Application of COX6A2 activator in preparation of heart failure related drugs

Abstract

The invention relates to the technical field of biological medicines, in particular to application of a COX6A2 activator in preparation of heart failure related medicines. According to the application disclosed by the invention, by activating the COX6A2 and improving the expression level of the COX6A2, pathological cardiac hypertrophy caused by pressure overload (such as hypertension and aortic stenosis) can be prevented or treated, and the pathological process of heart failure (especially ejection fraction retention type heart failure and HFpEF) can be delayed or reversed; and myocardial mitochondrial dysfunction related diseases (such as diabetic cardiomyopathy and ischemic heart disease) can be improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a COX6A2 activator in the preparation of heart failure-related drugs. Background Art

[0002] Heart failure (HF) is a global public health issue that poses a serious threat to human health. Its core pathological characteristic is the progressive decline of the heart's pumping function, leading to inadequate peripheral tissue perfusion and multiple organ dysfunction. The development and progression of this disease involves complex pathological mechanisms, including cardiomyocyte apoptosis, myocardial fibrosis, and an imbalance in the oxidative stress response. These mechanisms intertwine to form a vicious cycle, significantly increasing the difficulty of clinical treatment.

[0003] Recent studies have confirmed that the progression of heart failure is closely linked to reprogramming of myocardial energy metabolism. While the normal myocardium primarily relies on fatty acid oxidation for energy production, heart failure undergoes a significant shift in energy metabolism, manifested by abnormally enhanced glucose metabolism, mitochondrial dysfunction, and decreased ATP production efficiency. This metabolic disturbance not only exacerbates myocardial contractile function but also accelerates ventricular remodeling by activating inflammatory signaling pathways.

[0004] Current standard clinical treatments (such as angiotensin-converting enzyme inhibitors and beta-blockers) primarily work by modulating neuroendocrine hyperactivation. However, these drugs suffer from a single mechanism of action and are prone to developing drug resistance with long-term use. Data show that while existing treatments can slow disease progression, they only improve patients' five-year survival rates to a limited extent (falling short of the clinical need for a significant improvement in prognosis). Therefore, developing novel therapeutic strategies targeting abnormal myocardial energy metabolism and mitochondrial dysfunction has become a pressing need for breakthroughs in the field of heart failure. Summary of the Invention

[0005] Based on this, the present invention provides the use of a COX6A2 activator in the preparation of heart failure-related drugs, which at least solves one problem in the prior art.

[0006] In a first aspect, the present invention provides use of a COX6A2 activator in the preparation of a medicament for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction.

[0007] By activating the COX6A2 gene and regulating its expression, myocardial mitochondrial function can be improved and pathological myocardial hypertrophy or heart failure can be inhibited. Therefore, COX6A2 activators can be used to prepare drugs for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction.

[0008] In a second aspect, the present invention provides a drug for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction, which comprises a COX6A2 activator.

[0009] Due to the adoption of the above technical solution, the embodiments of the present invention have at least the following beneficial effects: providing an application of the natural plant endogenous bioactive ingredient isoliensinine in targeted activation of COX6A2 to improve ventricular remodeling and mitochondrial oxidative stress and thus treat heart failure, broadening the medical use of isoliensinine, providing a new option for improving the survival prognosis of heart failure patients, and having important clinical significance for the treatment of heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Statistical graph of ultrasound detection results of each group of mice in the embodiment of the present invention.

[0011] Figure 2 Statistical graph of the content of atrial natriuretic peptide or brain natriuretic peptide in each group of mice in the examples of the present invention.

[0012] Figure 3 The relative expression levels of hypertrophy-related gene mRNA in the myocardial tissue of each group of mice in the examples of the present invention.

[0013] Figure 4 These are the results of WGA staining and Masson staining of myocardial tissue of each group of mice in the examples of the present invention.

[0014] Figure 5 These are the results of Tunel staining of myocardial tissue of each group of mice in the examples of the present invention.

[0015] Figure 6 Graph showing the Western blot results of cardiomyocytes of mice in each group in the examples of the present invention.

[0016] Figure 7 Graph showing the ROS detection results of H9C2 cardiomyocytes in each group in the examples of the present invention.

[0017] Figure 8 Quantitative statistical graph of ROS detection results of H9C2 cardiomyocytes in each group in the embodiment of the present invention.

[0018] Figure 9 This is the transcriptomics volcano map in the embodiment of the present invention.

[0019] Figure 10 Graph showing the Western blot results of H9C2 cells in each group in the examples of the present invention.

[0020] Figure 11 This is a graph showing the results of a phalloidin staining experiment in an embodiment of the present invention.

[0021] Figure 12 This is a quantitative statistical graph of the phalloidin staining experiment in the examples of the present invention.

[0022] Figure 13 Graph showing the results of detecting reactive oxygen species in myocardial cells in an embodiment of the present invention.

[0023] Figure 14 This is a diagram of WB experimental results in an embodiment of the present invention.

[0024] Figure 15 This is a quantitative statistical graph of the WB experimental results in the embodiment of the present invention. DETAILED DESCRIPTION

[0025] The following is a clear and complete description of the concept of the present invention and the technical effects produced, so as to fully explain the purpose, scheme and effects of the present invention.

[0026] Mitochondria, as the core of cellular energy metabolism, not only provide essential ATP for cardiomyocytes but also regulate various physiological processes, including cellular redox status, calcium homeostasis, and apoptosis. COX6A2 (cytochrome c oxidase subunit 6A2), a key subunit of complex IV of the mitochondrial electron transport chain, regulates oxidative phosphorylation and ATP production, and is closely linked to cardiac energy metabolism.

[0027] The inventors have discovered that by activating COX6A2 and increasing its expression, it is possible to prevent or treat pathological myocardial hypertrophy caused by pressure overload (such as hypertension and aortic stenosis), delay or reverse the pathological progression of heart failure (especially heart failure with preserved ejection fraction, HFpEF), and improve diseases associated with myocardial mitochondrial dysfunction (such as diabetic cardiomyopathy and ischemic heart disease). The inventors have also discovered that isoliensinine (IL) is a COX6A2 activator that effectively activates COX6A2. The structural formula of isoliensinine is as follows: Accordingly, the present invention provides the use of a COX6A2 activator in the preparation of heart failure-related drugs, and provides drugs for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction.

[0028] In a first aspect, the present invention provides use of a COX6A2 activator in the preparation of a medicament for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction.

[0029] By activating the COX6A2 gene and regulating its expression, myocardial mitochondrial function can be improved and pathological myocardial hypertrophy or heart failure can be inhibited. Therefore, COX6A2 activators can be used to prepare drugs for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction.

[0030] In some optional embodiments, the COX6A2 activator includes isoliensinine.

[0031] Isoliensinine plays a core role in inhibiting myocardial hypertrophy, fibrosis and oxidative stress by upregulating COX6A2 gene expression. Therefore, it can be used as an active ingredient in drugs for the treatment of myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction.

[0032] In some optional embodiments, the above-mentioned drug for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction is a drug for improving cardiac remodeling and ejection function.

[0033] In some optional embodiments, the above-mentioned drug for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction is a drug that inhibits myocardial cell apoptosis, a drug that resists myocardial fibrosis or a drug that resists myocardial cell oxidative stress.

[0034] In a second aspect, the present invention provides a drug for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction, which comprises a COX6A2 activator.

[0035] In some optional embodiments, the COX6A2 activator includes isoliensinine.

[0036] In some optional embodiments, the above-mentioned drugs for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction also include SGLT2 inhibitors, beta-blockers or antioxidants.

[0037] When isoliensinine is used in combination with existing cardiovascular drugs (such as SGLT2 inhibitors, beta-blockers or antioxidants), the therapeutic effects of the above drugs for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction can be enhanced.

[0038] In some optional embodiments, the above-mentioned drug for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction further includes a pharmaceutically acceptable excipient.

[0039] In some optional embodiments, the pharmaceutically acceptable excipient is at least one of a solvent and a diluent.

[0040] In some optional embodiments, the pharmaceutically acceptable excipient is water or physiological saline.

[0041] The technical solutions and technical effects of the present invention can be demonstrated through the following animal experiments and experimental results.

[0042] First, C57BL / 6J male mice were randomly divided into the following groups (with equal numbers of mice in each group) and treated accordingly: Control group (Control): sham surgery (only thoracotomy without aortic constriction); Isoliensinine treatment group: sham operation was performed and isoliensinine (20 mg / kg / d) was administered orally. Aortic arch coarctation surgery group (Control+TAC): aortic arch coarctation surgery was performed; The aortic arch coarctation surgery group combined with isoliensinine treatment group (Isoliensinine+TAC): aortic arch coarctation surgery was performed and isoliensinine (20 mg / kg / d) was gavage administered.

[0043] After the mouse model was successfully established, M-mode ultrasound was performed on each group of mice using the Visual Sonics Vevo 2100 small animal ultrasound detection system to record heart rate, left ventricular ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter at end-systolic (LVID;d), left ventricular internal diameter at end-systolic (LVID;s) and other indicators. Figure 1 As shown in the data, compared with the Control+TAC group, the EF and FS indicators of the mice in the Isoliensinine+TAC group were increased, while the LVID;d and LVID;s indicators were decreased, indicating that Isoliensinine can improve the ejection function and myocardial hypertrophy of the mice heart.

[0044] Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of mouse atrial natriuretic peptide (ANP) or brain natriuretic peptide (BNP) in serum samples. Figure 2 As shown in the results, compared with the Control+TAC group, the levels of atrial natriuretic peptide or brain natriuretic peptide in mice in the Isoliensinine+TAC group were decreased. This result indicates that Isoliensinine can improve the abnormal cardiac stress secretion caused by aortic arch stenosis by inhibiting the synthesis and release of ANP and BNP.

[0045] Real-time fluorescence quantitative polymerase chain reaction (qPCR) was used to detect the mRNA expression levels of hypertrophy-related genes (ANP, BNP and β-MHC) in myocardial tissue. Figure 3As shown in the results, compared with the control + TAC group, the mRNA expression levels of ANP, BNP, and β-MHC in the myocardial tissue of mice treated with isoliensinine (isoliensinine + TAC group) were significantly reduced. This result suggests that isoliensinine can alleviate the pathological process of myocardial hypertrophy by inhibiting the transcriptional activation of hypertrophy marker genes induced by TAC surgery.

[0046] The mice were anesthetized and then killed. The weight of the mice was recorded. The heart and lungs were quickly removed and the weight of the heart and lungs was recorded. A part of the myocardial tissue was embedded in OCT and frozen sectioned, and then stained with WGA and Tunel; the other part of the myocardial tissue was fixed with 4% paraformaldehyde and paraffin sectioned, and then stained with Masson. Figure 4 and Figure 5 As shown in the results, WGA staining results showed that the cross-sectional area of ​​myocardial cells in the isoliensinine+TAC group was significantly reduced compared with the control+TAC group. MASSON staining results showed that the percentage of collagen fiber deposition area in the myocardial tissue of the isoliensinine+TAC group was significantly lower than that of the control+TAC group. TUNEL staining results showed that the number of apoptotic cells in the myocardial tissue of the isoliensinine+TAC group was significantly lower than that of the control+TAC group. These results indicate that isoliensinine can inhibit myocardial cell hypertrophy and fibrosis and reduce myocardial cell apoptosis in mice.

[0047] Secondly, H9C2 cardiomyocytes were randomly divided into the following groups and treated accordingly: Control group (CON): untreated control group; Isoliensinine group (ILS): treated with 5 μM isoliensinine for 24 h; Isoprenaline group (ISO): 40 μM isoproterenol treatment for 24 h; Isoliensinine+Isoprenaline group (ILS+ISO): 40 μM isoproterenol and 5 μM isoliensinine were co-treated for 24 h.

[0048] Western blot was used to detect the expression of pan-apoptosis-related molecules such as Bax, Bcl-2, Caspase-3 and their active components in H9C2 cardiomyocytes. Figure 6As shown in the results, compared with the CON group, the expression level of Bax protein in the ISO group was significantly increased, the expression of Bcl-2 protein and the Bcl-2 / Bax ratio were significantly decreased, and the expression of activated Caspase-3 (Cleaved-Caspase-3) was significantly increased; compared with the ISO group, the above changes were significantly reversed in the ISO+ILS group, as shown by decreased Bax expression, increased Bcl-2 expression and the Bcl-2 / Bax ratio, and significantly suppressed Cleaved-Caspase-3 activation, indicating that isoliensinine can inhibit mouse cardiomyocyte apoptosis.

[0049] H9C2 cells were seeded on confocal culture dishes and treated according to grouping. The culture medium was discarded and the cells were washed with PBS. They were incubated in the dark for 20-30 minutes with DHE or MitoSOX probes. After washing away the unbound probes, the fluorescence intensity was observed using a fluorescence microscope. The average fluorescence intensity was calculated using ImageJ to reflect the ROS level in cells and mitochondria. The stronger the fluorescence, the higher the ROS level. Figure 7 and Figure 8 As shown in the figure, DHE detection showed that the overall red fluorescence intensity of H9C2 cells in the ISO group was significantly higher than that in the CON group, while the fluorescence intensity in the ISO+ILS group was significantly lower than that in the ISO group; MitoSOX detection found that the green fluorescence derived from mitochondria in the ISO group was significantly enhanced, while the fluorescence intensity in the ISO+ILS group was significantly weakened compared with the ISO group, indicating that isoliensinine can inhibit oxidative stress in H9C2 cardiomyocytes.

[0050] Combined transcriptomics and Western blot were used to detect the expression levels of COX6A2 and β-actin in cardiomyocytes. Figure 9 and Figure 10 As shown, isoliensinine can activate COX6A2, resulting in an increase in the expression level of COX6A2.

[0051] Cell hypertrophy was detected by phalloidin. After H9C2 cells were treated according to grouping, the culture medium was discarded and washed with PBS. They were fixed with 4% paraformaldehyde for 15 minutes. After washing with PBS, 0.1% Triton X-100 was added for permeabilization for 10 minutes. Then, 5 μg / mL fluorescently labeled phalloidin was incubated in the dark for 30 minutes to bind to F-actin in the cytoskeleton. After washing, the nucleus was stained and the slides were sealed. The cell morphology was observed under a fluorescence microscope, and the cell area was measured using software to evaluate the hypertrophy. Figure 11 and Figure 12 As shown in the results, after knocking down COX6A2 expression, the ILS+ISO group failed to alleviate the signs of cell hypertrophy, indicating that isoliensinine inhibits cardiomyocyte hypertrophy by activating COX6A2.

[0052] After H9C2 cells were grouped and treated, the culture medium was discarded and washed with PBS. 5-10 μM DHE probe was added and incubated in the dark for 20-30 minutes. After incubation, unbound probe was washed away with PBS. Red fluorescence was observed under a fluorescence microscope at an excitation light of 510-560 nm and an emission light of 590 nm. The fluorescence intensity reflects the level of intracellular reactive oxygen species. Figure 13 As shown in Figure 3, after knocking down COX6A2 expression, the ILS+ISO group failed to effectively reduce the generation of cellular reactive oxygen species, indicating that isoliensinine inhibits myocardial oxidative stress by activating COX6A2. Figure 14 and Figure 15 As shown in the results, after knocking down COX6A2 expression, the ILS+ISO group failed to effectively downregulate the expression of apoptosis-related proteins, indicating that isoliensinine inhibits cell apoptosis by activating COX6A2.

[0053] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any method that achieves the technical effects of the present invention by the same or equivalent means shall fall within the scope of protection of the present invention. Within the scope of protection of the present invention, various modifications and variations of the technical solutions and / or implementation methods are possible.

Claims

1. Use of a COX6A2 activator in the preparation of a drug for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction.

2. The use according to claim 1, characterized in that The COX6A2 activator includes isoliensinine.

3. The use according to claim 1, characterized in that The drug for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction is a drug for improving cardiac remodeling and ejection function.

4. The use according to claim 1, characterized in that The drug for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction is a drug for inhibiting myocardial cell apoptosis, a drug for resisting myocardial fibrosis or a drug for resisting myocardial cell oxidative stress.

5. A drug for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction, characterized in that: Including COX6A2 activators.

6. The drug for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction according to claim 5, characterized in that: The COX6A2 activator includes isoliensinine.

7. The drug for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction according to claim 6, characterized in that: The drugs for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction also include SGLT2 inhibitors, beta-receptor blockers or antioxidants.

8. The drug for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction according to claim 5, characterized in that: The drug for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction further comprises a pharmaceutically acceptable excipient.

9. The drug for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction according to claim 8, characterized in that: The pharmaceutically acceptable excipient is at least one of a solvent and a diluent.

10. The drug for preventing or treating myocardial hypertrophy, heart failure or myocardial mitochondrial dysfunction according to claim 8, characterized in that: The pharmaceutically acceptable excipient is water or physiological saline.

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