RAGE targeted drug screening method based on RAGE overexpression and SPR technology

Abstract

The invention discloses a RAGE targeted drug screening method based on RAGE overexpression and an SPR (Surface Plasmon Resonance) technology. An RAGE overexpressed osteoblast model is constructed through a lentiviral vector, and an inhibitor with high affinity is screened out by combining SPR real-time detection of interaction between a traditional Chinese medicine extract and RAGE. The method comprises the steps of lentivirus packaging of RAGE genes, preparation of SPR chips of stabilized RAGE membrane protein, construction of a traditional Chinese medicine ingredient library, analysis of inhibitor binding kinetics, SPR screening and cell model function verification, RAGE inhibitor molecules with high affinity can be rapidly screened, and active ingredients obtained through screening can remarkably improve the cell activity of high-glucose damaged cells, promote proliferation and differentiation and improve the activity of the RAGE inhibitor molecules. The invention provides a new strategy for developing natural drugs targeting RAGE. The method not only can accurately simulate the disease state, but also can efficiently screen and verify the traditional Chinese medicine components with potential therapeutic effects, significantly improves the efficiency and success rate of drug research and development, and has important scientific value and clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the cross-field of biomedicine and traditional Chinese medicine modernization, and specifically relates to a RAGE targeted drug screening method based on RAGE overexpression and SPR technology. Background Art

[0002] Diabetic osteoporosis (DOP) is a serious complication of diabetes, and its onset is closely related to the imbalance of bone metabolism caused by the overactivation of the advanced glycation end products (AGEs)-receptor for advanced glycation end products (RAGE) signaling pathway. Competitive binding with AGEs to RAGE and blocking the AGEs / RAGE pathway are considered to be the main means of treating diabetic osteoporosis. At present, there is a lack of specific drugs targeting RAGE in clinical practice. Traditional Chinese medicines such as Rehmannia glutinosa, Epimedium, Rubus idaeus, Ligustrum lucidum, and Polygonatum sibiricum have shown the potential to regulate bone metabolism in the "kidney tonifying" effect, but the interaction mechanism between their active ingredients and RAGE has not yet been clarified. RAGE is a transmembrane protein that participates in pathological processes such as inflammation, oxidative stress, and apoptosis, and is an important target for the treatment of various diseases. The existing technology has the following technical problems:

[0003] 1) As a membrane protein, RAGE is easily inactivated in in vitro experiments. Traditional screening methods (such as ELISA, fluorescence polarization, molecular docking, and animal models) are inefficient and difficult to accurately reflect the ligand binding properties of RAGE in its natural conformation.

[0004] 2) Existing technologies have defects such as low throughput, high false positive rate, and inability to monitor kinetic parameters in real time. Although surface plasmon resonance (SPR) technology can detect molecular interactions in real time, its application in traditional Chinese medicine screening is limited by the complex composition of traditional Chinese medicine, poor stability of membrane proteins, and difficulty in immobilization.

[0005] Therefore, it is of great value to develop an efficient and stable RAGE inhibitor screening method. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention discloses a RAGE targeted drug screening method based on RAGE overexpression and SPR technology, constructs a RAGE overexpression osteoblast model through a lentiviral vector, combines SPR to detect the interaction between Chinese medicine extracts and RAGE membrane proteins in real time, and screens out targeted drugs with high affinity. The method includes lentiviral packaging of RAGE gene, preparation of SPR chip for stabilizing RAGE membrane protein, construction of Chinese medicine component library, SPR screening, and analysis of targeted drug binding kinetics, and can quickly screen RAGE targeted drugs with high affinity. The present invention provides a new strategy for the development of natural drugs targeting RAGE.

[0007] The present invention is achieved through the following technical solutions:

[0008] The present invention discloses a RAGE targeted drug screening method based on RAGE overexpression and SPR technology, comprising the following steps:

[0009] a) Lentiviral vector was used to infect osteoblasts MC3T3-E1 to establish a cell model with high RAGE expression;

[0010] b) Extracting RAGE membrane protein from a cell model with high RAGE expression;

[0011] c) Construction of SPR protein chip: fix the RAGE membrane protein on the surface of the SPR chip to obtain the SPR chip modified with the RAGE membrane protein, investigate the stability of the SPR chip modified with the RAGE membrane protein, and obtain the RAGE membrane protein chip after stability verification;

[0012] d) Screening of active compounds: Extract active ingredients from traditional Chinese medicine and construct a library of traditional Chinese medicine ingredients; detect the KD value, a binding kinetic parameter of the active ingredients of traditional Chinese medicine with RAGE, through the RAGE membrane protein chip, and rank the affinity of the active ingredients of traditional Chinese medicine with RAGE protein according to the KD value. According to the relationship between KD value and affinity, obtain RAGE targeting components, the smaller the KD value, the higher the affinity.

[0013] As a further improvement, in step a) of the present invention, the lentiviral vector infects osteoblasts MC3T3-E1, and the expression level of RAGE is significantly increased.

[0014] As a further improvement, in step c) of the present invention, the SPR protein chip detection adopts the Open SPR system, and the RAGE membrane protein is directionally fixed on the carboxyl chip by the amino coupling method. The density of RAGE membrane protein immobilized on the chip surface is 500-1500 RU, chip channel 1 is the control channel, and chip channel 2 is the coupled RAGE protein detection channel.

[0015] As a further improvement, in step c) of the present invention, the RAGE membrane protein is fixed on the surface of the SPR chip, and the effect of different buffer pH values ​​or RAGE membrane protein concentrations on the directional fixation of the RAGE membrane protein on the carboxyl chip is investigated.

[0016] As a further improvement, the Chinese medicine in step c) of the present invention is any one of Rehmannia glutinosa, Epimedium, Rubus idaeus, Ligustrum lucidum, and Polygonatum sibiricum.

[0017] As a further improvement, the binding kinetic parameter KD value in step d) of the present invention is calculated from the response signals of the active ingredients of the traditional Chinese medicine and the RAGE membrane protein by fitting the kinetic curve.

[0018] As a further improvement, in step d) of the present invention, the smaller the value of the binding kinetic parameter KD is, the higher the affinity between the active ingredients of the traditional Chinese medicine and RAGE is, and the stronger the targeting is.

[0019] As a further improvement, the RAGE-targeted Chinese medicine based on RAGE overexpression and SPR technology screened by the present invention has a component of flavonoids, phenylethanol, saponins, or terpenes, and its KD value with RAGE is 3.90×10 -9 -8.52×10 -7 M range.

[0020] As a further improvement, the flavonoids described in the present invention are one of icariin, icariin I, epimedin B, epimedin C, baohuoside I, dehydrated icariin, and kaempferol-3-O-rutinoside; the phenethyl alcohol is one of pyroreptitol phenylethanol glycoside A1 and pyroreptitol phenylethanol glycoside B1; the saponins are smilax china sapogenin and diosgenin; the terpenes are one of catalpol, privet glycoside, and rehmannia glutinosa D.

[0021] The beneficial effects of the present invention are as follows:

[0022] 1. A lentiviral vector-mediated RAGE high-expression cell model was used to construct an osteoblast cell line with stable overexpression of RAGE, accurately simulate the pathological environment of diabetic osteoporosis, and improve the clinical relevance of screening results. This model can not only highly restore the molecular mechanism under the disease state, but also significantly improve the clinical relevance of drug screening results, providing a more reliable experimental basis for subsequent drug development. Compared with conventional transfection methods, lentiviral vectors have higher transfection efficiency and continuous expression characteristics, providing sufficient biological material guarantee for subsequent RAGE membrane protein extraction.

[0023] 2. Stabilization of RAGE membrane protein and directional fixation with SPR: The amino coupling method was combined with the SPR system to achieve directional fixation of RAGE membrane protein on the carboxyl chip. By systematically examining key parameters such as buffer pH (affecting protein charge distribution) and protein concentration (controlling coupling density), the fixation density was precisely controlled within the range of 500-1500 RU. Compared with the random fixation method, this technology effectively maintains the natural conformation of RAGE membrane protein, significantly improves the biological relevance of the test results, greatly reduces nonspecific adsorption, improves the sensitivity and accuracy of the test, and provides solid technical support for subsequent high-throughput screening.

[0024] 3. Based on the Open SPR system, a dual-channel detection system (channel 1 control / channel 2 detection) is constructed to realize the automatic calculation of KD value by dynamically fitting the kinetic curve through real-time monitoring of the response signal. Compared with traditional endpoint detection methods such as ELISA, the binding rate and dissociation rate can be obtained simultaneously, and the drug-target interaction can be quantitatively evaluated in multiple dimensions.

[0025] 4. Precision screening of multi-components of traditional Chinese medicine: Through UPLC-MS technology, a standardized kidney-tonifying traditional Chinese medicine component library (including flavonoids, saponins, organic acids and terpenes, etc.) was established. Combined with SPR screening, active ingredients with high affinity to RAGE were quickly and accurately screened out. Through cell function verification, highly active ingredients with significant bone protective effects were identified, providing a scientific basis for the modernization of traditional Chinese medicine research.

[0026] 5. Significantly improved screening efficiency: SPR protein chip technology enables the detection throughput to reach the screening capacity of multiple samples per day, which greatly improves the screening efficiency compared with the experimental cycle of traditional animal models or cell experiments. Combined with the screening of active ingredients of traditional Chinese medicine, it can achieve rapid analysis of complex systems of traditional Chinese medicine.

[0027] 6. Universality of the screening method: The screening method has successfully verified 14 active ingredients from 5 types of Chinese medicinal materials (Rehmannia glutinosa, Epimedium, Rubus idaeus, Ligustrum lucidum, Polygonatum sibiricum), covering 4 types of compound structures. The system can be quickly transformed into a universal drug screening platform by replacing the target protein.

[0028] 7. Breakthrough in the research and development of drugs against diabetes or diabetic complications: RAGE is a key target for diseases such as diabetes or diabetic complications. The high-affinity components screened can be used as targeted drugs for RAGE.

[0029] 8. In vitro efficacy verification: In the high glucose damaged osteoblast model, the bone protective effect of the candidate ingredients was verified by MTT and alkaline phosphatase (ALP) staining. The promoting effect of the candidate ingredients on osteoblast proliferation and differentiation was evaluated.

[0030] Through the above technical scheme, the present invention can not only accurately simulate the disease state, but also efficiently screen and verify Chinese medicine ingredients with potential therapeutic effects, significantly improving the efficiency and success rate of drug research and development, and has important scientific value and clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 :Results of lentivirus infection of osteoblasts;

[0032] Figure 2 :RAGE membrane protein expression in osteoblasts infected with lentivirus;

[0033] Figure 3:Effect of buffer pH on RAGE membrane protein coupling;

[0034] Figure 4 :The effect of RAGE membrane protein concentration on RAGE membrane protein coupling;

[0035] Figure 5 :Results of the RAGE membrane protein stability study;

[0036] Figure 6 :Activity characterization diagram of SPR protein chip;

[0037] Figure 7 : The results of the kinetic determination of dehydrated icariin on the RAGE membrane protein chip;

[0038] Figure 8 : Fitting curve of dehydrated icariin in RAGE membrane protein chip kinetic assay;

[0039] Fig. 9 : The results of kinetic determination of dehydrated icariin on recombinant RAGE protein chip;

[0040] Fig.10 : The fitting curve of the kinetic determination of dehydrated icariin in the recombinant RAGE protein chip;

[0041] Fig.11 :The effect of Baohuoside I on the proliferation activity of osteoblasts damaged by high glucose;

[0042] Fig.12 :The effect of Baohuoside I on osteoblast differentiation damaged by high glucose;

[0043] Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Fig. 9 , Fig.10 Signal (RU) in Chinese means: signal (signal response unit); Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Fig. 9 Time (Seconds) in Chinese means: time (seconds); Figure 8 , Fig.10 Concentration (M) (10^-9) is explained in Chinese as: concentration (unit is 10^-9M); Fig.11Cell viability(%) is explained in Chinese as: cell viability (percentage). DETAILED DESCRIPTION

[0044] The present invention discloses a RAGE targeted drug screening method based on RAGE overexpression and SPR technology, comprising the following steps:

[0045] a) Lentiviral vector was used to infect osteoblasts MC3T3-E1 to establish a cell model with high RAGE expression; Lentiviral vector was used to infect osteoblasts MC3T3-E1, and the expression of RAGE was significantly increased.

[0046] b) Extracting RAGE membrane protein from a cell model with high RAGE expression;

[0047] c) Construction of SPR protein chip: RAGE membrane protein was fixed on the surface of SPR chip to obtain SPR chip modified with RAGE membrane protein, and the stability of SPR chip modified with RAGE membrane protein was investigated to obtain RAGE membrane protein chip after stability verification; Open SPR system was used for SPR protein chip detection, and RAGE membrane protein was fixed on carboxyl chip by amino coupling method, and the density of RAGE membrane protein fixed on the chip surface was 500-1500 RU, chip channel 1 was the control channel, and chip channel 2 was the coupled RAGE protein detection channel. RAGE membrane protein was fixed on the surface of SPR chip, and the effect of different buffer pH value or RAGE membrane protein concentration on the fixed orientation of RAGE membrane protein on carboxyl chip was investigated. The Chinese medicine was any one of Rehmannia glutinosa, Epimedium, Rubus idaeus, Ligustrum lucidum, and Polygonatum sibiricum.

[0048] d) Screening of active compounds: Extract active ingredients from traditional Chinese medicine and build a library of traditional Chinese medicine ingredients; detect the KD value of the binding kinetic parameter between the active ingredients of traditional Chinese medicine and RAGE through the RAGE membrane protein chip, sort the affinity of the active ingredients of traditional Chinese medicine and RAGE protein according to the KD value, and obtain the RAGE targeting component according to the relationship between the KD value and affinity. The smaller the KD value, the higher the affinity. The binding kinetic parameter KD value is calculated by fitting the kinetic curve from the response signal of the active ingredients of traditional Chinese medicine and RAGE membrane protein.

[0049] The smaller the binding kinetic parameter KD value, the higher the affinity of the active ingredients of traditional Chinese medicine to RAGE and the stronger the targeting.

[0050] The RAGE-targeted Chinese medicine drugs screened based on RAGE overexpression and SPR technology are composed of flavonoids, phenylethanol, saponins, or terpenes, and their KD values ​​with RAGE are between 3.90×10 -9 -8.52×10 -7M range. The flavonoids are one of icariin, icariin I, epimedin B, epimedin C, baohuoside I, and kaempferol-3-O-rutinoside; the phenylethanol is one of pyrodipine phenylethanol glycoside A1 and pyrodipine phenylethanol glycoside B1; the saponins are smilax glabra sapogenin and diosgenin; the terpenes are one of catalpol, privet glycoside, and rehmannia glycoside D.

[0051] The technical solution of the present invention is further described below by means of specific embodiments in conjunction with the accompanying drawings:

[0052] 1. Construction of RAGE overexpression cell model by lentivirus

[0053] (1) Osteoblast culture: Osteoblasts MC3T3-E1 were placed in DMEM medium containing double antibodies and 10% fetal bovine serum and cultured in an incubator at 37°C and 5% CO2. Cells in the logarithmic growth phase were taken for subsequent experiments.

[0054] (2) Lentiviral vector infection of osteoblasts: The RAGE lentiviral vector designed by Genema Biotechnology was used to infect MC3T3-E1 cells to establish a RAGE high-expression cell model. After 24 hours of infection, the cell infection efficiency was observed using a fluorescence microscope. The results are shown in Figure 1 .

[0055] like Figure 1 As shown, after the cells were infected with the lentivirus, the fluorescence intensity of the RAGE overexpression group was significantly higher than that of the negative control group, indicating that the lentivirus-infected osteoblast model established osteogenesis.

[0056] (3) Determination of RAGE membrane protein content: After the cells are infected with lentivirus, the cells are collected to prepare RAGE membrane protein. The concentration of RAGE membrane protein is detected using a BCA kit.

[0057] (4) Characterization of RAGE membrane protein: Immunoblotting was used to observe the expression of RAGE membrane protein. The RAGE membrane protein was separated by 10% SDS-PAGE gel electrophoresis. After the electrophoresis, the RAGE membrane protein on the gel was transferred to the PVDF membrane by electrophoresis, and the PVDF membrane was blocked with 0.5% BSA at room temperature for 0.5 h. After blocking, the PVDF membrane was incubated with primary antibodies RAGE and GAPDH (target protein RAGE, 1:2000; internal reference protein GAPDH, 1:2000), respectively, at 4°C overnight. The membrane was then washed with PBST and the PVDF membrane was incubated with secondary antibodies for 2 h (1:5000). After incubation, the PVDF membrane was washed with PBST to remove excess antibodies. Grayscale value analysis was performed using a gel imager and ImageJ software. The results are shown in Figure 2 .

[0058] like Figure 2As shown in the figure, compared with the negative control group, the RAGE expression level in osteoblasts infected with lentivirus was significantly higher than that in the control group (P < 0.01), indicating that RAGE was overexpressed after lentivirus infection of osteoblasts.

[0059] 2. RAGE membrane protein coupling

[0060] (1) RAGE membrane protein was coupled using a carboxyl chip. The carboxyl groups on the surface of the carboxyl chip were cross-linked with the RAGE membrane protein using an amino coupling kit. Channel 1 without RAGE membrane protein coupling was used as the control channel, and channel 2 with RAGE membrane protein coupling was used as the detection channel. The coupling time was 400 s, the flow rate was 20 μL / min, and the running buffer solution was PBST + 5% DMSO.

[0061] (2) Effect of different buffer pH values ​​on RAGE membrane protein coupling: The physical adsorption response signals of RAGE membrane protein on the carboxyl chip were detected under different buffer pH conditions, pH values ​​of 4.0, 4.5, 5.0, and 5.5, to find the optimal buffer pH condition. The results are shown in Figure 3 .

[0062] like Figure 3 As shown, when the pH value of the buffer was 4.0, the RAGE membrane protein had the highest physical adsorption response signal on the carboxyl chip.

[0063] (3) Effect of RAGE membrane protein concentration on RAGE membrane protein coupling: The physical adsorption response signals of RAGE membrane protein at different concentrations of 20, 30, 40 and 50 μg / mL on the carboxyl chip were detected to find the optimal coupling concentration of RAGE membrane protein. The results are shown in Figure 4 .

[0064] like Figure 4 As shown, when the concentration of RAGE membrane protein is 40 μg / mL, RAGE membrane protein has a better physical adsorption response signal on the carboxyl chip.

[0065] (4) Stability study of SPR protein chip: The same concentration of ellagic acid was injected into the SPR chip modified with RAGE membrane protein at 0h and 48h to detect the response signal. Figure 5 .

[0066] like Figure 5 As shown in the figure, at 0 h and 48 h after coupling, ellagic acid can generate binding response signals with the SPR chip modified with RAGE membrane protein, indicating that the SPR chip modified with RAGE membrane protein has good stability within 48 h.

[0067] (5) Characterization of the activity of the SPR protein chip: Different concentrations of RAGE antibody solutions (1, 5 and 10 μg / mL) were injected into the SPR chip modified with RAGE membrane protein to detect the response signal. The binding time was 400 s. After each detection, 10 mM glycine-HCl solution (pH = 2.5) was used to remove the residual antibodies on the carboxyl chip to regenerate the chip. The results are shown in Figure 6 .

[0068] like Figure 6 As shown, the response signals of different concentrations of RAGE antibodies on the RAGE membrane protein chip system increased with increasing concentrations.

[0069] 3. SPR screening of active ingredients in traditional Chinese medicine

[0070] (1) Recovery of active ingredients of traditional Chinese medicine bound to RAGE membrane protein: Inject the traditional Chinese medicine extract sample into the SPR system, recover the components bound to the RAGE membrane protein chip, repeat 10 cycles, freeze-dry, re-dissolve in 50% methanol water, and perform UPLC-QTOF-MS analysis to construct the active ingredients of traditional Chinese medicine bound to RAGE membrane protein.

[0071] (2) UPLC-QTOF-MS analysis conditions: The recovered samples were analyzed using an AB Sciex X500R QTOF liquid phase mass spectrometer system. Chromatographic conditions: ACQUITY UPLC BEH C18 (150×2.1 mm, 1.7 μm) column, mobile phase 0.1% formic acid acetonitrile (A)-0.1% formic acid water (B), gradient elution program: 0-14 min, 99%-50% B; 14-15 min, 50%-40% B; 15-15.5 min, 40%-1% B; 15.5-18 min, 1% B; flow rate: 0.3 mL / min; injection plate temperature: 8°C; column temperature: 40°C; injection volume: 4 μL. Mass spectrometry conditions: Electrospray ionization (ESI) source, positive and negative ion scanning mode. The specific conditions were as follows: ion source nebulizer gas (Ion Source Gas1, Gas1): 55, auxiliary gas pressure (Ion Source Gas2, Gas2): 55, curtain gas (CUR): 35, source temperature (Source Temperature): 600℃, spray voltage (Ion Sapary Voltage Floating, ISVF): 5500 V / -4500 V (positive and negative ion modes); TOF MS scan m / z range: 50-1500 Da, production scan m / z range: 25-1000 Da, TOF MS scan accumulation time 0.25 s / spectra, production scan accumulation time 0.035 s / spectra; the secondary mass spectrum was acquired in information dependent acquisition (IDA) mode, and high sensitivity mode was used, Declustering potential (DP): ± 60 V (positive and negative ion modes), Collision Energy: ± 35 ± 15 eV, and IDA settings were as follows: Exclude isotopes within 4 Da, Candidate ions to monitor per cycle: 12.

[0072] (3) Mass spectrometry analysis results: RAGE-targeted drugs in Rehmannia glutinosa include: catalpol, rehmannia glycoside D, pyroreptitol phenylethanoid glycoside A1, pyroreptitol phenylethanoid glycoside B1; RAGE-targeted drugs in Epimedium include: icariin, icariin I, epimedin B, epimedin C, baohuoside I, dehydrated icariin; RAGE-targeted drugs in Rubus idaeus include: kaempferol-3-O-rutinoside; RAGE-targeted drugs in Ligustrum lucidum include: terunguisu glycoside; RAGE-targeted drugs in Polygonatum sibiricum include: smilaxone and diosgenin.

[0073] (4) Affinity test: The active ingredients of traditional Chinese medicine that respond on the SPR protein chip were injected into the SPR chip modified with RAGE membrane protein at different concentrations to detect their response signals. The affinity data were processed using TraceDrawer 1.5 software to calculate the KD value of the binding kinetic parameter between the active ingredients of traditional Chinese medicine and RAGE. Among them, the KD value of catalpol was 1.45×10 - 7 The KD value of M and rehmannia glycoside D was 8.52×10 -7 M, the KD value of pyrodipine phenylethanol glycoside A1 was 8.15×10 -8 M, the KD value of pyrodipine phenylethanol glycoside B1 was 1.05×10 -7 M. The KD value of icariin is 3.90×10 -9 M, the KD value of icariin I was 8.78×10 -8 The KD value of epimedin B was 2.27×10 -7 The KD value of epimedin C was 9.25×10 -8 The KD value of M and Baohuoside I was 1.39×10 -7 M. The KD value of dehydrated icariin is 8.59×10 -9 The KD value of M, kaempferol-3-O-rutinoside was 5.07×10 -8 The KD value of M and terephthaloside was 5.81×10 -9 M, the KD value of sarsaparilla sapogenin was 1.32×10 -8 M, the KD value of diosgenin is 1.00×10 -7 M; According to the relationship between KD value and affinity, the affinity strength of the above active ingredients to RAGE is as follows: icariin>terivirgoside>dehydrated icariin>smilaxone>kaempferol-3-O-rutinoside>pyroremycin phenylethanol glycoside A1>icariin I>epimedin C>diosgenin>pyroremycin phenylethanol glycoside B1>bauroside I>catalpol>epimedin B>rehmannioside D.

[0074] 4. Method Comparison

[0075] (1) Recombinant RAGE protein SPR chip: The recombinant RAGE protein was coupled to the carboxyl chip using the RAGE membrane protein SPR chip coupling method. Channel 1 that was not coupled to the recombinant RAGE protein was used as the control channel, and channel 2 that was coupled to the recombinant RAGE protein was used as the detection channel. The coupling time was 400 s and the flow rate was 20 μL / min.

[0076] (2) Differences between different RAGE protein SPR chips: The active ingredient of traditional Chinese medicine, dehydrated icariin, was injected into the SPR chip system modified with RAGE membrane protein and the SPR chip system modified with recombinant RAGE protein. The response values ​​of dehydrated icariin on the two SPR chips were measured, and kinetic analysis was performed to determine the binding kinetic parameters. The results are shown in Figure 7-10 .

[0077] like Figure 7-10 As shown in the figure, dehydrated icariin can generate binding response signals with both chips. After kinetic determination and fitting curve analysis, the binding dissociation constants were 236 μM and 8.59×10 -3 μM, compared with recombinant RAGE protein, anhydroicariin has high affinity and binding specificity to RAGE membrane protein.

[0078] 5. Cell Model Validation

[0079] (1) Establishment of a high-glucose-damaged osteoblast model: MC3T3-E1 osteoblasts were cultured in a culture medium containing 25 mM / L glucose to establish a high-glucose-damaged osteoblast model.

[0080] (2) Cell proliferation activity: When osteoblasts were cultured to a confluency of about 80-90%, the cells were digested, centrifuged, the supernatant was discarded, and DMEM medium was used to mix them evenly, and then they were evenly inoculated into a 96-well plate with a volume of 100 μL per well. After the cells adhered to the wall for 24 hours, the original culture medium was discarded, and the cells were divided into a blank group, a high glucose group, and a high glucose + different doses of Baohuoside I groups (0.01, 0.1, 1 μM). After incubation for 24 hours, the cells were collected and the cell viability was detected by MTT method. The results are shown in Fig.11 .

[0081] like Fig.11 As shown, compared with the blank group, the activity of osteoblasts damaged by high glucose was significantly reduced (P < 0.01). Compared with the model group, the activity of osteoblasts damaged by high glucose was significantly enhanced when the dosage of Baohuoside I was 0.1 and 1 μM (P < 0.01), and it could promote the proliferation of osteoblasts damaged by high glucose.

[0082] (3) Alkaline phosphatase (ALP) staining: When osteoblasts were cultured to a confluency of about 80-90%, the cells were digested, centrifuged, the supernatant was discarded, and DMEM medium was used to mix evenly, and then evenly inoculated into a 6-well plate with a volume of 2 mL per well. After the cells adhered to the wall for 24 hours, the original culture medium was discarded, and the cells were divided into a blank group, a high glucose group, and a high glucose + different doses of Baohuoside I groups (0.1, 1 μM). After incubation for 24 hours, the cells were collected and ALP staining was performed using an ALP staining kit. The results are shown in Fig.12 .

[0083] like Fig.12 As shown, compared with the blank group cells, the production of alkaline phosphatase in osteoblasts damaged by high glucose was significantly reduced. Compared with the model group, different doses of Baohuoside I groups could promote the production of alkaline phosphatase in osteoblasts damaged by high glucose. The results showed that Baohuoside I could promote the differentiation of osteoblasts damaged by high glucose.

[0084] Finally, it should be noted that the above examples are only specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A RAGE-targeted drug screening method based on RAGE overexpression and SPR technology, characterized in that: The following steps are involved: a) Lentiviral vector was used to infect osteoblasts MC3T3-E1 to establish a cell model with high RAGE expression; b) Extracting RAGE membrane protein from a cell model with high RAGE expression; c) Construction of SPR protein chip: fix the RAGE membrane protein on the surface of the SPR chip to obtain the SPR chip modified with the RAGE membrane protein, investigate the stability of the SPR chip modified with the RAGE membrane protein, and obtain the RAGE membrane protein chip after stability verification; d) Screening of active compounds: extract active ingredients from traditional Chinese medicine and construct a library of traditional Chinese medicine ingredients; detect the KD value, a kinetic parameter for the binding between the active ingredients of traditional Chinese medicine and RAGE membrane protein, using a RAGE membrane protein chip, rank the affinity of the active ingredients of traditional Chinese medicine and RAGE protein according to the KD value, and obtain RAGE targeting components based on the relationship between the KD value and affinity, wherein the smaller the KD value, the higher the affinity.

2. The RAGE targeted drug screening method based on RAGE overexpression and SPR technology according to claim 1, characterized in that: In step c), the SPR protein chip detection uses the Open SPR system, and the RAGE membrane protein is directionally fixed on the carboxyl chip by the amino coupling method. The density of RAGE membrane protein immobilized on the chip surface is 500-1500 RU. Chip channel 1 is the control channel, and chip channel 2 is the coupled RAGE membrane protein detection channel.

3. The RAGE targeted drug screening method based on RAGE overexpression and SPR technology according to claim 2, characterized in that: In step c), the RAGE membrane protein is fixed on the surface of the SPR chip, and the effects of different buffer pH values ​​or RAGE membrane protein concentrations on the directional fixation of the RAGE membrane protein on the carboxyl chip are investigated.

4. The RAGE targeted drug screening method based on RAGE overexpression and SPR technology according to claim 3, characterized in that: In step c), the Chinese medicine is any one of Rehmannia glutinosa, Epimedium, Rubus idaeus, Fructus Ligustri Lucidi and Polygonatum sibiricum.

5. The RAGE targeted drug screening method based on RAGE overexpression and SPR technology according to claim 1, 2, 3 or 4, characterized in that: The binding kinetic parameter KD value in step d) is calculated from the response signals of the active ingredients of the traditional Chinese medicine and the RAGE membrane protein by fitting the kinetic curve.

6. The RAGE targeted drug screening method based on RAGE overexpression and SPR technology according to claim 5, characterized in that: The smaller the KD value of the binding kinetic parameter in step d), the higher the affinity between the active ingredient of the traditional Chinese medicine and RAGE, and the stronger the targeting.

7. The RAGE targeted drug screening method based on RAGE overexpression and SPR technology according to claim 1, 2, 3, 4 or 6, characterized in that: The screened RAGE-targeted Chinese medicine based on RAGE overexpression and SPR technology has a component of flavonoids, phenylethanol, saponins, or terpenes, and its KD value with RAGE membrane protein is 3.90×10 -9 -8.52×10 -7 M range.

8. The RAGE targeted drug screening method based on RAGE overexpression and SPR technology according to claim 7, characterized in that: The flavonoids are one of icariin, icariin I, epimedin B, epimedin C, baohuoside I, dehydrated icariin, and kaempferol-3-O-rutinoside; the phenylethanol is one of pyroreptitol phenylethanol glycoside A1 and pyroreptitol phenylethanol glycoside B1; the saponins are smilax glabra sapogenin and diosgenin; the terpenes are one of catalpol, privet glycoside, and rehmannia glutinosa D.

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