Application of dehydroandrographolide in preparation of medicine for treating diabetic cardiomyopathy

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of dehydroandrographolide in preparation of a medicine for treating diabetic cardiomyopathy. It is found that DA can significantly reduce the weight and blood sugar of diabetic mice and improve oral glucose tolerance tests and insulin tolerance tests. Meanwhile, cardiac hypertrophy is relieved, and cardiac output is increased. Liver metabolism and transcriptomics prompt that DA affects obesity and diabetes related metabolites, especially tyrosine metabolism. Cell tests show that DA promotes sugar absorption, and an insulin signal channel and a metabolic process are enhanced by inhibiting PTPRF protein. Actinomycete ketone and immunoprecipitation prompt that PTPRF is oxidized and degraded, and magnesium ions need to participate in the process. Cardiac transcriptome analysis shows that DA can inhibit a diabetes pathway and activate tricarboxylic acid circulation, an insulin pathway and carbon and fat metabolism.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of dehydroandrographolide in preparing a medicine for treating diabetic cardiomyopathy. Background Art

[0002] Metabolic diseases include not only obesity, diabetes, non-alcoholic fatty liver disease but also other health-threatening diseases. These diseases are closely related to inflammatory factors. 1 The pro-inflammatory cytokine TNF-α not only reduces insulin sensitivity through signaling pathways such as JNK, leading to glucose intolerance, but also upregulates intracellular signaling molecules (such as IKKβ), leading to impaired insulin function. A similar inflammatory factor, interleukin 1-β (IL-1β), can also weaken the effects of insulin, but the more prominent role of IL-1β is to inhibit pancreatic β-cell function. 2 In addition, macrophages accumulate in adipose tissue, and polarized macrophages lead to the release of many inflammatory cytokines and other factors that reduce insulin sensitivity. 3 , thereby promoting the progression of diabetes.

[0003] Diabetic cardiomyopathy refers to a myocardial disease that occurs in diabetic patients and cannot be explained by hypertension, coronary atherosclerosis, valvular heart disease or other heart diseases. It is an important cause of heart failure and death in some diabetic patients. 4 Among them, diabetic cardiomyopathy is mainly manifested by myocardial hypertrophy and ventricular reduction, with limited early diastolic function. It is different from ischemic cardiomyopathy represented by myocardial infarction, which is mainly manifested by myocardial thinning and ventricular enlargement. Heart failure is a clinical syndrome with a very high mortality rate, representing the terminal stage of most cardiovascular diseases. 5 There is a close relationship between diabetes and heart failure, which can increase the risk of heart failure by 2-5 times. 6 .

[0004] Diabetes and heart failure are linked through metabolic disorders and inflammation caused by insulin resistance. In diabetic hearts, the NF-κB signaling pathway and the renin-angiotensin-aldosterone system are activated, thereby promoting myocardial inflammation. Advanced glycation products and injury-related factors are also triggers of inflammation. The mediators produced by the inflammatory process regulate specific intracellular signaling mechanisms in myocardial cells, thereby promoting the development of diabetic cardiomyopathy. 7 .

[0005] Although a large number of glucose-lowering drugs have been developed, most of them are ineffective in controlling blood sugar. In addition, blood sugar control alone is not enough to prevent long-term complications of diabetes. 8. In fact, only three classes of glucose-lowering drugs have shown a preventive effect on complications: metformin, glucagon-like peptide 1 (GLP-1) receptor agonists, and sodium-glucose cotransporter 2 (SGLT-2 inhibitors. While GLP-1 receptor agonists can alleviate cardiovascular disease and SGLT-2 inhibitors can prevent heart failure and kidney disease, none of these drugs have shown effectiveness against other complications such as retinopathy, non-alcoholic fatty liver disease, and diabetes-related inflammatory diseases. Most importantly, with the exception of metformin, existing drugs do not slow the progression of diabetes 1 .

[0006] Therefore, it is necessary to develop new drugs for diabetic cardiomyopathy (with myocardial hypertrophy as the main manifestation).

[0007] 1.Kenny,HC&Abel,EDHeart Failure in Type 2Diabetes Mellitus.CircRes 124,121-141(2019).https: / / doi.org:10.1161 / CIRCRESAHA.118.311371.

[0008] 2. Neeland, I Jet al. Metabolic syndrome. Nat Rev Dis Primers 10, 77 (2024). https: / / doi.org:10.1038 / s41572-024-00563-5.

[0009] 3. Rohm, TV, Meier, DT, Olefsky, JM & Donath, MYInflammation inobesity, diabetes, and related disorders. Immunity 55, 31-55 (2022). https: / / doi.org:10.1016 / j.immuni.2021.12.013.

[0010] 4. Ying, W., Fu, W., Lee, YS & Olefsky, JM The role of macrophages inobesity-associated islet inflammation and beta-cell abnormalities. Nat RevEndocrinol 16, 81-90 (2020). https: / / doi.org:10.1038 / s41574-019-0286-3.

[0011] 5.Dillmann,WHDiabetic Cardiomyopathy.Circ Res 124,1160-1162(2019).https: / / doi.org:10.1161 / CIRCRESAHA.118.314665

[0012] 6. Zhang, Y., Bauersachs, J. & Langer, HFImmune mechanisms in heart failure. Eur J Heart Fail 19, 1379-1389 (2017). https: / / doi.org:10.1002 / ejhf.942.

[0013] 7. Frati, G. et al. An overview of the inflammatory signaling mechanisms in the myocardium underlying the development of diabeticcardiomyopathy. Cardiovasc Res 113, 378-388 (2017). https: / / doi.org:10.1093 / cvr / cvx011.

[0014] 8. Piuri, G. et al. Magnesiumin Obesity, Metabolic Syndrome, and Type2Diabetes. Nutrients13 (2021). https: / / doi.org:10.3390 / nu13020320.

[0015] 9. Tsutsumi, R. et al. Assay to visualize specific protein oxidation reveals spatio-temporal regulation of SHP2. Nat Commun 8, 466 (2017). https: / / doi.org:10.1038 / s41467-017-00503-w. Summary of the Invention

[0016] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0017] The present invention provides application of dehydroandrographolide (DA) in preparing medicine for treating diabetic cardiomyopathy.

[0018] In some embodiments of the present invention, the structure of dehydroandrographolide is shown in Formula I:

[0019]

[0020] In some embodiments of the present invention, the diabetic cardiomyopathy refers to a myocardial disease occurring in diabetic patients that cannot be explained by hypertensive heart disease, coronary atherosclerotic heart disease and other heart diseases, and has clinical manifestations of myocardial hypertrophy and ventricular reduction.

[0021] In some embodiments of the present invention, the dehydroandrographolide includes at least one of a pharmaceutically acceptable salt and a pharmaceutically acceptable modification.

[0022] In some embodiments of the present invention, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0023] In some embodiments of the present invention, the metal salt includes an alkali metal salt or an alkaline earth metal salt.

[0024] In some embodiments of the present invention, the alkali metal salt includes at least one of a sodium salt and a potassium salt.

[0025] In some embodiments of the present invention, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt, and aluminum salt.

[0026] In some embodiments of the present invention, the salt formed with the organic base includes a salt formed with at least one of the following organic bases: trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0027] In some embodiments of the present invention, the salt formed with an inorganic acid includes a salt formed with at least one of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0028] In some embodiments of the present invention, the salt formed with the organic acid includes a salt formed with at least one of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0029] In some embodiments of the present invention, the salt formed with a basic amino acid includes a salt formed with at least one of the following basic amino acids: arginine, lysine, and ornithine.

[0030] In some embodiments of the present invention, the salt formed with the acidic amino acid includes a salt formed with at least one of the following acidic amino acids: aspartic acid and glutamic acid.

[0031] In some embodiments of the present invention, the pharmaceutically acceptable modification comprises at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation.

[0032] In some embodiments of the present invention, the product comprises a pharmaceutical.

[0033] In some embodiments of the present invention, the drug includes pharmaceutically acceptable excipients, and / or any one or more other active ingredients.

[0034] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

[0035] Furthermore, in order to facilitate medication, the active ingredient dehydroandrographolide or its derivatives can be processed into a specific dosage form with any one or more pharmaceutically acceptable excipients. These excipients can be diluents (such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol and microcrystalline cellulose, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, light magnesium oxide and calcium carbonate, etc.), wetting agents (such as water and ethanol, etc.), binders (such as hydroxypropyl methylcellulose, povidone, starch slurry and syrup, etc.), disintegrants (such as dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants and cross-linked polyvinylpyrrolidone, etc.), lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol and micropowder), etc. silica gel, etc.), colorants (such as titanium dioxide, sunset yellow, methylene blue and pharmaceutical iron oxide, etc.), coating materials (such as acrylic resin, hydroxypropyl methylcellulose and povidone, etc.), solvents (such as water for injection, ethanol, propylene glycol and glycerol, etc.), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid and sodium tartrate, etc.), antioxidants (such as sodium sulfite, sodium metabisulfite and sodium thiosulfate, etc.), antibacterial agents (such as phenol, benzyl alcohol and thimerosal, etc.), and may also be isotonic regulators (such as sodium chloride and glucose, etc.).

[0036] In some embodiments of the present invention, the dosage form of the product includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

[0037] In some embodiments of the present invention, the gastrointestinal dosage form includes at least one of powders, tablets, granules, capsules, sustained-release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[0038] Furthermore, the dosage forms for administration through the gastrointestinal tract include but are not limited to enteric-coated tablets, coated tablets, film-coated tablets, sugar-coated tablets, dispersible tablets, suckable tablets, chewable tablets, effervescent tablets, scratched tablets, sustained-release and controlled-release dosage forms such as sustained-release tablets, sustained-release coated tablets, controlled-release tablets, orally disintegrating tablets, lozenges, oral patches, etc.

[0039] In some embodiments of the present invention, the non-gastrointestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

[0040] Furthermore, the injectable dosage forms include but are not limited to injection solutions, injection solutions, intravenous injection solutions, injection suspensions, sterile powders for injection, intravenous injections, water injections, injection emulsions, powder injections, injections, sterile powder injections, freeze-dried powder injections, etc.

[0041] In some embodiments of the present invention, the product is administered to a mammal.

[0042] In some embodiments of the invention, the mammal comprises a human.

[0043] The beneficial effects of the present invention are:

[0044] The present invention discovered that dehydroandrographolide has the effect of treating diabetic cardiomyopathy. DA can significantly reduce the body weight and blood sugar of diabetic mice, and improve oral glucose tolerance test and insulin tolerance test. At the same time, it reduces myocardial hypertrophy and increases cardiac output. Liver metabolism and transcriptomics suggest that DA affects obesity and diabetes-related metabolites, especially the metabolism of tyrosine. Cell experiments show that DA promotes sugar absorption and enhances the insulin signaling pathway and metabolic process by inhibiting PTPRF protein. Actinomycin and immunoprecipitation suggest that PTPRF is oxidatively degraded, and this process requires the participation of magnesium ions. Cardiac transcriptome analysis suggests that DA can inhibit the diabetes pathway and activate the tricarboxylic acid cycle, insulin pathway and carbon and fat metabolism. DA can significantly reduce the blood sugar of KK mice and increase cardiac output, providing a potential drug for the treatment of diabetic cardiomyopathy and partially explaining the importance of magnesium ions to cardiovascular disease and diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0046] Figure 1 The figure shows the therapeutic effect of DA on diabetic mice, wherein: A is the experimental flow chart of the present invention; B is the weight change results of mice in different treatment groups; C is the blood glucose change results of mice in different treatment groups; D is the insulin tolerance test results of different treatment groups; E is the glucose tolerance test results of different treatment groups.

[0047] Figure 2 The results of DA improving diabetic myocardial function, where: A is the cardiac ultrasound measurement results of mice in different treatment groups; B is the cardiac ultrasound measurement results of mice in different treatment groups.

[0048] Figure 3 Non-targeted metabolic sequencing results for mouse liver samples from different treatment groups. A is the analysis result in the Human Metabolome Database (HMDB); B is the analysis result in the KEGG database.

[0049] Figure 4 mRNA sequencing was performed on mouse heart samples from different treatment groups. A is a volcano plot of the test results; B is the analysis results in the KEGG database.

[0050] Figure 5The results of DA promoting glucose absorption by HepG2 cells: A is the CCK-8 reagent for detecting cell viability; B is the LDH reagent for detecting cell death rate; C is the glucose (GO) detection kit for detecting the glucose content in cell culture medium; D is the 2-NBDG kit for detecting the ability of HepG2 cells to absorb glucose.

[0051] Figure 6 After HepG2 cells were treated with 10 μM DA, miRNA sequencing analysis was performed. A is the KEGG database analysis result; B is the GO database analysis result.

[0052] Figure 7 Figure 3 shows the degradation effect of DA on PTPRF protein: A: HepG2 cells were treated with DA at different time points, B: different concentrations of DA, and then Western Blot analysis was performed; C: HepG2 cells were treated with cycloheximide and then DA, and then protein expression was detected; D: HepG2 cells treated with 5,5-dimethyl-1,3-cyclohexanedione and then immunoprecipitated to detect protein expression.

[0053] Figure 8 The results of the effect of DA on oxidative free radicals in HepG2 cells: A shows the detection of oxygen free radicals using DHE after HepG2 cells were treated with different concentrations of DA; B shows the detection of oxygen free radicals using DHE after HepG2 cells were treated with 10 μM DA at different times; C shows the detection of intracellular calcium ions using Fluo-3; D shows the detection of oxygen free radicals and calcium ions using different reagents; E shows the detection of peroxynitrite using HPF reagent; F shows the effect of 10 μM DA on oxygen free radicals in HepG2 cells detected by DHE after pretreatment with different inhibitors.

[0054] Figure 9 The results of DA inhibiting the expression of PTPRF mediated by magnesium ions: A is the detection of protein expression of HepG2 cells by 10 μM DA after pretreatment with different inhibitors; B is the detection using flow cytometry; C is the detection of intracellular magnesium ion status using Mag-Fura-2; D is the detection of protein expression of HepG2 cells by 10 μM DA after pretreatment with different inhibitors. DETAILED DESCRIPTION

[0055] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0056] Example 1 The therapeutic effect of DA on diabetic mice

[0057] Male KK.Cg-Ay / J mice aged 6-8 weeks were selected and divided into three groups: control group: normal saline; low-dose group: DA 30mg / kg; high-dose group: DA 60mg / kg. Intraperitoneal injection was performed daily for 4 weeks. Three days after DA withdrawal, glucose tolerance test and insulin tolerance test were performed. Afterwards, cardiac ultrasound examination was performed. After the above tests were completed, the mice were killed and samples were taken for mRNA analysis of the liver and heart. The experimental process is as follows Figure 1 As shown in A.

[0058] After 4 weeks of drug treatment, it was found that the low-dose group had improved the body weight of mice ( Figure 1 Middle B), the trend of lowering blood sugar ( Figure 1 C), but not statistically significant. High doses of DA can significantly improve the body weight of mice, lower blood sugar, and improve insulin tolerance and glucose tolerance results ( Figure 1 These results suggest that high-dose DA has potential effects in the treatment of diabetes.

[0059] The results of DA on improving diabetic myocardial function are as follows Figure 2 As shown in Figure A, the high-dose DA group significantly improved the cardiac function of diabetic mice. Although there was no statistically significant change in ejection fraction ( Figure 2 Middle B), but cardiac output and left ventricular end-diastolic diameter improved significantly after DA treatment, and DA can reduce the thickness of the ventricular septum and left ventricular free wall, and has a significant improvement effect on early diabetic cardiomyopathy.

[0060] After completing cardiac ultrasound, perfusion sampling was performed. Liver tissue was extracted for non-targeted metabolomics analysis. The results were as follows: Figure 3 As shown in Figure A. A total of 2193 metabolites were analyzed. The results showed that compared with the control group, a total of 308 metabolites in the high-dose group were statistically changed, of which 141 were upregulated and 167 were downregulated. The differential metabolites were analyzed in the Human Metabolome Database (HMDB), and the diseases were concentrated in obesity and diabetes ( Figure 3 KEGG database analysis suggests that DA mainly affects metabolic processes, especially glycerol phospholipid metabolism. Metabolic pathway enrichment analysis showed that tyrosine metabolism was the most significantly altered ( Figure 3 Middle B).

[0061] After the mice were killed, heart tissue was extracted for mRNA analysis ( Figure 4 Middle A), the high-dose DA group inhibited diabetes and infection, activated the insulin pathway, tricarboxylic acid cycle and metabolism ( Figure 4Middle B). Gene set enrichment analysis (GSEA) also showed that DA promoted the tricarboxylic acid cycle, fat metabolism, the insulin pathway, and carbon metabolism, and suppressed diabetes. These results indicate that DA indeed improves obesity and diabetes at the metabolite level.

[0062] Example 2 DA promotes glucose absorption in HepG2 cells

[0063] 1. Experimental methods

[0064] HepG2 cells were pretreated with DA at different concentrations (0.1, 1, and 10 μM) for 24 hours. The cell number and cell death rate were detected using a CCK-8 kit and an LDH kit. The glucose content in the cell culture medium was detected using a glucose (GO) detection kit. The glucose uptake ability of HepG2 cells was detected using a 2-NBDG kit.

[0065] 2. Experimental results

[0066] Cell viability and lactate dehydrogenase release assays indicated that DA 10 μM had no significant cytotoxicity and did not inhibit growth ( Figure 5 The extracellular glucose residual test indirectly suggested that DA could promote the glucose uptake of HepG2 cells ( Figure 5 (C). 2-NBDG is a glucose analog with immunofluorescence. Flow cytometry was used to detect the glucose uptake capacity of cells. DA significantly increased the glucose uptake capacity of cells. Phloretin was used as a negative control to demonstrate that glucose uptake of cells could be inhibited ( Figure 5 Middle D).

[0067] Example 3DA promotes the degradation of PTPRF protein

[0068] 1. Experimental methods

[0069] Some HepG2 cells were pretreated with 10 μM DA for 24 hours and then treated with 100 nM insulin for 10 minutes. Other HepG2 cells were pretreated with 5 μg / ml cycloheximide for 1 hour to inhibit protein synthesis and then treated with 10 μM DA for 2 or 4 hours. Afterwards, miRNA sequencing and Western blot analysis were performed. PTPRF, a member of the protein tyrosine phosphatase family (PTPs), has a conserved active site: -[I / V]HCSXGXGR[S / T]G-. It is characterized by an unusually acidic catalytic cysteinyl (Cys) residue. Cys residues in PTPs can be oxidized to a sulfonic acid state (SOH). Depending on the enzyme, this Cys-SOH rapidly reacts with adjacent backbone amide nitrogens to form intramolecular sulfonamide bonds (SN) or with adjacent cysteinyl residues to form intramolecular or intermolecular disulfide bonds (SS). Dimethyl ketone (5,5-dimethyl-1,3-cyclohexanedione) is a cell-permeable, mild nucleophile that labels cysteine sulfonic acids but not cysteine in its thiol, disulfide, sulfinic, or sulfonic acid states. 9 HepG2 cells were pretreated with 10 μM DA for 3 hours and then treated with 100 nM insulin for 10 minutes. They were then treated with 5 mM dimethicone in PBS for 5 minutes. After immunoprecipitation with a PTPRF antibody, Western blotting was performed using an anti-dimethicone-cysteine antibody (Anti-Cys-Sul) to examine PTPRF protein oxidation.

[0070] 2. Experimental results

[0071] DA 10 μM was selected as the treatment concentration for HepG2. miRNA analysis was performed, and the results showed that in the KEGG pathway enrichment, in addition to lysosome and endocytosis, it mainly affected insulin resistance. GO enrichment analysis also suggested that DA mainly affected metabolic processes ( Figure 6 In HepG2 cells, DA was found to reduce the expression of PTPRF, increase the expression of insulin receptor phosphorylation, and simultaneously increase the phosphorylation of downstream AKT. Moreover, this phenomenon was concentration-dependent and time-dependent ( Figure 7 In this example, cycloheximide (CHX) was used to inhibit protein synthesis. It was shown that after 4 hours of DA treatment, DA promoted the degradation of PTPRF protein compared with the CHX alone group. This process was confirmed by immunoprecipitation that PTPRF protein was oxidized by DA ( Figure 7 Medium CD).

[0072] Example 4 DA-induced oxidative free radicals in HepG2 cells showed a concentration-dependent

[0073] 1. Experimental methods

[0074] HepG2 cells were pretreated with 0.1μM, 1μM, and 10μM DA for 12 hours, 10μM DA for 30 minutes, 1 hour, 3 hours, 6 hours, and 9 hours for some cells, and 10μM DA for 1 hour before treatment with 2mM EDTA, 10μM BAPTA-AM, 5mM NAC, and 10μM VC for 1 hour. DHE, DCFDA, Fluo-3, Rhod-2, and HPF were then used for corresponding detections.

[0075] 2. Experimental results

[0076] The oxidative free radicals in HepG2 cells induced by DA showed a concentration-dependent ( Figure 8 A), and reached a peak value after 3 hours of treatment ( Figure 8 Middle B). Fluo-3 detects endogenous calcium ions and also shows concentration dependence ( Figure 8 However, DA does not cause an increase in intracellular hydroxide, and endogenous calcium ions do not cause mitochondrial calcium overload. This proves its safety ( Figure 8 HPF can produce strong green fluorescence by reacting with the hydroxyl radical of peroxynitrite in cells. The figure above also proves that DA does not cause changes in peroxynitrite ( Figure 8 In addition, the oxygen free radical scavengers NAC and VC and the exogenous calcium ion chelator EDTA can reduce the oxygen free radicals induced by DA. However, the intracellular calcium ion chelator BAPTA is ineffective ( Figure 8 Middle F).

[0077] Example 5 DA inhibits the expression of PTPRF through magnesium ion mediation, thereby enhancing insulin sensitivity

[0078] 1. Experimental methods

[0079] HepG2 cells were pretreated for 1 hour with 2mM EDTA, 0.2mM EGTA, 0.4mM EGTA, 10μM BAPTA-AM, 5mM NAC, and 10μM VC, respectively. The cells were then treated with 10μM DA for 3 hours and finally stimulated with 100nM insulin for 10 minutes. Protein expression was analyzed by Western blot and flow cytometry. Magnesium ion was detected using Mag-Fura-2.

[0080] 2. Experimental results

[0081] The results showed that EDTA could restore DA to reduce PTPRF, while EGTA had no such effect ( Figure 9A), we used flow cytometry to examine the expression of phosphorylated proteins and found similar effects. EDTA could inhibit the expression of p-ERK and p-AKT elevated by DA, while BAPTA had the opposite effect ( Figure 9 The most obvious difference between EDTA and EGTA is that EDTA can chelate magnesium ions, while EGTA has no significant effect. Furthermore, Fluo-3 staining is affected by magnesium ions, and the calcium influx caused by DA is not necessarily solely due to calcium ions. Using Mag-Fura-2 staining, it was found that DA can cause an increase in intracellular magnesium ions, which is enhanced by BAPTA, while EDTA reduces magnesium influx ( Figure 9 Interestingly, the ERK inhibitor U0126 also has an effect similar to EDTA ( Figure 9 In the literature (Ikari, A. et al. TRPM6 expression and cell proliferation are up-regulated by phosphorylation of ERK1 / 2 in renal epithelial cells. BiochemBiophys Res Commun 369, 1129-1133 (2008). https: / / doi.org:10.1016 / j.bbrc.2008.03.002), U0126 has been reported to affect the calcium-magnesium divalent cation channel TRPM6. Therefore, it is speculated that DA inhibits PTPRF expression through magnesium ion mediation, thereby enhancing insulin sensitivity.

[0082] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. Application of dehydroandrographolide in the preparation of a drug for treating diabetic cardiomyopathy; The structure of the dehydroandrographolide is shown in Formula I:

2. The diabetic cardiomyopathy according to claim 1, wherein: The diabetic cardiomyopathy has clinical manifestations of myocardial hypertrophy and ventricular reduction.

3. The use according to claim 1, characterized in that: The dehydroandrographolide includes at least one of a pharmaceutically acceptable salt and a pharmaceutically acceptable modification.

4. The use according to claim 3, characterized in that: The pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

5. The use according to claim 3, characterized in that: The pharmaceutically acceptable modification includes at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation.

6. The use according to claim 1, characterized in that: The products include pharmaceuticals.

7. The use according to claim 6, characterized in that: The drug includes pharmaceutically acceptable excipients.

8. The use according to claim 7, characterized in that: The pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

9. The use according to claim 6, characterized in that: The dosage form of the product includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

10. The use according to claim 9, characterized in that: The dosage form for administration via the gastrointestinal tract includes at least one of powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet; The non-gastrointestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

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