Application of alpinetin in preparation of PPAR gamma phosphorylation inhibitor

Abstract

The invention discloses application of alpinetin in preparation of a PPAR gamma phosphorylation inhibitor, and belongs to the technical field of medicines. Starting from the action mode of a compound and PPAR gamma, network pharmacology and biological experiments verify that the alpinetin can inhibit the phosphorylation level of PPAR gamma, and a new medicine and a new way are provided for treating diseases or symptoms caused by metabolic disorders.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to the application of shanpin in the preparation of a PPARγ phosphorylation inhibitor. Background Art

[0002] Peroxisome proliferator-activated receptor (PPAR) is a ligand-regulated transcription factor that belongs to the nuclear receptor protein superfamily and plays an important role in various physiological processes in the human body. There are three subtypes of PPAR: PPARα, PPARβ / δ and PPARγ, which differ in ligand specificity, transcriptional activity and tissue expression profile. Among them, PPARγ has become a research focus due to its key role in various diseases.

[0003] As a master regulator, PPARγ regulates the transcription of many genes (e.g., adipogenesis, inflammation, osteogenesis, immunity, glucose homeostasis, etc.), ensuring that these genes work synergistically to maintain metabolic homeostasis. Posttranslational modifications (PTMs) of PPARγ, such as phosphorylation, deacetylation, ubiquitination, and SUMO (Smallubiquitin-like modifier), increase the complexity of its regulation. In particular, the specific site phosphorylation of PPARγ has important clinical significance for the regulation of various physiological processes. The most clinically relevant phosphorylation site of PPARγ is Ser273, which is located in the ligand-binding domain (LBD) of PPARγ. High phosphorylation levels of Ser 273 (pSer 273) are common in patients with obesity and insulin resistance. For example, thiazolidinediones (TZDs), represented by rosiglitazone, play an important role in lowering blood sugar and improving insulin resistance. Their mechanism of action is to activate PPARγ and promote the sensitivity of peripheral tissues to insulin, including increased muscle uptake and utilization of glucose, inhibition of liver gluconeogenesis, inhibition of fatty acid decomposition and increased synthesis in adipose tissue, and promotion of adipocyte remodeling. In addition, TZDs also have the effect of inhibiting PPARγ phosphorylation levels. However, TZDs are high-affinity PPARγ ligands that can more widely affect the transcriptional activation of PPARγ, thereby increasing the risk of osteoporosis, cardiovascular risks, and weight gain, limiting their applicability for long-term use. SR1664 has basically no transcriptional activation effect on PPARγ, but has a high affinity for PPARγ, and is a non-agonist PPARγ ligand. Its action site with PPARγ is serine 273 of PPARγ. SR1664 can play a role by blocking CDK5-mediated phosphorylation of PPARγ at Ser 273, and will not interfere with bone formation (Nature. 2011 Sep 4;477 (7365): 477-81. doi: 10.1038 / nature10383). Therefore, the evaluation of the activity of PPARγ targeted drugs cannot be based solely on PPARγ transcriptional activity. The mode of action of the compound and PPARγ is the key to the activity of PPARγ drugs. The mode of action of the compound and PPARγ determines the intensity and breadth of PPARγ transcriptional activation.

[0004] In recent years, more and more studies have shown that natural plants, especially flavonoids, have a wide range of pharmacological activities. However, alpinetin, a flavonoid isolated from the ginger plant Alpinia officinalis, has no related research or patent invention on its mode of action in PPARγ. Therefore, the present invention provides the use of alpinetin in the preparation of PPARγ phosphorylation inhibitors, which provides a new therapeutic drug and approach for treating diseases or symptoms caused by metabolic disorders, and also provides a new application direction for alpinetin. Summary of the invention

[0005] In view of the above deficiencies in the prior art, the present invention provides the use of shanpin in the preparation of a PPARγ phosphorylation inhibitor, providing a new therapeutic drug and approach for treating diseases caused by metabolic disorders.

[0006] To achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0007] In a first aspect, the present invention provides the use of shanpin in the preparation of a PPARγ phosphorylation inhibitor.

[0008] In a second aspect, the present invention provides a PPARγ phosphorylation inhibitor, the raw materials of which include shanpin.

[0009] Furthermore, the effective dosage of the alpinia officinalis is 2.2 to 4.5 mg / kg per day.

[0010] Furthermore, the raw material also includes a pharmaceutically acceptable carrier and / or excipient.

[0011] Furthermore, the dosage form of the PPARγ phosphorylation inhibitor includes but is not limited to tablets, granules, capsules, pills, sustained-release preparations, oral liquid preparations, and injections.

[0012] In a third aspect, the present invention provides use of the PPARγ phosphorylation inhibitor in PPARγ-mediated diseases or conditions.

[0013] Furthermore, the diseases or conditions include diabetes, hypercholesterolemia, cardiovascular disease obesity, metabolic syndrome, and hyperlipidemia. In the animal model of type 2 diabetes (T2DM), a specific dose of shanjiangsu can inhibit the phosphorylation level of PPARγ Ser273 in 3T3-L1 cells induced by adipogenic differentiation, activate the PI3K / AKT pathway, and promote the expression and membrane translocation of GLUT4, thereby effectively alleviating the hyperglycemia and insulin resistance symptoms of diabetes, and is dose-dependent; if the dosage of shanjiangsu is too small, the inhibitory effect on the phosphorylation of PPARγ Ser273 is not obvious.

[0014] Compared with the prior art, the present invention is beneficial in that:

[0015] 1. The present invention is based on network pharmacology and biological experimental studies to prove that shanjiangsu can inhibit the phosphorylation level of PPARγ, providing a new drug and approach for treating diseases caused by metabolic disorders.

[0016] 2. The PPARγ phosphorylation inhibitor of the present invention can be used to treat PPARγ-mediated diseases or conditions, such as diabetes, hypercholesterolemia, cardiovascular disease obesity, metabolic syndrome, hyperlipidemia, etc., wherein, in an animal model of type 2 diabetes, a specific dose of sanshool can inhibit the phosphorylation level of PPARγ Ser273 in 3T3-L1 cells induced by adipogenic differentiation, thereby activating the PI3K / AKT pathway, promoting the expression and membrane translocation of GLUT4, thereby effectively alleviating the hyperglycemia and insulin resistance symptoms of diabetes, and showing dose-dependency. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a molecular dynamics simulation analysis of galangin and PPARγ; Figure 1 A is a comparison of the conformations of the sansingin-PPARγ complex at five different time points: 0, 25, 50, 75, and 100 ns; Figure 1 B is the calculation result of the root mean square deviation (RMSD) of the PPARγ protein backbone; Figure 1 C is the calculated result of the root mean square fluctuation (RMSF) of PPARγ protein residues; Figure 1 D is the calculated result of the radius of gyration (Rg) of the PPARγ-alpinia officinalis complex; Figure 1 E is the calculated result of average solvent accessible surface area (SASA); Figure 1 F is the hydrogen bonding diagram of shanjiangsu-PPARγ;

[0018] Figure 2 This is the research result of Example 1 that shanjiangsu exhibits partial agonist activity by blocking the phosphorylation of PPARγ; wherein, Figure 2 A is the molecular docking diagram of shanjiangsu and PPARγ; Figure 2 B is the dual luciferase reporter gene method to identify the agonist activity of galangin; Figure 2 C is a cell thermal migration assay to detect the binding efficiency of shanjiangsuin and PPARγ; Figure 2 D is the result of Western blot, which showed that shanjiangsuan reduced the phosphorylation level of PPARγ Ser273; Figure 2 E is the grayscale analysis result of PPARγ protein phosphorylation level;

[0019] Figure 3 The results of the network pharmacology analysis of shanjiangsu-type 2 diabetes in Example 2; wherein, Figure 3A is the Venn diagram of the action targets of galangin and the disease targets of T2DM; Figure 3 B is the PPI network analysis result of drug-disease intersection targets;

[0020] Figure 4 The results of GO and KEGG enrichment analysis of shanjiangsu-type 2 diabetes targets in Example 2; wherein, Figure 4 A is the GO enrichment analysis result of key targets of shanjiangsu in treating T2DM; Figure 4 B is the KEGG enrichment analysis result of key targets of shanjiangsu in treating T2DM;

[0021] Figure 5 The effect of shanjiangsu intervention on STZ-induced T2DM mouse model; Figure 5 A is the experimental design flow chart; Figure 5 B is a photo of mouse epididymal adipose tissue; Figure 5 C is a statistical graph of epididymal fat weight in mice; Figure 5 E and 5D are representative H&E staining sections and average cell size results of eWAT, respectively; ***p<0.001, ****p<0.0001;

[0022] Figure 6 The effect of shanjiangsu intervention on STZ-induced T2DM mouse model; Figure 6 A is a statistical graph of fasting blood glucose values ​​of mice; Figure 6 B is the statistical chart of insulin resistance index for mouse homeostasis model assessment; Figure 6 C and 6D are the glucose tolerance test (GTT) curve and the area under the curve (AUC), respectively; Figure 6 E and 6F are the insulin tolerance test (ITT) glucose curve and the area under the curve, respectively;

[0023] Figure 7 The effect of shanjiangsu intervention on insulin-related signaling pathways in T2DM mice; Figure 7 A-7B are immunofluorescence staining used to analyze the expression changes of GLUT4 in epididymal adipose tissue sections; 7C-7F are the expression level changes of proteins related to the PI3K / AKT-GLUT4 signaling pathway in epididymal adipose; 7G-7H are the phosphorylation level changes of PPARγ Ser273 in epididymal adipose;

[0024] Figure 8 This is the result of a study on the insulin resistance 3T3-L1 adipocyte model induced by TNFα, and the restoration of cell insulin sensitivity by shanjiangsu. Figure 8 A is the effect of shanjiangsu on the expression of proteins related to the insulin signaling pathway detected by Western blot; Figure 8B-8E are the grayscale analysis results of p-PI3K, p-AKT, p-AMPK, and GLUT4 proteins, respectively; Figure 8 F: Alpinia officinalis promoted the glucose uptake of cells; **p<0.01, ***p<0.001, ****p<0.0001;

[0025] Fig. 9 Immunofluorescence was used to detect the expression of GLUT4 on the cell membrane. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] In the following specific embodiments, the alpinol can be directly purchased from the market or prepared by conventional methods in the art; the alpinol can be derived from the mature seeds of the ginger plant Alpinia cardamomum, and its English name is: Alpinetin, CAS No. 36052-37-6, and its molecular formula is C 16 H 14 O4, the chemical structure is shown below:

[0028]

[0029] In the following specific embodiments, the sanpin working solution used is prepared by the following method: take 20 mg of purchased sanpin drug powder, add 740 μL of sterile DMSO according to the instructions for preparing the mother solution to prepare a sanpin mother solution with a concentration of 100 mM, wait until the mother solution is fully dissolved, divide it into EP tubes and seal them, and freeze them at -20°C for use. When using, dilute the sanpin mother solution to the required working concentration (25~100 μM).

[0030] Example 1

[0031] Study on the action mode of galangin on PPARγ

[0032] 1. Molecular dynamics analysis of galangin and its target protein PPARγ

[0033] In this experiment, Gromacs software was used to perform molecular dynamics simulations on the complex of PPARG protein and Alpinetin. In the molecular dynamics simulation, Amber14sb was selected as the protein force field, Gaff2 was selected as the ligand force field, and the SPC / E water model was used to add solvent to the protein-ligand system and establish a water box with a periodic boundary of 1.2 nm, in which an appropriate number of Na+ or Cl Maintain the electrical neutrality of the system and complete the system construction. The constructed system is energy minimized under the amber99sb force field to eliminate the unreasonable interatomic contacts in the system and prevent the dynamic simulation process from collapsing. After completing the minimization of the system energy, NVT and NPT pre-equilibrium are performed. After equilibrium, the system is set to simulate under the NPT ensemble with a simulation time of 100 ns. The temperature is set to 300 K, the pressure is set to 1.01325 bar, and the SHAKE algorithm is used to restrict the stretching vibration of all hydrogen-containing chemical bonds in the system. The step size is set to 2fs. Finally, the root mean square deviation (RMSD), root mean square fluctuation (RMSF), and gyration radius (Rg) in the molecular dynamics simulation trajectory of the PPARG protein and Alpinetin complex are analyzed, as well as the number of hydrogen bonds between the protein and the ligand in the complex and the structure comparison of the complex at five moments of 0, 25, 50, 75, and 100 ns. The analysis results are shown in Figure 1 .

[0034] Depend on Figure 1 A shows that at the five moments of 0, 25, 50, 75, and 100 ns, shanjiangsuin and PPARγ protein are in the same position without obvious changes. Figure 1 B-1D shows that the curve has no obvious fluctuation, the overall structure of the complex changes little, the addition of shanjiangsu has little effect on the stability of the amino acid residues in the PPARγ protein, and the structure of the complex is stable; Figure 1 E shows that the radius of gyration is very small, indicating that the system remains tight and stable in the dynamics simulation. Figure 1 F shows that the number of hydrogen bonds between PPARγ and shanjiangsu is 1-2, and the hydrogen bond disappears temporarily only at 50-60 ns, while the hydrogen bond curve fluctuates smoothly throughout the rest of the process.

[0035] 2. Molecular docking experiment of sansingin and target protein PPARγ

[0036] Ligand processing: The structure of galangin (PDB ID: 154279) was downloaded from the Pubchem database and processed using MGLTools1.5.6. After adding hydrogen, calculating charges, and merging non-polar hydrogens, the pdbqt format file was output.

[0037] Receptor processing: The protein structure of the receptor PPARγ (PDB ID: 7EFQ) was processed using MGLTools1.5.6, and the structure was saved as a pdbqt format file after hydrogenation, charge calculation, and merging of non-polar hydrogen.

[0038] Determination of active site: According to the original ligand position in PPARγ (PDB ID: 7EFQ), its active site was determined. The GridBox coordinates were set to (49.854, 3.881, 89.71), the box size was 30×30×30 grid points, and the distance between each small grid point was 0.1 nm.

[0039] Docking: Autodock Vina1.1.2 was used to dock small molecules with proteins, and the conformation with the highest score was taken for analysis and mapping.

[0040] Figure 2 A is the molecular docking diagram of shanjiangsu and PPARγ. The results show that shanjiangsu can bind to 342 Ser in the PPARγ sequence through hydrogen bonds with a bond length of 2.2Å.

[0041] 3. Dual luciferase reporter gene assay

[0042] (1) One day before transfection, plate 2×10 5 HEK293T cells, the cell confluence was controlled to be about 70%~80% on the second day;

[0043] (2) Balance LipofectaminerTM 2000, PPRE×3-luc reporter gene vector, PPARγ transcription factor vector, and serum-free culture medium to +15℃~25℃;

[0044] (3) For each well, dilute the corresponding reporter gene vector with 50 μL serum-free medium, dilute 1 μL Lipofectaminer TM2000 reagent with 50 μL serum-free medium, and incubate at room temperature for 5 min.

[0045] (4) Gently vortex the carrier solution and add the diluted Lipofectamine™ 2000 reagent dropwise. Mix thoroughly and let stand at room temperature for 10 min to 25 min to form a complex.

[0046] (5) Replace each well of a 24-well cell culture plate with 400 μL of serum-free medium and add the complex dropwise into the well while shaking.

[0047] (6) After culturing in an incubator at 37°C and 5% CO for 6 h, the culture medium was replaced with complete medium containing different doses of shanjiangsu and cultured for another 24 h.

[0048] (7) The fluorescence intensity was detected using a firefly luciferase reporter gene assay kit (RG010S, Shanghai Biotech Biotechnology Co., Ltd.).

[0049] Figure 2B is the dual luciferase reporter gene method to identify the agonist activity of shanjiangsu. The results showed that shanjiangsu induced the activation of PPARγ at concentrations of 25 μM and 50 μM, but the degree of activation was much lower than that of Rosi.

[0050] 4. Cell thermal shift technique verifies the binding effect of shanjiangsuin and PPARγ

[0051] (1) Take the induced differentiated mature 3T3-L1 adipocytes and inoculate them into two groups of 6-well plates until the cells reach 80%-90%;

[0052] (2) Add a 50 μM shanjiangsu solution to the 6-well plate and culture for 2 hours. This is the experimental group. The other group does not receive any treatment, which is the blank control group.

[0053] (3) Digest the cells with trypsin and collect the cells (including cells floating in the culture medium) and add them to a 15 mL centrifuge tube. Centrifuge at 1500 rpm for 3 min, discard the supernatant, and then wash the cells twice with PBS.

[0054] (4) Before the final wash, combine the two groups of cells into two 15 mL centrifuge tubes for the final wash. After the final wash, add 1 mL of PBS supplemented with PMSF to each centrifuge tube and carefully resuspend the cells.

[0055] (5) Divide each group of cell suspension into 6 different 0.2 mL PCR tubes, each containing 100 µl of cell suspension. Label each tube with a specified temperature (25, 40, 50, 60, 70, 80 °C) and equilibrate at room temperature (25 °C) for 20 min;

[0056] (6) Set the temperature on the PCR instrument and heat to the target temperature. Place the designated PCR tube in the PCR instrument and heat for 5 min. Take it out immediately after completion and continue incubating at room temperature for 5 min.

[0057] (7) After incubation, freeze and thaw the cell suspension twice in liquid nitrogen and 25°C to obtain cell lysate;

[0058] (8) Centrifuge the cell lysate at 15,000 rpm and 4 °C for 20 min. Collect 80 μL of the supernatant from each centrifuge tube and discard the cell debris pellet.

[0059] (9) Add 20 μL of 5× Loading buffer to each tube of lysate, mix well, and place in a 99°C metal bath for 20 min to denature.

[0060] (10) Western Blot was used to detect the changes in the target protein PPARγ at different temperatures, and the expression level-temperature curve was drawn.

[0061] Figure 2 C is a cell thermal migration experiment to detect the binding efficiency of shanjiange and PPARγ. The results show that compared with the DMSO group, the PPARγ protein in the shanjiange group has better thermal stability, indicating that shanjiange can bind to PPARγ and increase its thermal stability.

[0062] 5. Western Blot experiment verifies that galangin inhibits the phosphorylation of PPARγ Ser273

[0063] (1) Wash the cell culture medium in a centrifuge tube twice with PBS and collect it. Add cell lysis buffer and disrupt it by oscillation and ultrasonication at 4°C for 20 min.

[0064] (2) Prepare 10% concentration of SDS-PAGE gel (G2043-50T, Wuhan Saiweier Biotechnology Co., Ltd.);

[0065] (3) Determine protein concentration by BCA method to ensure consistent protein loading;

[0066] (4) Electrophoresis separation: Keep constant voltage at 200-250 V for 30 min. Stop electrophoresis when the phenol blue indicator reaches the bottom of the gel. For ease of comparison, use the same volume of each sample during electrophoresis.

[0067] (5) Transfer and antibody reaction: After the electrophoresis is completed, remove the gel and wash it with clean water. At the same time, soak the PVDF membrane and filter paper in ice-free rapid transfer buffer (G2148-1L, Wuhan Sevier Biotechnology Co., Ltd.) for 5 min, lay the gel and NC membrane in the order of cathode-sponge-filter paper-gel-NC membrane-filter paper-sponge-anode, put them into the electrophoresis tank, and electrophoresed at 400 mA for 40 min at low temperature;

[0068] (6) Remove the PVDF membrane and wash it with 1×PBST (PBS with 0.1% Tween-20) for 5 min. Add TBST blocking buffer containing 5% skim milk powder and incubate at 37°C for 2 h. Wash the PVDF membrane with TBST three times, 10 min each time. Add rabbit anti-PPARγ, p-PPARγ (Ser 273) monoclonal antibody (1: 1000 dilution in TBST) and incubate at 4°C overnight. Wash the PVDF membrane thoroughly with TBST three times, 5 min each time. Add HRP-labeled goat anti-rabbit IgG antibody (BL003A, Biosharp LifeSciences, 1: 10000 dilution in TBST), incubate at 37°C for 1 h, and wash the PVDF membrane thoroughly with TBST 3-5 times, 10 min each time. Add ECL chemiluminescent substrate (BL520B, Biosharp Life Sciences) at room temperature and away from light for 3 min, and observe and record the results in the dark.

[0069] According to the molecular docking results, shanjiangsu can form a strong hydrogen bond with the 342 Ser amino acid, which is closely related to the phosphorylation of PPARγ273 Ser. Figure 2 D is the result of Western blot, which showed that shanjiangsuan reduced the phosphorylation level of PPARγSer 273; Figure 2 E is the grayscale analysis result of PPARγ protein phosphorylation level; Figure 2 The results of D-2E showed that galangin could significantly inhibit the phosphorylation level of PPARγ-273 Ser in a dose-dependent manner.

[0070] Example 2

[0071] Network pharmacology study of shanjiangsuan on T2DM

[0072] 1. Target screening of sansingin

[0073] The standard structural formula and molecular conformation of galangin were obtained from the Pubchem database, and the structure was imported into the Swiss Target Prediction database (http: / / www.swisstargetprediction.ch) to predict targets and screen drug targets (select targets with prediction scores greater than 0). Finally, the relevant target proteins of the chemical components were integrated and annotated using the Uniprot (https: / / www.uniprot.org) database.

[0074] 2. Type 2 diabetes target screening

[0075] Using "T2DM", "insulin resistance" and "type 2 diabetes" as keywords, potential targets for type 2 diabetes were mined in the following databases: GeneCards database, OMIM database (Gene Map), TTD database (Therapeutic Target Database), and DRUGBANK database. Disease-related targets were obtained after merging and removing duplicates.

[0076] 3. Construction of the PPI network of sanshinol-type 2 diabetes target

[0077] The targets related to sanshin and those related to type 2 diabetes were input into Venny 2.1.0 software, and the Venn diagram was drawn to obtain the intersection. The intersection targets were uploaded to the STRING11.0 database to establish a protein-protein interaction (PPI) network model. In STRING11.0, the biological species was set to "Homo sapiens", the minimum interaction threshold was set to "medium confdence" (>0.4), and the rest were set to the original parameters to obtain the PPI network. The obtained network relationship data was imported into Cytoscape3.7.2 software to draw the PPI network diagram. Among them, the color of the node and its shade change and the size of the node represent the size of the Degree value.

[0078] Figure 3 A is the Venn diagram of the targets of galangin and T2DM disease targets; Figure 3 A shows that there are 100 effective targets of shanjiangsu and 228 targets related to type 2 diabetes. After taking the intersection, 11 common targets were obtained; Figure 3 B is the PPI network analysis result of drug-disease intersection target visualization; Figure 3 As can be seen from B, the number of nodes is 11, the number of edges is 20, and the average node degree is 3.636; according to the Degree value sorting, PPARγ is its key node.

[0079] 4. GO and KEGG enrichment analysis of galangin-type 2 diabetes targets

[0080] The GO (gene ontology) function and KEGG (Kyoto encyclopedia of genes and genomes) pathway enrichment analysis were performed using the Metascape (http: / / metascape.org / ) platform. The obtained intersection targets were imported into the gene list, the object was set to "human", personalized analysis was selected, the minimum overlap (MinOverlap) was set to 3, the p value was set to 0.05, and the minimum enrichment was set to 1.5. After obtaining the results, the data were visualized using R software.

[0081] Figure 4 A is the GO enrichment analysis result of key targets of shanjiangsu in treating T2DM; Figure 4 As shown in A, the biological process (BP) in the GO enrichment analysis is mainly related to the phosphatidylinositol-3-phosphate biosynthetic process; in the cellular component (CC), it is mainly related to the phosphatidylinositol 3-kinase complex, class IA; and in the molecular function (MF), it is mainly related to the 1-phosphatidylinositol-4-phosphate 3-kinase activity. Figure 4 B is the KEGG enrichment analysis result of key targets of shanjiangsu in treating T2DM; Figure 4 B shows that the main signaling pathways of galangin in the treatment of T2DM include the AGE-RAGE signaling pathway in diabetic complications, non-alcoholic fatty liver disease, and the regulation of lipolysis in adipocytes.

[0082] Example 3

[0083] Experimental study on the treatment of T2DM model mice with galangin

[0084] Six-week-old male C57BL / 6J mice were selected and fed with normal feed for 2 days. Ten mice were randomly selected as the normal control group and given normal feed. The rest of the animals were given high-fat and high-sugar feed (Research diet D12492, 60% kcal%). After 6 weeks of feeding (12 weeks of age), low-dose streptozotocin (STZ, 30 mg / kg, Sigma) was injected intraperitoneally every day for 3 consecutive days, and fasted for 5 h before each injection. Fasting blood glucose levels were measured two weeks after the first injection of STZ (14 weeks of age), and mice with blood glucose levels below 190 mg / dL (10.5mmol / L) were excluded. The experiment was divided into control group (Control), model group (T2DM), low-dose shanjiangsu administration group (25 mg / kg) (equivalent to 2.2 mg / kg for humans), high-dose shanjiangsu administration group (50 mg / kg) (equivalent to 4.5 mg / kg for humans), and rosiglitazone administration group (5 mg / kg). The drug-treated group was gavaged with appropriate doses of shanjiangsu and rosiglitazone every day, while the control group and the model group were gavaged with corresponding volumes of solvent (the solvent was saline containing 10% DMSO, 5% Tween80 and 35% PEG). The mice continued to eat high-fat and high-sugar diets, and their body weights were recorded every week until they were collected at 22 weeks of age (the experimental design flow chart is shown in the figure). Figure 5 A), detect relevant indicators, the specific detection methods and indicators are as follows:

[0085] (1) Recording of mouse body weight and epididymal fat weight

[0086] Figure 5 B is a comparison of mouse epididymal adipose tissue; Figure 5 C is a statistical graph of epididymal fat weight in mice; Figure 5 E and 5D are representative H&E-stained sections and average cell size results of eWAT, respectively; Figure 5 The results of B-5E showed that the high-dose galangin administration group (50 mg / kg) could effectively reduce the body weight, epididymal fat weight and adipocyte volume of mice.

[0087] (2) Determination of serum chemotherapeutic indicators in mice

[0088] The mice were fasted for 12 hours, and then the eyeballs were removed to collect blood. After standing at room temperature for 2 hours, the blood was centrifuged at 15°C and 3000 rpm for 10 minutes. The upper serum was collected and the fasting blood glucose was measured by an automatic biochemical analyzer. The serum insulin content was measured by ELISA kit, and the homeostasis model assessment insulin resistance (HOMA-IR) was calculated according to the following formula:

[0089] HOMA-IR = mouse fasting blood glucose value (mmol / L) × mouse fasting insulin value (μU / mL) / 22.5

[0090] Figure 6 A is a statistical graph of fasting blood glucose values ​​of mice; Figure 6 B is the statistical chart of insulin resistance index for mouse homeostasis model assessment; Figure 6 As can be seen from A-6B, the fasting blood glucose and HOMA-IR values ​​of the T2DM group were significantly higher than those of the control group. Treatment with galangin can effectively reduce the fasting blood glucose and HOMA-IR values ​​of T2DM mice.

[0091] (3) Glucose tolerance test (GTT) in mice

[0092] After 6 weeks of administration, mice were fasted for 12 h at 8:30 pm the previous day, and the GTT experiment was performed at 8:30 am the next day. Weigh 4 g of glucose and dissolve it in 20 mL of water, filter it with a 0.22 μm filter membrane to sterilize it, and prepare a 20% glucose solution. Weigh the body weight, measure the fasting blood glucose of mice as the blood glucose value at 0, and give mice a 20% glucose solution by gavage. The final glucose dose was 2 g / kg. The blood glucose values ​​were measured at 15 min, 30 min, 60 min, 90 min, and 120 min after glucose gavage, and the GTT curve was drawn.

[0093] Figure 6 C. Figure 6 D is the glucose tolerance test and the area under the curve statistical graph; Figure 6 C-6D shows that compared with the T2DM group, sansevieriaceae can significantly improve the glucose tolerance of T2DM mice, and there is no significant difference compared with the rosiglitazone group.

[0094] (4) Mouse insulin tolerance test (ITT)

[0095] At 8:30 in the morning, mice were fasted for 6 hours, and the blood glucose value of mice after fasting for 6 hours was measured as the blood glucose value at 0 point of the ITT experiment. At the same time, the weight of mice after fasting for 6 hours was weighed, and then recombinant human insulin injection was given by intraperitoneal injection according to body weight, and the insulin concentration was 0.5 U / g body weight. The blood glucose concentration was measured at 15 min, 30 min, 60 min, 90 min, and 120 min after insulin injection, and the ITT curve was drawn.

[0096] Figure 6 E. Figure 6 F is the insulin tolerance test and the area under the curve statistical chart; Figure 6 E-6F It can be seen that compared with the T2DM group, galangin can significantly improve the insulin sensitivity of T2DM mice, and there is no significant difference compared with the rosiglitazone group.

[0097] (5) Immunofluorescence staining

[0098] After xylene dewaxing, gradient alcohol dehydration, antigen retrieval, and 5% bovine serum albumin blocking, the epididymal tissue sections were incubated with the primary antibody (GLUT4, dilution ratio 1:200) at 4 °C overnight. The next day, the sections were incubated with the secondary antibody (dilution ratio 1:1000) at room temperature in the dark for 1 h, and the nuclei were counterstained with DAPI for 10 min. After sealing with antifade mounting medium, the sections were observed and photographed under an Eclipse Ti fluorescence microscope (Leica, Wetzlar, Germany). For the negative control group, only 5% BSA was used instead of the primary antibody, and other conditions and steps were the same.

[0099] The experimental results are shown in Figure 7 A, 7B; Figure 7 A and 7B show that galangin promoted the membrane translocation and expression of GLUT4 protein.

[0100] (6) Western blot experiment of epididymal adipose tissue

[0101] Epididymal white adipose tissue (eWAT) was collected and snap-frozen in liquid nitrogen. Tissue lysis buffer (Beyotime, Shanghai, China) was added and sonicated at 4 °C for 20 min. The cells were centrifuged at 12,000 rpm for 10 min at 4 °C. The protein concentration in the lysate was determined using a BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts of protein (20 µg) were loaded onto a 10% SDS-PAGE gel and transferred to a 0.45 µm PVDF membrane. The membrane was blocked with 5% skim milk in TBST at 37 °C for 2 h and then incubated with primary antibodies overnight at 4 °C. After washing, the membrane was incubated with HRP-conjugated secondary antibodies (Abbkine, Hubei, China). Protein bands were visualized using an ECL luminescence system (Millipore, MA, USA) and analyzed using Image J software. The results are shown in Figure 2. Figure 7 C-7H.

[0102] Figure 7 C-7F showed that shanjiangsu can promote the expression of PI3K / AKT-GLUT4 signaling pathway, and, Figure 7 G-7H showed that shanjiangsuan could inhibit the phosphorylation level of PPARγ Ser273 site.

[0103] Example 4

[0104] Cell experiment on the prevention and treatment of T2DM by alpinin

[0105] 1. 3T3-L1 adipocyte culture and intervention

[0106] (1) 3T3-L1 cells were seeded into 48-well plates (1×10 5 / well), after 24 h of contact inhibition, the medium was replaced with adipogenic induction medium A: DMEM culture medium with 10% fetal bovine serum containing 1 μM dexamethasone, 1.5 μg / mL insulin, and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX); after 3 days of induction, the medium was replaced with adipogenic induction medium B: DMEM culture medium with 10% fetal bovine serum containing 1.5 μg / mL insulin; after 3 days, the medium was replaced with normal culture medium;

[0107] (2) TNF-α (final concentration 10 ng / mL) was added to the treatment group and incubated for 24 h;

[0108] (3) The shanjiangsu treatment group was added with shanjiangsu solution with a final concentration of 25, 50, and 100 μM, respectively, and incubated for 24 h;

[0109] (4) The medium was replaced with DMEM. After 24 h of culture, the glucose content in the medium was measured using a glucose oxidase assay kit (E1010-200, Beijing Pulilai Gene Technology Co., Ltd.). The amount of glucose absorbed by adipocytes is equal to the difference in glucose concentration before and after uptake.

[0110] Figure 8 F represents that shanjiangin promotes glucose uptake in cells. The results show that shanjiangin can promote glucose uptake in 3T3-L1 adipocytes under insulin resistance.

[0111] 2. Western Blot Experiment

[0112] (1) Wash the cell culture medium in a centrifuge tube twice with PBS and collect it. Add cell lysis buffer and disrupt it by oscillation and ultrasonication at 4°C for 20 min.

[0113] (2) Prepare 10% concentration of SDS-PAGE gel (G2043-50T, Wuhan Saiweier Biotechnology Co., Ltd.);

[0114] (3) Determine protein concentration by BCA method to ensure consistent protein loading;

[0115] (4) Electrophoresis separation: Keep constant voltage at 200-250 V for 30 min. Stop electrophoresis when the phenol blue indicator reaches the bottom of the gel. For ease of comparison, use the same volume of each sample during electrophoresis.

[0116] (5) Transfer and antibody reaction: After the electrophoresis is completed, remove the gel and wash it with clean water. At the same time, soak the PVDF membrane and filter paper in ice-free rapid transfer buffer (G2148-1L, Wuhan Sevier Biotechnology Co., Ltd.) for 5 min, lay the gel and NC membrane in the order of cathode-sponge-filter paper-gel-NC membrane-filter paper-sponge-anode, put them into the electrophoresis tank, and electrophoresed at 400 mA for 40 min at low temperature;

[0117] (6) Remove the PVDF membrane and wash it with 1× PBST (PBS with % Tween-20) for 5 min, add TBST blocking buffer containing 5% skim milk powder, and incubate at 37°C for 2 h; wash the PVDF membrane with TBST for 3 times, 10 min each time; add rabbit anti-PI3K, p-PI3K, AMPK, p-AMPK, AKT, p-AKT monoclonal antibodies (1:1000 TBST dilution), mouse anti-GLUT4 monoclonal antibodies (1:1000 TBST dilution), and incubate at 4°C overnight; wash the PVDF membrane with TBST for 3 times, 5 min each time; add HRP-labeled goat anti-rabbit IgG antibody (BL003A, Biosharp Life Sciences, 1:10000 dilution in TBST), HRP-labeled goat anti-mouse IgG antibody (BL001A, Biosharp Life Sciences, 1:10000 dilution in TBST) and incubate at 37°C for 1 h, wash the PVDF membrane thoroughly with TBST 3-5 times, 10 min each time, add ECL chemiluminescent substrate (BL520B, Biosharp LifeSciences) at room temperature and avoid light for 3 min, and observe and record the results in the dark.

[0118] Figure 8 A is the effect of shanjiangsu on the expression of proteins related to the insulin signaling pathway detected by Western blot; Figure 8 B-8E are the grayscale analysis results of p-PI3K, p-AKT, p-AMPK, and GLUT4 proteins, respectively; Figure 8 A-8E results showed that under the action of galangin, the levels of PI3K, AKT, and AMPK protein phosphorylation were upregulated and the expression of GLUT4 protein was increased in 3T3-L1 adipocytes in the insulin-resistant state.

[0119] 3. Immunofluorescence staining of cells

[0120] (1) Preparation of 3T3-L1 adipocyte cell slides: Wash the sterilized cell slides (BS-18-RC, Biosharp Life Sciences) three times with PBS, place them in a 12-well plate and let them dry, then moisten the 12-well plate with complete culture medium (to make the cells adhere more evenly), and finally inoculate 3T3-L1 cells in the 12-well plate containing the slides, and start processing after the cells are fused;

[0121] (2) Fixation: Remove the culture medium and wash once with PBS. Fix with paraformaldehyde fixative for 20-30 min at room temperature and wash three times with PBS, 5 min each time.

[0122] (3) Permeabilization: Treat with PBS containing 0.5% Triton-X-100 at room temperature for 15 min (250 μL Triton + 50 mL PBS). Wash with PBS buffer for 5 min × 3 times and spin dry;

[0123] (4) Blocking: 3% BSA (prepared in deionized water) at room temperature for 30 min;

[0124] (5) Incubation with primary antibody: discard the blocking solution and dry at room temperature for 2 min. Prepare GLUT4 (sc-53566, Santa Cruz Biotechnology) antibody at a dilution of 1:200 with 3% BSA and incubate at 4°C overnight. Wash the antibody the next day with PBS buffer for 5 min × 3 times and spin dry.

[0125] (6) Apply fluorescent secondary antibody: Prepare fluorescent secondary antibody with 3% BSA, incubate at room temperature in the dark for 1 hour, then wash with PBS for 5 minutes × 3 times and spin dry.

[0126] (7) Observe and collect images under a laser scanning confocal microscope in a wet state.

[0127] The results are as follows Fig. 9 As shown, from Fig. 9 It can be seen that under the action of alpinia officinalis, the cell membrane translocation of GLUT4 is significantly enhanced.

[0128] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. Application of shanjiangsu in the preparation of PPARγ phosphorylation inhibitors.

2. A PPARγ phosphorylation inhibitor, characterized in that The raw materials include shanjaroin.

3. A PPARγ phosphorylation inhibitor according to claim 2, characterized in that: The effective dosage of the alpinia officinalis is 2.2 to 4.5 mg / kg per day.

4. A PPARγ phosphorylation inhibitor according to claim 2, characterized in that: The raw materials also include pharmaceutically acceptable carriers and / or excipients.

5. A PPARγ phosphorylation inhibitor according to claim 2, characterized in that: The dosage forms of the PPARγ phosphorylation inhibitor include, but are not limited to, tablets, granules, capsules, pills, sustained-release preparations, oral liquid preparations, and injections.

6. Use of the PPARγ phosphorylation inhibitor according to any one of claims 2 to 5 in PPARγ-mediated diseases or conditions.

7. Use of the PPARγ phosphorylation inhibitor according to claim 6 in PPARγ-mediated diseases or conditions, characterized in that: The diseases or conditions include diabetes, hypercholesterolemia, cardiovascular disease obesity, metabolic syndrome, hyperlipidemia.

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