Classification method for verifying burden-reducing and transsaccharide hypoglycemic drugs

Abstract

The invention discloses a classification method for verifying burden-reducing and transsaccharide hypoglycemic drugs, and belongs to the technical field of drug relationship classification, male mice are selected, are freely fed with common feed and then are replaced with high-fat feed, STZ injection is carried out, then the mice are selected and grouped, and the drug administration volume is adjusted according to the body weight; metabolism cage detection, OGTT / IRT and dynamic blood glucose detection are carried out alternately; carrying out sample collection according to a sequence of heart, kidney, liver, quadriceps femoris muscle, aorta and pancreas; establishing a database for data analysis; detecting a tissue morphological structure, a kidney SGLT2 protein expression quantity, a liver glycogen content and muscle GLUT4 membrane translocation efficiency; detecting the sensitivity of serum beta-hydroxybutyric acid (ketone body) and insulin; and pathological analysis is carried out through aorta oil red O and pancreas beta cell morphological staining. According to the method, a'burden-reducing type-transsaccharide 'classification standard based on a physiological mechanism is created for the first time, the problem that traditional chemical structure / target point classification is disjointed with drug effects is solved, and the classification accuracy reaches 92.3% (n is equal to 60 animal verification).

Description

Technical Field

[0001] The present invention belongs to the technical field of drug relationship classification, and specifically relates to a classification method for verifying burden-reducing and sugar-converting hypoglycemic drugs. Background Art

[0002] In the process of atherosclerosis, the aggregation of macrophages in the blood vessel wall and the formation of foam cells are closely related to cholesterol metabolism disorders and inflammatory responses; modified lipoproteins and cholesterol crystals induce NLRPS inflammasome activation, that is, atherosclerosis integrates cholesterol metabolism and inflammatory response. The current classification of glucose-lowering drugs mainly relies on chemical structure (such as sulfonylureas and biguanides) or molecular targets (such as SGLT2 inhibitors and DPP-4 inhibitors). This approach has significant flaws: ① Disconnection between mechanism and classification - drugs with the same physiological effects are separated and classified separately, for example, SGLT2 inhibitors promote glucose excretion and GLP-1 receptor agonists suppress appetite, which both belong to the "load-reducing" mechanism but belong to different categories; ② One-sided verification indicators - over-reliance on endpoint parameters such as blood glucose / HbA1c, ignoring the dynamic distribution of glucose load (urinary glucose excretion) and organ-specific glucose disposal (muscle GLUT4 translocation rate); ③ Lack of combination drug mechanism - existing models cannot verify the synergistic effect of "load-reducing + glucose-transforming" combination preparations; ④ Weak association with comorbidity improvement - there is a lack of a classification-oriented correlation evaluation system between the improvement of arteriosclerosis by load-reducing drugs (such as SGLT2i) and the protection of β cells by glucose-transforming drugs (such as sulfonylureas).

[0003] Therefore, it is urgent to establish a classification verification method based on the "load reduction-sugar conversion" physiological pathway, and to solve the fundamental limitations of the existing classification system in mechanism interpretation and clinical transformation by integrating dynamic load monitoring (urinary sugar excretion, blood sugar fluctuation MAGE value), terminal mechanism detection (liver glycogen content, GLUT4 membrane translocation rate) and comorbid organ assessment (aortic plaque area, β-cell score). Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a classification method for verifying blood sugar-lowering drugs of the burden-reducing type and the sugar-converting type, so as to solve the above problems.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a classification method for verifying blood sugar-lowering drugs of the burden-reducing type and the blood sugar-converting type, comprising the following steps:

[0007] S1. Male mice were selected and fed with a normal diet for one week. The normal diet was then replaced with a high-fat diet for another week. In the third week, STZ was injected. Mice with blood glucose levels ≥11.1 mmol / L for two consecutive times were selected.

[0008] S2. The selected mice were divided into groups and the administration volume was adjusted according to their body weight;

[0009] S3, interspersed with metabolic cage testing, OGTT / IRT, and dynamic blood glucose testing;

[0010] S4. Collect samples in the order of heart, kidney, liver, quadriceps, aorta, and pancreas;

[0011] S5. Establish a database for data analysis;

[0012] S6. Detect tissue morphology and structure, renal SGLT2 protein expression, liver glycogen content, and muscle GLUT4 membrane translocation efficiency;

[0013] S7. Detect serum β-hydroxybutyrate (ketone bodies) and insulin sensitivity;

[0014] S8, pathological analysis was performed by staining aorta with Oil Red O and pancreatic β-cell morphology;

[0015] S9. Conduct theoretical verification.

[0016] Furthermore, in step S1, the selected mice were 8-week-old mice, and 6 groups were set up, with 10 mice in each group, and a 12h / 12h light-dark cycle was maintained during feeding;

[0017] The fat content of the high-fat feed is 60%;

[0018] The STZ was injected intraperitoneally using a citric acid buffer solution with a pH value of 4.5 and STZ powder to prepare STZ, and was injected at a dose of 40 mg / kg / day for 5 days.

[0019] Furthermore, in step S2, the groups are HC group, DM / VC group, UR group, TR group, PC group, and VC group;

[0020] The HC group was gavaged with 0.1 ml / 10 g of normal saline.

[0021] The DM / VC group was gavaged with 0.5% carboxymethyl cellulose.

[0022] The UR group was administered with either 10 mg / kg of empagliflozin or 30 mg / kg of acarbose by oral gavage.

[0023] The TR group was administered with either 200 mg / kg of metformin or 0.5 U / kg of insulin by oral gavage.

[0024] The PC group was administered metformin + empagliflozin by gavage.

[0025] The VC group was intragastrically administered with carboxymethylcellulose.

[0026] Furthermore, in step S3, the metabolic cage detection method is to first transfer the animals to the metabolic cage for 12 hours of adaptation, collect urine for 24 hours, measure urine and urine volume, and record food intake and water intake;

[0027] The OGTT / IRT test method is to fast for 12 hours the night before, then measure fasting blood sugar at 9 am, gavage 20% glucose solution 5 minutes later, and then measure blood sugar at the tail tip every 15 minutes;

[0028] The dynamic blood glucose monitoring method is to implant a CGM probe subcutaneously in the groin and continuously record blood glucose for 72 hours.

[0029] Furthermore, in step S5, blood glucose dynamics, urine glucose excretion, and histological scores are recorded in the database, and then statistics are performed using inter-group comparison and correlation analysis.

[0030] Furthermore, in step S6, when detecting renal SGLT2 protein expression, heat-induced antigen retrieval is first performed at 95°C using a sodium citrate buffer with a pH of 6.0, and then incubated overnight at 4°C using an Anti-SGLT2 antibody at a dilution ratio of 1:200. The DAB color development time is then controlled in real time by a microscope, with the appearance of specific brown deposits on the renal tubular brush border as the endpoint. The percentage of renal tubular brush border-positive area is then quantified using ImageJ software;

[0031] To detect the liver glycogen content, 8 μm frozen sections were first mounted on anti-slip slides, then oxidized with periodic acid for 10 minutes and washed with distilled water, stained with Schiff reagent for 15 minutes, rinsed with running water for 5 minutes, and counterstained with hematoxylin to differentiate and return to blue.

[0032] When detecting muscle GLUT4 membrane translocation, quadriceps femoris tissue was first taken and the cytoplasm and membrane protein components were separated using a membrane protein separation kit. After quantification by BCA method, equal amounts of membrane protein were taken for Western Blot detection. The primary antibodies used were anti-GLUT4 antibody and membrane marker antibody anti-Na + / K + ATPase, anti-GAPDH antibody was used as the internal control for cytoplasm, and then incubated with HRP-labeled secondary antibody and developed by ECL. The grayscale ratio of membrane fraction GLUT4 and cytoplasmic GLUT4 was calculated using Image Lab software.

[0033] Furthermore, in step S7, when detecting serum β-hydroxybutyrate (ketone bodies), first take 10,000g of serum from mice that have been fasted for 6 hours and centrifuge for 5 minutes to remove protein, take 50 μl of the supernatant and mix it with 200 μl of the working solution, incubate at 37°C for 5 minutes, and then immediately measure the absorbance at a wavelength of 450 nm. A standard curve is drawn with a known concentration of β-hydroxybutyrate standard to calculate the serum ketone body content;

[0034] When testing insulin sensitivity, serum was collected from mice that had fasted for 6 hours, and the fasting insulin concentration was measured using an ELISA kit. At the same time, fasting blood glucose was measured using the glucose oxidase method, and the insulin resistance index was calculated.

[0035] Furthermore, in step S8, when staining the aorta with Oil Red O, the frozen aorta sections were first embedded in OCT, then rinsed with 60% isopropanol and stained with Oil Red O for 15 minutes, and then color-separated with differentiation solution (60% isopropanol) and counterstained with hematoxylin;

[0036] For pancreatic β cell morphology staining, paraffin sections were first sliced, and then the nuclei were stained with hematoxylin for 5 min, after which the nuclei were differentiated and returned to blue. The cytoplasm was then stained with eosin for 1 min, followed by dehydration with graded ethanol.

[0037] The beneficial effects of the present invention are:

[0038] 1. The present invention pioneered a "burden-reducing type-glycogen conversion type" classification standard based on physiological mechanisms, which is defined by quantitative indicators: ① Burden-reducing type: urine sugar excretion ≥20mg / 24h (a 5-fold increase in the basal value of the diabetic model) and a ≥15% decrease in food intake; ② Glycogen conversion type: liver glycogen content ≥3.5mg / g tissue (a 1.8-fold increase in the normal value) and a ≥25% increase in the muscle GLUT4 membrane translocation rate; this solves the problem of disconnection between traditional chemical structure / target classification and drug efficacy, with a classification accuracy rate of 92.3% (n=60 animal verification).

[0039] 2. The present invention constructs a multi-dimensional verification system to simultaneously achieve: 1. Dynamic load monitoring: CGM captures MAGE values ​​(sensitivity of blood glucose fluctuation assessment increases by 3.2 times) + metabolic cage quantification of urine glucose excretion (detection efficiency increases by 80%); 2. Terminal mechanism detection: WB / immunohistochemistry simultaneous analysis of renal SGLT2, liver glycogen, and muscle GLUT4 (data dimension expanded by 200%); 3. Comorbidity association evaluation: Oil red O staining of aortic plaques (area decreases ≥45%) + HE scoring of β-cell damage (improvement rate increases by 60%); breaking through the traditional single blood glucose endpoint verification model, the experimental cost is reduced by 37%.

[0040] 3. The present invention designed a "burden-reducing + sugar-converting compound drug control group" to analyze the synergistic mechanism: ① The compound group (such as metformin + empagliflozin) significantly improved the efficacy compared with the single drug group (FPG decreased by 18.2% vs. a single drug decrease of 9.3-11.7%, p<0.01); ② Revealed organ protection synergy: arterial plaque area decreased by 52.4% (single drug decreased by 28.6-36.1%) + β-cell damage score improved by 2.1 levels (single drug increased by 0.8-1.3 levels).

[0041] 4. The present invention establishes a clinical translation decision-making model, which realizes ① individualized medication matching: weight-reducing drugs are preferred for obesity (weight loss ≥ 10%); sugar conversion drugs are preferred for β-cell failure (functional recovery rate increased by 65%); ② metabolic risk warning: serum β-hydroxybutyrate detection (threshold > 2.8mM) is used to predict the ketosis risk of weight-reducing drugs in advance; clinical treatment efficacy is increased by 35%, and adverse reaction rate is reduced by 50%.

[0042] 5. The present invention has technical compatibility and universality: ① It is compatible with conventional experimental platforms (WB / IHC / CGM, etc.) and does not require special equipment; ② It covers the verification of 12 types of antidiabetic drugs (including SGLT2i, GLP-1RA, sulfonylureas, etc.) with an accuracy rate of >90%; and forms a "dual-pathway verification model + synergistic effect evaluation" methodological barrier.

[0043] Other advantages, objectives and features of the present invention will be described in the following description and will be apparent to those skilled in the art to some extent, or those skilled in the art can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to make the purpose, technical solutions and beneficial effects of the invention clearer, the present invention is described with the following drawings:

[0045] Figure 1 This is a schematic diagram of the establishment of the animal model and grouping and drug administration of the present invention;

[0046] Figure 2 It is a schematic diagram of the dynamic monitoring process of the present invention;

[0047] Figure 3 This is a schematic diagram of the tissue mechanism detection of the present invention;

[0048] Figure 4 This is a schematic diagram of the classification standard and verification of the present invention. DETAILED DESCRIPTION

[0049] like Figure 1-4 As shown, the present invention provides a classification method for verifying burden-reducing and sugar-converting hypoglycemic drugs.

[0050] Example 1

[0051] S1. Eight-week-old male mice were selected and divided into six groups of 10 mice each. After being fed a standard diet ad libitum for one week, the standard diet was replaced with a high-fat diet containing 60% fat for another week (maintaining a 12-h / 12-h light / dark cycle). In the third week, STZ was intraperitoneally injected at 40 mg / kg / day (STZ powder was prepared by mixing citric acid buffer with pH 4.5) for 5 days. Mice with blood glucose levels ≥11.1 mmol / L for two consecutive times were selected.

[0052] S2. The selected mice were divided into HC group, DM / VC group, UR group, TR group, PC group, and VC group. The HC group was gavaged with 0.1 ml / 10 g normal saline, the DM / VC group was gavaged with 0.5% carboxymethylcellulose, the UR group was gavaged with either 10 mg / kg empagliflozin or 30 mg / kg acarbose, the TR group was gavaged with either 200 mg / kg metformin or 0.5 U / kg insulin, the PC group was gavaged with metformin + empagliflozin, and the VC group was gavaged with carboxymethylcellulose;

[0053] S3, interspersed with metabolic cage testing, OGTT / IRT testing, and dynamic blood glucose testing;

[0054] The metabolic cage test method is to first transfer the animals to the metabolic cage to adapt for 12 hours, collect urine for 24 hours, measure urine volume, and record food intake and water intake;

[0055] The OGTT / IRT test method is to fast for 12 hours the night before, then measure fasting blood sugar at 9 am, gavage 20% glucose solution 5 minutes later, and then collect blood sugar at the tail tip every 15 minutes;

[0056] The dynamic blood glucose monitoring method is to implant a CGM probe subcutaneously in the groin to continuously record blood glucose for 72 hours;

[0057] S4. Collect samples in the order of heart, kidney, liver, quadriceps, aorta, and pancreas;

[0058] S5. Establish a database to analyze blood glucose dynamics, urine glucose excretion, and histological scores;

[0059] S6. Detect tissue morphology and structure, renal SGLT2 protein expression, liver glycogen content, and muscle GLUT4 membrane translocation efficiency;

[0060] When detecting renal SGLT2 protein expression, the antigen was first retrieval was induced by heat at 95°C using sodium citrate buffer at pH 6.0, and then incubated with Anti-SGLT2 (Abcam ab14687) antibody at a dilution ratio of 1:200 at 4°C overnight. The DAB color development time was then controlled in real time by a microscope, with the appearance of specific brown deposits on the renal tubular brush border as the endpoint. The percentage of renal tubular brush border positive area was then quantified using ImageJ software. The positive rate (%) = (SGLT2 + Pixel area / total renal tubular area) × 100, used to verify the characteristics of the burden-reducing hypoglycemic drugs: the positive rate of renal SGLT2 in the UR group was significantly higher than that in the DM group (p < 0.01), while there was no statistical difference in the TR group;

[0061] To detect the liver glycogen content, 8 μm frozen sections were first mounted on anti-slip slides, then oxidized with periodic acid for 10 minutes and washed with distilled water, stained with Schiff reagent for 15 minutes, rinsed with running water for 5 minutes, and counterstained with hematoxylin to differentiate and return to blue.

[0062] When detecting muscle GLUT4 membrane translocation, quadriceps femoris tissue was first taken and the cytoplasmic and membrane protein components were separated using a membrane protein separation kit (ThermoFisher89842). After quantification by BCA method, equal amounts of membrane protein were taken for Western Blot detection. The primary antibodies used were anti-GLUT4 antibody and membrane marker antibody anti-Na + / K + ATPase (Abcam ab76020, 1:2000) and anti-GAPDH antibody were used as internal control for cytoplasm. After incubation with HRP-conjugated secondary antibody, ECL development was performed. Image Lab software was used to calculate the grayscale ratio of membrane fraction GLUT4 to cytoplasmic GLUT4 (GLUT4-mem / GLUT4-cyto). An increase in this ratio indicates enhanced translocation of GLUT4 to the cell membrane.

[0063] S7. Detect serum β-hydroxybutyrate (ketone bodies) and insulin sensitivity;

[0064] When testing serum β-hydroxybutyrate (ketone bodies), first take the serum of mice fasting for 6 hours at 10000g and centrifuge for 5 minutes to remove protein, then take 50μl of supernatant and 200μl of working solution (containing β-hydroxybutyrate dehydrogenase and NAD +, Cayman Chemical #700190) and incubated at 37°C for 5 minutes. The absorbance at 450 nm (OD value) was immediately measured. A standard curve was drawn using β-hydroxybutyrate standards of known concentrations according to the formula: β-hydroxybutyrate concentration (mM) = (sample OD value / standard OD value) × standard concentration. Serum ketone body levels were calculated. A significant increase in this indicator (>1.0 mM) indicates the characteristic effect of weight-reducing drugs in promoting lipid metabolism.

[0065] To test insulin sensitivity, serum was collected from mice fasting for 6 hours, and fasting insulin concentration (μIU / ml) was measured using an ELISA kit (Mercodia 10-1249-01). Fasting blood glucose (mmol / L) was simultaneously measured using the glucose oxidase method. The insulin resistance index (HIMA-IR) was calculated using the formula: [fasting blood glucose * fasting insulin] / 22.5. A significant decrease in this value (<3.0) indicates the core effect of glucose-transferring drugs in improving insulin sensitivity.

[0066] S8, pathological analysis was performed by staining aorta with Oil Red O and pancreatic β-cell morphology;

[0067] When using aorta oil red O staining, first embed the aorta frozen section with OCT, then rinse with 60% isopropanol and stain with oil red O for 15 minutes, then use differentiation solution (60% isopropanol) for color separation and counterstain with hematoxylin;

[0068] When using pancreatic β-cell morphological staining, paraffin sections were first sliced, then the nuclei were stained with hematoxylin for 5 minutes and then differentiated and returned to blue, and then the cytoplasm was stained with eosin for 1 minute and then dehydrated with graded ethanol;

[0069] S9. Conduct theoretical verification and find that weight-reducing drugs can significantly improve blood sugar homeostasis indicators, intestinal sugar absorption, and body weight, and arteriosclerosis, with plaques decreasing by 50%, while urine sugar and serum ketone bodies increase.

[0070] In the case of glucose-converting drugs, basal blood sugar decreases, liver glycogen, muscle glucose uptake, and insulin sensitivity increase, body weight remains constant, and pancreatic β-cell function is partially restored.

[0071] The present invention is based on the dual-pathway regulation theory of the body's glucose metabolism balance:

[0072] 1. Sugar load unloading pathway (load reduction)

[0073] Directly reduce circulating blood glucose load by promoting glucose excretion (renal SGLT2-mediated increase in urine glucose) and reducing glucose intake (intestinal α-glucosidase inhibition).

[0074] Its core verification indicators are:

[0075] ① Increased urine sugar excretion;

[0076] ② Increased expression of SGLT2 in renal tubules;

[0077] ③Increase in serum ketone bodies.

[0078] 2. Sugar conversion and utilization pathway (conversion to sugars)

[0079] By enhancing sugar storage (increased liver glycogen synthesis) and sugar uptake (increased muscle GLUT4 membrane translocation), it improves the sugar utilization rate of peripheral tissues. Its core verification indicators are:

[0080] ① Increased liver glycogen content;

[0081] ② The GLUT4 membrane / plasma ratio increased;

[0082] ③HOMA-IR decreases.

[0083] 3. Scientific Basis for Classification Verification

[0084]

[0085] 4. Principles of detection methodology

[0086] ① Dynamic blood glucose fluctuation (MAGE value): Continuous CGM monitoring for 72 hours, calculating the average blood glucose fluctuation amplitude, and obtaining a specific indicator for weight reduction (due to direct blood glucose clearance, the fluctuation suppression rate is greater than 30%);

[0087] ② Membrane protein separation GLUT4 detection: Thermo Fisher 89842 kit was used to separate the cell membrane components, and Na + / K + ATPase is used as a membrane marker to accurately quantify the GLUT4 membrane translocation efficiency of transglycosylation drugs;

[0088] ③ Oil Red O staining to quantify plaques: 60% isopropyl alcohol selectively dissolves nonspecific lipids, resulting in only atherosclerotic plaques remaining red (step S8), objectively reflecting the vascular protective effect of the weight-reducing type.

[0089] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A classification method for verifying blood sugar-lowering drugs of the burden-reducing and sugar-transforming type, characterized by: The following steps are included: S1. Male mice were selected and fed with a normal diet for one week. The normal diet was then replaced with a high-fat diet for another week. In the third week, STZ was injected. Mice with blood glucose levels ≥11.1 mmol / L for two consecutive times were selected. S2. The selected mice were divided into groups and the administration volume was adjusted according to their body weight; S3, interspersed with metabolic cage testing, OGTT / IRT, and dynamic blood glucose testing; S4. Collect samples in the order of heart, kidney, liver, quadriceps, aorta, and pancreas; S5. Establish a database for data analysis; S6. Detect tissue morphology and structure, renal SGLT2 protein expression, liver glycogen content, and muscle GLUT4 membrane translocation efficiency; S7. Detect serum β-hydroxybutyrate (ketone bodies) and insulin sensitivity; S8, pathological analysis was performed by staining aorta with Oil Red O and pancreatic β-cell morphology; S9. Conduct theoretical verification.

2. A classification method for verifying blood sugar-lowering drugs of the burden-reducing and glucose-converting types according to claim 1, characterized in that: In step S1, the selected mice were 8-week-old mice, and 6 groups were set up, with 10 mice in each group, and the feeding was maintained on a 12h / 12h light-dark cycle; The fat content of the high-fat feed is 60%; The STZ was injected intraperitoneally using a citric acid buffer solution with a pH value of 4.5 and STZ powder to prepare STZ, and was injected at a dose of 40 mg / kg / day for 5 days.

3. A classification method for verifying blood sugar-lowering drugs of the burden-reducing and glucose-converting types according to claim 1, characterized in that: In step S2, the groups are HC group, DM / VC group, UR group, TR group, PC group, and VC group; The HC group was gavaged with 0.1 ml / 10 g of normal saline. The DM / VC group was gavaged with 0.5% carboxymethyl cellulose. The UR group was administered with either 10 mg / kg of empagliflozin or 30 mg / kg of acarbose by oral gavage. The TR group was administered with either 200 mg / kg of metformin or 0.5 U / kg of insulin by oral gavage. The PC group was administered metformin + empagliflozin by gavage. The VC group was intragastrically administered with carboxymethylcellulose.

4. A classification method for verifying blood sugar-lowering drugs of the burden-reducing and glucose-converting types according to claim 1, characterized in that: In step S3, the metabolic cage detection method is to first transfer the animal to the metabolic cage to adapt for 12 hours, collect urine for 24 hours, measure urine and urine volume, and record food intake and water intake; The OGTT / IRT test method is to fast for 12 hours the night before, then measure fasting blood sugar at 9 am, gavage 20% glucose solution 5 minutes later, and then measure blood sugar at the tail tip every 15 minutes; The dynamic blood glucose monitoring method is to implant a CGM probe subcutaneously in the groin and continuously record blood glucose for 72 hours.

5. A classification method for verifying blood sugar-lowering drugs of the burden-reducing and glucose-converting types according to claim 1, characterized in that: In step S5, blood glucose dynamics, urine glucose excretion, and histological scores are recorded in the database, and then statistics are performed using inter-group comparison and correlation analysis.

6. A classification method for verifying blood sugar-lowering drugs of the burden-reducing and glucose-converting types according to claim 1, characterized in that: In step S6, when detecting renal SGLT2 protein expression, first use sodium citrate buffer with a pH value of 6.0 at 95°C for heat-induced antigen retrieval, then use anti-SGLT2 antibody at a dilution ratio of 1:200 and incubate at 4°C overnight, then the DAB color development time is controlled in real time by a microscope, and the appearance of specific brown deposits on the renal tubular brush border is used as the endpoint, and then the proportion of the renal tubular brush border positive area is quantified by ImageJ software; To detect the liver glycogen content, 8 μm frozen sections were first mounted on anti-slip slides, then oxidized with periodic acid for 10 minutes and washed with distilled water, stained with Schiff reagent for 15 minutes, rinsed with running water for 5 minutes, and counterstained with hematoxylin to differentiate and return to blue. When detecting muscle GLUT4 membrane translocation, quadriceps femoris tissue was first taken and the cytoplasm and membrane protein components were separated using a membrane protein separation kit. After quantification by BCA method, equal amounts of membrane protein were taken for Western Blot detection. The primary antibodies used were anti-GLUT4 antibody and membrane marker antibody anti-Na + / K + ATPase, anti-GAPDH antibody was used as the internal control for cytoplasm, and then incubated with HRP-labeled secondary antibody and developed by ECL. The grayscale ratio of membrane fraction GLUT4 and cytoplasmic GLUT4 was calculated using Image Lab software.

7. A classification method for verifying blood sugar-lowering drugs of the burden-reducing and glucose-converting types according to claim 1, characterized in that: In step S7, when detecting serum β-hydroxybutyrate (ketone bodies), first take 10,000g of serum from mice that have been fasted for 6 hours and centrifuge it for 5 minutes to remove protein. Then, take 50 μl of the supernatant and mix it with 200 μl of the working solution. Incubate at 37°C for 5 minutes, and then immediately measure the absorbance at a wavelength of 450 nm. A standard curve is drawn with a known concentration of β-hydroxybutyrate standard to calculate the serum ketone body content. When testing insulin sensitivity, serum was collected from mice that had fasted for 6 hours, and the fasting insulin concentration was measured using an ELISA kit. At the same time, fasting blood glucose was measured using the glucose oxidase method, and the insulin resistance index was calculated.

8. A classification method for verifying blood sugar-lowering drugs of the burden-reducing and glucose-converting types according to claim 1, characterized in that: In step S8, when staining the aorta with Oil Red O, first embed the frozen aorta section with OCT, then rinse with 60% isopropanol and stain with Oil Red O for 15 minutes, then use differentiation solution (60% isopropanol) for color separation and counterstain with hematoxylin; For pancreatic β cell morphology staining, paraffin sections were first sliced, and then the nuclei were stained with hematoxylin for 5 min, after which the nuclei were differentiated and returned to blue. The cytoplasm was then stained with eosin for 1 min, followed by dehydration with graded ethanol.

Images