Application of HIF-2alpha specific inhibitor in preparation of medicine for treating diabetic cardiomyopathy

Abstract

The invention is applicable to the technical field of biological medicines, and provides application of an HIF-2alpha specific inhibitor in preparation of a medicine for treating diabetic cardiomyopathy. The invention clarifies the copper death of human myocardial cells induced by combination of high glucose and high fat and copper and related molecular mechanisms, namely when the extracellular copper concentration is increased, copper transporter CTR1 is reduced, DMT1 compensatory is increased, copper is transported into AC16, HF + HG further promotes DMT1 expression by enhancing m6A methyltransferase WTAP expression to mediate HIF-2alpha m6A modification, and finally the copper ion concentration in AC16 is increased, so that the copper death of the human myocardial cells is induced. In order to solve the problem that the copper death is caused due to the fact that the HIF-2 alpha specific inhibitor PT2385 is used for inhibiting the HIF-2 alpha, the expression of DMT1 in AC16 cells is reduced, the copper death index is recovered to be normal, and then a new target is provided for treating DCM.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of HIF-2α specific inhibitors in the preparation of drugs for diabetic cardiomyopathy. Background Technology

[0002] Diabetic cardiomyopathy (DCM) is a leading cause of death in diabetic patients. It is well known that DCM patients typically have abnormal blood glucose and lipid levels, manifesting as hyperglycemia and hypertriglyceridemia. Furthermore, multiple pieces of evidence suggest that metabolic diseases, including diabetes, are often accompanied by impaired regulation of various metal ions. Copper, in particular, is central to many important biological processes, including mitochondrial respiration, antioxidant defense, and the synthesis of biocompounds, and is closely related to the severity and progression of diabetes.

[0003] Copper death is a novel form of programmed cell death discovered in recent years, with elevated intracellular copper ion concentration being a key trigger. This copper-induced cell death is characterized by: firstly, decreased lipoylation of DLAT and DLST induced by direct copper binding and oligomerization of lipoylated proteins in the tricarboxylic acid cycle; and secondly, instability and overall reduction of iron-sulfur (Fe-S) cluster proteins. These factors collectively lead to protein toxicity stress and mitochondrial dysfunction. In summary, regulating copper metabolism or inhibiting copper death may be a novel strategy for treating diabetic complications. Therefore, exploring the effects of HF+HG on intracellular copper ion concentration in AC16 cells and the molecular mechanisms of copper death may provide new targets for the treatment of diabetic complication (DCM). Summary of the Invention

[0004] The purpose of this invention is to provide the application of HIF-2α specific inhibitors in the preparation of drugs for diabetic cardiomyopathy, aiming to solve the problems mentioned in the background art.

[0005] The present invention is implemented as follows: the application of HIF-2α specific inhibitors in the preparation of drugs for diabetic cardiomyopathy.

[0006] Preferably, the HIF-2α specific inhibitor is PT2385.

[0007] Preferably, the DCM is a DCM caused by high sugar and high fat and / or copper ion metabolism disorder.

[0008] Preferably, the HIF-2α specific inhibitor reduces copper ion concentration and copper death index by inhibiting HIF-2α expression, downregulating DMT1 expression, and reducing copper ion concentration.

[0009] Another objective of this invention is to provide a drug targeting DCM, the drug including a HIF-2α specific inhibitor.

[0010] Preferably, the HIF-2α specific inhibitor is PT2385.

[0011] Preferably, the drug further includes one or more pharmaceutically acceptable carriers.

[0012] This invention clarifies the molecular mechanism by which high-sugar, high-fat (HF+HG) combined with copper induces copper death in human cardiomyocytes (AC16). Specifically, when extracellular copper concentration increases, the copper transporter CTR1 decreases, and DMT1 compensates by increasing, transporting copper to AC16 cells. HF+HG, however, enhances the effect of high-sugar, high-fat (HF+HG) combined with copper, leading to copper death in AC16 cells. 6 A-methyltransferase WTAP expression mediates HIF-2α m 6 Modification A further promotes DMT1 expression, ultimately leading to an increase in copper ion concentration in AC16 cells and causing copper death. However, after inhibiting HIF-2α with the HIF-2α-specific inhibitor PT2385, DMT1 expression in AC16 cells is downregulated, and copper death indicators return to normal, thus providing a new target for the treatment of DCM. Attached Figure Description

[0013] Figures 1 to 4 The results provided in Embodiment 1 of the present invention regarding diabetic cardiomyopathy may be related to copper homeostasis imbalance (wherein) Figure 1 A volcano map. Figure 2 The results of the GO enrichment analysis are as follows. Figure 3 For clustering heatmaps, Figure 4 (For Venn diagrams) Figure 5 The results of copper-induced cardiomyocyte death induced by high glucose and high lipid combined with copper in Example 2 of this invention are shown below (where A is the copper ion concentration in AC16 cells; BD is the protein expression level and quantitative analysis of ACO2, LIAS, FDX1, Lipoy-DLAT and Lipoy-DLST in AC16 cells; E is the copper content in AC16 cells; FJ is the protein expression level and quantitative analysis of ACO2, LIAS, FDX1, Lipoy-DLAT and Lipoy-DLST in AC16 cells; P < 0.05, P < 0.01, P < 0.001, ****P < 0.0001). Figure 6The results of DMT1 upregulation mediating copper death in cardiomyocytes provided in Example 2 of this invention are as follows (where A represents cell viability; B and E represent the expression and quantitative analysis results of CTR1 and DMT1 proteins; C and FH represent the protein expression levels and quantitative analysis results of CTR1 and DMT1, as well as the expression results of DMT1 mRNA; D and JM represent the protein expression levels and quantitative analysis results of copper death marker molecules; I represents the changes in intracellular copper content; P < 0.05, P < 0.01, P < 0.001, ****P < 0.0001). Figure 7 The results for HIF-2α as a transcription factor of DMT1 provided in Example 3 of this invention are as follows (where A is the transcription factor of DMT1 predicted by the database; B and C are the protein expression and quantitative analysis results of HIF-2α; D is the expression result of HIF-2α mRNA; E and K are the protein expression and quantitative analysis results of HIF-2α, DMT1 and copper death marker molecules; L and M are the results of HIF-2α content differences; P < 0.05, P < 0.01, P < 0.001, ****P < 0.0001). Figures 8 to 10 The WTAP-mediated HIF2α m provided in Embodiment 4 of the present invention 6 The result of modification A (where) Figure 8 , Figure 9 Predict the m of HIF-2α for the database 6 Results of modification sites A; Figure 10 AE is the database prediction m 6 A shows the correlation between methyltransferase and HIF-2α; F shows the expression results of WTAP gene in normal individuals and patients with cardiomyopathy; G and I show the protein expression and quantitative analysis results of WTAP; J shows the expression results of WTAP mRNA; H and K show the protein expression and quantitative analysis results of HIF-2α; L shows the expression results of HIF-2α mRNA; P < 0.05, P < 0.01, P < 0.001, ****P < 0.0001) Figure 11 This is a diagram illustrating the molecular mechanism by which high sugar and high fat combined with copper induces copper death in human cardiomyocytes, as provided in an embodiment of the present invention. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0015] The main experimental reagents and other information used in the embodiments of this invention are as follows:

[0016] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0017] Example 1: Analysis of diabetic cardiomyopathy and copper homeostasis imbalance: The GSE179130 dataset of gene expression patterns related to cardiomyopathy in diabetic mice was extracted from the GEO database. After standardizing the high-throughput sequencing results, 143 differentially expressed genes (DGEs) were extracted using the limma package with p < 0.05 and Fold Change > 1.2 as cutoff values. A volcano plot was then generated, as shown below. Figure 1 As shown; GO enrichment analysis revealed that these significantly different genes are associated with metal ion metabolism at the molecular function (MF) level, such as Figure 2 As shown; genes associated with copper homeostasis were extracted from the Genecard database, and Venn diagrams were plotted by intersecting these genes with significantly differentially expressed genes in diabetic cardiomyopathy, as shown in the figure. Figure 3 , 4 As shown in the Venn diagram, there are 23 differentially expressed genes associated with diabetic cardiomyopathy; the above bioinformatics analysis results suggest that diabetic cardiomyopathy may be associated with copper homeostasis imbalance.

[0018] Example 2: Experimental analysis of copper-induced cardiomyocyte death induced by a combination of high glucose, high fat, and copper: To determine the effects of copper chloride (CuCl2) on CTR1 and DMT1, when the cell culture reached approximately 70%, cells were treated with different concentrations of CuCl2 (0, 10, 20, 40 μM) in serum-free medium (copper content less than 0.1 μM) for 48 h. The protein levels of CTR1 and DMT1 were detected by Western blot (WB). The results showed that when the extracellular copper concentration of AC16 cells increased, the copper transporter CTR1 decreased, while DMT1 increased compensatorily. Appropriate concentrations of CuCl2 (20 μM), HF+HG (33.5 mmol / L D-glucose and 100 μmol / L palmitate), and the DMT1-specific inhibitor DMT1 blocker 2 (10 μM) were selected. The experiment was divided into five groups: Control, CuCl2, HF+HG, HF+HG-CuCl2, and DMT1i-HF+HG-CuCl2 (DMT1i: DMT1 inhibitor). Cells in the first four groups were treated for 48 h. In the last group, cells were treated with CuCl2 combined with HF+HG for 24 h, followed by treatment with the DMT1-specific inhibitor DMT1 blocker 2 for another 24 h. Cell proliferation was assessed using a CCK8 assay, intracellular copper concentration was detected using a copper ion assay kit, and the levels of CTR1, DMT1, and copper death marker proteins were detected by Western blotting. The results are shown below. Figure 5 , Figure 6 As shown, specifically, compared with the control group, the intracellular copper ion content was increased in the ilismo + copper chloride group ( Figure 5 In A, n = 3), the levels of copper death markers ACO2, LIAS, FDX1, and Lipoy-DLAT / DLST decreased ( Figure 5 The protein expression levels and quantitative analysis results of ACO2, LIAS, FDX1, Lipoy-DLAT, and Lipoy-DLST in AC16 cells were detected by Western blot (n = 3), indicating that copper death occurred in the cells. Compared with the control group, the intracellular copper ion content and copper death indicators remained unchanged in the copper chloride group, while the intracellular copper ion content and copper death indicators decreased in the high-sugar, high-fat + copper chloride group, indicating that high-sugar, high-fat combined with copper induces copper death in cardiomyocytes. Figure 5 In the high-fat, high-sugar combined CuCl2 treatment, EJ represents the copper content in AC16 cells after treatment (n = 3); FJ represents the protein expression levels and quantitative analysis results of ACO2, LIAS, FDX1, Lipoy-DLAT, and Lipoy-DLST in AC16 cells as detected by Western blot (n = 3); the unpaired Student's t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups, followed by Tukey's post-hoc test. Cell viability was significantly lower in the high-sugar, high-fat combined CuCl2 treatment group compared to the CuCl2-treated group alone. Figure 6 (A) As copper ion concentration increases, the expression of the copper transporter CTR1 decreases, while DMT1 increases. High glucose and high lipid treatment increases both CTR1 and DMT1. After adding a DMT1 inhibitor, intracellular copper content decreases, and copper death markers return to normal. Figure 6In the study, AC16 cells were treated with CuCl2 at concentrations of 0, 10, 20, and 40 μM (B and E, respectively) by Western blot analysis of CTR1 and DMT1 protein expression and quantification (n = 3). C and FH were used for Western blot analysis of CTR1 and DMT1 protein expression levels and quantification, and RT-qPCR analysis of DMT1 mRNA expression (n = 3). D and JM were used for treatment with a 10 μM DMT1-specific inhibitor (DMT1i) combined with high glucose, high lipid, and CuCl2 stimulation. Western blot analysis of copper death marker molecules and quantification were performed (n = 3). I represented changes in intracellular copper content (n = 3). Comparisons between two groups were performed using an unpaired Student's t-test, and comparisons among multiple groups were performed using one-way ANOVA (subject to Tukey post-hoc test). The above confirms that HF+HG promotes DMT1 expression, which ultimately leads to an increase in copper ion concentration within AC16, resulting in copper death.

[0019] Example 3: Experimental analysis of how high sugar and high fat intake promotes DMT1 transcription via HIF-2α and induces copper death in cells: Appropriate concentrations of CuCl2 (20 μM), HF+HG (33.5 mmol / L D-glucose and 100 μmol / L palmitate), and the HIF-2α specific inhibitor PT2385 (10 μM) were selected. The experiment was divided into five groups: Control, CuCl2, HF+HG, HF+HG-CuCl2, and HF+HG-CuCl2+PT2385. The first four groups treated cells for 48 h, while the last group was treated with CuCl2 combined with HF+HG for 24 h, followed by treatment with the HIF-2α specific inhibitor PT2385 (10 μM) for another 24 h. Western blotting was used to detect HIF-2α protein levels, and real-time quantitative PCR (RT-qPCR) was used to detect HIF-2α mRNA levels. ChIP experiments were used for verification. A website was used to predict the DMT1 transcription factor. Western blotting was used to detect copper death markers. The results are shown below. Figure 7 As shown, specifically, the transcription factor prediction website indicates that HIF-2α is one of the DMT1 transcription factors. Figure 7 In the control group, HIF-2α expression remained unchanged after copper chloride treatment, but increased after high-sugar and high-fat treatment. Figure 7In the middle section (BD), B and C represent the protein expression and quantitative analysis results of HIF-2α detected by Western blot (n = 3); D represents the expression of HIF-2α mRNA (n = 3). After inhibiting HIF-2α, DMT1 expression in AC16 cells was downregulated, and copper death markers returned to normal. Figure 7 In the study, EK was used for Western blot analysis of protein expression and quantification of HIF-2α, DMT1, and copper death marker molecules (n = 3). ChIP results indicated that HIF-2α is a transcription factor of DMT1. Figure 7 The difference in HIF-2α binding to the DMT1 promoter region in AC16 cells between the control group and the high-glucose, high-fat, and copper chloride combined group was analyzed by ChIP-PCR and ChIP-qPCR (n = 3). Unpaired Student's t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups, followed by Tukey's post-hoc test. The above confirms that HIF-2α is a transcription factor for DMT1. High sugar and high fat intake promotes DMT1 transcription through HIF-2α, inducing copper death in cells.

[0020] Example 4: High sugar and high fat intake mediates HIF-2α m via WTAP 6 Experimental analysis of how A-modification promotes HIF-2α translation: Suitable concentrations of CuCl2 (20 μM) and HF+HG (33.5 mmol / L D-glucose and 100 μmol / L palmitate) were selected. The experiment was divided into six groups: Control, CuCl2, HF+HG, HF+HG-CuCl2, HF+HG-CuCl2+shNC (negative control), and HF+HG-CuCl2+shWTAP (WTAP knockdown). Cells in the first four groups were treated for 48 h, while the latter two groups were treated with CuCl2 combined with HF+HG. When the cells reached 80% confluence, the supernatant was discarded, and a plasmid mixture was added and cultured for another 48 h. [The last sentence appears to be unrelated and refers to a website predicting HIF-2α m.] 6 A-modified sites, online database prediction of m in the left ventricle 6 The correlation between A methyltransferase and HIF-2α was analyzed. Data set analysis revealed WTAP expression in cardiomyopathy patients. Western blotting (WB) was used to detect WTAP protein levels, and real-time quantitative PCR (RT-qPCR) was used to detect WTAP mRNA levels. After transfecting AC16 cells with a WTAP knockdown plasmid, HIF-2α protein levels were detected by WB, and HIF-2α mRNA levels were detected by RT-qPCR. The results are as follows: Figure 8-10 As shown, specifically, m6 Website A's prediction results show that HIF-2α has m 6 A modification site ( Figure 8 , 9 The database prediction results suggest that m 6 The A-methyltransferase WTAP showed the strongest correlation with HIF-2α, and the correlation was positive. Figure 10 In the dataset GSE120895 (AE), the expression of the WTAP gene in patients with cardiomyopathy was significantly increased compared with that in normal individuals. Figure 10 In the middle F), after high sugar and high fat treatment, the expression of HIF-2α and WTAP increased, and after knocking down WTAP, HIF-2α returned to normal levels. Figure 10 In the table, G and I represent the results of Western blot analysis of WTAP protein expression and quantification (n = 3); J represents the results of WTAP mRNA expression (n = 3); H and K represent the results of WTAP knockdown and Western blot analysis of HIF-2α protein expression and quantification (n = 3); L represents the results of HIF-2α mRNA expression (n = 3). Comparisons between two groups were performed using the unpaired Student's t-test, and comparisons among multiple groups were performed using one-way ANOVA, followed by Tukey's post-hoc test. The above confirms that WTAP mediates HIF2α m 6 A-modification, high sugar and high fat intake mediates HIF-2α m via WTAP. 6 A modification promotes HIF-2α translation.

[0021] In summary, the embodiments of this invention demonstrate that when extracellular copper concentration increases, CTR1 decreases, the copper transporter CTR1 decreases, and DMT1 compensates by increasing, transporting copper to AC16; HF+HG can enhance m 6 A-methyltransferase WTAP expression mediates HIF-2α m 6 Modification A further promotes the expression of the copper ion transporter DMT1, ultimately leading to copper ion accumulation within AC16 and resulting in copper death, such as... Figure 11 As shown.

[0022] Based on the above, this invention proposes new ideas for therapeutic targets and drugs targeting DCM. It suggests that WTAP and HIF-2α can be used as therapeutic targets, and the HIF-2α specific inhibitor PT2385 can be used as an active ingredient. One or more carriers can also be added according to actual needs. By combining with different carriers, drugs with different formulations can be prepared.

[0023] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of HIF-2α specific inhibitors in the preparation of drugs for diabetic cardiomyopathy.

2. The application of the HIF-2α specific inhibitor according to claim 1 in the preparation of drugs for diabetic cardiomyopathy, characterized in that, The specific inhibitor of HIF-2α is PT2385.

3. The application of the HIF-2α specific inhibitor according to claim 1 in the preparation of drugs for diabetic cardiomyopathy, characterized in that, The diabetic cardiomyopathy mentioned above is a diabetic cardiomyopathy caused by hyperglycemia, hyperlipidemia, and / or copper ion metabolism disorders.

4. The application of the HIF-2α specific inhibitor according to claim 1 in the preparation of drugs for diabetic cardiomyopathy, characterized in that, The HIF-2α specific inhibitor reduces copper ion concentration and copper death index by inhibiting HIF-2α expression, downregulating DMT1 expression, and reducing copper ion concentration.

5. A targeted drug for diabetic cardiomyopathy, characterized in that, The drugs include HIF-2α specific inhibitors.

6. The targeted drug for diabetic cardiomyopathy according to claim 5, characterized in that, The specific inhibitor of HIF-2α is PT2385.

7. The targeted drug for diabetic cardiomyopathy according to claim 5, characterized in that, The drug also includes one or more pharmaceutically acceptable carriers.

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