1 to Nrf2 promotes the ubiquitination and degradation of Nrf2, reducing the expression of Nrf2; drugs promoting Keap1 autophagy can reduce the binding of Keap1 to Nrf2, thereby promoting the expression of Nrf2.

[0009] Preferably, the drug treats diabetic cardiomyopathy by at least one of the following means:

[0010] (1) Reduce myocardial hypertrophy;

[0011] (2) Reduce myocardial fibrosis;

[0012] (3) Improve the systolic and diastolic function of diabetic heart.

[0013] Compared with the prior art, the advantages of the present invention are: the present invention discovers and verifies that fibroblast growth factor 9 can promote the interaction between Keap1 and p62, thereby leading to the autophagic degradation of Keap1, thereby reducing the binding of Keap1 to Nrf2 and promoting the expression of Nrf2. Therefore, drugs containing fibroblast growth factor 9 can be used to treat diabetic cardiomyopathy, especially to reduce myocardial fibrosis and improve the contractile and diastolic function of diabetic heart. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 To analyze the expression of FGF9 in diabetic hearts;

[0015] Figure 2 Schematic diagram of the test of the effect of FGF9 on primary cardiomyocytes induced by high glucose and palmitic acid;

[0016] Figure 3 Schematic diagram of the effect of FGF9 on diabetic cardiac remodeling and dysfunction;

[0017] Figure 4 Schematic diagram of the functional test of Nrf2 in cardiomyocytes;

[0018] Figure 5 Schematic diagram of the p62-Keap1 synergistic test. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below with reference to the accompanying drawings.

[0020] In the embodiments of the present invention, all the raw materials and reagents used can be purchased from the market.

[0021] db / db mice are leptin receptor-deficient mice; db / dm mice are control mice; WT are wild-type mice; MAN is mannitol; Nrf2-CKO are cardiomyocyte Nrf2 knockout mice; Lacz is the control virus; WGA is wheat germ agglutinin staining; DHE is oxidative stress staining; Troponin-T is the cardiomyocyte cytoskeletal protein.

[0022] Example 1 Expression of FGF9 in diabetic hearts

[0023] Figure 1 Figure 2 is the expression analysis of FGF in diabetic heart; A is the analysis of FGF transcription level; B is the analysis of FGF9 gene ontology (GO); C is the relative expression level of FGF9 mRNA in heart; D is the relative expression level of FGF9 mRNA in db / db mice under high glucose (HG) and palmitic acid (PA) treatment; (1) and (2) of E are the expression of FGF9 under high glucose and palmitic acid treatment.

[0024] a) Transcriptional level analysis

[0025] High-fat diet / streptozotocin (HFD / STZ)-induced mice and db / db leptin receptor-deficient mice are animal models of type 2 diabetes and its related complications. The transcription level of FGF in db / db mice was analyzed. Figure 1 As shown in the left three groups and the middle three groups in A, the expression of FGF5, FGF7, FGF9, FGF11, FGF16, FGF18 and FGF21 in the myocardium of db / db mice was significantly decreased (e.g. Figure 1 A).

[0026] The db / db mice were injected with a virus that specifically overexpressed FGF9 (rAAV9-cTnT-FGF9) in cardiomyocytes through the tail vein. Figure 1 As shown in the three groups on the right in A, the above-mentioned viruses not only lead to specific overexpression of FGF9 in cardiomyocytes, but also lead to increased levels of FGF1, FGF2, FGF18 and FGF21.

[0027] b) Gene ontology analysis

[0028] like Figure 1 As shown in B, gene ontology analysis showed that overexpression of FGF9 in db / db mice improved the oxidative stress response, tissue remodeling signaling pathways, and positive regulation of protein metabolic processes, while increasing fibroblast growth factor receptor (FGFR) signaling.

[0029] c) Expression of FGF9 in cardiomyocytes

[0030] During embryonic development (from embryonic day 10.5 [E10.5] to E12.5), the FGF9 gene is highly expressed in the lateral regions of the left and right ventricles and near the apical interventricular groove. However, this expression gradually decreases after E14.5, with the lowest expression observed in the ventricular myocardium of the adult heart. The expression of FGF9 in neonatal rat cardiomyocytes (NRCMs) and db / db mice was examined, and the results were shown in Figure 2. Figure 1 As shown in C, it can be seen that the expression of FGF9 in db / db mice was significantly downregulated.

[0031] Under high glucose and palmitic acid treatment, the mRNA expression of FGF9 in NRCMs (e.g. Figure 1 D) and protein (as Figure 1 E) levels were significantly decreased.

[0032] In summary, the expression of FGF9 is reduced in diabetic hearts, so it is speculated that FGF9 plays a key role in the regulation of diabetic cardiomyopathy.

[0033] Example 2 Effects of FGF9 on NRCMs Induced by High Glucose and Palmitic Acid

[0034] Figure 2 The figure is a schematic diagram of the test of the effect of FGF9 on primary cardiomyocytes induced by high glucose and palmitic acid; wherein A is a photograph of the size of the cells; B is a schematic diagram of the cell size test results; C (1) is the relative expression level of atrial natriuretic peptide (ANP) mRNA, (2) is the relative expression level of brain natriuretic peptide (BNP) mRNA, (3) is the relative expression level of collagen I mRNA, and (4) is the relative expression level of collagen III mRNA; D is a photo of the dihydroethidium (DHE) fluorescence intensity test, and E is a dihydroethidium fluorescence intensity test; F is a photo of the TUNEL-positive cell test; H is the relative expression level of collagen III, Nrf2 protein, HO1 and β-actin under different conditions (MAN is the control group using mannitol); G is a schematic diagram of the TUNEL-positive cell content; I is the ratio of Nrf2 to β-actin; J is the ratio of heme oxygenase 1 to β-actin; K is the ratio of collagen III to β-actin.

[0035] a) Effects of FGF9 treatment on cells induced by high glucose and palmitic acid

[0036] like Figure 2 As shown in A and B, FGF9 treatment significantly reduced HG+PA-induced cell hypertrophy. Figure 2As shown in C, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), collagen I and collagen III were down-regulated after FGF9 treatment. ANP, BNP, collagen I and collagen III are hypertrophy and fibrosis-related marker genes. The down-regulation of hypertrophy and fibrosis-related marker genes further proves that FGF9 treatment significantly reduces high glucose and palmitic acid-induced cell hypertrophy. b) Effects of FGF9 on NRCMs under high glucose and palmitic acid conditions

[0037] like Figure 2 As shown in D and E, after high sugar and palmitic acid treatment, the fluorescence intensity of dihydroethidium (DHE) increased. The change in the fluorescence intensity of dihydroethidium is closely related to the generation of reactive oxygen species (ROS). It can be seen that high sugar and palmitic acid treatment lead to the rapid generation of reactive oxygen species. Figure 2 As shown in F and G, the decrease in TUNEL-positive cells indicates that FGF9 treatment significantly attenuated cardiomyocyte apoptosis under high glucose and palmitic acid conditions.

[0038] like Figure 2 As shown in H and I, FGF9 treatment significantly restored Nrf2 protein levels in NRCMs exposed to high glucose and palmitic acid. Figure 2 As shown in J, the expression of Nrf2 target gene heme oxygenase 1 (HO1) was significantly decreased after high glucose and palmitic acid treatment, but significantly upregulated after FGF9 treatment. Nrf2 plays a role in regulating oxidative stress in diabetic complications, therefore, FGF9 treatment regulates oxidative stress by restoring Nrf2 protein levels.

[0039] like Figure 2 As shown in H and K, FGF9 improved HG+PA-induced type III collagen overexpression, further supporting its role in alleviating myocardial fibrosis.

[0040] In summary, FGF9 exerts a protective effect on cardiomyocytes through inflammatory pathways, regulating oxidative stress and fibrotic remodeling.

[0041] Example 3 FGF9 improves diabetic cardiac remodeling and dysfunction

[0042] like Figure 3As shown, it is a schematic diagram of the effect of FGF9 on diabetic heart remodeling and dysfunction; wherein, AC is a schematic diagram of left ventricular systolic function test; D is a photo of heart size; E is a photo of cross-section of cardiomyocytes; F is the cross-sectional area of ​​cardiomyocytes; G is a photo of collagen volume test in the heart; H is the collagen volume in the heart; I (1) is the relative expression level of atrial natriuretic peptide (ANP) mRNA, (2) is the relative expression level of brain natriuretic peptide (BNP) mRNA, (3) is the relative expression level of collagen I mRNA, and (4) is the relative expression level of collagen III mRNA; J is a schematic diagram of the test of collagen III, Nrf2 protein, HO1 and β-actin content in the heart under different conditions; K is a schematic diagram of the ratio of nrf2 to β-actinNrf2 to β-actin in the heart; L is the ratio of heme oxygenase 1 to β-actin; M is the ratio of collagen III to β-actin.

[0043] a) FGF9 improves the systolic and diastolic function of diabetic hearts

[0044] Recombinant adeno-associated virus serotype 9 expressing FGF9 (rAAV9-ctnt-FGF9) was injected into 6-week-old db / db mice via the tail vein to overexpress FGF9 specifically in cardiomyocytes; control animals were injected with rAAV9-null. Mice were monitored for 8 weeks and echocardiographic evaluation was performed at 14 weeks of age. Figure 3 AC and Table 1.

[0045] Echocardiography was used to evaluate cardiac function, and it was found that db / db mice had significant left ventricular (LV) systolic dysfunction, increased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS). Therefore, FGF9 overexpression can improve left ventricular systolic function, indicating that FGF9 can improve both systolic and diastolic function in diabetic hearts.

[0046] Table 1 Echocardiographic analysis of left ventricular function in 14-week-old db / dm or db / db mice

[0047]

[0048] b) Effects of FGF9 on cardiac structural remodeling

[0049] Detection of cardiac hypertrophy in db / db mice. Figure 3 As shown in D, after FGF9 overexpression, the heart size was reduced, indicating that FGF9 overexpression can reduce cardiac hypertrophy.

[0050] like Figure 3 As shown in E and F, wheat germ agglutinin (WGA) staining showed that the cross-sectional area of ​​cardiomyocytes in FGF9-overexpressing db / db mice was significantly smaller than that in control db / db mice. Figure 3 As shown in G and H, FGF9 treatment also attenuated left ventricular collagen deposition, indicating that collagen volume was reduced in FGF9-treated hearts. Figure 3 Real-time fluorescence quantitative PCR confirmed this effect, that is, the mRNA levels of hypertrophy and fibrosis markers were significantly reduced in the case of FGF9 overexpression.

[0051] like Figure 3 JL showed that, at the molecular level, FGF9 treatment significantly restored Nrf2 protein expression and upregulated the Nrf2 target gene heme oxygenase 1 in the hearts of db / db mice, while untreated db / db mice downregulated the expression of the above genes.

[0052] like Figure 3 As shown in J and M, FGF9 treatment reduced the accumulation of collagen III in hearts exposed to high glucose and high fat conditions, thereby alleviating myocardial fibrosis.

[0053] In summary, FGF9 improves the systolic and diastolic function of diabetic hearts and has beneficial effects on cardiac remodeling and dysfunction in diabetic hearts by regulating oxidative stress, fibrosis, and myocardial hypertrophy, as well as restoring Nrf2-mediated protective signaling pathways.

[0054] Example 4 Function of Nrf2 in cardiomyocytes

[0055] Figure 4 Schematic diagram of the functional test of Nrf2 in cardiomyocytes; A is a picture of mouse cardiomyocytes; B is a schematic diagram of the test of the cross-sectional area of ​​mouse cardiomyocytes; C is a picture of the test of mouse myocardial fibrosis; D is a schematic diagram of the test results of myocardial fibrosis; E is a schematic diagram of the test of cardiac contraction and diastolic function; F is a schematic diagram of the test results of left ventricular shortening fraction (LVFS); G is a schematic diagram of the test results of ejection fraction (LVEF); H is a schematic diagram of the test of relative production of hydrogen peroxide; I (1) is the relative expression level of atrial natriuretic peptide (ANP) mRNA, (2) is the relative expression level of brain natriuretic peptide (BNP) mRNA, (3) is the relative expression level of collagen I mRNA, and (4) is the relative expression level of collagen III mRNA. Construction of cardiomyocyte-specific induced Nrf2 knockout mice (Myh6-CreEsr1, Nrf2 f / f ; Nrf2-CKO).

[0056] Table 2 Nrf2 expression after 12 weeks of T2DM induced by streptozotocin and high-fat diet f / f Echocardiographic analysis of WT and Nrf2-CKO mice

[0057]

[0058]

[0059] like Figure 4 As shown in Table 2, FGF9 treatment significantly reduced cardiomyocyte hypertrophy, myocardial fibrosis, and cardiac systolic and diastolic dysfunction in db / db mice.

[0060] However, these protective effects were significantly attenuated in cardiomyocyte-specific Nrf2-deficient mice. Figure 3 As shown in Figure 3, the ability of FGF9 to reduce hydrogen peroxide levels was significantly reduced in Nrf2-CKO mice. Figure 3 As shown in Figure 1, FGF9 treatment failed to effectively suppress the mRNA levels of hypertrophy and fibrosis marker genes in Nrf2-CKO mice.

[0061] It can be seen from this that the lack of cardiomyocyte-specific Nrf2 hinders the protective effect of FGF9 on diabetic cardiomyopathy. Therefore, Nrf2 plays a key role in mediating the treatment and protection of diabetic cardiomyopathy by FGF9.

[0062] Example 5 In NRCMs, FGF9 promotes Nrf2 expression by promoting the synergistic action of p62-Keap1

[0063] Figure 5 Schematic diagram of p62-Keap1 co-action test; wherein, A is the co-immunoprecipitation analysis of MG132 co-treatment; BD is a schematic diagram of MG132 co-immunoprecipitation test results; E is the co-immunoprecipitation analysis of autophagy inhibitor Baf-A1 co-treatment; FH is a schematic diagram of autophagy inhibitor Baf-A1 co-immunoprecipitation test results; I is the co-immunoprecipitation analysis of selective AMPK inhibitor co-treatment; JL is a schematic diagram of the co-immunoprecipitation test results of the selective AMPK inhibitor, and compound C is a selective AMPK inhibitor.

[0064] a) FGF9 regulates Keap1 through a non-proteasomal degradation mechanism

[0065] FGF9 treatment restored the expression of Nrf2 protein in NRCMs exposed to high glucose and palmitic acid. Figure 5 As shown in A and C, treatment with the proteasome inhibitor MG132 enhanced FGF9-induced Nrf2 protein accumulation.

[0066] like Figure 5 As shown in A and D, under high glucose and palmitic acid conditions, FGF9 treatment reduced Keap1 protein levels, but co-treatment with the proteasome inhibitor MG132 did not further affect Keap1 protein levels, indicating that FGF9 regulates the stability of Keap1 through a non-proteasome degradation mechanism.

[0067] like Figure 5 As shown in AD, co-immunoprecipitation analysis showed that high glucose and palmitic acid treatment promoted the interaction between Nrf2 and Keap1, leading to Nrf2 ubiquitination and subsequent degradation. FGF9 treatment disrupted the interaction between Nrf2 and Keap1 and prevented Nrf2 degradation. However, in MG132-treated cells, FGF9 did not significantly increase the binding of Nrf2 to Keap1, indicating that FGF9 regulates Keap1 degradation through a proteasome-independent mechanism.

[0068] b) FGF9 affects the expression of Nrf2 through autophagy regulation

[0069] Keap1 is known to interact with p62 and release Nrf2 for nuclear translocation. Figure 5 As shown in E and F, high glucose and palmitic acid treatment increased the interaction between Keap1 and p62 in NRCMs, and, as Figure 5 As shown in E and F, the above interactions returned to baseline levels after FGF9 treatment.

[0070] like Figure 5 As shown in Figures E, G, and H, co-treatment with the autophagy inhibitor bafilomycin A (Baf-A1) enhanced the interaction between Keap1 and Nrf2, regardless of whether FGF9 was treated or not. This indicates that Baf-A1 inhibited the autophagic degradation of Keap1 and p62 induced by FGF9. Therefore, FGF9 treatment promotes the interaction between Keap1 and p62 and their autophagic degradation.

[0071] The accumulation of Keap1 protein increases the probability of Keap1 binding to Nrf2, thereby promoting the ubiquitination and degradation of Nrf2. FGF9 induces the autophagic degradation of Keap1 and p62, thereby reducing Keap1 protein and further reducing the binding of Keap1 protein to Nrf2. Therefore, FGF9 promotes the expression of Nrf2 through autophagy regulation.

[0072] c) FGF9 regulates autophagy through AMPK

[0073] like Figure 5 As shown in IL, selective energy sensing kinase (AMPK) inhibitors can block the effects of FGF9 on AMPK phosphorylation, autophagy induction, and Nrf2 nuclear translocation, indicating that FGF9 regulates autophagy through the energy sensing kinase AMPK. Therefore, FGF9 improves HG+PA-induced cardiomyocyte dysfunction by promoting Keap1 degradation, promoting Keap1 autophagy through the AMPK / p62-dependent autophagy pathway, and thus promoting Nrf2 nuclear translocation.

[0074] In summary, FGF9 regulates autophagy through AMPK, thereby affecting the expression of Nrf2 through autophagy regulation of Keap1 and p62, further treating diabetic cardiomyopathy.

Claims

1. Application of fibroblast growth factor 9 in drugs for the treatment of diabetic cardiomyopathy.

2. The use of fibroblast growth factor-9 according to claim 1 in a drug for treating diabetic cardiomyopathy, characterized in that: The drug promotes the expression of Nrf2 protein.

3. The use of fibroblast growth factor-9 in a drug for treating diabetic cardiomyopathy according to claim 2, characterized in that: The drug promotes Keap1 autophagy.

4. The use of fibroblast growth factor-9 in a drug for treating diabetic cardiomyopathy according to claim 1, characterized in that: The drug treats diabetic cardiomyopathy by at least one of the following means: (1) Reduce myocardial hypertrophy; (2) Reduce myocardial fibrosis; (3) Improve the systolic and diastolic function of diabetic heart.

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