Experimental method for influence of iridium complex on FGF21 protein function

Abstract

The invention belongs to the field of iridium complexes, and particularly relates to an experimental method for influence of iridium complexes on FGF21 protein function, 1, the method comprises the following steps: S1, preparing iridium complexes, proteins, cells and reagents: preparing iridium complexes, camel, mouse and human FGF21 proteins, mouse kidney fibroblasts BHK21 and bovine mammary epithelial cells MAC-T; s2, combining characteristics of the iridium complexes and the protein: dissolving the iridium complexes with dimethyl sulfoxide to prepare mother liquor with the concentration of 10mg / ml, then diluting the mother liquor with a PBS buffer solution in a gradient manner to enable the final concentration of the iridium complexes to be 0.05 mg / ml, 0.1 mg / ml, 0.5 mg / ml and 1mg / ml, and adding the iridium complexes with different concentrations into 20mu g of camel FGF21 protein with a His label. According to the method, the binding of the iridium complex and the camel FGF21 protein, the binding specificity of the iridium complex and the FGF21 protein and the cytotoxicity of the iridium complex can be efficiently verified.

Description

Technical Field

[0001] The invention relates to the technical field of iridium complexes, and in particular to an experimental method for 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 formed an intersection. Iridium (III) complexes, as metal organic compounds, can bind to biological macromolecules for labeling and tracing, and have become a current research hotspot. Fibroblast growth factor 21 (FGF21) is a member of the FGF gene family. It has the function of regulating sugar, fat and protein metabolism and plays an important role in biological processes such as cell growth, differentiation and energy metabolism.

[0003] Iridium complexes are a class of metal organic compounds with unique chemical and physical properties and potential functions. They have broad application prospects in materials science, biology, medicine and other fields. Research in the past decade has shown that iridium complexes can interact with a variety of biological molecules, including DNA, RNA and proteins. These interactions regulate many related physiological processes by affecting the structure and partial functions of biological molecules. However, the current research on the binding of iridium complexes to proteins is still in its infancy, especially the interaction with FGF21 protein has not been fully studied. Therefore, we proposed an experimental method for the effect of iridium complexes on the function of FGF21 protein to solve the above problems. 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: An experimental method for the effect of iridium complexes on FGF21 protein function comprises the following steps: S1: Preparation of iridium complexes, proteins, cells and reagents: Iridium complexes (such as IrCN, etc.), camel, mouse, human FGF21 proteins, mouse kidney fibroblasts BHK21 and bovine mammary epithelial cells MAC-T were all preserved by our laboratory; S2: Binding characteristics of iridium complexes with proteins: Iridium complexes were dissolved in dimethyl sulfoxide to prepare a mother solution with a concentration of 10 mg / ml, and then the mother solution was gradiently diluted with PBS buffer to make the final concentration of iridium complexes 0.05, 0.1, 0.5, and 1 mg / ml. Iridium complexes of different concentrations were added to 20 μg of camel FGF21 protein with a His tag, and the changes in fluorescence intensity at different times were recorded by a chemiluminescence imaging system, and the fluorescence intensity was analyzed using ImageJ software; S3: Binding specificity of iridium complexes to FGF21 protein: labeling and mixing, the FGF21 protein labeled with the iridium complex probe was mixed with histidine-containing serum albumin (BSA), and different iridium complex to FGF21 protein concentration ratios (1:100, 1:200, 1:300, 1:400) and different ratios of BSA to FGF21 protein were set; Gel electrophoresis and detection: After 24 hours, gel electrophoresis was performed to separate the BSA and the fluorescence was detected by chemiluminescence imaging system. The binding specificity between the iridium complex and FGF21 protein was determined by analyzing the results of different lanes. S4: Cytotoxicity test of iridium complexes: Cell inoculation and treatment: At 37°C and 5% CO2, subcultured cells were inoculated in a 96-well cell culture plate. When the cells grew to a density of about 85%, the culture medium in the wells was removed and the culture medium solution containing different concentrations of iridium complex probes prepared in advance was added to make the final concentrations of iridium complex probes 0, 0.01, 0.02, 0.05, 0.1, 0.2 mg / ml, etc., respectively. A control group was set up at the same time, and 3 parallel experiments were set up in each group. Toxicity detection: After 24 hours of co-culture with cells, 10 μl of CCK8 solution was added to each well and incubated in the incubator for another 2 hours. The absorbance of the solution in each well at a wavelength of 450 nm was measured with an ELISA reader. The cell survival rate was calculated based on the absorbance value to evaluate the effect of the iridium complex on cell viability. S5: Determination of hemolytic activity of iridium complex probe: Take blood from healthy mice, add 10 times the amount of 0.9% sodium chloride solution, centrifuge and wash, remove the supernatant, make the obtained red blood cells into a 4% suspension with 0.9% sodium chloride solution, prepare the sample solution in a centrifuge tube, set up positive and negative controls, add 200μl of red blood cell suspension to each tube, centrifuge at 1000g for 3min, observe the hemolysis phenomenon with the naked eye, aspirate 100μL of supernatant into a new 96-well plate, and detect OD540nm; S6: Live cell imaging test: BHK21 cells were inoculated in a confocal dish. When the cells grew to a density of about 50%, different concentrations of iridium complex probe IrCN solution were added to the dish. After co-culture with the cells for 10 minutes, the dish was washed twice with phosphate buffered saline (PBS) and used directly for imaging. S7: Immunofluorescence experiment: BHK21 cells were seeded in a confocal dish. When the cells grew to a density of about 50%, they were washed twice with pre-cooled PBS, fixed with 4% paraformaldehyde for 30 minutes at room temperature, washed 3 times with PBS, permeabilized in 0.2% TritonX-100 for 10 minutes, washed 3 times with PBS, and stained with 40,6-diaminophenylindole (DAPI) for 10 minutes. After washing 3 times with PBS, different concentrations of iridium complex probe IrCN were added to the dish, 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 cDNA were detected by UV spectrophotometer. 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 (PVDF) membrane (Biosharp, Beijing, China). The membrane was blocked with blocking buffer for 20 min and then incubated with anti-tubulin antibody (1:10000), anti-PI3K antibody (1:1000), anti-AKT antibody (1:1000), and anti-mTOR antibody (1:1000) at 4 °C overnight. After washing with PBS three times, the membrane was incubated with enzyme-labeled goat anti-rabbit IgG (1:5000) at room temperature for 2 h. Finally, the protein bands were visualized using ECL chemiluminescence detection and captured by a gel imaging system (Tannon, Shanghai, China). S10. Statistical analysis: The results were analyzed using GraphPad Prism (version 9.3.1) software (San Diego, CA, USA). The data were expressed as mean ± SEM. Unpaired Student's t test was used for comparison between the two groups. Two-way ANOVA was used for multiple hippocampal measurement groups. P < 0.05 was considered statistically significant.

[0006] Preferably, the S1 also needs to prepare DMEM culture medium, phosphate buffered saline (PBS, pH=7.2), trypsin-EDTA solution (0.25%), penicillin and streptomycin (100 times), collagenase IV, RIPA protein lysis buffer, TRIzol, CCK-8, propidium iodide (PI), BCA protein detection kit, ECL chemiluminescence detection kit, reverse transcription kit, SYBR Premix Ex Taq II kit, G418, protein markers, fetal bovine serum (FBS), 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.

[0007] Preferably, S2 also includes a 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.

[0008] 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 dark box three-purpose UV analyzer. Observe 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.

[0009] 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%.

[0010] Preferably, the S6 imaging and analysis uses ImageJ software to perform fluorescence intensity analysis, and observes the intracellular fluorescence distribution through a confocal microscope to evaluate the detection ability of IrCN for intracellular histidine and the changes in intracellular fluorescence signals under treatment with different concentrations of IrCN.

[0011] 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.

[0012] Preferably, S8 also includes qPCR reaction and analysis: the qPCR reaction system is prepared according to the protocol of SYBR Premix Ex Taq II Kit, and the reaction is performed on a LightCycler instrument (Roche, Indianapolis, IN, USA). The relative expression of genes is analyzed by the 2-DDCt method, and the number of transcripts in each sample is normalized using ACTIN as an internal reference to evaluate the expression levels of genes such as PI3K, Akt, and mTOR.

[0013] Beneficial effects of the present invention: 1. The iridium complex was combined with camel FGF21 protein, and the optimal concentration ratio of the binding was 1:100, the optimal binding time was ten minutes, and it had strong binding stability and a long binding half-life; the protein was separated by gel electrophoresis. When the concentration ratio of the iridium complex to the FGF21 protein was 1:100, the free probe would specifically bind 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 combined with the protein, and no free probe would specifically bind to other proteins.

[0014] 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 iridium complex probes 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 markers were 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. As time went on, the fluorescence intensity did not weaken, indicating that IrCN has strong stability.

[0015] 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 FGF21 after labeling with iridium complexes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the experimental result diagram of the binding ratio of iridium complex to protein; Figure 2 This is the experimental result diagram of the binding time of iridium complex and protein; Figure 3 This is the experimental result diagram of the stability of the binding between iridium complex and protein; Figure 4 This is a diagram showing the experimental results of the binding specificity between the iridium complex and the FGF21 protein; Figure 5 Figure 1 is the experimental result of cytotoxicity; Figure 6 The figure is the experimental result of the effect of iridium complex on hemolytic activity; Figure 7 This is the experimental result of live cell fluorescence imaging of probe IrSN1; Figure 8-10 This is the result of immunofluorescence experiment; 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

[0017] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] Reference Figure 1-12 , an experimental method for 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 complexes (such as IrCN, etc.), camel, mouse, human FGF21 proteins, mouse kidney fibroblasts BHK21 and bovine mammary epithelial cells MAC-T were all preserved by our laboratory; S2: Binding characteristics of iridium complexes with proteins: Iridium complexes were dissolved in dimethyl sulfoxide to prepare a mother solution with a concentration of 10 mg / ml, and then the mother solution was gradiently diluted with PBS buffer 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 camel FGF21 protein with a His tag, and the changes in fluorescence intensity at different times were recorded by a chemiluminescence imaging system, and 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 in a chemiluminescence instrument. The results are shown in Figure 2. Figure 1 , the fluorescence intensity gradually increased with the increase of concentration, and the fluorescence intensity reached the highest in the test tube with 0.2μg iridium complex and 20μg FGF21 protein added, which indicated that the optimal binding concentration ratio of iridium complex and FGF21 protein was 1:100; 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 action 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; More specifically, the binding stability and half-life of iridium complexes and FGF21 proteins. In this experiment, the same concentration of iridium complexes and FGF21 proteins were placed in -20℃, 4℃ and room temperature environments, and the fluorescence intensity was recorded at different times using an intelligent dark box three-purpose UV analyzer. The results are shown in Figure 2. Figure 3 As the action time increases, the fluorescence intensity of the iridium complex fluorescent probe binding to FGF21 protein at room temperature is 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. After 22 days of reaction, the iridium complex and the FGF21 protein still have green fluorescence in the test tube, which preliminarily considers that the binding half-life of the iridium complex and the FGF21 protein is relatively long.

[0019] S3: Binding specificity of iridium complexes to FGF21 protein: labeling and mixing, the FGF21 protein labeled with the iridium complex probe was mixed with histidine-containing serum albumin (BSA), and different iridium complex to FGF21 protein concentration ratios (1:100, 1:200, 1:300, 1:400) and different ratios of BSA to FGF21 protein were set; 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, and the binding specificity of the iridium complex and the FGF21 protein was determined by analyzing the results of different lanes; 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 chemiluminescence imaging analysis system was used to detect whether BSA showed fluorescence. The results are as follows: Figure 4 In Figure A, lanes 1, 2, 3, and 4 have an obvious bright band at 66 kDa compared with lane 9. In Figure B, the brightness of the band 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, there would be free probes that specifically bind 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 was completely bound to the protein, and there would be no free probes that specifically bind to other proteins. Moreover, whether the iridium complex fluorescent probe dissociates after the complete reaction with the FGF21 protein has nothing to do with the amount of BSA protein added. S4: Cytotoxicity test of iridium complexes: Cell inoculation and treatment: At 37°C and 5% CO2, subcultured cells were inoculated in a 96-well cell culture plate. When the cells grew to a density of about 85%, the culture medium in the wells was removed and the culture medium solution containing different concentrations of iridium complex probes prepared in advance was added to make the final concentrations of iridium complex probes 0, 0.01, 0.02, 0.05, 0.1, 0.2 mg / ml, etc., respectively. A control group was set up at the same time, and 3 parallel experiments were set up in each group. Toxicity detection: After 24 hours of co-culture with cells, 10 μl of CCK8 solution was added to each well and incubated in the incubator for another 2 hours. The absorbance of the solution in each well at a wavelength of 450 nm was measured with an ELISA reader. The cell survival rate was calculated based on the absorbance value to evaluate the effect of the iridium complex on cell viability. 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 them by the CCK8 method. The results are as follows 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. S5: Determination of hemolytic activity of iridium complex probe: Take blood from healthy mice, add 10 times the amount of 0.9% sodium chloride solution, centrifuge and wash, remove the supernatant, make the obtained red blood cells into a 4% suspension with 0.9% sodium chloride solution, prepare the sample solution in a centrifuge tube, set up positive and negative controls, add 200μl of red blood cell suspension to each tube, centrifuge at 1000g for 3min, observe the hemolysis phenomenon with the naked eye, aspirate 100μL of supernatant into a new 96-well plate, and detect OD540nm; 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 degree of hemolysis. Figure 6 The hemolytic activity of iridium complexes at concentrations of 0.01, 0.02, and 0.05 mg / ml was relatively 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 was significantly reduced; S6: Live cell imaging test: BHK21 cells were inoculated in a confocal dish. When the cells grew to a density of about 50%, different concentrations of iridium complex probe IrCN solution were added to the dish. After co-culture with the cells for 10 minutes, the dish was washed twice with phosphate buffered saline (PBS) and used directly for imaging. More specifically, the cytotoxicity of the probe IrCN to cells was investigated by using the CCK8 method, showing that when the concentration of the iridium complex 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, indicating that the probe IrSN1 had low cytotoxicity at this concentration and was suitable for live cell imaging studies. The ability of the probe IrCN to detect histidine in BHK21 cells was evaluated using confocal microscopy. The results are as follows: Figure 7 , the fluorescence in the cells mainly comes from the cytoplasm region, indicating that IrCN can successfully penetrate the cell membrane and enter the cell; we also studied the treatment of cells with different concentrations of IrCN. Compared with 0.01mg / ml IrCN, a stronger green fluorescence signal can be observed in the entire cell region when the IrCN concentration is 0.02mg / ml. This shows that as the concentration increases, the probe IrSN1 binds more to the intracellular histidine, resulting in an increase in the intracellular green fluorescence signal; S7: Immunofluorescence experiment: BHK21 cells were seeded in a confocal dish. When the cells grew to a density of about 50%, they were washed twice with pre-cooled PBS, fixed with 4% paraformaldehyde for 30 minutes at room temperature, washed 3 times with PBS, permeabilized in 0.2% TritonX-100 for 10 minutes, washed 3 times with PBS, and stained with 40,6-diaminophenylindole (DAPI) for 10 minutes. After washing 3 times with PBS, different concentrations of iridium complex probe IrCN were added to the dish, and images were captured and recorded using a laser confocal microscope. More specifically, after treating cells with different concentrations of iridium complexes, the cell nuclei were stained with DAPI. Fluorescence images were taken using a confocal microscope, and the results are shown in Figure 2. Figure 8-10 , very weak fluorescence was observed in the cell nucleus, and most of the labeling was located 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 weaken, indicating that the iridium complex has strong stability; 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 cDNA were detected by UV spectrophotometer. 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 (PVDF) membrane (Biosharp, Beijing, China). The membrane was blocked with blocking buffer for 20 min and then incubated with anti-tubulin antibody (1:10000), anti-PI3K antibody (1:1000), anti-AKT antibody (1:1000), and anti-mTOR antibody (1:1000) at 4 °C overnight. After washing with PBS three times, the membrane was incubated with enzyme-labeled goat anti-rabbit IgG (1:5000) at room temperature for 2 h. Finally, the protein bands were visualized using ECL chemiluminescence detection and captured by a gel imaging system (Tannon, Shanghai, China). More specifically, in order to confirm the effect of iridium complex IrCN on the expression of downstream signaling pathway marker molecules 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 proteins of various species and FGF21 proteins labeled with iridium complexes were significantly upregulated. The results showed that FGF21 protein specifically regulates the expression of marker molecules in downstream signaling pathways, and iridium complexes have no effect on regulating the expression of marker molecules in downstream signaling pathways. S10. Statistical analysis: The results were analyzed using GraphPad Prism (version 9.3.1) software (San Diego, CA, USA). The data were expressed as mean ± SEM. Unpaired Student's t test was used for comparison between the two groups. Two-way ANOVA was used for multiple hippocampal measurement groups. P < 0.05 was considered statistically significant.

[0020] In this embodiment, S1 also needs to prepare DMEM culture medium, phosphate buffered saline (PBS, pH=7.2), trypsin-EDTA solution (0.25%), penicillin and streptomycin (100 times), collagenase IV, RIPA protein lysis buffer, TRIzol, CCK-8, propidium iodide (PI), BCA protein detection kit, ECL chemiluminescence detection kit, reverse transcription kit, SYBR Premix Ex Taq II kit, G418, protein marker, fetal bovine serum (FBS), 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.

[0021] In this embodiment, S2 also includes a 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. S2 also includes: Binding stability determination: the same concentration of iridium complex and FGF21 protein are placed in -20°C, 4°C and room temperature environments, and the fluorescence intensity is recorded at different times using an intelligent dark box three-purpose ultraviolet analyzer. Observe the change in fluorescence intensity of the iridium complex fluorescent probe binding to the FGF21 protein with the extension of the action time at different temperatures to determine the binding stability of the iridium complex and the FGF21 protein at different temperatures.

[0022] In this embodiment, S5 also uses the hemolysis rate calculation formula: hemolysis rate = (OD sample test group-OD negative control group) / (OD positive control group-OD negative control group) × 100% to evaluate the hemolytic activity of the iridium complex. S6 imaging and analysis uses ImageJ software to perform fluorescence intensity analysis, and observes the intracellular fluorescence distribution through a confocal microscope to evaluate the detection ability of IrCN for intracellular histidine and the changes in intracellular fluorescence signals under different concentrations of IrCN treatment. S7 imaging and analysis: 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. S8 also includes qPCR reaction and analysis: preparing the qPCR reaction system according to the SYBR Premix Ex Taq II Kit protocol, and the reaction is performed on a LightCycler instrument (Roche, Indianapolis, IN, USA). The 2-DDCt method was used to analyze the relative expression of genes, and ACTIN was used as an internal reference to normalize the number of transcripts in each sample to evaluate the expression levels of genes such as PI3K, Akt, and mTOR.

[0023] In the present invention, the iridium complex is combined with the camel FGF21 protein to obtain an optimal concentration ratio of 1:100, an optimal binding time of ten minutes, strong binding stability and a long binding half-life; the protein is separated by gel electrophoresis, when the iridium complex and the FGF21 protein are at a concentration ratio of 1:100, the iridium complex will bind to the protein and there will be free probes that specifically bind to other proteins, and when the iridium complex and the FGF21 protein are at a concentration ratio of 1:200 and lower, the iridium complex is completely combined with the protein, and there will be no free probes that specifically bind to other proteins. The cytotoxicity of the iridium complex is detected by the CCK8 method. When the concentration of the iridium complex is greater than 0.05 mg / ml, the cell survival rate is significantly reduced. When the concentration of the iridium complex is lower than 0.05 mg / ml, the cytotoxicity of the iridium complex probe is low. The hemolytic toxicity of the iridium complex is evaluated by a hemolysis test. When the concentration of the iridium complex is higher than 0.1 mg / ml, the hemolytic activity is significantly reduced. Cell imaging analysis of IrCN showed very weak fluorescence in the nucleus, and most of the markers were located in the cytoplasm, indicating that IrCN can successfully penetrate the cell membrane and enter the cell. As the concentration increases, more IrCN enters the cell, and the fluorescence intensity increases accordingly. As time goes by, the fluorescence intensity does not decrease, indicating that IrCN has strong stability. 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 regulating the expression of marker molecules in the downstream signaling pathway, which can provide a basis for subsequent exploration of the function and tracing effect of FGF21 labeled with iridium complexes.

[0024] The above is a detailed introduction to the experimental method of the effect of an iridium complex on the function of FGF21 protein provided by the present invention. Specific examples are used herein to illustrate the principles and implementation methods of the present invention, and the description of the above examples is only used to help understand the method of the present invention and its core idea. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present invention, the present invention can also be improved and modified in several ways, and these 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 complexes (such as IrCN), camel, mouse, human FGF21 proteins, mouse kidney fibroblasts BHK21 and bovine mammary epithelial cells MAC-T were all preserved by our laboratory; S2: Binding characteristics of iridium complexes with proteins: Iridium complexes were dissolved in dimethyl sulfoxide to prepare a mother solution with a concentration of 10 mg / ml, and then the mother solution was gradiently diluted with PBS buffer to make the final concentration of iridium complexes 0.05, 0.1, 0.5, and 1 mg / ml. Iridium complexes of different concentrations were added to 20 μg of camel FGF21 protein with a His tag, and the changes in fluorescence intensity at different times were recorded by a chemiluminescence imaging system, and the fluorescence intensity was analyzed using ImageJ software; S3: Binding specificity of iridium complexes to FGF21 protein: labeling and mixing, the FGF21 protein labeled with the iridium complex probe was mixed with histidine-containing serum albumin (BSA), and different iridium complex to FGF21 protein concentration ratios (1:100, 1:200, 1:300, 1:400) and different ratios of BSA to FGF21 protein were set; Gel electrophoresis and detection: After 24 hours, gel electrophoresis was performed to separate the BSA and the fluorescence was detected by chemiluminescence imaging system. The binding specificity between the iridium complex and FGF21 protein was determined by analyzing the results of different lanes. S4: Cytotoxicity test of iridium complexes: Cell inoculation and treatment: At 37°C and 5% CO2, subcultured cells were inoculated in a 96-well cell culture plate. When the cells grew to a density of about 85%, the culture medium in the wells was removed and the culture medium solution containing different concentrations of iridium complex probes prepared in advance was added to make the final concentrations of iridium complex probes 0, 0.01, 0.02, 0.05, 0.1, 0.2 mg / ml, etc., respectively. A control group was set up at the same time, and 3 parallel experiments were set up in each group. Toxicity detection: After 24 hours of co-culture with cells, 10 μl of CCK8 solution was added to each well and incubated in the incubator for another 2 hours. The absorbance of the solution in each well at a wavelength of 450 nm was measured with an ELISA reader. The cell survival rate was calculated based on the absorbance value to evaluate the effect of the iridium complex on cell viability. S5: Determination of hemolytic activity of iridium complex probe: Take blood from healthy mice, add 10 times the amount of 0.9% sodium chloride solution, centrifuge and wash, remove the supernatant, make the obtained red blood cells into a 4% suspension with 0.9% sodium chloride solution, prepare the sample solution in a centrifuge tube, set up positive and negative controls, add 200μl of red blood cell suspension to each tube, centrifuge at 1000g for 3min, observe the hemolysis phenomenon with the naked eye, aspirate 100μL of supernatant into a new 96-well plate, and detect OD540nm; S6: Live cell imaging test: BHK21 cells were inoculated in a confocal dish. When the cells grew to a density of about 50%, different concentrations of iridium complex probe IrCN solution were added to the dish. After co-culture with the cells for 10 minutes, the dish was washed twice with phosphate buffered saline (PBS) and used directly for imaging. S7: Immunofluorescence experiment: BHK21 cells were seeded in a confocal dish. When the cells grew to a density of about 50%, they were washed twice with pre-cooled PBS, fixed with 4% paraformaldehyde for 30 minutes at room temperature, washed 3 times with PBS, permeabilized in 0.2% TritonX-100 for 10 minutes, washed 3 times with PBS, and stained with 40,6-diaminophenylindole (DAPI) for 10 minutes. After washing 3 times with PBS, different concentrations of iridium complex probe IrCN were added to the dish, 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 cDNA were detected by UV spectrophotometer. 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 (PVDF) membrane (Biosharp, Beijing, China). The membrane was blocked with blocking buffer for 20 min and then incubated with anti-tubulin antibody (1:10000), anti-PI3K antibody (1:1000), anti-AKT antibody (1:1000), and anti-mTOR antibody (1:1000) at 4 °C overnight. After washing with PBS three times, the membrane was incubated with enzyme-labeled goat anti-rabbit IgG (1:5000) at room temperature for 2 h. Finally, the protein bands were visualized using ECL chemiluminescence detection and captured by a gel imaging system (Tannon, Shanghai, China). S10. Statistical analysis: The results were analyzed using GraphPad Prism (version 9.3.1) software (San Diego, CA, USA). The data were expressed as mean ± SEM. Unpaired Student's t test was used for comparison between the two groups. Two-way ANOVA 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 needs to prepare DMEM culture medium, phosphate buffered saline (PBS, pH=7.2), trypsin-EDTA solution (0.25%), penicillin and streptomycin (100 times), collagenase IV, RIPA protein lysis buffer, TRIzol, CCK-8, propidium iodide (PI), BCA protein detection kit, ECL chemiluminescence detection kit, reverse transcription kit, SYBR Premix Ex Taq II kit, G418, protein markers, fetal bovine serum (FBS), 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.

3. The experimental method for the effect of an iridium complex on the function of FGF21 protein according to claim 1, characterized in that: The S2 also includes a 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.

4. The experimental method for the effect of an iridium complex on the function of FGF21 protein according to claim 1, characterized in that: The S2 also includes: Binding stability determination: placing the iridium complex and FGF21 protein at the same concentration in -20°C, 4°C and room temperature respectively, and recording the fluorescence intensity at different times using an intelligent dark box type three-purpose UV analyzer. Observe 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.

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 is evaluated according to the hemolytic rate calculation formula: hemolytic 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, evaluate the detection ability of IrCN for intracellular histidine, and evaluate the changes in intracellular fluorescence signals under different concentrations of IrCN treatment.

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: S8 also includes qPCR reaction and analysis: the qPCR reaction system was prepared according to the protocol of SYBR Premix Ex Taq II Kit, and the reaction was performed on a LightCycler instrument (Roche, Indianapolis, IN, USA). The relative expression of genes was analyzed by the 2-DDCt method, and the number of transcripts in each sample was normalized using ACTIN as an internal reference to evaluate the expression levels of genes such as PI3K, Akt, and mTOR.

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