Multi-dimensional evaluation method for insulin resistance hepatocytes

Abstract

The invention provides a multi-dimensional evaluation method for insulin-resistant hepatocytes, which specifically comprises the following steps: performing phenotype screening on an insulin-resistant hepatocyte model, constructing a cell model and setting experimental groups; the influence on an insulin signal channel is verified through western blot; and finally performing comprehensive evaluation: when the phenotypic index is not matched with the mechanism index, further verifying the inspection range of time gradient, mitochondrial dynamics related protein or insulin signal pathway key protein so as to determine the subsequent research direction, and the method realizes in-situ correlation analysis of morphology, function and molecular pathways, is comprehensive in evaluation dimension, and is suitable for large-scale popularization and application. And in-depth research can be efficiently guided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical detection and evaluation, and particularly relates to a multi-dimensional evaluation method for insulin-resistant hepatocytes. BACKGROUND

[0002] The core pathology of type 2 diabetes is insulin resistance, which is directly related to intracellular oxidative stress and mitochondrial dysfunction. In a high-sugar and high-fat environment, key metabolic sites such as hepatocytes will produce excess reactive oxygen species, which will damage mitochondrial function and inhibit the insulin signaling pathway, ultimately leading to glucose uptake disorders. This mechanism requires simultaneous and in situ observation of mitochondrial function morphology and correlation with insulin signal changes in a cell model. However, the existing technology has obvious limitations: 1) traditional biochemical methods can detect signaling pathway proteins, but require cell lysis, which loses spatial information and cannot be correlated with imaging; 2) conventional microscopic imaging techniques have insufficient resolution, making it difficult to clearly distinguish the fine structure of mitochondria and their colocalization with ROS; 3) existing membrane potential-dependent functional imaging techniques use probes for imaging, but this method has reliability challenges in pathological models because the membrane potential itself decreases in insulin resistance models, which can cause distortion of signals from such probes, affecting the accuracy of quantification; 4) the observation dimensions of various technologies are fragmented, and there is a lack of integrated in situ multi-dimensional evaluation means for morphology, function, and molecular pathways.

[0003] The existing technology, such as Chinese patent application number CN202510158322.7, published on April 8, 2025, discloses a liver cancer target detection method based on staining and sequencing, but this method focuses on morphological observation and gene enrichment analysis.

[0004] However, the above documents are mainly used for morphological observation. Although current structured light illumination microscopy and other super-resolution techniques can improve resolution to about 100 nm, they have not yet been effectively integrated for simultaneous quantitative mitochondrial function indicators and in situ correlation analysis with insulin signaling pathways. The mitochondrial function indicators can be used for preliminary positioning and directional analysis, and the insulin signaling pathway can be determined based on the preliminary positioning and directional analysis results for further verification analysis, thereby providing direction for subsequent detection and evaluation from the aspects of morphology and signaling pathways, improving detection efficiency and accuracy. SUMMARY

[0005] The present application provides a multi-dimensional evaluation method for insulin-resistant hepatocytes, which combines structured light super-resolution imaging technology with fluorescent semi-quantitative analysis, and further verifies the insulin-related pathway using intracellular protein immunoblotting. If the results are inconsistent, further clarify the mechanism through time gradient, protein detection, or pathway experiments. This method has good universality and stable reproducibility, and improves detection efficiency and accuracy.

[0006] To achieve the above object, the technical scheme of the present application is: a multi-dimensional evaluation method for insulin-resistant hepatocytes, comprising the following specific steps: S1 phenotype screening: providing an insulin-resistant hepatocyte model, setting up experimental groups, including a normal group, a model group, and a gradient concentration of test compound treatment group; quantitatively analyzing the mitochondrial morphology and active oxygen level of the experimental groups, and screening at least one test compound with improved effect on insulin resistance as a lead compound; S2 mechanism verification step: detecting the influence of insulin signaling pathway key proteins in treated cells by intracellular protein immunoblotting for the lead compound screened in step S1; S3 comprehensive evaluation and giving the next evaluation direction: when any two of the mitochondrial morphology and active oxygen level in step S1 and the influence of insulin signaling pathway key proteins do not match, and under the condition that the normal group and the model group are normal and abnormal, respectively, according to the mismatching condition, further verify the time gradient, mitochondrial dynamics related protein or insulin signaling pathway key protein inspection range.

[0007] The above method first groups hepatocytes into normal, model and test drug groups, then tests the morphology and active oxygen content of mitochondria by fluorescence probe staining on different groups, to determine the test compound with improved effect on insulin resistance, then determines the improvement of insulin signaling pathway by intracellular protein immunoblotting for the determined lead compound, then compares the morphology and active oxygen content of mitochondria with the improvement of insulin signaling pathway, if the results of any two do not match, and through the correct results of the normal group and the model group, the next test plan can be given. This way, through phenotype screening, the test compound with good effect can be determined, and the main functional direction can be preliminarily determined, then further insulin pathway detection is performed on the compound, which can further verify and determine the time gradient, mitochondrial dynamics related protein or insulin key protein direction in the case of any two detection results not matching, so as to improve the detection efficiency and reliability and universality of the test compound effect.

[0008] Further, step S1 further comprises: S11 extracting target cells, the target cells being HepG2 cells or THLE-2 cells, constructing experimental groups, the experimental groups including a normal group formed after the target cells are extracted, a model group formed by using a metabolic stress inducer on cells of one normal group, a drug treatment group formed by using a gradient concentration of a to-be-tested drug on cells of another normal group, and a positive control group formed by using a metabolic stress inducer and a positive drug on cells of still another normal group; S12 performing cell viability detection, glucose consumption detection, and intracellular AGEs content determination on cells in the experimental groups by using a fluorescence probe, and determining the to-be-tested compound as a lead compound in combination with test results of the positive control group; S13 performing staining on the to-be-tested compound in the cells in the experimental groups after using the lead compound by using an active oxygen probe, a mitochondrial morphology probe, and a cell nucleus probe, performing image acquisition on mitochondrial membrane structures, active oxygen accumulation signals, and cell nucleus positioning after the staining, determining active oxygen fluorescence intensity and cell nucleus fluorescence intensity, and determining a trend of a ratio of the active oxygen fluorescence intensity to the cell nucleus fluorescence intensity based on the images; S14 determining that a to-be-tested compound corresponding to a concentration at which the ratio of the active oxygen fluorescence intensity to the cell nucleus fluorescence intensity is obviously reduced relative to the active oxygen fluorescence intensity and the cell nucleus fluorescence intensity corresponding to the normal group and the model group is the lead compound.

[0009] The above settings achieve detection of the active oxygen fluorescence intensity and the cell nucleus fluorescence intensity by using the active oxygen and other fluorescence probes, and determine the ratio. For a to-be-tested compound corresponding to a concentration at which the ratio of the active oxygen fluorescence intensity to the cell nucleus fluorescence intensity is obviously reduced, it is proved that the to-be-tested compound has a good effect on active oxygen and morphology, so that the to-be-tested compound is locked as the lead compound.

[0010] Further, the grouping formation process in step S11 comprises: extracting target cells, the target cells including human hepatoma cell line HepG2 and normal human hepatocyte line THLE-2, the target cells being cultured in a MEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2 to form a normal group, the target cells being inoculated into another culture vessel, a DMEM culture medium containing high sugar and dexamethasone being used to induce the target cells for 48 hours to form a model group, and different concentrations of to-be-tested compounds being used to treat the target cells to form to-be-tested compound treatment groups, the to-be-tested compounds including nomilin, and the drugs including metformin.

[0011] The above setup involves first extracting target cells, HepG2 cells or IR-THLE-2 cells. HepG2 cells are insulin-resistant human liver cancer HepG2 cells. These cells are then seeded into suitable cell culture vessels, such as culture flasks and multi-well plates. Once the cells have grown to a suitable density and stabilized, a uniformly growing cell population is selected as the normal group. This group of cells is not subjected to subsequent stress induction or drug treatment; only the basic culture conditions are maintained, serving as a physiological baseline reference for subsequent experiments. The model group was constructed by taking a portion of cells from the normal group A, replacing them with high glucose and dexamethasone containing metabolic stress inducers, and continuing to culture them in a 37 ºC, 5% CO2 incubator for 48 hours. High glucose simulated the pathological environment of hyperglycemia and dexamethasone induced abnormal insulin signaling pathway, thus jointly constructing a stable insulin-resistant cell model to reflect abnormal cell function under pathological conditions. To construct the test compound treatment group, another portion of cells from the normal group were taken and the test compound nomiline was added at gradient concentrations, such as 8 μM, 16 μM, 32 μM, and 64 μM, to evaluate the improvement effect of different concentrations of nomiline on insulin-resistant cells. The positive control group was constructed by taking a portion of cells from the normal group and the positive drug, which served as a standard reference for verifying drug efficacy and was used to determine whether the experimental system was effective and whether the effect of the drug was statistically significant.

[0012] Furthermore, S13 also includes: the experimental groups were co-stained with 5 μM reactive oxygen species probe, 0.1 μM mitochondrial morphology probe and 1 μM nuclear probe at 37℃ for 30 minutes, and then image acquisition was performed using structured light microscopy. The channels were set to blue channel, green channel and red channel, and the green fluorescence signal intensity in the green channel was determined to be the reactive oxygen species fluorescence intensity, the blue fluorescence signal intensity in the blue channel was determined to be the nuclear fluorescence intensity, and the trend of the ratio of reactive oxygen species fluorescence intensity to nuclear fluorescence intensity was determined.

[0013] The above method, after identifying the lead compound to be tested, determines the trend of the ratio of reactive oxygen species fluorescence intensity to nuclear fluorescence intensity by using reactive oxygen species and nuclear probes in experimental groups, and further assists in testing by combining the morphology detected by mitochondrial morphology probes, thereby enabling further target visualization and quantitative verification of the lead compound.

[0014] Furthermore, step S2 also includes: fixing, permeabilizing, and blocking the cells in the experimental group, followed by incubation with primary and secondary antibodies in sequence, and finally using an imaging system to collect fluorescence signals, and determining the ratios of p-PI3K / PI3K, p-Akt / Akt, p-GSK3β / GSK3β, and GLUT4 / β-actin in IR-THLE-2 cells containing the test compound based on image analysis.

[0015] The above method, after processing the experimental groups, uses an imaging system to measure various ratios of insulin signals in IR-THLE-2 cells, thereby determining that the anti-diabetic effect is exerted by activating the PI3K / Akt / GLUT4 signaling pathway.

[0016] Furthermore, step S3 also includes: S31 determining whether the indicators of the normal control group and the model group are the expected normal and abnormal, respectively; if so, proceeding to step S32. S32 If step S1 confirms that mitochondrial morphology has improved, but reactive oxygen species have not decreased, then a time gradient experiment and other oxidative stress indicators should be performed. If step S1 determines that the decrease in reactive oxygen species has not improved, then the detection of mitochondrial dynamics-related proteins and mitochondrial biogenesis will be performed. If reactive oxygen species and their morphology are improved in step S1, but the insulin signaling pathway is not activated in step S2, the detection range of the pathway is expanded.

[0017] The above setup ensures the reliability of the internal control by first performing normal and abnormal tests on the normal and model groups. Then, it determines the direction of further testing based on whether the three aspects of mitochondrial morphology, whether reactive oxygen species are reduced, and whether key protein pathways are opened match. This ensures that even when there is no match, a more in-depth and specific direction can be found.

[0018] Furthermore, the time gradient experiment in step S32 includes: extending the treatment time of the test compound and observing whether the fluorescence intensity of reactive oxygen species improves with a delay; Other indicators of oxidative stress include detecting the activity of superoxide dismutase (SOD) and glutathione (GSH) endogenous antioxidant systems to determine whether the test compound works indirectly by activating the endogenous defense system.

[0019] The above settings, when reactive oxygen species are not matched, are used to detect the treatment time of the test compound and other endogenous oxidation systems to determine whether the failure of reactive oxygen species to decrease is due to the endogenous defense system.

[0020] Furthermore, in step S32, mitochondrial dynamics-related proteins are detected: the expression or phosphorylation levels of Drp1, MFN1 / 2, and OPA1 proteins are detected by intracellular protein immunoblotting to verify whether the test compound has no direct effect on mitochondrial division / fusion. Detection of mitochondrial biogenesis: detection of the expression of key factors such as PGC-1α and TFAM.

[0021] The above settings determine the effect of the tested compound on mitochondrial separation or fusion by detecting the protein expression or phosphorylation levels of other proteins in mitochondria, and identify morphologically unchanged factors by detecting mitochondrial biogener.

[0022] Furthermore, step S32 expands the scope of pathway detection: the method of step S2 is used to screen the performance of other signaling pathways activated by the compound under test.

[0023] The above settings, when the pathway results do not match the reactive oxygen species results, further expand the pathway detection range to determine whether the specificity of the pathway affects the detection results.

[0024] Furthermore, in step S13, the excitation wavelength used for the green channel is 488nm and the emission wavelength is 495-550nm, the excitation wavelength used for the blue channel is 405nm and the emission wavelength is 420-488nm, and the excitation wavelength used for the red channel is 643nm and the emission wavelength is 655-1000nm.

[0025] The above settings allow different cellular parameters to be used as signals after fluorescent staining of the target cells, and the imaging parameters can be distinguished, making the observation and judgment more obvious. Attached Figure Description

[0026] Figure 1 This is a flowchart illustrating the process of this invention.

[0027] Figure 2 This is a comparative diagram showing the effects of nomiline in step S1 of the present invention on significant toxicity to HepG2 cells, glucose consumption, reduction of AGEs accumulation, and improvement of cellular reactive oxygen species accumulation.

[0028] Figure 3 This is a comparative diagram showing the effect of nomiline on improving mitochondrial morphology and reactive oxygen species levels in HepG2 cells in step S2 of the present invention.

[0029] Figure 4 This is a comparison chart of the p-PI3K / PI3K, p-Akt / Akt, p-GSK3β / GSK3β, and GLUT4 / β-actin ratios of THLE-2 cells in step S3 of the present invention. Detailed Implementation

[0030] like Figures 1-4 As shown, a multidimensional assessment method for insulin-resistant hepatocytes includes the following steps: S1. Phenotypic screening of insulin-resistant hepatocyte models: Provide insulin-resistant hepatocyte models and set up experimental groups, including normal group, model group and treatment groups with gradient concentrations of test compounds; image live cells of the experimental groups after co-staining with mitochondrial morphology probes and reactive oxygen species probes, perform quantitative analysis of mitochondrial morphology and reactive oxygen species levels, and screen at least one test compound that has an ameliorative effect on insulin resistance as a lead compound. S2 mechanism verification step: The lead compounds selected in step S1 are screened and the expression or phosphorylation level of key proteins in the insulin signaling pathway in the treated cells is detected by intracellular protein immunoblotting. S3 provides a comprehensive assessment and directions for the next step: When any two of the following two items in step S1—mitochondrial morphology and reactive oxygen species levels—are mismatched with the expression or phosphorylation levels of key proteins in the insulin signaling pathway, and while ensuring that the normal group and the model group are normal and abnormal, further verification is conducted on the time gradient, mitochondrial dynamics-related proteins, or the scope of key proteins in the insulin signaling pathway, based on the mismatch.

[0031] like Figure 1 As shown, step S1 includes: S11 Extracting target cells, which are HepG2 cells or THLE-2 cells; constructing experimental groups, which include a normal group formed by extracting target cells; a model group formed by using a metabolic stress inducer on cells from one normal group; a drug treatment group formed by using gradient concentrations of the test drug on cells from another normal group; and a positive control group formed by using a metabolic stress inducer and a positive drug on cells from yet another normal group. The gradient concentrations include 8, 16, 32, and 64 μM, and the test compound is nomiline.

[0032] The specific groupings were as follows: Target cells were extracted, including the human hepatocellular carcinoma cell line HepG2 and the normal human hepatocellular carcinoma cell line THLE-2. The target cells were cultured in MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2 to form the normal group. The target cells were then seeded into another culture dish and induced for 48 hours in DMEM medium containing high glucose and dexamethasone to form the model group. The target cells were then treated with different concentrations of the test compound to form the test compound treatment group. The test compound was nomiline, and the drug was metformin.

[0033] S12 used fluorescent probes to detect cell viability, glucose consumption, and intracellular AGEs content in cells within the experimental group. Combined with the test results of the positive control group, the test compound was identified as a lead compound. Cell viability assays included: seeding cells from experimental groups at 5 × 10³ cells / well in 96-well plates, treating with nomiline (0-128 μM) for 24 hours, measuring absorbance at 450 nm using CCK-8 reagent, and calculating cell viability.

[0034] The glucose consumption determination included: seeding cells from the experimental groups at 5×10³ cells / well in 96-well plates; after 48 hours of cell modeling and group intervention, collecting the supernatant and detecting the residual glucose at 510 nm using a glucose oxidase-peroxidase assay kit, and calculating the glucose consumption level of each group.

[0035] The determination of intracellular AGEs content included: seeding cells from the experimental groups at 5×10³ cells / well in black 96-well plates; 48 hours after cell modeling and grouping intervention, directly measuring the intracellular AGEs-related fluorescence intensity using a fluorescence microplate reader (excitation / emission: 355 / 405 nm).

[0036] Intracellular reactive oxygen species (ROS) detection was performed using a microplate reader. Specifically, cells from the experimental groups were seeded at 5 × 10³ cells / well in black 96-well plates. After 48 hours of cell modeling and grouping intervention, the culture medium was discarded, the cells were washed three times with PBS, and 5 μM Carboxy-H2DCFDA staining solution was added. The cells were incubated at 37°C in the dark for 30 minutes. After washing with PBS, the fluorescence intensity was measured using a fluorescence microplate reader (excitation / emission: 488 / 515 nm). The results were normalized to the cell count.

[0037] like Figure 2 As shown, Figure 2 -A、 Figure 2 -C、 Figure 2 -E、 Figure 2 -G represents a comparison of cell viability, AGEs-related fluorescence intensity, glucose consumption, and reactive oxygen species content in HepG2 cells. Figure 2 -B、 Figure 2 -D、 Figure 2 -F、 Figure 2 -H represents a comparison of cell viability, AGEs-related fluorescence intensity, glucose consumption, and reactive oxygen species content in THLE-2 cells.

[0038] The experimental results show that nomiline has no significant toxicity to either type of hepatocyte in the 64 μM range. Figure 2 A, 2B). Compared with the model group, nomiline increased glucose consumption in a concentration-dependent manner (Figure 2 C, 2D), reduce AGEs accumulation ( Figure 2 E, 2F) and intracellular reactive oxygen species accumulation (E, 2F) and Figure 2 G, 2H), and showed a consistent trend of improvement in both cell models.

[0039] In S13, after using the lead compound, the cells in the experimental group were stained with reactive oxygen species (ROS) probes, mitochondrial morphology probes, and nuclear probes. Images of the stained mitochondrial membrane structure, ROS accumulation signals, and nuclear localization were acquired to determine the ROS fluorescence intensity and nuclear fluorescence intensity. Based on the image analysis, the trend of the ratio of ROS fluorescence intensity to nuclear fluorescence intensity was determined.

[0040] In this embodiment, the specific testing process is as follows: the cells in the experimental groups are divided into 6×10 4 Cells were seeded in confocal culture dishes with glass bottoms, and IR models were constructed and grouped according to the method in Example 1. After the intervention, Carboxy-H2DCFDA (5 μM, reactive oxygen species probe), HBmito Crimson mitochondrial morphology probe (0.1 μM, membrane potential insensitive mitochondrial morphology probe), and Hoechst 33342 (1 μM, nuclear probe) were co-stained at 37°C for 30 minutes.

[0041] Image acquisition was performed using structured lighting microscopy, with the following main parameters: Objective lens: 63x oil immersion lens; Environmental control: 37℃, 5% CO2; Channel settings: Blue channel (Hoechst): 405 nm excitation, 420-488 nm reception; Green Channel (Carboxy-H2DCFDA): 488 nm excitation, 495-550 nm reception; Red channel (HBmito Crimson): 643 nm excitation, 655-1000 nm reception; Exposure time: 30 milliseconds for each channel; S14 identifies the test compounds whose ratio of reactive oxygen species fluorescence intensity to nuclear fluorescence intensity is significantly lower than that of the normal group and the model group at the corresponding concentrations as lead compounds.

[0042] like Figure 3 This is a super-resolution imaging display image. Figure 3 -A、 Figure 3 -B and Figure 3-C represents the distribution of reactive oxygen species fluorescence intensity corresponding to HepG2, the distribution of nuclear fluorescence intensity, and the bar chart of the ratio of reactive oxygen species fluorescence intensity to nuclear fluorescence intensity. Figure 3 -D、 Figure 3 -E and Figure 3 -F represents the distribution of reactive oxygen species fluorescence intensity for THLE-2, the distribution of nuclear fluorescence intensity, and the bar chart showing the ratio of reactive oxygen species fluorescence intensity to nuclear fluorescence intensity.

[0043] Consistent improvement was observed in both cell models: mitochondria in the model group exhibited significant morphological distortions such as fragmentation and swelling, and the internal green fluorescence signal (reactive oxygen species) was significantly enhanced. Figure 3 -A, Figure 3 -D), after treatment with the test compound -nomilin, the mitochondrial morphology returned to the normal reticular structure, and the intensity of green fluorescence decreased in a dose-dependent manner. Figure 3 -B, Figure 3 Semi-quantitative analysis further confirmed that nomiline can significantly reduce the ratio of reactive oxygen species to the fluorescence intensity of the cell nucleus (-E). Figure 3 -C, Figure 4 -F).

[0044] Steps S1 and S2 verification: Simultaneous visualization and quantitative analysis of mitochondrial morphology and reactive oxygen species were achieved. The results showed that nomiline can effectively alleviate mitochondrial dysfunction in insulin-resistant cells.

[0045] like Figure 4 As shown, step S2 includes: fixing, permeabilizing, and blocking the cells in the experimental group, followed by incubation with primary and secondary antibodies in sequence, and finally using an imaging system to collect fluorescence signals. Based on image analysis, the ratios of p-PI3K (phosphatidylinositol-3-kinase) / PI3K (intracellular phosphatidylinositol kinase), p-Akt (phosphorylated protein kinase B) / Akt, p-GSK3β / GSK3β (glycogen synthase kinase-3β), and GLUT4 (glucose transporter 4) / β-actin (β-actin) in IR-THLE-2 cells containing the test compound are determined.

[0046] like Figure 4 As shown, the test compound, nomiline, significantly increased the p-PI3K / PI3K ratio in THLE-2 cells. Figure 4 AB), p-Akt / Akt ( Figure 4 CD), p-GSK3β / GSK3β ( Figure 4 EF) and GLUT4 / β-actin ​The ratio of GH indicates that it can exert an anti-diabetic effect by activating the PI3K / Akt / GLUT4 signaling pathway. In this embodiment, in all statistical charts, the English letters (a, b, c, d) above the bars represent statistical differences after one-way ANOVA and Tukey's multiple comparison test (p<0.05); there is no significant difference between groups sharing the same letter (p ≥ 0.05); there is a significant difference between groups without any shared letters (p<0.05).

[0047] Step S3 includes: S31 determining whether the indicators of the normal control group and the model group are as expected (normal and abnormal), respectively; if so, proceeding to step S32. S32 If step S1 confirms that mitochondrial morphology has improved, but reactive oxygen species have not decreased, then a time gradient experiment and other oxidative stress indicators are performed. The time gradient experiment includes extending the treatment time of the test compound and observing whether the reactive oxygen fluorescence intensity improves with a delay. Other indicators of oxidative stress include detecting the activity of superoxide dismutase (SOD) and glutathione (GSH) endogenous antioxidant systems to determine whether the test compound works indirectly by activating the endogenous defense system.

[0048] If step S1 determines that the decrease in reactive oxygen species has not improved, then the detection of mitochondrial dynamics-related proteins and mitochondrial biogenesis will be performed. Detection of mitochondrial dynamics-related proteins: The expression or phosphorylation levels of Drp1, MFN1 / 2, and OPA1 proteins will be detected by intracellular protein immunoblotting to verify whether the test compound has no direct effect on mitochondrial division / fusion. Detection of mitochondrial biogenesis: detection of the expression of key factors such as PGC-1α and TFAM.

[0049] If reactive oxygen species and their speciation are improved in step S1, but the insulin signaling pathway is not activated in step S2, the pathway detection scope is expanded. Expanding the pathway detection scope involves using the method in step S2 to screen for the performance of other signaling pathways activated by the test compound.

[0050] The working principle of this invention is as follows: First, hepatocytes are divided into normal, model, and test drug groups. Then, fluorescent probes are used to stain different groups to test mitochondrial morphology and reactive oxygen species (ROS) content, thereby identifying test compounds that improve insulin resistance. Next, the improvement of the insulin signaling pathway is determined by intracellular protein immunoblotting of the identified lead compounds. The mitochondrial morphology and ROS content are then compared with the improvement of the insulin signaling pathway. If any two results are inconsistent, and the results of the normal and model groups are correct, a further testing plan can be proposed. This phenotypic screening identifies compounds with better effects among the test compounds and preliminarily determines their main functional direction. Further insulin pathway testing is then conducted on these compounds for further verification. If any two test results are mismatched, the temporal gradient, mitochondrial compound kinetics-related proteins, or the direction of expanding key insulin proteins are further determined. This step-by-step approach improves detection efficiency and reliably confirms the effect of the test compounds, thus enhancing the reliability of the detection.

Claims

1. A multidimensional assessment method for insulin-resistant hepatocytes, characterized by: steps include: S1. Phenotypic screening of insulin-resistant hepatocyte models: Provide insulin-resistant hepatocyte models and set up experimental groups, including normal group, model group and treatment group with gradient concentrations of test compound; Quantitative analysis of mitochondrial morphology and reactive oxygen species levels in the experimental groups was performed to screen at least one test compound that could improve insulin resistance as a lead compound. S2 Mechanism Validation Steps: The lead compounds screened in step S1 were used to detect the effects of treatment on key proteins in the insulin signaling pathway in cells by intracellular protein immunoblotting. S3 provides a comprehensive assessment and directions for the next step: When any two of the factors in S1—mitochondrial morphology and reactive oxygen species levels—are mismatched with the effects of key proteins in the insulin signaling pathway, and while ensuring that the normal group and the model group are normal and abnormal, further verification should be conducted on the time gradient, mitochondrial dynamics-related proteins, or the scope of key proteins in the insulin signaling pathway, depending on the mismatch.

2. The multidimensional assessment method for insulin-resistant hepatocytes according to claim 1, characterized in that: Step S1 further includes: S11 Extracting target cells, which are HepG2 cells or THLE-2 cells; constructing experimental groups, which include a normal group formed by extracting target cells; a model group formed by applying a metabolic stress inducer to cells in one normal group; a drug treatment group formed by applying gradient concentrations of the test drug to cells in another normal group; and a positive control group formed by applying a metabolic stress inducer and a positive drug to cells in yet another normal group. S12 used fluorescent probes to detect cell viability, glucose consumption, and intracellular AGEs content in cells within the experimental group. Combined with the test results of the positive control group, lead compounds were preliminarily screened. S13 used a seed compound to stain the cells of the test compound in the experimental group, followed by staining with reactive oxygen species (ROS) probes, mitochondrial morphology probes, and nuclear probes. Images of the stained mitochondrial membrane structure, ROS accumulation signals, and nuclear localization were acquired to determine the ROS fluorescence intensity and nuclear fluorescence intensity. Based on the image analysis, the trend of the ratio of ROS fluorescence intensity to nuclear fluorescence intensity was determined. S14 identifies the test compounds whose ratio of reactive oxygen species (ROS) fluorescence intensity to nuclear fluorescence intensity is significantly lower than that of the normal and model groups at corresponding concentrations as lead compounds.

3. The multidimensional assessment method for insulin-resistant hepatocytes according to claim 2, characterized in that: The grouping process in step S11 includes: extracting target cells, which include the human hepatocellular carcinoma cell line HepG2 and the normal human hepatocellular carcinoma cell line THLE-2. The target cells are cultured in MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2 to form a normal group. The target cells are seeded into another culture dish and induced for 48 hours in DMEM medium containing high glucose and dexamethasone to form a model group. The target cells are then treated with different concentrations of the test compound to form the test compound treatment group. The test compound includes nomiline, and the drug includes metformin.

4. The multidimensional assessment method for insulin-resistant hepatocytes according to claim 2, characterized in that: S13 also includes: 5 μM reactive oxygen species (ROS) probe, 0.1 μM HBmito Crimson mitochondrial morphology probe and 1 μM nuclear probe were co-stained at 37℃ for 30 minutes in the experimental groups, and then images were acquired using structured light microscopy. The channels were set to blue, green and red channels, and the green fluorescence signal intensity in the green channel was determined to be the ROS fluorescence intensity, and the blue fluorescence signal intensity in the blue channel was determined to be the nuclear fluorescence intensity. The trend of the ratio of ROS fluorescence intensity to nuclear fluorescence intensity was also determined.

5. A multidimensional assessment method for insulin-resistant hepatocytes according to claim 1, characterized in that: Step S2 also includes: fixing, permeabilizing, and blocking the cells in the experimental group, followed by incubation with primary and secondary antibodies in sequence, and finally using an imaging system to collect fluorescence signals. Based on image analysis, the ratios of p-PI3K / PI3K, p-Akt / Akt, p-GSK3β / GSK3β, and GLUT4 / β-actin in IR-THLE-2 cells containing the test compound are determined.

6. The multidimensional assessment method for insulin-resistant hepatocytes according to claim 1, characterized in that: Step S3 further includes: S31 determining whether the indicators of the normal control group and the model group are as expected (normal and abnormal), respectively; if so, proceeding to step S32. S32 If step S1 confirms that mitochondrial morphology has improved, but reactive oxygen species have not decreased, then a time gradient experiment and other oxidative stress indicators should be performed. If step S1 determines that the decrease in reactive oxygen species has not improved, then the detection of mitochondrial dynamics-related proteins and mitochondrial biogenesis will be performed. If reactive oxygen species and their morphology are improved in step S1, but the insulin signaling pathway is not activated in step S2, the detection range of the pathway is expanded.

7. A multidimensional assessment method for insulin-resistant hepatocytes according to claim 6, characterized in that: The time gradient experiment in step S32 includes: extending the treatment time of the compound to be tested and observing whether the fluorescence intensity of reactive oxygen species improves with a delay; Other indicators of oxidative stress include detecting the activity of superoxide dismutase (SOD) and glutathione (GSH) endogenous antioxidant systems to determine whether the test compound works indirectly by activating the endogenous defense system.

8. A multidimensional assessment method for insulin-resistant hepatocytes according to claim 6, characterized in that: In step S32, mitochondrial dynamics-related proteins were detected: the expression or phosphorylation levels of Drp1, MFN1 / 2, and OPA1 proteins were detected by intracellular protein immunoblotting to verify whether the test compound had no direct effect on mitochondrial division / fusion. Detection of mitochondrial biogenesis: detection of the expression of key factors such as PGC-1α and TFAM.

9. A multidimensional assessment method for insulin-resistant hepatocytes according to claim 6, characterized in that: In step S32, the scope of pathway detection is expanded: the method in step S2 is used to screen the performance of other signaling pathways activated by the compound under test.

10. A multidimensional assessment method for insulin-resistant hepatocytes according to claim 2, characterized in that: In step S13, the excitation wavelength used for the green channel is 488nm and the emission wavelength is 495-550nm; the excitation wavelength used for the blue channel is 405nm and the emission wavelength is 420-488nm; and the excitation wavelength used for the red channel is 643nm and the emission wavelength is 655-1000nm.

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