An experimental method for the effect of iridium complexes on FGF21 protein function

Abstract

The present invention belongs to the field of iridium complexes, and in particular, an experimental method for studying the effect of iridium complexes on the function of FGF21 protein, comprising the following steps: S1: preparation of iridium complexes, proteins, cells, and reagents: iridium complex IrCN, FGF21 protein, mouse kidney fibroblasts BHK21, and bovine mammary epithelial cells MAC‑T; S2: binding characteristics of iridium complexes with proteins: the iridium complex is dissolved in dimethyl sulfoxide to prepare a mother solution with a concentration of 10 mg / ml, and then the mother solution is gradiently diluted with PBS buffer to give a final concentration of iridium complex of 0.05, 0.1, 0.5, and 1 mg / ml, and different concentrations of iridium complex are added to 20 μg of camel FGF21 protein with a His tag. The present invention can efficiently verify the binding of iridium complexes to camel FGF21 protein, the binding specificity of iridium complexes to FGF21 protein, and the cytotoxicity of iridium complexes.

Description

Technical Field

[0001] The present invention relates to the technical field of iridium complexes, and in particular to an experimental method for studying the influence of iridium complexes on the function of FGF21 protein. Background Art

[0002] With the rapid development of science and technology, biomedicine and inorganic chemistry have intersected. Iridium (III) complexes, as metal-organic compounds, can bind to biomacromolecules for labeling and tracing, becoming a current research hotspot. Fibroblast growth factor 21 (FGF21), a member of the FGF gene family, regulates sugar, fat, and protein metabolism, playing a vital role in biological processes such as cell growth, differentiation, and energy metabolism.

[0003] Iridium complexes are a class of organometallic compounds with unique chemical and physical properties and potential functionalities, offering broad application prospects in materials science, biology, and medicine. Research over the past decade has demonstrated that iridium complexes can interact with a variety of biomolecules, including DNA, RNA, and proteins. These interactions regulate numerous physiological processes by influencing the structure and partial function of these biomolecules. However, research on the binding of iridium complexes to proteins is still in its infancy, and their interaction with the FGF21 protein, in particular, remains understudied. Therefore, we developed an experimental method to investigate the effects of iridium complexes on FGF21 protein function to address this issue. Summary of the Invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art and to propose an experimental method for the effect of iridium complexes on the function of FGF21 protein.

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

[0006] An experimental method for the effect of an iridium complex on the function of FGF21 protein comprises the following steps:

[0007] S1: Preparation of iridium complexes, proteins, cells, and reagents: iridium complex IrCN, FGF21 protein, mouse kidney fibroblasts BHK21, and bovine mammary epithelial cells MAC-T;

[0008] S2: Binding characteristics of iridium complexes to proteins: The iridium complex was dissolved in dimethyl sulfoxide to prepare a 10 mg / ml stock solution. The stock solution was then graded diluted with PBS buffer to give final iridium complex concentrations of 0.05, 0.1, 0.5, and 1 mg / ml. Iridium complexes of varying concentrations were added to 20 μg of His-tagged camel FGF21 protein. Fluorescence intensity changes at different times were recorded using a chemiluminescence imaging system and analyzed using ImageJ software.

[0009] S3: Binding specificity of iridium complexes to FGF21 protein: labeling and mixing, FGF21 protein labeled with iridium complex probe was mixed with serum albumin containing histidine, and different iridium complex to FGF21 protein concentration ratios were set to 1:100, 1:200, 1:300, 1:400, as well as different ratios of BSA to FGF21 protein;

[0010] Gel electrophoresis and detection: After 24 hours, gel electrophoresis separation was performed and a chemiluminescence imaging system was used to detect whether BSA showed fluorescence. By analyzing the results of different lanes, the binding specificity of the iridium complex to the FGF21 protein was determined.

[0011] S4: Cytotoxicity test of iridium complexes: Cell inoculation and treatment: At 37°C and 5% CO2, subcultured cells were inoculated into 96-well cell culture plates. When the cells grew to a density of about 85%, the culture medium in the wells was removed and pre-prepared culture medium solutions containing different concentrations of iridium complex probes were added to make the final concentrations of iridium complex probes 0, 0.01, 0.02, 0.05, 0.1, and 0.2 mg / ml, respectively. A control group was set up at the same time, and 3 parallel experiments were set up for each group.

[0012] Toxicity assay: After 24 hours of co-culture with cells, add 10 μl of CCK8 solution to each well and continue incubation in the incubator for 2 hours. Measure the absorbance of the solution in each well at a wavelength of 450 nm using a microplate reader. Calculate the cell viability based on the absorbance to evaluate the effect of the iridium complex on cell viability.

[0013] S5: Determination of hemolytic activity of iridium complex probe: Blood was collected from healthy mice, and 10 times the amount of 0.9% sodium chloride solution was added. The blood was washed by centrifugation and the supernatant was removed. The obtained red blood cells were made into a 4% suspension with 0.9% sodium chloride solution. The sample solution was prepared in a centrifuge tube. Positive and negative controls were set up. 200 μl of red blood cell suspension was added to each tube. The tube was centrifuged at 1000 g for 3 minutes. The hemolysis phenomenon was observed with the naked eye. 100 μl of supernatant was aspirated into a new 96-well plate and the OD540nm was detected.

[0014] S6: Live cell imaging test: BHK21 cells were seeded in a confocal microplate. When the cells grew to a density of about 50%, different concentrations of iridium complex probe IrCN solution were added to the microplate. After co-incubation with the cells for 10 minutes, the microplate was washed twice with phosphate buffer and used directly for imaging.

[0015] S7: Immunofluorescence experiment: BHK21 cells were seeded in confocal microplates. When the cells grew to a density of about 50%, they were washed twice with pre-cooled PBS and fixed with 4% paraformaldehyde for 30 minutes at room temperature. After washing three times with PBS, the cells were permeabilized in 0.2% TritonX-100 for 10 minutes, washed three times with PBS, and stained with 4,0,6-diaminophenylindole for 10 minutes. After washing three times with PBS, different concentrations of the iridium complex probe IrCN were added to the microplates, and images were captured and recorded using a laser confocal microscope.

[0016] S8: RNA extraction and cDNA synthesis: Total RNA was extracted from camel fibroblasts using the TRIzol method, and cDNA was synthesized according to the PrimeScript RT Reagent Kit instructions. The concentration and purity of the cDNA were determined by UV spectrophotometry.

[0017] S9: Protein extraction and concentration determination: Total cell protein was extracted with RIPA protein lysis buffer, and the protein concentration was determined using a BCA protein assay kit. Equal amounts of total protein were separated by 12% SDS-PAGE gel electrophoresis and then electrotransferred to a 0.45 µm polyvinylidene difluoride membrane. The membrane was blocked with blocking buffer for 20 minutes and then incubated with anti-tubulin, anti-PI3K, anti-AKT, and anti-mTOR antibodies at 4°C overnight. After washing three times with PBS, the membrane was incubated with enzyme-labeled goat anti-rabbit IgG at room temperature for 2 hours. Finally, the protein bands were visualized using ECL chemiluminescence detection and captured by a gel imaging system.

[0018] S10. Statistical analysis: GraphPad Prism software was used to analyze the results. Data are expressed as mean ± SEM. Unpaired Student's t test was used for comparison between two groups. Two-way analysis of variance was used for multiple hippocampal measurement groups. P < 0.05 was considered statistically significant.

[0019] Preferably, the S1 also needs to prepare DMEM culture medium, phosphate buffered saline (PBS, pH = 7.2), trypsin-EDTA solution, penicillin and streptomycin, collagenase IV, RIPA protein lysis buffer, TRIzol, CCK-8, propidium iodide, BCA protein detection kit, ECL chemiluminescence detection kit, reverse transcription kit, SYBR Premix Ex Taq II kit, G418, protein marker, fetal bovine serum, rabbit anti-PI3K polyclonal antibody, rabbit anti-AKT polyclonal antibody, rabbit anti-mTOR polyclonal antibody, rabbit anti-β-Tubulin antibody, enzyme-labeled goat anti-rabbit IgG, PCR primers and various reagents.

[0020] Preferably, S2 also includes binding time determination: after the iridium complex is combined with the FGF21 protein, the fluorescence intensity at different action times is also recorded by a chemiluminescence imaging system, and the result graph analyzes the change in protein fluorescence intensity with the extension of the action time to determine the optimal binding time of the iridium complex and the FGF21 protein.

[0021] Preferably, S2 also includes: binding stability determination: placing the iridium complex and FGF21 protein of the same concentration in -20°C, 4°C and room temperature environments, respectively, and recording the fluorescence intensity at different times using an intelligent darkbox three-purpose UV analyzer; observing the change in the fluorescence intensity of the iridium complex fluorescent probe binding to the FGF21 protein as the action time increases at different temperatures to determine the binding stability of the iridium complex and the FGF21 protein at different temperatures.

[0022] Preferably, in S5, the hemolytic activity of the iridium complex is evaluated according to the hemolysis rate calculation formula: hemolysis rate = (OD sample test group-OD negative control group) / (OD positive control group-OD negative control group)×100%.

[0023] Preferably, the S6 imaging and analysis uses ImageJ software to perform fluorescence intensity analysis, and the intracellular fluorescence distribution is observed by confocal microscopy to evaluate the detection ability of IrCN for intracellular histidine and the changes in intracellular fluorescence signals under treatment with different concentrations of IrCN.

[0024] Preferably, the imaging and analysis in S7: using a laser confocal microscope to capture and record images, using ImageJ software to perform fluorescence intensity analysis, and observing the fluorescence distribution and intensity changes in the cell nucleus and cytoplasm after treatment with different concentrations of iridium complexes.

[0025] Preferably, S8 also includes qPCR reaction and analysis: a qPCR reaction system is prepared according to the SYBR Premix Ex Taq II Kit protocol, and the reaction is performed on a LightCycler instrument. The relative gene expression levels are analyzed using the 2-DDCt method, and the number of transcripts in each sample is normalized using ACTIN as an internal reference to assess the expression levels of PI3K, Akt, and mTOR genes.

[0026] Beneficial effects of the present invention:

[0027] 1. The iridium complex was combined with the camel FGF21 protein, and the optimal binding concentration ratio was 1:100. The optimal binding time was ten minutes, with strong binding stability and a long binding half-life. Proteins were separated by gel electrophoresis. When the concentration ratio of the iridium complex to the FGF21 protein was 1:100, free probes would bind specifically to other proteins after the iridium complex bound to the protein. When the concentration ratio of the iridium complex to the FGF21 protein was 1:200 or lower, the iridium complex was completely bound to the protein, and no free probes would bind specifically to other proteins.

[0028] 2. The cytotoxicity of iridium complexes was detected by the CCK8 method. When the concentration of iridium complexes was greater than 0.05 mg / ml, the cell survival rate was significantly reduced. When the concentration of iridium complexes was lower than 0.05 mg / ml, the cytotoxicity of the iridium complex probe was low. The hemolytic toxicity of iridium complexes was evaluated by hemolysis test. When the concentration of iridium complexes was higher than 0.1 mg / ml, the hemolytic activity was significantly reduced. Cell imaging analysis of IrCN showed very weak fluorescence in the cell nucleus, and most of the labeling was located in the cytoplasm, indicating that IrCN can successfully penetrate the cell membrane and enter the cell. As the concentration increased, more IrCN entered the cell, and the fluorescence intensity was observed to increase accordingly. The fluorescence intensity did not decrease over time, indicating that IrCN has strong stability.

[0029] 3. FGF21 can activate the PI3K / AKT / mTOR signaling pathway. The expression levels of PI3K, Akt, and mTOR were evaluated by qPCR and Western Blot. The results showed that FGF21 protein specifically regulates the expression of marker molecules in the downstream signaling pathway, and iridium complexes have no effect on the expression of marker molecules in the downstream signaling pathway. This can provide a basis for subsequent exploration of the function and tracing effect of iridium complex-labeled FGF21. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the experimental result diagram of the binding ratio of iridium complex to protein;

[0031] Figure 2 This is the experimental result diagram of the binding time of iridium complexes and proteins;

[0032] Figure 3 This is the experimental result of the stability of the binding between iridium complex and protein;

[0033] Figure 4 This is a diagram showing the experimental results of the binding specificity between the iridium complex and the FGF21 protein;

[0034] Figure 5 Figure 1 is the experimental result of cytotoxicity;

[0035] Figure 6 This is the experimental result diagram of the effect of iridium complex on hemolytic activity;

[0036] Figure 7 This is the experimental result of live cell fluorescence imaging of the probe IrSN1;

[0037] Figure 8-10 Figure 1 is the experimental result of immunofluorescence;

[0038] Figure 11-12 This figure shows the experimental results of the effect of IrCN-labeled FGF21 protein on mRNA expression and protein secretion. DETAILED DESCRIPTION

[0039] The technical solutions of the present invention will be described clearly and completely below with reference to specific embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0040] Reference Figure 1-12 , an experimental method for the effect of iridium complexes on the function of FGF21 protein, comprising the following steps:

[0041] S1: Preparation of iridium complexes, proteins, cells, and reagents: iridium complex IrCN, FGF21 protein, mouse kidney fibroblasts BHK21, and bovine mammary epithelial cells MAC-T;

[0042] S2: Binding characteristics of iridium complexes to proteins: Iridium complexes were dissolved in dimethyl sulfoxide to prepare a stock solution with a concentration of 10 mg / ml, and then the stock solution was diluted with PBS buffer in a gradient manner to make the final concentration of iridium complexes 0.05, 0.1, 0.5, and 1 mg / ml. Different concentrations of iridium complexes were added to 20 μg of His-tagged camel FGF21 protein, and the changes in fluorescence intensity at different times were recorded by a chemiluminescence imaging system. The fluorescence intensity was analyzed using ImageJ software. In this experiment, different concentrations of iridium complexes were used to bind to FGF21 protein, and the fluorescence intensity was observed by a chemiluminescence instrument. The results are as follows: Figure 1 , the fluorescence intensity gradually increased with the increase of concentration, and reached the highest level in the test tube with 0.2 μg iridium complex and 20 μg FGF21 protein added, indicating that the optimal binding concentration ratio of iridium complex to FGF21 protein was 1:100;

[0043] More specifically, the optimal binding time of the iridium complex and the FGF21 protein is as shown in Figure 2. After the iridium complex is combined with the FGF21 protein, the fluorescence intensity gradually increases with the extension of the reaction time, and reaches the highest at 10 minutes of reaction, indicating that the optimal binding time of the iridium complex and the FGF21 protein is 10 minutes;

[0044] More specifically, the binding stability and half-life of iridium complexes to FGF21 protein were investigated. In this experiment, iridium complexes and FGF21 protein of the same concentration were placed at -20°C, 4°C, and room temperature, and the fluorescence intensity was recorded at different times using an intelligent darkroom three-purpose UV analyzer. The results are shown in Figure 2. Figure 3 As the reaction time increased, the fluorescence intensity of the iridium complex fluorescent probe bound to the FGF21 protein at room temperature was significantly higher than that at 4°C and -20°C, indicating that the binding of the iridium complex to the FGF21 protein is most stable at room temperature. Even after 22 days of reaction, green fluorescence was still present in the test tube, suggesting that the binding half-life of the iridium complex to the FGF21 protein is relatively long.

[0045] S3: Binding specificity of iridium complexes to FGF21 protein: labeling and mixing, FGF21 protein labeled with iridium complex probe was mixed with serum albumin containing histidine, and different iridium complex to FGF21 protein concentration ratios were set to 1:100, 1:200, 1:300, 1:400, as well as different ratios of BSA to FGF21 protein;

[0046] More specifically, gel electrophoresis and detection: gel electrophoresis separation was performed after 24 hours, and a chemiluminescence imaging system was used to detect whether BSA showed fluorescence. By analyzing the results of different lanes, the binding specificity of the iridium complex to the FGF21 protein was determined;

[0047] More specifically, the specific binding rules of iridium complexes and FGF21 protein. In this experiment, FGF21 protein labeled with iridium complex fluorescent probe at different concentrations was mixed with BSA protein, and gel electrophoresis was performed after 24 hours. The fluorescence of BSA was detected by chemiluminescence imaging analysis system. The results are as follows: Figure 4In Figure A, lanes 1, 2, 3, and 4 have a distinct bright band at 66 kDa compared to lane 9. In Figure B, the brightness of the bands at 66 kDa in lanes 1-6 did not increase. When the concentration ratio of the iridium complex to the FGF21 protein was 1:100, free probes would bind specifically to other proteins after the iridium complex binds to the protein. When the concentration ratio of the iridium complex to the FGF21 protein was 1:200 or lower, the iridium complex completely bound to the protein, and no free probe would bind specifically to other proteins. Moreover, whether the iridium complex fluorescent probe dissociates after the reaction with the FGF21 protein is complete has nothing to do with the amount of BSA protein added.

[0048] S4: Cytotoxicity test of iridium complexes: Cell inoculation and treatment: At 37°C and 5% CO2, subcultured cells were inoculated into 96-well cell culture plates. When the cells grew to a density of about 85%, the culture medium in the wells was removed and pre-prepared culture medium solutions containing different concentrations of iridium complex probes were added to make the final concentrations of iridium complex probes 0, 0.01, 0.02, 0.05, 0.1, and 0.2 mg / ml, respectively. A control group was set up at the same time, and 3 parallel experiments were set up for each group.

[0049] Toxicity assay: After 24 hours of co-culture with cells, add 10 μl of CCK8 solution to each well and continue incubation in the incubator for 2 hours. Measure the absorbance of the solution in each well at a wavelength of 450 nm using a microplate reader. Calculate the cell viability based on the absorbance to evaluate the effect of the iridium complex on cell viability.

[0050] More specifically, in order to detect the effect of iridium complexes on cell viability, we cultured BHK21 and MAC-T cells, co-cultured the cells with iridium complex solutions of different concentrations for 24 hours, and then measured the results using the CCK8 method. Figure 5 When the concentration of iridium complex is greater than 0.05 mg / ml, the cell survival rate is significantly reduced. When the concentration of iridium complex is lower than 0.05 mg / ml, the cytotoxicity of iridium complex probe is low.

[0051] S5: Determination of hemolytic activity of iridium complex probe: Blood was collected from healthy mice, and 10 times the amount of 0.9% sodium chloride solution was added. The blood was washed by centrifugation and the supernatant was removed. The obtained red blood cells were made into a 4% suspension with 0.9% sodium chloride solution. The sample solution was prepared in a centrifuge tube. Positive and negative controls were set up. 200 μl of red blood cell suspension was added to each tube. The tube was centrifuged at 1000 g for 3 minutes. The hemolysis phenomenon was observed with the naked eye. 100 μl of supernatant was aspirated into a new 96-well plate and the OD540nm was detected.

[0052] More specifically, hemolysis refers to the rupture or destruction of red blood cells, resulting in the release of hemoglobin and other cell contents into the surrounding environment. The purpose of the hemolysis test is to determine the effect of the reagent on the red blood cells in the blood by observing whether hemolysis occurs and the extent of hemolysis. Figure 6 The hemolytic activity of iridium complexes at concentrations of 0.01, 0.02, and 0.05 mg / ml was high, with hemolysis rates of 0.8%, 1.2%, and 3.2%, respectively. When the concentration of iridium complexes was higher than 0.1 mg / ml, the hemolytic activity decreased significantly.

[0053] S6: Live cell imaging test: BHK21 cells were seeded in a confocal microplate. When the cells grew to a density of about 50%, different concentrations of iridium complex probe IrCN solution were added to the microplate. After co-incubation with the cells for 10 minutes, the microplate was washed twice with phosphate buffer and used directly for imaging.

[0054] More specifically, the cytotoxicity of the probe IrCN on cells was investigated using the CCK8 assay. The results showed that when the iridium complex concentration was 0.01 and 0.02 mg / ml, the cell survival rate was high after the probe IrCN solution was co-cultured with the cells. This indicates that the probe IrSN1 has low cytotoxicity at this concentration and is suitable for live cell imaging studies. The ability of the probe IrCN to detect histidine in BHK21 cells was evaluated using confocal microscopy. Figure 7 The fluorescence in the cells mainly originated from the cytoplasm, indicating that IrCN can successfully penetrate the cell membrane and enter the cell. We also studied the cells treated with different concentrations of IrCN. Compared with 0.01mg / ml IrCN, a stronger green fluorescence signal was observed in the entire cell area when the IrCN concentration was 0.02mg / ml. This shows that as the concentration increases, the probe IrSN1 binds more to the histidine in the cell, resulting in an increase in the green fluorescence signal in the cell.

[0055] S7: Immunofluorescence experiment: BHK21 cells were seeded in confocal microplates. When the cells grew to a density of about 50%, they were washed twice with pre-cooled PBS and fixed with 4% paraformaldehyde for 30 minutes at room temperature. After washing three times with PBS, the cells were permeabilized in 0.2% TritonX-100 for 10 minutes, washed three times with PBS, and stained with 4,0,6-diaminophenylindole for 10 minutes. After washing three times with PBS, different concentrations of the iridium complex probe IrCN were added to the microplates, and images were captured and recorded using a laser confocal microscope.

[0056] More specifically, cells were treated with different concentrations of iridium complexes and then the nuclei were stained with DAPI. Fluorescence images were taken using a confocal microscope. Figure 8-10Very weak fluorescence was observed in the cell nucleus, and most of the labeling was localized in the cytoplasm. According to fluorescence detection, as the concentration increased, more IrCN entered the cell, and the fluorescence intensity was observed to increase accordingly; and after three hours of incubation, the fluorescence intensity did not decrease, indicating that the iridium complex has strong stability;

[0057] S8: RNA extraction and cDNA synthesis: Total RNA was extracted from camel fibroblasts using the TRIzol method, and cDNA was synthesized according to the PrimeScript RT Reagent Kit instructions. The concentration and purity of the cDNA were determined by UV spectrophotometry.

[0058] S9: Protein extraction and concentration determination: Total cell protein was extracted with RIPA protein lysis buffer, and the protein concentration was determined using a BCA protein assay kit. Equal amounts of total protein were separated by 12% SDS-PAGE gel electrophoresis and then electrotransferred to a 0.45 µm polyvinylidene difluoride membrane. The membrane was blocked with blocking buffer for 20 minutes and then incubated with anti-tubulin, anti-PI3K, anti-AKT, and anti-mTOR antibodies at 4°C overnight. After washing three times with PBS, the membrane was incubated with enzyme-labeled goat anti-rabbit IgG at room temperature for 2 hours. Finally, the protein bands were visualized using ECL chemiluminescence detection and captured by a gel imaging system.

[0059] More specifically, in order to confirm the effect of iridium complex IrCN on the expression of downstream signaling pathway markers in BHK21 cells, FGF21 proteins from different species and FGF21 proteins from different species labeled with iridium complexes were used to treat BHK21 cells, and the expression levels of PI3K, Akt, and mTOR were evaluated by qPCR and Western Blot. Figure 11-12 As shown, at the mRNA level, compared with the control group, the expression levels of PI3K, Akt, and mTOR signaling molecules in various species were significantly upregulated, and the expression level of FGF21 protein labeled with iridium complexes also increased significantly. Consistent with this, the expression levels of PI3K, Akt, and mTOR signaling marker molecules in FGF21 protein of various species and FGF21 protein labeled with iridium complexes were significantly upregulated. The results show that FGF21 protein specifically regulates the expression of marker molecules in downstream signaling pathways, and iridium complexes have no effect on the expression of marker molecules in downstream signaling pathways.

[0060] S10. Statistical analysis: GraphPad Prism software was used to analyze the results. Data are expressed as mean ± SEM. Unpaired Student's t test was used for comparison between two groups. Two-way analysis of variance was used for multiple hippocampal measurement groups. P < 0.05 was considered statistically significant.

[0061] In this example, S1 also needs to prepare DMEM culture medium, phosphate-buffered saline (PBS, pH=7.2), trypsin-EDTA solution, penicillin and streptomycin, collagenase IV, RIPA protein lysis buffer, TRIzol, CCK-8, propidium iodide, BCA protein detection kit, ECL chemiluminescence detection kit, reverse transcription kit, SYBR Premix Ex Taq II kit, G418, protein marker, fetal bovine serum, rabbit anti-PI3K polyclonal antibody, rabbit anti-AKT polyclonal antibody, rabbit anti-mTOR polyclonal antibody, rabbit anti-β-Tubulin antibody, enzyme-labeled goat anti-rabbit IgG, PCR primers and other reagents.

[0062] In this embodiment, S2 also includes a binding time determination: after the iridium complex is bound to the FGF21 protein, the fluorescence intensity at different exposure times is also recorded using a chemiluminescence imaging system. The results are analyzed graphically to determine the optimal binding time of the iridium complex and the FGF21 protein by analyzing the change in protein fluorescence intensity with the extension of the exposure time. S2 also includes a binding stability determination: the iridium complex and FGF21 protein at the same concentration are placed at -20°C, 4°C, and room temperature, and the fluorescence intensity is recorded at different times using an intelligent darkroom three-in-one UV analyzer. The change in fluorescence intensity of the iridium complex fluorescent probe bound to the FGF21 protein with the extension of the exposure time at different temperatures is observed to determine the binding stability of the iridium complex and the FGF21 protein at different temperatures.

[0063] In this example, S5 also evaluated the hemolytic activity of the iridium complex according to the hemolysis rate calculation formula: Hemolysis rate = (OD sample test group - OD negative control group) / (OD positive control group - OD negative control group) × 100%. S6 imaging and analysis used ImageJ software for fluorescence intensity analysis. Confocal microscopy was used to observe the intracellular fluorescence distribution to evaluate the ability of IrCN to detect intracellular histidine and the changes in intracellular fluorescence signals after treatment with different concentrations of IrCN. S7 imaging and analysis involved capturing and recording images using a laser confocal microscope and analyzing the fluorescence intensity using ImageJ software to observe the fluorescence distribution and intensity changes in the cell nucleus and cytoplasm after treatment with different concentrations of iridium complex. S8 also included qPCR reaction and analysis: qPCR reaction system was prepared according to the SYBR Premix Ex Taq II Kit protocol and the reaction was performed on a LightCycler instrument. The relative gene expression levels were analyzed using the 2-DDCT method. ACTIN was used as an internal reference to normalize the number of transcripts in each sample and evaluate the expression levels of PI3K, Akt, and mTOR genes.

[0064] In the present invention, an iridium complex was bound to camel FGF21 protein, achieving an optimal binding concentration ratio of 1:100 and an optimal binding time of ten minutes, demonstrating strong binding stability and a long binding half-life. Protein separation by gel electrophoresis revealed that when the iridium complex was bound to FGF21 at a concentration ratio of 1:100, free probes specifically bound to other proteins. At concentrations of 1:200 or lower, the iridium complex was completely bound to the protein, with no free probes specifically binding to other proteins. Cytotoxicity of the iridium complex was assessed using the CCK8 assay, revealing a significant decrease in cell viability when the iridium complex concentration was greater than 0.05 mg / ml. Cytotoxicity of the iridium complex probe was also low when the iridium complex concentration was below 0.05 mg / ml. Hemolytic activity of the iridium complex was also assessed using a hemolysis assay, revealing a significant decrease in hemolytic activity when the iridium complex concentration was greater than 0.1 mg / ml. Cell imaging analysis of IrCN revealed very weak fluorescence in the nucleus, with the majority of the labeling localized in the cytoplasm, indicating that IrCN can successfully penetrate the cell membrane and enter the cell. Furthermore, as the concentration increased, more IrCN entered the cell, and the fluorescence intensity increased accordingly. The fluorescence intensity did not decrease over time, indicating the strong stability of IrCN. FGF21 can activate the PI3K / AKT / mTOR signaling pathway. PI3K, Akt, and mTOR expression levels were assessed by qPCR and Western Blot. The results showed that FGF21 protein specifically regulates the expression of marker molecules in downstream signaling pathways, and that iridium complexes had no effect on this regulation. This provides a foundation for further exploration of the function and tracing effects of iridium complex-labeled FGF21.

[0065] The above describes in detail the experimental method for the effect of an iridium complex on the function of the FGF21 protein provided by the present invention. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above examples is only intended to help understand the method and core concept of the present invention. It should be noted that for those skilled in the art, various improvements and modifications may be made to the present invention without departing from the principles of the present invention, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. An experimental method for the effect of iridium complexes on the function of FGF21 protein, characterized in that: The following steps are involved: S1: Preparation of iridium complexes, proteins, cells, and reagents: iridium complex IrCN, FGF21 protein, mouse kidney fibroblasts BHK21, and bovine mammary epithelial cells MAC-T; S2: Binding characteristics of iridium complexes to proteins: The iridium complex was dissolved in dimethyl sulfoxide to prepare a 10 mg / ml stock solution. The stock solution was then graded diluted with PBS buffer to give final iridium complex concentrations of 0.05, 0.1, 0.5, and 1 mg / ml. Iridium complexes of varying concentrations were added to 20 μg of His-tagged camel FGF21 protein. Fluorescence intensity changes at different times were recorded using a chemiluminescence imaging system and analyzed using ImageJ software. S3: Binding specificity of iridium complexes to FGF21 protein: labeling and mixing, FGF21 protein labeled with iridium complex probe was mixed with serum albumin containing histidine, and different iridium complex to FGF21 protein concentration ratios were set to 1:100, 1:200, 1:300, 1:400, as well as different ratios of BSA to FGF21 protein; Gel electrophoresis and detection: After 24 hours, gel electrophoresis separation was performed and a chemiluminescence imaging system was used to detect whether BSA showed fluorescence. By analyzing the results of different lanes, the binding specificity of the iridium complex to the FGF21 protein was determined. S4: Cytotoxicity test of iridium complexes: Cell inoculation and treatment: At 37°C and 5% CO2, subcultured cells were inoculated into 96-well cell culture plates. When the cells grew to a density of about 85%, the culture medium in the wells was removed and pre-prepared culture medium solutions containing different concentrations of iridium complex probes were added to make the final concentrations of iridium complex probes 0, 0.01, 0.02, 0.05, 0.1, and 0.2 mg / ml, respectively. A control group was set up at the same time, and 3 parallel experiments were set up for each group. Toxicity assay: After 24 hours of co-culture with cells, add 10 μl of CCK8 solution to each well and continue incubation in the incubator for 2 hours. Measure the absorbance of the solution in each well at a wavelength of 450 nm using a microplate reader. Calculate the cell viability based on the absorbance to evaluate the effect of the iridium complex on cell viability. S5: Hemolytic activity assay of iridium complex probe: Blood was collected from healthy mice, 10 times the volume of 0.9% sodium chloride solution was added, and the blood was centrifuged and washed. The supernatant was removed, and the resulting red blood cells were made into a 4% suspension in 0.9% sodium chloride solution. Prepare sample solutions in centrifuge tubes, set up positive and negative controls, add 200 μl of red blood cell suspension to each tube, centrifuge at 1000 g for 3 min, observe hemolysis with the naked eye, pipette 100 μl of supernatant into a new 96-well plate, and measure OD540nm; S6: Live cell imaging test: BHK21 cells were seeded in a confocal microplate. When the cells grew to a density of about 50%, different concentrations of iridium complex probe IrCN solution were added to the microplate. After co-incubation with the cells for 10 minutes, the microplate was washed twice with phosphate buffer and used directly for imaging. S7: Immunofluorescence experiment: BHK21 cells were seeded in confocal microplates. When the cells grew to a density of about 50%, they were washed twice with pre-cooled PBS and fixed with 4% paraformaldehyde for 30 minutes at room temperature. After washing three times with PBS, the cells were permeabilized in 0.2% TritonX-100 for 10 minutes, washed three times with PBS, and stained with 4,0,6-diaminophenylindole for 10 minutes. After washing three times with PBS, different concentrations of the iridium complex probe IrCN were added to the microplates, and images were captured and recorded using a laser confocal microscope. S8: RNA extraction and cDNA synthesis: Total RNA was extracted from camel fibroblasts using the TRIzol method, and cDNA was synthesized according to the PrimeScript RT Reagent Kit instructions. The concentration and purity of the cDNA were determined by UV spectrophotometry. S9: Protein extraction and concentration determination: Total cell protein was extracted with RIPA protein lysis buffer, and the protein concentration was determined using a BCA protein assay kit. Equal amounts of total protein were separated by 12% SDS-PAGE gel electrophoresis and then electrotransferred to a 0.45 µm polyvinylidene difluoride membrane. The membrane was blocked with blocking buffer for 20 minutes and then incubated with anti-tubulin, anti-PI3K, anti-AKT, and anti-mTOR antibodies at 4°C overnight. After washing three times with PBS, the membrane was incubated with enzyme-labeled goat anti-rabbit IgG at room temperature for 2 hours. Finally, the protein bands were visualized using ECL chemiluminescence detection and captured by a gel imaging system. S10. Statistical analysis: GraphPad Prism software was used to analyze the results. Data are expressed as mean ± SEM. Unpaired Student's t test was used for comparison between two groups. Two-way analysis of variance was used for multiple hippocampal measurement groups. P < 0.05 was considered statistically significant.

2. The experimental method for the effect of an iridium complex on the function of FGF21 protein according to claim 1, characterized in that: The S1 also requires preparation of DMEM culture medium, phosphate-buffered saline, trypsin-EDTA solution, penicillin and streptomycin, collagenase IV, RIPA protein lysis buffer, TRIzol, CCK-8, propidium iodide, BCA protein detection kit, ECL chemiluminescence detection kit, reverse transcription kit, SYBR Premix Ex Taq II kit, G418, protein markers, fetal bovine serum, rabbit anti-PI3K polyclonal antibody, rabbit anti-AKT polyclonal antibody, rabbit anti-mTOR polyclonal antibody, rabbit anti-β-Tubulin antibody, enzyme-labeled goat anti-rabbit IgG, PCR primers and various reagents.

3. The experimental method for the effect of an iridium complex on the function of FGF21 protein according to claim 1, characterized in that: Said S2 also includes binding time determination: after the iridium complex is combined with the FGF21 protein, the fluorescence intensity at different action times is also recorded by the chemiluminescence imaging system, and the result graph analyzes the change in protein fluorescence intensity with the extension of the action time to determine the optimal binding time of the iridium complex and the FGF21 protein.

4. The experimental method for the effect of an iridium complex on the function of FGF21 protein according to claim 1, characterized in that: S2 also includes a binding stability test: the iridium complex and FGF21 protein at the same concentration are placed at -20°C, 4°C, and room temperature, and the fluorescence intensity is recorded at different times using an intelligent darkroom three-in-one UV analyzer. The change in fluorescence intensity of the iridium complex fluorescent probe binding to the FGF21 protein is observed over time at different temperatures to determine the binding stability of the iridium complex and FGF21 protein at different temperatures.

5. The experimental method for the effect of an iridium complex on the function of FGF21 protein according to claim 1, characterized in that: In S5, the hemolytic activity of the iridium complex was evaluated according to the hemolysis rate calculation formula: hemolysis rate = (OD sample test group - OD negative control group) / (OD positive control group - OD negative control group) × 100%.

6. The experimental method for the effect of an iridium complex on the function of FGF21 protein according to claim 1, characterized in that: The S6 imaging and analysis used ImageJ software to perform fluorescence intensity analysis, and confocal microscopy was used to observe the intracellular fluorescence distribution to evaluate the detection ability of IrCN for intracellular histidine and the changes in intracellular fluorescence signals under treatment with different concentrations of IrCN.

7. The experimental method for the effect of an iridium complex on the function of FGF21 protein according to claim 1, characterized in that: Imaging and analysis in S7: using a laser confocal microscope to capture and record images, using ImageJ software to perform fluorescence intensity analysis, and observing the fluorescence distribution and intensity changes in the cell nucleus and cytoplasm after treatment with different concentrations of iridium complexes.

8. The experimental method for the effect of an iridium complex on the function of FGF21 protein according to claim 1, characterized in that: Said S8 also includes qPCR reaction and analysis: preparing the qPCR reaction system according to the SYBR Premix Ex Taq II Kit protocol, and performing the reaction on a LightCycler instrument; using the 2-DDCt method to analyze the relative expression of genes, and using ACTIN as an internal reference to normalize the number of transcripts in each sample, and evaluating the expression levels of PI3K, Akt, and mTOR genes.

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