Active substance screening platform and application thereof

Abstract

The invention relates to the technical field of medicine screening, in particular to an active substance screening platform and application thereof. The active substance screening platform comprises a cell combination; the cell combination comprises immunoregulation screening cells and antioxidant screening cells; the immunoregulation screening cell is a mouse mononuclear macrophage RAW 264.7 carrying an NF-kappa B element conserved sequence and a reporter gene; the antioxidant screening cell is a porcine small intestine epithelial cell IPEC-J2 cell carrying an ARE element conserved sequence and a reporter gene. The invention provides a screening platform based on a specific cell combination, which can quickly obtain an active substance with immune regulation, antioxidation or two functions from a large number of candidate active substances, has the advantages of low cost and high efficiency, and has important application value.

Description

Technical Field

[0001] The present invention relates to the technical field of drug screening, and in particular to an active substance screening platform and applications thereof. Background Art

[0002] In the current biomedicine and related fields, health faces numerous challenges. The immune system, as a key line of defense against foreign pathogens, is of undeniable importance. Immune deficiency or dysregulation of the immune response can lead to various diseases and, in severe cases, cancer. Among them, Toll-like receptors (TLRs), TLR4 / MD2, play a central role in the innate immune system's pathogen recognition. They recognize agonist molecules such as lipopolysaccharide (LPS) to initiate an immune response. Therefore, targeting TLR4 / MD2 to enhance immunity has become a key strategy for strengthening immune defenses.

[0003] At the same time, oxidative stress is also a major issue affecting human health. When the body's redox homeostasis is disrupted by excessive reactive oxygen species (ROS), cellular DNA, proteins, and lipids are damaged, leading to cellular dysfunction and a range of diseases such as cancer, atherosclerosis, and rheumatoid arthritis. The Keap1-Nrf2-ARE pathway, a major antioxidant defense mechanism, stimulates ROS signals to dissociate Nrf2 from Keap1 and translocate to the cell nucleus. There, Nrf2 binds to the antioxidant response element (ARE), activating the expression of downstream antioxidant genes, restoring redox homeostasis, and protecting cells.

[0004] In recent years, functional active substances that can simultaneously regulate immunity and oxidative stress have received widespread attention. However, research on functional active substances that can both enhance immunity and combat oxidative stress is still limited. These substances have great potential to solve related problems in medicine, food, agriculture, and other fields, and are expected to provide more efficient and economical solutions. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides an active substance screening platform and its application.

[0006] In a first aspect, the present invention provides a cell combination comprising: immunomodulatory screening cells and / or antioxidant screening cells; The immunoregulatory screening cells are mouse mononuclear macrophage RAW 264.7 cells carrying the conservative sequence of NF-κB elements and the reporter gene; the antioxidant screening cells are porcine intestinal epithelial cells IPEC-J2 cells carrying the conservative sequence of ARE elements and the reporter gene.

[0007] The reporter gene described in the present invention is a tool gene used to detect and monitor gene expression, regulatory element activity, or cellular physiological status. In this invention, it is primarily used to monitor the activity of NF-κB and ARE elements. It can be a fluorescent reporter gene, such as GFP or its variants, and the screening results can be directly determined by fluorescence intensity. Alternatively, it can be a luciferase reporter gene, and the screening results can be determined by adding luciferase detection buffer and measuring the luminescence intensity. Alternatively, it can be β-galactosidase (LacZ), which produces a detectable dye signal through an enzymatic reaction, and the screening results can be determined by the intensity of the dye signal.

[0008] The process of constructing a cell combination for accurate and efficient screening of antioxidant or immunomodulatory functions faces many challenges. The core challenges stem from the inherent complexity of antioxidant and immunomodulatory mechanisms: (1) Multiple pathways and synergistic effects: When the body produces antioxidant or immunomodulatory responses, it activates a large number of signaling pathways (such as immunomodulatory-related interferon and MAPK pathways; antioxidant-related GSH and NOX pathways, etc.). At the same time, a single pathway often involves the coordinated regulation of multiple functional elements. (2) Functional limitations of the elements themselves: When used alone, most of these functional elements are difficult to accurately and comprehensively reflect the complex antioxidant or immunomodulatory picture, limiting their effectiveness as screening indicators. (3) Consideration of biological background: The selection of elements also needs to fully consider their biological characteristics, including key factors such as functional specificity in different cell types, compatibility with the metabolic pathways of target cells, and stability within cells. After a large number of systematic studies, the present invention successfully constructed an optimized cell combination. The functional elements and cell lines used in this combination not only achieve excellent compatibility and synergistic effects, ensuring the effective performance of the screening function, but also show high accuracy and stability in screening applications, thus having significant practical value.

[0009] Furthermore, the NF-κB element conserved sequence includes the nucleotide sequence shown in SEQ ID NO.1; and / or, the ARE element conserved sequence includes the nucleotide sequence shown in SEQ ID NO.2.

[0010] Furthermore, the immunomodulatory screening cells are prepared by the following method: The NF-κB element conserved sequence, the promoter and the reporter gene are constructed on a vector to obtain a plasmid; and the plasmid is introduced into the mouse mononuclear macrophage RAW 264.7 via lentiviral transfection.

[0011] Furthermore, the antioxidant screening cells are prepared by the following method: The ARE element conserved sequence, promoter and reporter gene are constructed on a vector to obtain a plasmid; and the plasmid is introduced into the porcine small intestinal epithelial cell IPEC-J2 cell through lentiviral transfection.

[0012] Furthermore, the promoter is minP; and the reporter gene is luciferase.

[0013] In a second aspect, the present invention provides an active substance screening platform, comprising the aforementioned cell combination.

[0014] In a third aspect, the present invention provides a method for screening active substances, comprising: incubating the active substances to be screened with the immunomodulatory screening cells and antioxidant screening cells in the aforementioned cell combination under conditions of 4-8% carbon dioxide and 35-40°C for 10-24 hours, respectively, and screening the active substances to be screened according to the detection results of the reporter gene.

[0015] Furthermore, the screening of active substances based on the detection results of the reporter gene includes: When the level of the reporter gene in the immunomodulatory screening cells increases, it is judged that the drug to be screened has immunomodulatory activity; and / or, when the level of the reporter gene in the antioxidant screening cells increases, it is judged that the active substance to be screened has antioxidant activity.

[0016] In a fourth aspect, the present invention provides the use of the aforementioned cell combination or the aforementioned active substance screening platform in screening active substances having any of the following functions: (1) Immunomodulatory activity; (2) Antioxidant activity.

[0017] In a fifth aspect, the present invention provides use of a polypeptide in preparing a product for immunomodulation or anti-oxidation; The polypeptide comprises one or more of YGPSSYGYGRKDVY, RKDVYYGPSSYGYG, YGPSSYGYGKEKKEVVE, VLPVPQKDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG, RKDVYRGPPP, QLNWDRKDVY, RGPPPRKDVY, EVVEEAYGPSSYGYG, KEKKEVVEYGPSSYGYG, RKDVY, KEKKEVVE, EVVEEA, DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG, RGPPP, YGPSSYGYG, QLNWD or VLPVPQK; The product is a medicine, a health product, a food, a food additive, a feed or a feed additive.

[0018] Among them, RKDVYRGPPP, RGPPPRKDVY, YGPSSYGYGKEKKEVVE, EVVEEAYGPSSYGYG, VLPVPQKDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG, RKDVY, DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG, KEKKEVVE and EVVEEA can be used to prepare immunomodulatory products.

[0019] RKDVYYGPSSYGYG, YGPSSYGYGRKDVY, QLNWDRKDVY, KEKKEVVEYGPSSYGYG, VLPVPQKDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG, RGPPP, YGPSSYGYG, QLNWD and VLPVPQK can be used to prepare antioxidant products.

[0020] The present invention has the following beneficial effects: The present invention constructs NF-κB conserved sequence elements and ARE conserved sequence elements using fluorescein reporter genes to obtain a screening platform that can simultaneously screen functional active substances with immunomodulatory activity and antioxidant activity. The screening platform has high efficiency and accuracy, can be used as an ideal functional active substance screening platform, and is widely used in the fields of medicine, food, health care, feed, nutrition, etc. for humans and animals, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Schematic diagram of the construction of the RAW 264.7-Lucia-NF-κB stable cell line provided in Example 1 of the present invention; wherein, a is the construction map of the plasmid elements of the stable cell line; b is the sensitivity and stability test results of the stable cell line; **** indicates that there is a very significant difference between the two (p<0.0001).

[0023] Figure 2 Schematic diagram of the construction of the IPEC-J2-Lucia-ARE stable cell line provided in Example 2 of the present invention; wherein, a is the construction map of the plasmid elements of the stable cell line; b is the sensitivity and stability test results of the stable cell line; **** indicates that there is a very significant difference between the two (p<0.0001).

[0024] Figure 3 3 are the results of LIAH screening of candidate polypeptides provided in Example 3 of the present invention; wherein, a is the result of screening candidate polypeptides with immunomodulatory activity using the RAW 264.7-Lucia-NF-κB stably transfected cell line; b is the result of screening candidate polypeptides with antioxidant activity using the IPEC-J2-Lucia-ARE stably transfected cell line; ** represents a highly significant difference between the two (p<0.01), *** represents a very significant difference between the two (p<0.001), and **** represents a very significant difference between the two (p<0.0001).

[0025] Figure 4 is the functional activity verification result of the candidate polypeptide provided in Example 3 of the present invention; wherein, ac is the verification result of the immunomodulatory activity of the candidate polypeptide, that is, the stimulation level of the candidate polypeptide on RAW 264.7 cytokines (TNF-α, IL-6, IL-1β); d is the verification result of the antioxidant activity of the candidate polypeptide, that is, the protective effect of the candidate polypeptide on hydrogen peroxide-induced oxidative damage of HepG2 cells; * represents a significant difference between the two (p<0.5), ** represents a very significant difference between the two (p<0.01), *** represents a very significant difference between the two (p<0.001), and **** represents a very significant difference between the two (p<0.0001). DETAILED DESCRIPTION

[0026] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0027] Unless otherwise specified, the experimental methods involved in the following examples are all conventional methods in the art. For example, reference can be made to experimental manuals in the art, or the conditions recommended by the manufacturer's instructions.

[0028] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0029] Example 1 Construction of RAW 264.7-Lucia-NF-κB Stable Cell Line Based on Luciferase Reporter Gene The minimal promoter minP and luciferase Lucia were ligated into pCMV to generate the pCMV-minP-Lucia plasmid. The conserved sequence of the NF-κB element was ligated into the pCMV-minP-Lucia plasmid and transformed into competent Escherichia coli DH5α cells to generate the pCMV-NF-κB-minP-Lucia plasmid. Using a three-plasmid packaging system, pCMV-NF-κB-minP-Lucia, pMD2.G, and psPAX2 were transfected into 293T cells to produce virus. The viral fluid was then transfected into RAW 264.7 cells, and positive cells were selected with puromycin to obtain a stable transfected cell line.

[0030] After the stable cell line was constructed, it was stimulated with 100 ng / mL LPS in a 5% carbon dioxide, 37°C cell culture incubator for 12 h. The sensitivity and stability of the stable cell line were then tested using luciferase assay buffer (50 mM HEPES, pH 7.0; 50 mM NaCl; 0.05% CHAPS; 10 mM EDTA; and 1 μM coelenterazine).

[0031] The NF-κB sequence information is shown in SEQ ID NO.1, and the plasmid map and stable cell line test results are shown in Figure 1 The cell line successfully constructed using LPS stimulation increased the activation level of luciferase by nearly 15 times compared with the control group, and the trait remained stable after 20 generations of passages, indicating that there were no problems with the sensitivity and stability of the RAW 264.7-Lucia-NF-κB stably transfected cell line.

[0032] Example 2 Construction of IPEC-J2-Lucia-ARE Stable Cell Line Based on Luciferase Reporter Gene The conserved sequence of the ARE element was ligated into the pCMV-minP-Lucia plasmid and transformed into Escherichia coli DH5α competent cells to obtain the pCMV-ARE-minP-Lucia plasmid. The pCMV-ARE-minP-Lucia, pMD2.G, and psPAX2 were transfected into 293T cells using a three-plasmid packaging system to produce the virus. The viral liquid was then transfected into IPEC-J2 cells and positive cells were selected using puromycin to obtain a stable cell line.

[0033] After the stable cell line was constructed, it was stimulated with 10 μM tBHQ in a 5% carbon dioxide, 37°C cell culture incubator for 12 h. The sensitivity and stability of the stable cell line were then tested using luciferase assay buffer (50 mM HEPES, pH 7.0; 50 mM NaCl; 0.05% CHAPS; 10 mM EDTA; and 1 μM coelenterazine).

[0034] The sequence information of ARE is shown in SEQ ID NO.2, and the plasmid map and stable cell line test results are shown in Figure 2 The cell line successfully constructed using tBHQ stimulation increased the activation level of luciferase by nearly 5 times compared with the control group, and the trait remained stable after 20 generations of passages. The results showed that there were no problems with the sensitivity and stability of the IPEC-J2-Lucia-NF-κB stably transfected cell line.

[0035] Example 3 Validation of a high-throughput screening platform for immunomodulatory and antioxidant active substances based on synthetic peptides Eighteen candidate peptides were synthesized by chemical synthesis and stimulated RAW 364.7-Lucia-NF-κB and IPEC-J2-Lucia-ARE stably transfected cell lines at a concentration of 100 μg / mL in a 5% CO2, 37°C cell culture incubator for 12 h. Luciferase assay buffer (50 mM HEPES, pH 7.0; 50 mM NaCl; 0.05% CHAPS; 10 mM EDTA; and 1 μM coelenterazine) was then used to detect and calculate the stimulation results. Simultaneously, RAW 264.7 cells were used to detect the activation level of cytokines in RAW 264.7 cells to evaluate the immunomodulatory activity of the candidate peptides. The hydrogen peroxide-induced oxidative damage model of HepG2 cells was used to evaluate the antioxidant activity of the candidate peptides.

[0036] The polypeptide sequence is as follows: Table 1 Synthesized candidate peptides

[0037] The results of stimulation of the stably transfected cell lines RAW 264.7-Lucia-NF-κB and IPEC-J2-Lucia-ARE with the candidate peptides are shown in Figure 3 , the results showed that some peptides could activate NF-κB and ARE signaling pathways respectively.

[0038] Among them, PR, RP, YTa, TbY and VA, as well as P, A, Ta, Tb showed significance in the immunomodulatory screening, and PY, YP, MP, TaY, VA and R, Y, M, V showed significance in the antioxidant screening.

[0039] To verify the authenticity of this result, the present invention further used commonly used immune stimulation cell models and oxidative damage cell models to verify the functional activity of the candidate hybrid peptides, as follows: For immune stimulation models: RAW 264.7 was plated at 2 × 10 6 Cells were plated into 6-well plates and incubated for 12 hours. The candidate peptide was then added to a final concentration of 100 μg / mL, and 100 ng / mL LPS was used as a positive control. The cells were incubated in a cell culture incubator for 24 hours. Cell supernatants were collected and assayed for TNF-α, IL-6, and IL-1β levels using ELISA kits.

[0040] For the oxidative damage model, HepG2 cells were plated at 2 × 10 5 Cells were plated into 96-well plates and incubated for 12 hours. The medium from each well was aspirated and replaced with the candidate peptide solution (final concentration 100 μg / mL, dissolved in basal DMEM) for an additional 2 hours. Hydrogen peroxide (final concentration 850 μM) was then added to each well and incubated for 6 hours. Cytotoxicity was assessed using a CCK-8 assay.

[0041] See the results Figure 4 The cell model verification results were compared with the screening results of the screening cell line constructed by the present invention. The results showed that the data of the two were highly consistent, that is, the candidate polypeptides that can activate NF-κB or ARE in the screening cell line also have immunomodulatory activity or antioxidant activity in the cell model, which shows that the results of the screening platform constructed by the present invention are real and can be used for the rapid screening of functional active substances.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A cell combination, characterized in that: include: Immunomodulatory screening cells and / or antioxidant screening cells; The immunoregulatory screening cells are mouse mononuclear macrophage RAW 264.7 cells carrying the conservative sequence of NF-κB elements and the reporter gene; the antioxidant screening cells are porcine intestinal epithelial cells IPEC-J2 cells carrying the conservative sequence of ARE elements and the reporter gene.

2. The cell combination according to claim 1, characterized in that The NF-κB element conserved sequence includes the nucleotide sequence shown in SEQ ID NO.1; and / or, the ARE element conserved sequence includes the nucleotide sequence shown in SEQ ID NO.

2.

3. The cell combination according to claim 1 or 2, characterized in that The immunomodulatory screening cells are prepared by the following method: The NF-κB element conserved sequence, the promoter and the reporter gene are constructed on a vector to obtain a plasmid; and the plasmid is introduced into the mouse mononuclear macrophage RAW 264.7 via lentiviral transfection.

4. The cell combination according to claim 1 or 2, characterized in that The antioxidant screening cells are prepared by the following method: The ARE element conserved sequence, promoter and reporter gene are constructed on a vector to obtain a plasmid; and the plasmid is introduced into the porcine small intestinal epithelial cell IPEC-J2 cell through lentiviral transfection.

5. The cell combination according to claim 3 or 4, characterized in that The promoter is minP; the reporter gene is luciferase.

6. An active substance screening platform, characterized in that: The cell combination comprises any one of claims 1 to 5.

7. A method for screening active substances, characterized in that: include: The active substance to be screened is incubated with the immunomodulatory screening cells and the antioxidant screening cells in the cell combination according to any one of claims 1 to 5 under 4-8% carbon dioxide and 35-40° C. for 10-24 hours, and the active substance to be screened is screened according to the detection results of the reporter gene.

8. The active substance screening method according to claim 7, characterized in that: The method of screening active substances according to the detection results of the reporter gene includes: When the level of the reporter gene in the immunomodulatory screening cells increases, it is judged that the active substance to be screened has immunomodulatory activity; and / or when the level of the reporter gene in the antioxidant screening cells increases, it is judged that the active substance to be screened has antioxidant activity.

9. Use of the cell combination according to any one of claims 1 to 5, or the active substance screening platform according to claim 6, in screening active substances having any of the following functions: (1) Immunomodulatory activity; (2) Antioxidant activity.

10. Application of polypeptides in the preparation of products for immunomodulation or anti-oxidation; The polypeptide comprises: One or more of YGPSSYGYGRKDVY, RKDVYYGPSSYGYG, YGPSSYGYGKEKKEVVE, VLPVPQKDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG, RKDVYRGPPP, QLNWDRKDVY, RGPPPRKDVY,EVVEEAYGPSSYGYG, KEKKEVVEYGPSSYGYG, RKDVY, KEKKEVVE, EVVEEA, DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG, RGPPP, YGPSSYGYG, QLNWD, or VLPVPQK; The product is a medicine, a health product, a food, a food additive, a feed or a feed additive.

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