Application of liquiritin in preparation of PPAR gamma receptor partial agonist

Abstract

The invention provides an application of liquiritin in preparation of a PPAR gamma receptor partial agonist. Through a structure-based high-throughput virtual screening technology, it is found that liquiritin can be used as a partial agonist of a PPAR gamma receptor, and the relative activation efficiency of liquiritin is 35.9% of that of rosiglitazone; in-vitro experiments prove that liquiritin can remarkably promote glucose uptake and consumption of HepG2 cells and does not induce adipocyte differentiation; in-vivo experiments prove that liquiritin can effectively improve the blood glucose level of an insulin resistance mouse model induced by high fat diet and reduce the level of serum proinflammatory factors. Compared with thiazolidinedione compounds, liquiritin not only can relieve and treat insulin resistance or related metabolic diseases, but also does not cause adverse reactions such as weight gain, fat accumulation and myocardial hypertrophy, provides a new candidate compound for developing safe and effective anti-diabetic drugs, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of liquiditin in the preparation of peroxisome proliferator-activated receptor γ (PPARγ) partial agonists. Background Technology

[0002] Insulin resistance refers to the weakening of the physiological function of insulin in the body. It is a common pathological mechanism in the development of many metabolic-related diseases, especially the core pathological mechanism of type 2 diabetes mellitus (T2DM). At the same time, insulin resistance is also a common breeding ground for the occurrence and development of many chronic metabolic diseases, which can lead to a series of clinical syndromes such as diabetes, obesity, metabolic syndrome, cardiovascular and cerebrovascular diseases, and fatty liver, causing serious harm.

[0003] Peroxisome proliferator-activated receptors (PPARs) regulate the gene expression of proteins involved in glucose and lipid metabolism, modulating various physiological processes, including lipid homeostasis, adipogenesis, inflammation, and wound healing. PPARs include three subtypes: α, β / δ, and γ. PPARγ is primarily distributed in adipose tissue. Upon recognition, binding, and activation by endogenous and exogenous ligands, it regulates the expression of target genes involved in numerous biological effects such as cellular energy metabolism, material metabolism, and cell proliferation. PPARγ activation increases the sensitivity of peripheral tissues to insulin, including increased glucose uptake and utilization in muscles, inhibition of hepatic gluconeogenesis, inhibition of fatty acid breakdown and increased synthesis in adipose tissue, and promotion of adipocyte remodeling. Therefore, PPARγ agonists are used as insulin sensitizers in the treatment of diabetes with proven efficacy. Existing complete PPARγ agonists (such as thiazolidinediones and rosiglitazone) can improve insulin sensitivity, but they have serious side effects such as weight gain, edema, and myocardial hypertrophy. To maximize the preservation of PPARγ-mediated insulin sensitization efficacy and minimize related adverse reactions, the concept of selective PPARγ modulators (SPPARMs) emerged. Compared to existing complete PPARγ agonists, SPPARMs have similar or different PPARγ receptor binding sites, differ in affinity or specificity for recruiting receptor cofactors, or exhibit a biased and selective approach to a series of target genes regulated by PPARγ, with varying levels of transcriptional activity. The currently widely accepted view is that conformational differences between ligand-receptor-cofactor groups affect the selective transcriptional regulation of target genes, further influencing the therapeutic effects and adverse reactions of PPARγ-targeted drugs.

[0004] Partial PPARγ agonists, as selective PPARγ modulators, include INT131, GW1929, and MBX-102. INT131, for example, exhibits significantly weaker transcriptional agonistic activity against PPARγ than the full agonist rosiglitazone. While it shares a similar binding site with PPARγ, its ability to recruit cofactors is significantly reduced. In terms of efficacy, INT131 retains insulin-sensitizing effects similar to rosiglitazone, reducing hyperglycemia, but without inducing lipogenesis or weight gain, effectively avoiding or reducing adverse events associated with full PPARγ agonists. Developing novel partial PPARγ agonists with enhanced therapeutic efficacy and reduced side effects remains a key focus in this field. Summary of the Invention

[0005] This invention provides the application of glycyrrhizin in the preparation of PPARγ receptor partial agonists. Glycyrrhizin significantly improves glucose and lipid metabolism disorders by targeting and activating PPARγ and regulating downstream signaling pathways, while avoiding the side effects of traditional PPARγ full agonists.

[0006] In a first aspect, the present invention provides the use of glycyrrhizin or a pharmaceutically acceptable salt thereof in the preparation of PPARγ receptor partial agonists.

[0007] As described above, the chemical structural formula of the glycyrrhizin is shown in Formula 1:

[0008]

[0009] As described above, the relative activation efficiency of glycyrrhizin on the PPARγ receptor is lower than that of a complete PPARγ agonist on the PPARγ receptor; further, the complete PPARγ agonist is a thiazolidinedione compound; even further, the complete PPARγ agonist is rosiglitazone; even further, the relative activation efficiency of 120 μM glycyrrhizin on the PPARγ receptor is 35.9% of the relative activation efficiency of 10 μM rosiglitazone on the PPARγ receptor, and the relative activation efficiency (Relative Transcriptional Activity) can be obtained by dual-luciferase reporter gene assay.

[0010] As described above, glycyrrhizin specifically binds to the PPARγ ligand-binding domain with an equilibrium dissociation constant (KD) of 5.696 μM.

[0011] In a second aspect, the present invention provides the use of glycyrrhizin or a pharmaceutically acceptable salt thereof in the preparation of medicaments for relieving and / or treating insulin resistance or related metabolic diseases.

[0012] As described above, the relevant metabolic diseases include at least one of type 2 diabetes, obesity, and metabolic syndrome.

[0013] As described above, relief and treatment specifically refer to administering the glycyrrhizin of the present invention to a subject in need of it, which can alleviate, relieve, alter, improve, enhance, or influence a condition (e.g., diabetes), one or more symptoms of the condition (e.g., hyperglycemia), or a tendency toward the condition. For example, "relief and treatment of type 2 diabetes" generally means maintaining blood glucose levels near normal levels and may include increasing or decreasing blood glucose levels as appropriate in a given situation.

[0014] Thirdly, the present invention provides the use of glycyrrhizin or a pharmaceutically acceptable salt thereof in the preparation of products that improve the sensitivity of subjects to insulin.

[0015] As described above, insulin sensitivity refers to the degree of insulin resistance in a subject. Higher insulin resistance results in lower insulin sensitivity, less effective insulin per unit, and reduced carbohydrate breakdown.

[0016] Fourthly, the present invention provides a PPARγ receptor partial agonist, comprising glycyrrhizin or a pharmaceutically acceptable salt thereof.

[0017] As described above, PPARγ receptor partial agonists also include pharmaceutically acceptable carriers.

[0018] The dosage form of the PPARγ receptor partial agonist as described above can be selected from any one of the following dosage forms: tablets, capsules, granules, oral liquids, syrups, sprays, inhalers, ointments, powders, injections, or lyophilized powder for injection.

[0019] Fifthly, the present invention also provides a method for alleviating and / or treating insulin resistance or related metabolic diseases, comprising administering to a subject a therapeutically effective amount of glycyrrhizin or a pharmaceutically acceptable salt thereof.

[0020] As described above, the dosage of PPARγ receptor modulators used to alleviate and treat insulin resistance or related metabolic diseases is variable, depending on the method of administration, route of administration, individual age and / or weight, and the individual circumstances of the subject, and is ultimately determined by the attending physician. The dosage administered to an individual, in the case of this invention, should be sufficient for a period of time to elicit a beneficial response in the individual. This dosage is the "therapeuticly effective amount."

[0021] In this invention, a pharmaceutically acceptable salt refers to a salt of a compound that retains the biological activity of the parent compound. For example, pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. By way of example only, salts derived from inorganic bases include sodium, potassium, lithium, ammonium, calcium, zinc, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines; for example, pharmaceutically acceptable acid addition salts can also be prepared from inorganic and organic acids. Salts derived from inorganic acids include salts of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Salts derived from organic acids include salts of acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

[0022] In this invention, pharmaceutically acceptable carriers include any standard pharmaceutical carrier suitable for administration to or by an individual in need, such as phosphate-buffered saline solutions, water, emulsions (such as oil / water or water / oil emulsions), and various types of wetting agents.

[0023] In this invention, the subject suffers from insulin resistance or a related metabolic disease. The subject can be a mammal, selected from bovine, equine, feline, canine, lagomorph, suidae, camel, rodent, and primate animals, including but not limited to cattle, horses, goats, sheep, cats, rabbits, pigs, camels, alpacas, rats, mice, guinea pigs, non-human primates (such as apes, monkeys, baboons, and orangutans), and humans; more preferably, humans.

[0024] This invention utilizes structure-based high-throughput virtual screening technology to discover that glycyrrhizin can act as a partial agonist of the PPARγ receptor, with a relative activation efficiency of 35.9% that of rosiglitazone. Furthermore, in vitro experiments confirmed that glycyrrhizin significantly promotes glucose uptake and consumption in HepG2 cells without inducing adipocyte differentiation. In vivo experiments demonstrated that glycyrrhizin effectively improves blood glucose levels and reduces serum pro-inflammatory cytokine levels in a high-fat diet-induced insulin resistance mouse model. Compared to thiazolidinediones, glycyrrhizin not only alleviates and treats insulin resistance or related metabolic diseases but also does not induce adverse reactions such as weight gain, fat accumulation, and myocardial hypertrophy, providing a new candidate compound for the development of safe and effective antidiabetic drugs with broad application prospects. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the 3D structure of the PPARγ protein.

[0026] Figure 2The SPR results show the binding affinity of three small molecule compounds to PPARγ protein; where A is the SPR result of the binding affinity of glycyrrhizin (LIQ) to PPARγ protein, B is the SPR result of the binding affinity of Aurantiamide acetate (AA) to PPARγ protein, and C is the SPR result of the binding affinity of [(2R)-2-[[[(2R)-2-(benzoylamino)-3-phenylpropanoyl]amino]methyl]-3-phenylpropyl]acetate (BAMPA) to PPARγ protein.

[0027] Figure 3 This shows the docking of glycyrrhizin and PPARγ protein at docking sites with the lowest molecular docking energy.

[0028] Figure 4 The transcriptional activation effects of different concentrations of glycyrrhizin and rosiglitazone on PPARγ protein;

[0029] Figure 5 Oil Red O staining results of primary adipocytes of C57BL / 6J mice at different concentrations of glycyrrhizin and rosiglitazone;

[0030] Figure 6 The results of quantification of PPARγ and its downstream genes in primary adipocytes under different treatments are shown below. Among them, A is the quantitative result of PPARγ gene, B is the quantitative result of CD36 gene, C is the quantitative result of aP2 gene, D is the quantitative result of C / EBPα gene, E is the quantitative result of LPL gene, and F is the quantitative result of Fasn gene.

[0031] Figure 7 The regulatory effect of different concentrations of glycyrrhizin on glucose consumption in HepG2 cells;

[0032] Figure 8 The ability of different concentrations of glycyrrhizin to induce glucose uptake in HepG2 cells;

[0033] Figure 9 The effects of different treatment groups on insulin resistance in HepG2 cells;

[0034] Figure 10 The changes in body weight of each group of insulin resistance model mice;

[0035] Figure 11 Fasting blood glucose levels in each group of insulin resistance model mice;

[0036] Figure 12 The effect of glycyrrhizin on OGTT in insulin-resistant mouse model;

[0037] Figure 13 The area AUC of each group of insulin resistance model mice under OGTT;

[0038] Figure 14 The effect of glycyrrhizin on ITT in an insulin-resistant mouse model;

[0039] Figure 15 The area AUC under ITT in each group of insulin resistance model mice;

[0040] Figure 16 The effects of glycyrrhizin on TC, TG, LDL-c, HDL-c and blood glucose in insulin-resistant mice were investigated. Among them, A represents the effect on TC, B represents the effect on TG, C represents the effect on LDL-c, D represents the effect on HDL-c, and E represents the effect on blood glucose.

[0041] Figure 17 The effects of glycyrrhizin on AST and ALT in insulin-resistant mice; where A represents the effect on ALT and B represents the effect on AST.

[0042] Figure 18 The effects of glycyrrhizin on urea, uric acid, and creatinine in insulin-resistant mice were investigated. A represents the effect on urea, B on uric acid, and C on creatinine.

[0043] Figure 19 The effects of glycyrrhizin on the levels of inflammatory factors in insulin-resistant mice are shown in Figure 1. A represents the effect on TNF-α, B on IL-6, C on IL-1β, and D on CCL2.

[0044] Figure 20 The effects of different treatment groups on the tissue quality of major organs in insulin-resistant mice; where A is a photograph of the tissue of major organs in each treatment group, and BC is the tissue quality of the major organs in mice.

[0045] Figure 21 The images show the magnetic resonance imaging results of adipose tissue in insulin-resistant mice under different treatment groups; where A represents the imaging results and B represents the statistical results of fat content percentage.

[0046] Figure 22 H&E staining results of adipose tissue from insulin-resistant mouse models in different treatment groups;

[0047] Figure 23 The effects of different treatment groups on the risk of cardiotoxicity in insulin-resistant mice were investigated; where A represents the staining results of mouse heart tissue and B represents the statistical results of left ventricular wall thickness in mice.

[0048] In the figure, ns indicates no significant difference; # indicates model group vs. control group, P<0.05; ## indicates model group vs. control group, P<0.01; ### indicates model group vs. control group, P<0.001; * indicates drug treatment group vs. model group, P<0.05; ** indicates drug treatment group vs. model group, P<0.01; *** indicates drug treatment group vs. model group, P<0.001. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, embodiments of this invention, and should not be construed as limiting the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. In the description of this invention, it should be understood that the terminology used is for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0051] Example 1: Obtaining and Displaying the Structure of Human PPARγ Protein

[0052] Figure 1 The crystal structure of the human PPARγ ligand-binding domain (LBD) is shown (PDB ID: 7AWC). This structure was downloaded from the RCSBPDB database (https: / / www.rcsb.org / ) and is a complex co-crystallized with rosiglitazone, with a resolution of [resolution missing].

[0053] Example 2: Molecular docking of glycyrrhizin with PPARγ protein

[0054] The PPARγ crystal structure (7AWC) was imported into LeDock software. Water molecules were removed, hydrogen atoms were added, and energy optimization was performed. The pocket occupied by the protoligand rosiglitazone was designated as the active site. 14,249 small molecule compound structures were obtained from the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP). After data cleaning and deduplication, 13,729 unique small molecule compound chemical structures and their related information were obtained. In LeDock software, the pre-processed small molecule compound structures were aligned to the defined PPARγ active pocket, and the binding free energy (LeDock Score) was calculated. The top 2,000 small molecule compounds were screened using the Lipinski five-rule and ADMET feature filtering, resulting in three potential compounds. Information on the three potential compounds is shown in Table 1.

[0055] Table 1 Information on three compounds screened based on molecular docking

[0056]

[0057] Example 3: Surface plasmon resonance (SPR) detection of the binding affinity of the above three small molecule compounds to PPARγ

[0058] To overcome the potential shortcomings and limitations of computer-based virtual screening technology and to further confirm whether the above candidate compounds are ligands of PPARγ, surface plasmon resonance (SPR) technology was used to detect the interaction between the above candidate compounds and the PPARγ ligand binding domain. The specific steps are as follows:

[0059] 1. Chip Modification: The CM5 carboxymethyl dextran sensor chip (Cytiva) was selected. Purified human PPARγ protein (OriGene, purity ≥95%) was immobilized using EDC / NHS chemical coupling. 0.1 mg / mL PPARγ was dissolved in 10 mM sodium acetate buffer (pH 4.0) and injected into the chip channels at a flow rate of 10 μL / min until the response value reached 8000-10000 RU. Unreacted sites were blocked with 1 M ethanolamine (pH 8.5).

[0060] 2. Analyte preparation: The three candidate compounds shown in Table 1 were diluted with PBST buffer containing 5% DMSO to prepare six concentration gradients (0.1953 μM, 0.7813 μM, 3.125 μM, 12.5 μM, 50 μM, 200 μM).

[0061] 3. SPR detection: Candidate compound solutions of different concentrations were sequentially flowed over the chip surface at a flow rate of 25 μL / min, with a binding time of 120 seconds and a dissociation time of 300 seconds. The response signal (RU) was recorded in real time. After each round of detection, the chip surface was regenerated with 10 mM glycine-HCl (pH 2.5), and the process was repeated 3 times.

[0062] 4. Data Analysis: The Biacore 8K+ analysis software was used to subtract the reference channel signal. A 1:1 Langmuir model was used to fit the dynamic parameters, and the equilibrium dissociation constant (KD value) was calculated. The results are as follows: Figure 2 As shown, all three candidate compounds can interact with PPARγ protein to varying degrees, with equilibrium dissociation constants (KD values) of 5.696 μM, 173 μM, and 13.3 μM, respectively. Among them, the compound liquiritin (LIQ) has the lowest KD value and the strongest binding ability to PPARγ, exhibiting a typical concentration-dose dependent characteristic, that is, the binding ability of liquiritin to the PPARγ ligand binding domain increases with increasing liquiritin concentration.

[0063] 5. Combined with pattern analysis: The lowest-energy conformation in the docking results was analyzed to observe the interactions (hydrogen bonds and hydrophobic interactions) between glycyrrhizin and key amino acid residues of PPARγ (such as TYR473, TYR327, CYS285, GLY284, LEU330, ARG288, VAL339, ILE341). Particular attention was paid to the lack of direct hydrogen bond interactions between glycyrrhizin and the H12 helix (residues HIS323 and HIS449) that regulate AF2 function. Results were visualized using PyMOL 2.5, as shown below. Figure 3 As shown.

[0064] Example 4: Dual-luciferase reporter gene assay to detect the activation effect of glycyrrhizin on PPARγ transcription.

[0065] 1. Cell culture and transfection: 293T cells in good growth condition were seeded into 24-well plates (density 0.5-2 × 10⁶ cells / well). 5 / hole); 25μL of Opti-MEM TM Culture medium and 0.75 μL of Lipofectamine TM The first mixture was obtained by mixing 3000 reagents (Invitrogen), and 25 μL of Opti-MEM was added. TM Culture medium, 1 μg of co-transfected reporter gene plasmid pPPARγ-TA-Luc (Beyotime) and internal control plasmid pRL-TK (Beyotime), and 2 μL of P3000 TMThe reagents were mixed to obtain a second mixture. The first and second mixtures were incubated at room temperature for 10-15 minutes and then mixed to obtain a transfection complex. When the cell density reached 70%, the transfection complex was added to the cells. After 24 hours of transfection, the medium was replaced with DMEM containing 10% FBS.

[0066] 2. Drug Treatment: After changing the culture medium, 293T cells were divided into 8 groups. Group 1 was treated with 10 μM glycyrrhizin solution, Group 2 with 20 μM glycyrrhizin solution, Group 3 with 40 μM glycyrrhizin solution, Group 4 with 80 μM glycyrrhizin solution, Group 5 with 100 μM glycyrrhizin solution, Group 6 with 120 μM glycyrrhizin solution, and Group 7 with 10 μM rosiglitazone solution (Rosig) as a positive control (the solvent for Groups 1-8 was DMSO). Group 8 was treated with an equal volume of DMSO as a negative control. Cells were then cultured for another 24 hours.

[0067] 3. Fluorescence Detection: After culture, discard the culture medium and wash the cells twice with PBS. Add 100 μL of reporter gene cell lysis buffer to each well and vortex for 5 minutes. Collect the lysis buffer and centrifuge at 10,000g for 3-5 minutes. Take the supernatant and add 20 μL of the supernatant to a black 96-well plate according to the instructions of the Dual Luciferase Reporter Gene Detection Kit (Beyotime). Add 100 μL of firefly luciferase detection reagent, mix well, and immediately detect firefly luciferase activity (RLU1) using a microplate reader. Then add 100 μL of Renida luciferase detection reagent (working solution prepared by adding 100× substrate to Renida luciferase detection buffer at a ratio of 1:100), mix well, and detect Renida luciferase activity (RLU2). Calculate the relative luciferase activity = RLU1 / RLU2, using the DMSO group as the baseline (100%), and calculate the relative transcriptional activity of glycyrrhizin and rosiglitazone.

[0068] The results are as follows Figure 4As shown, at a treatment concentration of 120 μM, glycyrrhizin significantly induced PPARγ transcriptional activation, but its maximum activation effect was only 35.9% of that of the positive control rosiglitazone (Rosig), indicating that its activation effect on PPARγ was significantly weaker than that of classic full agonists. Further analysis revealed that the activation efficacy of glycyrrhizin exhibited a clear concentration-dependent effect; when the treatment concentration was reduced to 10 μM, its transcriptional activation effect was comparable to that of the blank control group. These results indicate that although glycyrrhizin can specifically bind to the PPARγ ligand-binding domain, it only exhibits the typical characteristics of a partial agonist, i.e., producing a limited activation effect at higher concentrations, but still lacking the activation ability of a full agonist. This suggests that glycyrrhizin is a novel partial agonist of the PPARγ receptor.

[0069] Example 5: Detection of the effect of glycyrrhizin on adipocyte differentiation using Oil Red O staining

[0070] A standardized primary adipocyte differentiation-inducing model was used, with different group treatments. Oil Red O staining was then employed to analyze the ability of glycyrrhizin to induce preadipocyte differentiation. Rosiglitazone was used as a positive control group. The specific steps included:

[0071] 1. Cell model establishment: Primary adipocytes from C57BL / 6J mice were extracted and seeded in 6-well plates (2×10⁶ cells / wells). 5 / well). After the cells have grown to confluence, replace the induction medium I (DMEM complete medium containing 0.5mM IBMX, 1μM DEX, and 10μg / mL Insulin) and culture for 48 hours. Replace the induction medium II (DMEM complete medium containing 10μg / mL Insulin) and culture for another 48 hours until maturity (lipid droplet formation).

[0072] 2. Drug Treatment: Matured cells were divided into 5 groups: Group 1 (Vehicle) was the blank control group, receiving an equal volume of complete culture medium; Group 2 (LIQ-80) received a mixture of induction solution I and 80 μM glycyrrhizin; Group 3 (LIQ-100) received a mixture of induction solution I and 100 μM glycyrrhizin; Group 4 (LIQ-120) received a mixture of induction solution I and 120 μM glycyrrhizin; and Group 5 (Rosig-10) was the positive control group, receiving a mixture of induction solution I and 10 μM rosiglitazone. After 48 hours of treatment, the cells were replaced with the corresponding differentiation medium (with or without induction solution II) and cultured for another 48 hours.

[0073] 3. Staining: After culture, discard the culture medium and wash the cells twice with PBS. Fix with 4% paraformaldehyde at 4°C for 60 minutes. Wash twice with PBS. Add freshly prepared Oil Red O working solution and stain at room temperature in the dark for 30 minutes. Wash three times with PBS. Cover with PBS and observe and photograph under an inverted optical microscope.

[0074] The results are as follows Figure 5 As shown, the cell staining areas of the blank control group (Vehicle), experimental groups (LIQ-80, LIQ-100, LIQ-120), and positive control group (Rosig-10) were 6.448%, 8.331%, 18.296%, 29.352%, and 65.907%, respectively. The proportion of lipid droplet staining area presented by high concentrations of glycyrrhizin was significantly lower than that of the rosiglitazone positive control group, indicating that glycyrrhizin had no significant promoting effect on the differentiation process of preadipocytes, only induced a small amount of lipid accumulation, and had a weak ability to promote adipocyte differentiation.

[0075] Example 6: RT-qPCR detection of the effect of glycyrrhizin on the expression of downstream genes of PPARγ

[0076] 1. Cell treatment: Primary adipocytes that have been induced to differentiate (method as in Example 5) were divided into Vehicle group (blank control), Rosig 10μM group (positive control), and LIQ group (120μM glycyrrhizin), and treated with the corresponding drugs for 48 hours.

[0077] 2. RNA Extraction: After culturing, discard the culture medium and add 1 mL of TRIZOL (Invitrogen) to each well, lysing on ice for 15 minutes. Add 200 μL of chloroform, vortex vigorously, and incubate on ice for 10 minutes. Centrifuge at 13,000 rpm for 20 minutes at 4°C. Collect the upper aqueous phase, add an equal volume of isopropanol to precipitate RNA, and incubate at -20°C for 5 minutes. Centrifuge at 13,000 rpm for 20 minutes at 4°C. Wash the precipitate with 1 mL of pre-chilled 75% ethanol (prepared with RNase-free water) (centrifuge at 7,500 rpm for 10 minutes at 4°C). Discard the ethanol, air dry, and dissolve in 20 μL of RNase-free water. Determine the concentration and purity using NanoDrop (OD260 / 280 = 1.8–2.0).

[0078] 3. cDNA Synthesis: The Takara reverse transcription kit was used. 1 μg of total RNA was added, and a 10 μL gDNA removal mixture was prepared according to the manufacturer's instructions. The mixture was incubated at 42°C for 2 minutes. The mixture was immediately placed on an ice pack and centrifuged after cooling. Then, 1.0 μL of PrimeScript RTEnzyme Mix I, 1.0 μL of RT Primer Mix, 4.0 μL of 5X PrimeScript Buffer 2, and 4.0 μL of RNase-Free dH2O were added sequentially to obtain a total reaction volume of 20.0 μL. After thorough mixing, the mixture was briefly centrifuged before reverse transcription. The reverse transcription program was set as follows: 37°C for 15 minutes, 85°C for 5 seconds, and 4°C for 10 minutes. The resulting cDNA was diluted 10-fold for later use.

[0079] 4. Real-time PCR: qPCR was performed using the Takara high-specificity qPCR kit. A total of 20 μL of reaction mixture was prepared by mixing 2.0 μL of DNA template, 10 μL of 2X TB Green Premix Ex Taq II (Tli RNaseH Plus), 0.8 μL of 10 μM forward primer, 0.8 μL of 10 μM reverse primer, and 6.4 μL of sterile water. The nucleotide sequences of the forward and reverse primers used are shown in Table 2. The total reaction mixture was then subjected to qPCR according to the reaction procedure shown in Table 3.

[0080] Table 2. Forward and reverse primers used in qPCR reactions

[0081]

[0082] Table 3. qPCR reaction procedure

[0083]

[0084]

[0085] 5. Data Analysis: Using 2 -ΔΔCt The relative expression levels of mRNA for genes (PPARγ, CD36, aP2, C / EBPα, LPL, Fasn) were calculated using a method with β-actin as an internal reference. Data are expressed as mean ± SEM (n = 3).

[0086] The results are as follows Figure 6As shown, glycyrrhizin only weakly promotes the expression of PPARγ and its downstream lipid metabolism-related genes CD36, aP2, LPL, C / EBPα, and Fasn, with a significantly lower effect than the positive control rosiglitazone. This result is consistent with the findings obtained in adipocyte differentiation experiments. Combining the activation experiments of the PPARγ receptor, the induction of primary adipocyte differentiation phenotypes, and the expression of downstream lipid metabolism-related genes, glycyrrhizin only exhibits a weak activation effect on PPARγ and does not induce significant lipid accumulation, suggesting that it may be further studied as a novel, low-side-effect PPARγ partial agonist.

[0087] Example 7: Detection of the effects of glycyrrhizin on glucose consumption and uptake in HepG2 cells

[0088] Using HepG2 cells as the research subject, an insulin-resistant HepG2 cell model was established in vitro. The GOD-POD method was used to evaluate the regulatory effect of the candidate compound glycyrrhizin on glucose consumption in HepG2 cells. The specific steps included:

[0089] 1. Glucose consumption (GOD-POD method):

[0090] Healthy HepG2 cells were separated into 96-well plates and cultured until the cell density reached 80%. The culture medium was then discarded, and the cells were divided into groups for drug treatment. Six groups were formed, with six wells per group. The first group consisted of a blank medium group (100 mL of phenol red-free incomplete medium), an insulin group (phenol red-free incomplete medium containing 10 nM insulin), and LIQ groups (phenol red-free incomplete medium containing 10 μM, 20 μM, 40 μM, and 80 μM LIQ, respectively). After culturing at 37°C for 24 hours, the absorbance at 505 nm was measured according to the GOD-POD kit procedure. Glucose consumption for each group was calculated using the GOD-POD method and the OD value of the blank group.

[0091] The results are as follows Figure 7 As shown, compared with the blank control group, 10 nM insulin significantly promoted glucose consumption in HepG2 cells. 10-80 μM glycyrrhizin significantly increased cellular glucose consumption in a concentration-dependent manner. The effect of 80 μM glycyrrhizin was comparable to that of 10 nM insulin.

[0092] 2. The 2-NBDG method was used to evaluate the ability of glycyrrhizin to inhibit glucose uptake in HepG2 cells:

[0093] HepG2 cells in logarithmic growth phase were treated with serum-free high-glucose DMEM medium and cultured to 70% confluence. The old medium was discarded, and the cells were washed three times with PBS, then cultured for another 24 hours in DMEM high-glucose medium containing 1 μM insulin to establish an insulin resistance model. Simultaneously, HepG2 cells were cultured in high-glucose medium without insulin as a blank control group. The insulin resistance model cells were divided into 6 groups (model group, positive control group, and different concentrations of LIQ groups), with 8 wells in each group. The blank control group and model group (IR) cells were washed three times with PBS and then cultured in DMEM high-glucose medium. The positive control group (IR+Rosig) was cultured in DMEM high-glucose medium containing 10 μM rosiglitazone, and the glycyrrhizin treatment groups (LIQ groups) were cultured in DMEM high-glucose medium containing 10 μM, 20 μM, 40 μM, and 80 μM LIQ, respectively. After 24 hours of intervention, the medium was discarded, and the cells were washed three times with PBS. Add DMEM low-glucose medium containing 50 μM 2-NBDG (MedChemExpress) to each well and incubate at 37°C in the dark for 30 minutes. Discard the medium and wash three times with PBS to remove free probes. Immediately measure fluorescence intensity using a microplate reader (excitation 485 nm, emission 535 nm). Calculate the 2-NBDG uptake ratio (normalized to the blank control group).

[0094] The results are as follows Figure 8 As shown, compared with the blank control group, the 2-NBDG glucose uptake rate of the model group cells was significantly reduced. Treatment with 20-80 μM glycyrrhizin could significantly increase the uptake capacity of insulin-resistant HepG2 cells for 2-NBDG. Among them, the glucose uptake capacity of 80 μM glycyrrhizin was comparable to that of the positive control rosiglitazone.

[0095] Example 8: Glycyrrhizin improves insulin resistance in HepG2 cells

[0096] Using an insulin-induced HepG2 cell model of insulin resistance, changes in glucose consumption in cells under different treatment groups were detected, specifically including:

[0097] 1. Model establishment and processing: Same as the method for establishing the insulin resistance model in Example 7.

[0098] 2. Insulin sensitivity testing:

[0099] After successfully establishing the insulin resistance model, the experiment was divided into a blank control group, a model group, a positive control group, and a LIQ group, with six replicates in each group. The blank control group and the model group were replaced with fresh phenol red-free basal medium. Simultaneously, one column of each of the blank control group and the model group was supplemented with 10 nM insulin. The positive control group was supplemented with phenol red-free incomplete medium containing 10 μM rosiglitazone, and the glycyrrhizin group was supplemented with phenol red-free incomplete medium containing gradient concentrations of 10 μM, 20 μM, 40 μM, and 80 μM LIQ. Cell culture supernatants were collected 24 hours after intervention in each group.

[0100] Glucose consumption was determined using the glucose oxidase-peroxidase (GOD-POD) colorimetric method: 2.5 μL of supernatant and 250 μL of working solution were added to a new 96-well plate, gently shaken, and incubated at 37°C for 10 minutes. The absorbance at OD 505 nm was measured using a multi-functional microplate reader. A blank control group and a standard group were also set up, with 2.5 μL of distilled water and 2.5 μL of standard added to each group, respectively.

[0101] 3. Detection and Analysis:

[0102] Glucose consumption was measured using the same GOD-POD method as in Example 7. The model group's response to 10 nM insulin was compared (i.e., the improvement in insulin resistance). Results are as follows: Figure 9 As shown, physiological doses of insulin (10 nM) significantly increased glucose consumption in normal HepG2 cells (P < 0.001), while the model group cells induced by 1 μM insulin showed no change in glucose consumption response to the same concentration of insulin, indicating insulin resistance. Pharmacological interventions showed that 10 μM rosiglitazone, a positive control agent, significantly improved insulin resistance in the model group cells. Treatment with 40 and 80 μM glycyrrhizin also significantly increased glucose consumption in a dose-dependent manner. The effect of 80 μM glycyrrhizin was comparable to that of rosiglitazone, indicating that glycyrrhizin has the effect of improving cellular insulin resistance.

[0103] Example 9: Effects of glycyrrhizin on body weight and fasting blood glucose in insulin-resistant mice

[0104] An insulin resistance mouse model was established by feeding C57BL / 6J mice a high-fat diet for 12 weeks. Simultaneously, glycyrrhizin or rosiglitazone (a positive control) was administered daily by gavage. Mouse body weight and blood glucose were monitored weekly. Specifically, 32 seven-week-old male C57BL / 6J mice were randomly divided into four groups of eight each: Group 1 was the control group (normal diet + saline gavage); Group 2 was the model group (high-fat diet + saline gavage); Group 3 was the LIQ group (high-fat diet + 20 mg / kg glycyrrhizin gavage); and Group 4 was the Rosig group (high-fat diet + 10 mg / kg rosiglitazone gavage). Weight was recorded weekly for 12 weeks, and fasting blood glucose (FBG) was measured using a portable Roche glucometer after fasting each week.

[0105] The results are as follows Figure 10-11 As shown, the body weight of the model group mice showed a continuous upward trend. Glycyrrhizin administered by gavage significantly inhibited the increase in body weight in insulin-resistant mice. Rosiglitazone treatment resulted in mice with higher body weight than the model group mice starting from week 13, and this weight continued to increase, indicating that rosiglitazone can induce a weight gain effect, consistent with previous reports. Simultaneously, insulin-resistant mice showed a significant increase in blood glucose levels, with a significant difference compared to the control group starting from week 9. Glycyrrhizin treatment significantly reduced fasting blood glucose levels in insulin-resistant mice, with an effect comparable to that of the positive control drug rosiglitazone.

[0106] Example 10: Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT)

[0107] OGTT: On the third day of the last week of the experiment, mice were fasted for 16 hours but allowed free access to water. They were then administered 2 g / kg D-glucose solution by gavage. Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 minutes via tail tip sampling. The area under the blood glucose curve (AUC) was calculated. Results are as follows: Figure 12-13 As shown, the model group mice showed a significant increase in blood glucose after glucose gavage. The basal blood glucose level in the glycyrrhizin group was lower than that in the model group. When glucose was administered orally for 15 minutes, glycyrrhizin could significantly reduce the blood glucose level in insulin-resistant mice, with an effect comparable to that of 10 mg / kg rosiglitazone.

[0108] ITT: Three days after the OGTT, mice were fasted but allowed free water for 6 hours. They were then injected intraperitoneally with 0.75 U / kg insulin solution. Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 minutes via tail tip sampling. The AUC of blood glucose was calculated. Results are as follows. Figure 14-15As shown, the blood glucose in the model group gradually recovered 60 minutes after insulin injection. The blood glucose in the glycyrrhizin or rosiglitazone groups was lower than that in the model group within 0-120 minutes, showing a good effect in improving insulin resistance. The above results indicate that glycyrrhizin can significantly improve the insulin sensitivity of insulin-resistant mice.

[0109] Example 11: Effects of glycyrrhizin on blood lipids, blood glucose, and liver and kidney function in insulin-resistant mice

[0110] Mice were fasted for 12 hours after the experiment, anesthetized with isoflurane, and blood was collected from the carotid artery. Serum was separated (3000 rpm, 10 minutes) and stored at -80℃. The following indicators were measured using a fully automated biochemical analyzer: total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), blood glucose (GLU), alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatinine (Scr), uric acid (UA), and urea (UREA). The results are as follows: Figure 16-18 As shown. According to Figure 16 It was found that, compared with the normal group, the levels of TC, TG, LDL-c, and GLU in the model group mice were significantly increased, and the differences were statistically significant. Compared with the model group, administration of glycyrrhizin or rosiglitazone significantly improved the levels of TC, TG, LDL-c, and GLU in insulin-resistant mice, but had no significant effect on HDL-c levels. Figure 17 It was found that the ALT and AST levels in the model group mice were significantly higher than those in the normal group, indicating that the model group mice had obvious liver damage. After intervention with glycyrrhizin or rosiglitazone, the ALT and AST levels decreased, showing a significant difference compared with the model group. Kidney damage is one of the most common long-term complications of chronic diseases such as diabetes. By detecting the renal function indicators of each group of mice, according to... Figure 18 It can be seen that, compared with the normal group, the serum urea, uric acid and creatinine levels of mice in the model group were significantly increased, indicating that insulin resistance induced by a high-fat diet may lead to kidney damage in mice, and administration of glycyrrhizin or rosiglitazone can improve the levels of renal function-related indicators in insulin-resistant mice.

[0111] Example 12: Effects of glycyrrhizin on serum inflammatory factor levels in insulin-resistant mice

[0112] The levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and chemokine ligand 2 (CCL2) in the serum collected in Example 11 were detected using an ELISA kit, following the specific procedures outlined in the kit instructions. Results are as follows: Figure 19As shown, compared with control mice, insulin-resistant mice generally exhibited elevated levels of TNF-α, IL-6, IL-1β, and CCL2 in their serum, while treatment with glycyrrhizin or rosiglitazone significantly reduced these inflammatory factors. These results indicate that glycyrrhizin possesses a strong anti-inflammatory effect in insulin resistance.

[0113] Example 13: Effects of glycyrrhizin on the quality of major organ tissues in insulin-resistant mice

[0114] After blood was collected from the mice at the end of the experiment in Example 11, the heart, liver, kidneys, spleen, pancreas, posterior abdominal wall fat, and epididymal adipose tissue were dissected and removed. The tissues were rinsed with physiological saline, blotted dry with filter paper, weighed, and recorded. Data are expressed as mean ± SEM (n = 8). Results are as follows: Figure 20 As shown, rosiglitazone induces an increase in the volume and mass of liver, kidney, posterior abdominal wall fat, and epididymal adipose tissue in mice. Unlike the effects of the full agonist rosiglitazone, treatment with glycyrrhizin did not result in pathological volume increases in these tissues. Further analysis revealed that glycyrrhizin significantly reduced the mass of posterior abdominal wall fat and epididymal adipose tissue compared to the insulin resistance model group. These results indicate that glycyrrhizin does not cause adverse effects such as increased weight in major organs and tissues associated with rosiglitazone.

[0115] Example 14: Effects of glycyrrhizin on adipose tissue content (MRI) and morphology (H&E) in insulin-resistant mice

[0116] Mice were anesthetized with 1-2% isoflurane and fixed in a BioSpec 70 / 20USR small animal MRI scanner. A three-dimensional fast gradient echo sequence was used (parameters: TR = 12 ms, TE = 2.5 ms, flip angle 15°, FOV 8x4x4 cm). 3 A full-body fat scan was performed using a 256x128x128 matrix (NSA = 2). The percentage of fat content was calculated using Bruker Paravision 6.0 software: Fat content (%) = (Total fat volume (TFV) / Total body volume) × 100%. Results are as follows... Figure 21 As shown, compared with the control group, the adipose tissue content of insulin-resistant mice was significantly increased. The rosiglitazone intervention group showed an increasing trend in adipose tissue content compared with the model group, consistent with the increase in body weight and adipose tissue weight induced by rosiglitazone. However, compared with the model group, the adipose tissue content in the glycyrrhizin group was significantly decreased. These results further demonstrate that glycyrrhizin, unlike the classic drug rosiglitazone, can effectively improve abnormal lipid accumulation in insulin-resistant mice.

[0117] Hematoxylin and eosin (H&E) staining was performed on mouse epididymal and subcutaneous adipose tissue. Specifically, white epididymal and subcutaneous adipose tissue were collected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Sections were dewaxed to water, stained with hematoxylin for 3-5 minutes, allowed to differentiate and turn blue, and counterstained with eosin for 15 seconds. The sections were dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin. The morphology and size of adipocytes were observed under a light microscope. Results are as follows: Figure 22 As shown, the epididymal and subcutaneous adipose tissues of insulin-resistant mice exhibited a typical increase in adipocyte volume. The morphology of adipose tissue in the rosiglitazone treatment group was not improved, while glycyrrhizin intervention could resist the continuous hypertrophy of adipocytes caused by insulin resistance. H&E staining results showed that glycyrrhizin significantly reduced the volume of adipocytes in the epididymal and subcutaneous adipose tissues.

[0118] Example 15: Effects of glycyrrhizin on heart tissue morphology (H&E) in insulin-resistant mice

[0119] Mouse heart tissue was collected, rinsed with physiological saline, and fixed with 4% paraformaldehyde. The heart tissue was stained using the same H&E staining procedure as in Example 14, and myocardial structure was observed under a light microscope, with a focus on assessing left ventricular wall thickness (measured and analyzed using ImageJ software). Data are presented as mean ± SEM (n = 8).

[0120] The results are as follows Figure 23 As shown, rosiglitazone significantly increased left ventricular wall thickness, while no significant pathological changes were observed in the myocardial structure of the glycyrrhizin-treated group. This result indicates that glycyrrhizin does not exhibit the cardiotoxicity risks associated with rosiglitazone, such as myocardial hypertrophy.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of glycyrrhizin or its pharmaceutically acceptable salts in the preparation of PPARγ receptor partial agonists.

2. The application according to claim 1, characterized in that, The relative activation efficiency of glycyrrhizin on PPARγ receptors is lower than that of complete PPARγ agonists on PPARγ receptors.

3. The application according to claim 2, characterized in that, The PPARγ complete agonist is a thiazolidinedione compound.

4. The application according to claim 2 or 3, characterized in that, The complete PPARγ agonist is rosiglitazone.

5. A PPARγ receptor partial agonist, characterized in that, This includes glycyrrhizin or its pharmaceutically acceptable salts.

6. The PPARγ receptor partial agonist according to claim 5, characterized in that, The agonist also includes a pharmaceutically acceptable carrier.

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