Application of dihydroberberine in preparation of medicine for treating lipotoxic myocardial injury

Abstract

The invention provides an application of dihydroberberine in preparation of a medicine for treating lipotoxic myocardial injury. The application proves that dihydroberberine can reduce inflammatory cell infiltration and / or fibrosis in heart tissues caused by myocardial cell lipid accumulation and lipotoxicity, and reduce myocardial cell death; and a novel medicine is provided for treating the lipotoxic myocardial injury.

Description

Technical Field

[0001] The present application belongs to the field of cardiac disease treatment. Specifically, the present application provides the use of dihydroberberine in the preparation of a drug for treating lipotoxic myocardial injury. Background Art

[0002] Fatty acids are the main energy source for the heart, but a high-fat diet or metabolic diseases can cause a significant increase in the level of free fatty acids in the body, which in turn leads to an imbalance in energy metabolism in myocardial cells and causes lipotoxic myocardial damage. Lipotoxic myocardial damage is common in metabolic diseases such as obesity and hyperlipidemia, and is accompanied by reduced energy uptake and utilization efficiency of myocardial cells, causing myocardial hypertrophy and fibrosis, damaging heart function, and ultimately causing heart failure. Currently, most of the drugs used in clinical practice are symptomatic treatments, which cannot completely improve the patient's prognosis and reverse myocardial cell damage. Therefore, drugs that alleviate lipotoxic damage to myocardial cells remain the focus and difficulty of clinical and basic research.

[0003] The Chinese herbal medicine Coptis chinensis has been used to treat diarrhea, diabetes, etc. since ancient times. Berberine is the main active ingredient of Coptis chinensis, which can promote insulin secretion, reduce insulin resistance, regulate glucose and lipid metabolism, relieve obesity and diabetic complications, and has a good protective effect on cardiovascular and cerebrovascular damage caused by ischemia. The bioavailability of berberine's metabolite dihydroberberine is significantly higher than that of berberine. However, whether dihydroberberine has a therapeutic effect on myocardial cell damage caused by lipotoxicity needs further verification, and its specific mechanism of action is still unclear. Summary of the invention

[0004] In one aspect, the present application provides the use of dihydroberberine in the preparation of a drug for treating lipotoxic myocardial injury.

[0005] Furthermore, the lipotoxic myocardial injury is lipotoxic myocardial injury caused by hyperlipidemia.

[0006] Other diseases or physiological conditions that may lead to fat accumulation in myocardial cells are also included in the scope of protection of this application, including but not limited to obesity, metabolic diseases, etc.

[0007] Further, the medicament reduces inflammatory cell infiltration and / or fibrosis in cardiac tissue.

[0008] Further, the drug reduces lipid accumulation in cardiomyocytes.

[0009] Further, the drug reduces cardiomyocyte cell death.

[0010] Furthermore, the drug increases the expression of Glut4, PDK4, PFKM, HK2, CS, ACOX and / or DGAT.

[0011] Furthermore, the drug reduces the expression of CD36.

[0012] Furthermore, the drug is an oral or injectable drug.

[0013] Furthermore, the drug is a tablet or a water injection.

[0014] Furthermore, the drug also contains a pharmaceutically acceptable carrier or excipient.

[0015] The drug of the present invention can be in any clinically acceptable dosage form, including various dosage forms for oral and parenteral administration. When used orally, it can be tablets, capsules, soft capsules, oral liquids, syrups, granules, dripping pills, orally disintegrating tablets, sustained-release tablets, sustained-release capsules, controlled-release tablets, controlled-release capsules; when used for parenteral administration, it can be water injections, freeze-dried powder injections, sterile powder injections, infusions. The drug composition of the present invention preferably uses tablet and water injection dosage forms.

[0016] For the above-mentioned drug composition, the pharmaceutically acceptable carrier or excipient can be selected from medicinal excipients suitable for oral preparations, including fillers, binders, lubricants, disintegrants, solubilizers, surfactants, adsorption carriers, etc.; or medicinal excipients suitable for injections, including solvents, antioxidants, solubilizers, adsorbents, osmotic pressure regulators, pH regulators.

[0017] The minimum unit of the drug refers to one tablet, one capsule, one bag of granules or one injection, etc.

[0018] The dosage forms of the present invention can be produced by any commonly used methods well-known to those skilled in the art of pharmaceutical technology and there is no particular limitation thereto. For example, the tablets of the present invention can be granulated, dried and sieved using suitable methods well-known in the art for the main drug, excipient, binder, etc., adding lubricants, etc. to the obtained mixture and then mixing and forming tablets. Granulation can be carried out by any suitable method well-known in the art, such as wet granulation, dry granulation or heat granulation. Suitable non-limiting examples include using a high-speed stirring granulator, a fluidized granulation dryer, an extrusion granulator or a roller compactor for these granulation methods. In addition, methods such as drying and sieving can be carried out according to the needs of granulation. The mixture of the main drug, excipient, binder, lubricant, etc. can also be directly formed into tablets. If film coating is required, any film coating device known in the art can be used, and as the film coating matrix, suitable examples include sugar coating base, hydrophilic film coating base, enteric film coating base and sustained-release film coating base. Description of the Drawings

[0019] Figure 1 Show the effect of berberrubine on the pathological morphology of the heart in high-fat-induced mice.

[0020] Figure 2 To show the protective effect of berberrubine on palmitic acid-induced H9c2 cardiomyocyte injury.

[0021] Figure 3 To show the effect of berberrubine on genes related to glycolipid metabolism in H9c2 cardiomyocytes.

[0022] Figure 4 To show the effect of berberrubine on the cardiac pathological morphology of db / db diabetic mice.

[0023] Figure 5 To show the effect of berberrubine on cardiac function of diabetic mice detected by echocardiography.

[0024] Figure 6 To show the effect of berberrubine on the ultrastructure of cardiomyocytes in diabetic mice.

[0025] Figure 7 To show the effect of berberrubine on the expression of genes related to glycolipid metabolism, mitochondrial function and apoptosis in myocardial tissue (partially).

[0026] Figure 8 To show the effect of berberrubine on the expression of genes related to glycolipid metabolism, mitochondrial function and apoptosis in myocardial tissue (partially). Specific implementation methods

[0027] Example 1 Effect of berberrubine on the cardiac pathological morphology of high-fat-induced mice

[0028] C57BL / 6 mice were divided into a control group, a model group and a berberrubine administration group. The model group was induced with a high-fat Western diet for 16 weeks. The berberrubine administration group was induced with a high-fat diet for 4 weeks and then simultaneously induced and gavaged once a day with 12.5 mg / kg and 25 mg / kg respectively. After 12 weeks, the mice were sacrificed by cervical dislocation, the hearts were taken, fixed with formalin, and sections were stained with HE, Masson and oil red.

[0029] As Figure 1 shown, compared with the model group, each dose of berberrubine could significantly reduce inflammatory cell infiltration, alleviate fibrosis and reduce lipid accumulation in cardiomyocytes.

[0030] Example 2 Protective effect of berberrubine on palmitic acid-induced H9c2 cardiomyocyte injury

[0031] H9c2 cardiomyocytes were incubated with 25, 50, 100, 200 μmol / L palmitic acid and 0.625, 1.25, 2.5, 5 μmol / L berberrubine for 48 h respectively to screen out the appropriate drug administration doses and modeling doses. The cells were divided into a control group, a palmitic acid-induced injury group, and a berberrubine treatment group. The cell viability was detected by the CCK-8 method. The normal control group (control) was given 100 μl of serum-free DMEM medium per well; the palmitic acid injury group (model) was given 100 μl of palmitic acid with a final concentration of 200 μM per well and acted for 48 h; after the berberrubine treatment group (0.625, 1.25, 2.5 μmol / L) was incubated for 48 h, the cell viability of each group was detected by the CCK-8 method.

[0032] As Figure 2 shown, compared with the control group, the cell viability in the palmitic acid 50, 100, 200 μmol / L treatment groups was significantly decreased, and the cell viability in the berberrubine 5 μmol / L treatment group was significantly decreased. Compared with the model group, the cell viability in the berberrubine 1.25, 2.5 μmol / L treatment groups was significantly increased, showing a dose-dependent relationship.

[0033] Example 3 Effect of Berberrubine on Genes Related to Glucose and Lipid Metabolism in H9c2 Cardiomyocytes

[0034] The experiment was randomly divided into 4 groups: a normal control group, a berberrubine single-drug addition group (2.5 μmol / L), a palmitic acid injury group (200 μmol / L), and a berberrubine treatment group. After incubation for 48 h, the cells were lysed with Trizol to extract RNA, the concentration was measured, and it was reverse-transcribed into cDNA according to the kit steps, and real-time fluorescence quantitative PCR experiments were performed to detect the expression of various genes related to glucose and lipid metabolism.

[0035] As Figure 3 shown, compared with the control group, the palmitic acid-induced injury group could significantly reduce the expression of genes related to cell glucose metabolism such as Glut4, PDK4, PFKM, and HK2, increase the expression of the lipid transport-related gene CD36, and reduce the expression of lipid metabolism-related genes CS, ACOX, and DGAT; compared with the model group, berberrubine incubation for 48 h could significantly improve the levels of the above-mentioned genes related to glucose and lipid metabolism, and the differences were significant.

[0036] Example 4 Effect of Berberrubine on the Heart Pathological Morphology of db / db Diabetic Mice.

[0037] Mice were divided into a control group, a model group, and a berberrubine dihydrochloride administration group. The model group used 6 - 8 - week - old db / db mice, and the berberrubine dihydrochloride administration group was given 50 mg / kg and 25 mg / kg respectively. After 12 weeks, the mice were sacrificed by cervical dislocation, the hearts were taken, fixed with formalin, and sections were stained with HE, Masson, and oil red.

[0038] As Figure 4 shown, compared with the control group, inflammatory cell infiltration appeared in the myocardial tissue of the model - group mice, and there was an increase in myocardial cell fibrosis and lipid accumulation. Each dose group of berberrubine dihydrochloride (50, 25 mg / kg) could improve the myocardial tissue morphology of the model - group mice to varying degrees.

[0039] Example 5: Detection of the effect of berberrubine dihydrochloride on the cardiac function of diabetic mice by echocardiography

[0040] After 12 weeks of administration, the mice were anesthetized with isoflurane, and cardiac function was detected by echocardiography. As Figure 5 shown, the ejection fraction, fractional shortening, left ventricular wall thickness, and left ventricular anterior wall thickness of the model - group mice were significantly reduced. Each dose group of berberrubine dihydrochloride could significantly increase the cardiac function of diabetic mice.

[0041] Example 6: Effect of berberrubine dihydrochloride on the ultrastructure of myocardial cells in diabetic mice

[0042] After 12 weeks of administration, the mice were anesthetized and sacrificed by cervical dislocation, and the hearts were cut into 1 - mm 3 pieces, fixed with glutaraldehyde, sectioned, stained, and observed by transmission electron microscopy scanning and photographing. As Figure 6 shown, compared with the normal control group, a large number of lipid droplets were visible in the myocardial cells of the model - group mice, and the mitochondrial morphology was irregular. Treatment with each dose of berberrubine dihydrochloride could significantly reduce the lipid droplets in myocardial cells and improve the mitochondrial morphology.

[0043] Example 7: Effect of berberrubine dihydrochloride on the expression of genes related to glycolipid metabolism, mitochondrial function, and apoptosis in myocardial tissue.

[0044] After the administration was completed, the hearts of the mice in the normal control group, model group, and berberrubine dihydrochloride administration group were taken. The cells were lysed with Trizol to extract RNA, the concentration was measured, and it was reversed to cDNA according to the steps of the kit, and real - time fluorescence quantitative PCR experiments were carried out to detect the expression of genes related to glycolipid metabolism, mitochondrial dynamics, and apoptosis.

[0045] As Figure 7 and Figure 8As shown, compared with the control group, the model group significantly down-regulated the expression of glycolipid metabolism-related genes GK, Glut4, PDK4, PFKM, HK2, CS, ACOX, and DGAT, while up-regulating the expression of the lipid transport-related gene CD36, promoting lipotoxic injury of cardiomyocytes; up-regulating the expression of the mitochondrial fission-promoting gene Drp1 and down-regulating the expression of the mitochondrial fusion-related genes OPA1 and Mfn2, resulting in abnormal mitochondrial morphology and function; up-regulating the expression of the apoptosis-promoting genes caspase3, caspase9, and Bax, while inhibiting the expression of the anti-apoptosis gene Bcl-2, promoting apoptosis; after administration of berberrubine, the expression of the above genes can be significantly improved, promoting glycolipid metabolism in cardiomyocytes, reducing lipotoxic injury, reducing mitochondrial dysfunction, and alleviating apoptosis.

[0046] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And these obvious changes or variations derived from the spirit of the present invention are still within the protection scope of the present invention.

Claims

1. Use of berberrubine in the preparation of a medicament for treating lipotoxic myocardial injury.

2. The use according to claim 1, wherein the lipotoxic myocardial injury is lipotoxic myocardial injury caused by hyperlipidemia.

3. The use according to claim 1 or 2, wherein the medicament reduces inflammatory cell infiltration and / or fibrosis in cardiac tissue.

4. The use according to claim 1 or 2, wherein the medicament reduces lipid accumulation in cardiomyocytes.

5. The use according to claim 1 or 2, wherein the medicament reduces cardiomyocyte death.

6. The use according to claim 1 or 2, wherein the medicament increases the expression of Glut4, PDK4, PFKM, HK2, CS, ACOX and / or DGAT.

7. The use according to claim 1 or 2, wherein the medicament reduces the expression of CD36.

8. The use according to claim 1, wherein the medicament is an oral or injectable medicament.

9. The use according to claim 8, wherein the medicament is a tablet or an aqueous injection.

10. The use according to claim 8, wherein the medicament further comprises a pharmaceutically acceptable carrier or excipient.

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