BON sensitized cell model as well as preparation method and application thereof

Abstract

The invention relates to the field of biomedicine, in particular to a BON sensitized cell model as well as a preparation method and application thereof. The invention provides an application of a BON sensitized cell model, which is characterized in that paeoniflorin is used for intervening the BON sensitized cell model, and a small molecule target of paeoniflorin is screened by a chemical proteome method of a limited enzyme digestion method; the problems that an existing BON cell line model is not sensitized or sensitization operation is complex, sensitization sensitivity is low, and stability and specificity are poor are solved. The BON-1 cells are treated with isovaleric acid, the sensitization state in intestinal diseases is simulated, and a reliable cell model is provided and can be used for studying the intestinal diseases; isovaleric acid and paeoniflorin have a synergistic effect, so that compared with unsensitized cells, the sensitivity and the specificity are higher; target screening is carried out by adopting a novel chemical proteomics method of coupling a limited enzyme digestion method with a mass spectrum (LiP-MS) technology, chemical probe preparation is not needed, and the method has the characteristics that the operation is simple and convenient, and a target sequence can be finely positioned.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a BON-sensitized cell model, a preparation method and applications thereof. Background Art

[0002] Chronic visceral pain is a core symptom of irritable bowel syndrome (IBS). Its essence is visceral hyperalgesia, which refers to the increased sensitivity of visceral tissue to stimulation, including hyperalgesia caused by noxious stimulation and allodynia caused by physiological stimulation [Ford AC, Vanner S, Kashyap PC, Nasser Y. Chronic Visceral Pain: New Peripheral Mechanistic Insights and Resulting Treatments[J]. Gastroenterology 2024; 166(6): 976-94]. Epidemiological studies have found that the incidence of visceral hypersensitivity in IBS is as high as 33% to 90%.

[0003] Visceral hypersensitivity is the result of a complex multifactorial mechanism and cascade of reactions, involving intestinal infection, dysbiosis, psychological stress, inflammation and immunity, and gut-brain interactions, leading to pathophysiological processes such as barrier disruption, immune activation, and neuroendocrine disturbances. However, compared with symptoms such as diarrhea and constipation, visceral hypersensitivity in irritable bowel syndrome (IBS) has received less attention, diagnosis, and treatment, and effective interventions or therapies are still lacking. Clinically, approximately 26.2% of patients receive symptomatic treatment with antispasmodics or analgesics. These agents are associated with a high incidence of adverse reactions, such as dry mouth, visual disturbances, and dizziness, and are prone to recurrence after discontinuation. Osmotic laxatives (such as polyethylene glycol), commonly used to treat irritable bowel syndrome (IBS), may worsen abdominal pain and bloating. For patients with more severe abdominal pain, neuromodulators (such as tricyclic antidepressants) should be considered as a second-line option. However, studies have shown that approximately 44% of patients discontinue treatment due to adverse events.

[0004] Therefore, visceral hyperalgesia is both a key and challenging aspect of IBS treatment, and controlling and improving visceral hypersensitivity is crucial. Existing research indicates that enterochromaffin cell sensitization and activation of 5-hydroxytryptamine (5-HT) signaling can cause intestinal hypersensitivity. By studying enterochromaffin cell sensitization models, we can analyze the mechanisms and influencing factors of visceral hypersensitivity and identify specific intervention targets and treatment options.

[0005] However, existing research methods have certain limitations when simulating the sensitized state of enterochromaffin cells. The isolation and in vitro culture of enterochromaffin cells are difficult, and there are currently no commercially available cell lines. Currently, human pancreatic neuroendocrine tumor (BON) cell lines, KRJ-1 cell lines, and rat insulinoma (RIN14B) cell lines are commonly used to simulate enterochromaffin cells. Among them, the BON cell line is highly recognized and widely used. However, existing technologies all use unsensitized BON cell lines as models, which have problems such as low sensitivity, specificity, and specificity in drug target screening, which is not conducive to the study of disease mechanisms, drug targets, and other treatment options.

[0006] Based on this, there is an urgent need for a simple, efficient and stable BON-sensitized cell model and its preparation method for screening drug targets for BON cell sensitization, so as to improve the sensitivity, specificity and stability of drug target screening. Summary of the Invention

[0007] This invention aims to provide a BON-sensitized cell model and its preparation method, and to use this model to screen targets for paeoniflorin intervention. This method involves sensitizing BON-1 cells with isovaleric acid (ISV), then intervening with paeoniflorin. Small molecule targets for paeoniflorin are screened using a chemoproteomic approach using limited enzymatic digestion.

[0008] The present invention provides a method for preparing a BON-sensitized cell model, comprising the following steps:

[0009] S1. Cell culture:

[0010] BON-1 cell line was used and cultured in culture medium;

[0011] S2. Sensitization treatment:

[0012] BON-1 cells were sensitized with isovaleric acid to obtain a BON-sensitized cell model.

[0013] Studies have shown that isovaleric acid has a neurosensitizing effect. This innovative method uses isovaleric acid as a sensitizer to sensitize BON cells, resulting in simple operation and effective sensitization. Sensitization treatments (such as inflammatory cytokine stimulation, oxidative stress induction, and the construction of specific disease models) may significantly increase the expression levels of certain potential targets (such as receptors, enzymes, or signaling pathway proteins) in cells, making them more susceptible to drug action and detection. Sensitization treatments simulate the cellular environment of disease states, making the selected targets more closely aligned with the actual pathological environment and facilitating the discovery of potential drug mechanisms of action in treatment. Sensitization treatments may also enrich specific targets through stress or pathological stimulation, reducing interference from nonspecific binding in normal cells and enhancing drug-target interactions. In contrast, unsensitized cells may experience low target activity due to physiological homeostasis and complex and redundant signaling pathways, making it difficult to distinguish between direct and indirect drug effects.

[0014] Furthermore, the culture medium described in step S1 consists of 90% DMEM, 10% FBS and 1% triple antibody.

[0015] As a preferred technical solution, step S1 specifically comprises: taking BON-1 cells in the logarithmic growth phase, digesting them with trypsin to form a single cell suspension, counting and inoculating them on a culture plate, and culturing them at 37°C and 5% CO2. Furthermore, the culture plate is a 6-well plate, a 12-well plate, or a 24-well plate; the cell inoculation density is 5×10 5 ~10×10 5 cells / well; the culture time is 4h to 24h, preferably 8h to 12h.

[0016] As a preferred technical solution, step S2 specifically comprises: taking out the well plate, removing the culture medium, adding a culture medium containing isovaleric acid, and sensitizing in a 37°C, 5% CO2 incubator.

[0017] Furthermore, the concentration of isovaleric acid in the culture medium containing isovaleric acid in step S2 is 0 to 1000 μM; for the sake of cell safety, it is preferably 10 μM to 200 μM; for the sake of sensitization dose effect, it is more preferably 12.5 μM to 50 μM, and most preferably 25 μM.

[0018] Furthermore, the preparation method of the culture medium containing isovaleric acid in step S2 is: first, isovaleric acid is prepared into an isovaleric acid stock solution with a concentration of 10M using DMSO, and then the isovaleric acid stock solution is diluted with culture medium according to different proportions to obtain culture medium containing different concentrations of isovaleric acid; further, the concentration of DMSO in the culture medium containing isovaleric acid is less than 1‰.

[0019] Furthermore, the sensitization time in step S2 is 18 h to 72 h, preferably 24 h to 48 h, or preferably 36 h.

[0020] The present invention also provides a BON-sensitized cell model prepared by the above-mentioned method for preparing a BON-sensitized cell model.

[0021] The present invention also provides an application of a BON-sensitized cell model, characterized in that paeoniflorin is used to intervene in the BON-sensitized cell model, and the small molecule target of paeoniflorin is screened by a chemical proteomics method of limited enzyme digestion.

[0022] Specifically, the method includes the following steps:

[0023] S1. Cell culture:

[0024] BON-1 cell line was used and cultured in medium;

[0025] S2, sensitization treatment and paeoniflorin intervention:

[0026] BON-1 cells were treated with isovaleric acid and paeoniflorin to obtain BON-sensitized cells induced by paeoniflorin.

[0027] S3. Target screening:

[0028] Restriction protein digestion-mass spectrometry (LiP-MS) technology was used to screen the binding targets of paeoniflorin and identify the protein sequences bound by paeoniflorin.

[0029] For the above scheme, chemical proteomics can reveal the direct binding effect between small molecules and proteins, and is often used to find the targets of small molecules such as drugs and endogenous metabolites. The traditional chemical proteomics method requires the target small molecule to be prepared into a chemical probe, and then incubated with a protein solution to fish for the target it can bind to, and finally the proteomics pair is used for identification and quantitative research. However, due to the certain difficulty in preparing chemical probes, the application of probe affinity method is limited. Limited enzyme cleavage is a new type of chemical proteomics method: after the drug binds to the target protein, it will block the enzyme cleavage site of the protein, thereby causing the target protein sequence to be "resistant" to enzyme cleavage, resulting in changes in the sequence and abundance of the peptide products produced by enzyme cleavage. Therefore, by performing sequence identification and quantitative analysis of the enzyme cleavage products through mass spectrometry, it is possible to distinguish which protein sequences are the binding targets of small molecules.

[0030] The present invention is based on a chemical proteomics method using limited enzyme digestion, which does not require the preparation of chemical probes, has high operability, and is capable of precisely locating target sequences. Peony was first recorded in the Shennong Bencao Jing, which says: "It tastes bitter and flat, and is used to treat abdominal pain caused by evil spirits, remove blood stasis, break up hard masses, cold and heat hernias, relieve pain, promote urination, and invigorate qi." In the late Tang and early Song dynasties, peony was divided into red and white. The predecessors said that "white peony nourishes and red peony purges, white astringes and red peony disperses." Although both can relieve pain, white peony is better at softening the liver and relieving pain, while red peony is better at promoting blood circulation, removing blood stasis, and relieving pain. White peony has the effects of nourishing blood, softening the liver, astringing yin, astringing sweat, relieving pain, etc. Since ancient times, it has been an important medicine for treating various pains, such as abdominal pain. The Compendium of Materia Medica says: "upper and lower diarrhoea, abdominal pain, heaviness in the back", and the Essentials of Materia Medica says: "It nourishes blood, purges the liver, benefits the spleen, astringes liver yin, and treats abdominal pain caused by blood deficiency." White peony root is mainly produced in Zhejiang, Anhui, and Sichuan. From its dried roots, a mixture of physiologically effective components such as paeoniflorin, hydroxypeoniflorin, paeoniflorin, and paeoniflorin lactone glycosides can be obtained, which is called total glycosides of white peony. Among them, the content of paeoniflorin accounts for more than 90%. The original components of compound prescriptions containing peony that enter the blood include paeoniflorin, oxidized paeoniflorin, and paeoniflorin lactone glycosides [Xu Yanli, Li Shaohong, Li Jian, et al., Study on the key quality attributes of the antispasmodic effect of the classic prescription Shaoyao Gancao Decoction, Chinese Journal of Modern Applied Pharmacy, 2023; 40(06): 721-9].

[0031] It can be seen that paeoniflorin has good anti-inflammatory, antispasmodic, analgesic and immunomodulatory effects. Although there is no concept of cell sensitization or visceral hypersensitivity in traditional Chinese medicine, peony or its active ingredients may have antispasmodic and analgesic effects, immune regulation, and sensitization improvement effects. The present invention uses paeoniflorin to intervene in target screening of BON sensitized cell models, pioneering the combination of traditional Chinese medicine theory and modern cytology. Cells sensitized with isovaleric acid can improve the sensitivity of target detection, activate related signaling pathways, reduce background interference, and improve specificity, thereby more accurately capturing the regulatory effect of paeoniflorin on the pathway. At the same time, it has a guiding role and far-reaching significance for the subsequent establishment of other cell models and target screening.

[0032] Furthermore, the culture medium described in step S1 consists of 90% DMEM, 10% FBS and 1% triple antibody.

[0033] As a preferred technical solution, step S1 specifically comprises: taking BON-1 cells in the logarithmic growth phase, digesting them with trypsin to form a single cell suspension, counting and inoculating them on a culture plate, and culturing them at 37°C and 5% CO2. Furthermore, the culture plate is a 6-well plate, a 12-well plate, or a 24-well plate; the cell inoculation density is 5×10 5 ~10×10 5 cells / well; the culture time is 4h to 24h, preferably 8h to 12h.

[0034] As a preferred technical solution, step S2 is specifically as follows: taking out the well plate, removing the culture medium, adding a culture medium containing isovaleric acid to sensitize the cells, aspirating the supernatant, and then adding a culture medium containing paeoniflorin, and incubating in a 37°C, 5% CO2 incubator.

[0035] Furthermore, the concentration of isovaleric acid in the culture medium containing isovaleric acid in step S2 is 0 to 1000 μM; for cell safety considerations, it is preferably 10 μM to 200 μM; for sensitization dose-effect considerations, it is more preferably 12.5 μM to 50 μM, and most preferably 25 μM.

[0036] Furthermore, the concentration of paeoniflorin in the culture medium containing paeoniflorin in step S2 is 50 μM to 2000 μM; for the sake of cell safety and therapeutic effect, it is preferably 100 μM to 1600 μM, more preferably 200 μM to 1000 μM, and most preferably 400 μM to 800 μM.

[0037] Furthermore, the preparation method of the culture medium containing isovaleric acid described in step S2 is: first, isovaleric acid is prepared into an isovaleric acid stock solution with a concentration of 10M using DMSO, and then different proportions of the isovaleric acid stock solution are added to the culture medium to obtain culture media containing different concentrations of isovaleric acid; further, the concentration of DMSO in the culture medium containing isovaleric acid is less than 1‰.

[0038] Furthermore, the preparation method of the culture medium containing paeoniflorin described in step S2 is: first dissolve paeoniflorin with ultrapure water to prepare a paeoniflorin stock solution with a concentration of 160mM, and then use the culture medium to dilute the paeoniflorin stock solution according to different proportions to obtain a culture medium containing paeoniflorin at different concentrations.

[0039] Furthermore, the sensitization time in step S2 is 18h to 72h, preferably 24h to 48h, or preferably 36h; the action time is 18h to 72h, preferably 24h to 48h, or preferably 36h.

[0040] It should be explained that the LiP-MS technology described in step S3 is based on the effect of protein conformational changes on protease accessibility. The principle is that when a protein binds to a small molecule (such as a drug, metabolite, etc.) or undergoes post-translational modification, its conformation changes, thereby changing the accessibility of the protease cleavage site. Through limited proteolysis and mass spectrometry analysis, these conformational changes can be detected, thereby identifying disease-related protein markers. If the abundance of a longer peptide segment of a certain sequence is detected to increase, and the abundance of its corresponding shorter peptide segment decreases, it indicates that the sequence may have blocked the cleavage site on the sequence due to drug binding. The possibility that the sequence is the target sequence is relatively high, and therefore it is considered a priority.

[0041] As a preferred technical solution, step S3 is specifically as follows:

[0042] S31, Limited proteolysis: Under mild conditions, a non-specific protease is used to partially hydrolyze the protein sample to generate protein fragments with structural specificity;

[0043] S32. Mass spectrometry analysis: Analyze the enzymatically digested peptides using liquid chromatography-mass spectrometry (LC-MS / MS) to obtain the protein cleavage map.

[0044] S33. Data analysis: Compare the differences in protein enzyme cleavage patterns under different conditions, identify proteins with conformational changes, and further determine their binding sites and sequence information.

[0045] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0046] 1. The present invention provides a method for preparing a BON-sensitized cell model. By treating BON-1 cells with isovaleric acid, the sensitized state in intestinal diseases is simulated, providing a reliable cell model for subsequent pharmacological studies. The model can be used for intestinal disease research, especially for drug screening for intestinal diseases. The innovative use of isovaleric acid as a sensitizer for sensitizing BON cells results in simple operation, good sensitization stability, and high sensitivity.

[0047] 2. The present invention utilizes an established BON-sensitized cell model to screen targets for paeoniflorin intervention. The synergistic effect of isovaleric acid and paeoniflorin improves the sensitivity of target detection, activates relevant signaling pathways, reduces background interference, and improves specificity compared to cells not sensitized with isovaleric acid, thereby more accurately capturing the regulatory effects of paeoniflorin on the pathways. The new chemical proteomics method of limited enzyme digestion coupled to mass spectrometry (LiP-MS) is used for target screening. This method does not require chemical probe preparation, is easy to operate, and can precisely locate target sequences.

[0048] 3. The BON-sensitized cell model provided by the present invention is not only suitable for the study of paeoniflorin, but can also be extended to the target screening and mechanism of action research of other drugs, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0050] Figure 1 Statistical histogram of the results of each group in Example 5 of the present invention;

[0051] Figure 2 is the characteristic sequence peptide segment of SLC25A5 in Example 6 of the present invention;

[0052] Figure 3 is the characteristic sequence peptide segment of DLD in Example 6 of the present invention;

[0053] Figure 4 is the characteristic sequence peptide segment of PSMB4 in Example 6 of the present invention;

[0054] Figure 5 is the characteristic sequence peptide segment of KRT9 in Example 6 of the present invention;

[0055] Figure 6 is the characteristic sequence peptide segment of RPS4X in Example 6 of the present invention;

[0056] Figure 7 is the characteristic sequence peptide segment of EIF5A in Example 6 of the present invention;

[0057] Figure 8 This is the characteristic sequence peptide segment of TUBA1C in Example 6 of the present invention. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the embodiments and drawings. The schematic embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention. The embodiments described below are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0059] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0060] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0061] The terms “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0062] In order to better illustrate the effect of the technical solution in this embodiment, the following specific examples are provided for illustration:

[0063] Example 1

[0064] A method for preparing a BON-sensitized cell model comprises the following steps:

[0065] S1. Cell culture:

[0066] BON-1 cells in the logarithmic growth phase were obtained, digested with trypsin to form a single cell suspension and counted. The inoculation density was 5 × 10 5 Cells / well were seeded in 12-well plates and cultured at 37°C in a 5% CO environment for 8 h;

[0067] S2. Sensitization treatment:

[0068] The well plate was taken out, the culture medium was removed, and culture medium containing isovaleric acid at different concentrations was added. BON-1 cells were sensitized for 24 h or 48 h in a 37°C, 5% CO2 incubator to obtain a BON-sensitized cell model.

[0069] In order to explore the safe dose of isovaleric acid, the CCK-8 method was used to test the safety of the BON-sensitized cell model obtained by treating with different isovaleric acid concentrations and different treatment times. The results are shown in Table 1.

[0070] Table 1 Screening of safe doses of isovaleric acid

[0071]

[0072] According to the results in Table 1, it can be seen that the concentration of the sensitizer isovaleric acid is safe at a concentration below 200 μM, which proves that the isovaleric acid-sensitized BON cell model of the present invention is highly safe.

[0073] Example 2

[0074] A method for preparing a BON-sensitized cell model comprises the following steps:

[0075] S1. Cell culture:

[0076] BON-1 cells in the logarithmic growth phase were obtained, digested with trypsin to form a single cell suspension and counted. The inoculation density was 5 × 10 5 Cells / well were seeded in 12-well plates and cultured at 37°C in a 5% CO environment for 8 h;

[0077] S2. Sensitization treatment:

[0078] After removing the plate and removing the culture medium, medium containing isovaleric acid was added at concentrations of 0, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM. BON-1 cells were sensitized for 48 hours in a 37°C, 5% CO2 incubator to generate a BON-sensitized cell model. ELISA was then used to measure 5-HT levels in the system. The results showed that 5-HT peaked at a sensitizer concentration of 25 μM, confirming that 25 μM isovaleric acid is the optimal concentration.

[0079] Example 3

[0080] An application of a BON sensitized cell model comprises the following steps:

[0081] S1. Cell culture:

[0082] BON-1 cells in the logarithmic growth phase were obtained, digested with trypsin to form a single cell suspension and counted. The inoculation density was 5 × 10 5 Cells / well were seeded in 12-well plates and cultured at 37°C in a 5% CO environment for 8 h;

[0083] S2, sensitization treatment and paeoniflorin intervention:

[0084] The well plate was taken out, the culture medium was removed, and the cells were sensitized with culture medium containing 25 μM isovaleric acid for 48 h. The supernatant was aspirated and culture medium containing different concentrations of paeoniflorin was added. The cells were incubated in a 37°C, 5% CO2 incubator.

[0085] In order to explore the safe concentration of paeoniflorin, the CCK-8 method was used to test the safety of the BON-sensitized cell model intervened by paeoniflorin obtained by different paeoniflorin concentrations and different treatment times in the above step S3. The results are shown in Table 2.

[0086] Table 2 Safe dose screening of paeoniflorin

[0087]

[0088] According to the results in Table 2, it can be seen that the concentration of paeoniflorin is safe at least within 1600 μM, which proves that the BON-sensitized cell model intervened by paeoniflorin is relatively safe and the cytotoxicity of paeoniflorin is relatively low.

[0089] Example 4

[0090] An application of a BON sensitized cell model comprises the following steps:

[0091] S1. Cell culture:

[0092] BON-1 cells in the logarithmic growth phase were obtained, digested with trypsin to form a single cell suspension and counted. The inoculation density was 5 × 10 5 Cells / well were seeded in 12-well plates and cultured at 37°C in a 5% CO environment for 8 h;

[0093] S2, sensitization treatment and paeoniflorin intervention:

[0094] The well plate was removed, the culture medium was removed, and the cells were sensitized with culture medium containing 25 μM isovaleric acid for 48 h. The supernatant was then aspirated and culture medium containing different concentrations of paeoniflorin, such as 0 μM, 50 μM, 100 μM, 200 μM, 400 μM, 800 μM, and 1600 μM, was added. The cells were incubated in a 37°C, 5% CO2 incubator for 48 h as the experimental group. A BON cell model without any treatment agent or drug was used as the blank control group. The specific concentrations are shown in Table 3.

[0095] Table 3 Dose screening of PAE intervention for BON cell sensitization

[0096]

[0097] The 5-HT concentration in the BON sensitized cell model obtained in step S2 of the experimental and control groups was detected by ELISA, and the following results were obtained:

[0098] 1) Compared with No. 2, the 5-HT concentration in groups 3-8 gradually decreased, that is, after the addition of paeoniflorin, the 5-HT concentration gradually decreased with the increase of paeoniflorin concentration; the 5-HT concentrations in groups 4-8 began to show significant differences compared with those in group 2 (P < 0.05), indicating that paeoniflorin has a significant effect when the concentration reaches 100 μM or above.

[0099] 2) Compared with No. 1, No. 2 showed a significant increase in 5-HT, indicating that 5-HT increased after the addition of isovaleric acid. No. 1 showed no statistical difference in 5-8, indicating that paeoniflorin concentrations of 200 μM and above can counteract the effects of isovaleric acid, reducing 5-HT to a level that was statistically insignificant compared to No. 1 (blank control group) while maintaining a significant difference compared to No. 2 (isovaleric acid model group). Based on this, paeoniflorin concentrations of 200 μM and above are theoretically suitable for paeoniflorin target screening.

[0100] Example 5

[0101] An application of a BON sensitized cell model comprises the following steps:

[0102] S1. Cell culture:

[0103] BON-1 cells in the logarithmic growth phase were obtained, digested with trypsin to form a single cell suspension and counted. The inoculation density was 5 × 10 5 Cells / well were seeded in 12-well plates and cultured at 37°C in a 5% CO environment for 8 h;

[0104] S2, sensitization treatment and paeoniflorin intervention:

[0105] The well plate was removed, the culture medium was removed, and the cells were sensitized with a culture medium containing isovaleric acid for 48 h according to the following groups. The supernatant was aspirated, and culture medium containing different concentrations of paeoniflorin was added, and the cells were incubated in a 37°C, 5% CO2 incubator for 48 h.

[0106] TPH1 is a type of tryptophan hydroxylase, responsible for catalyzing the hydroxylation of L-tryptophan to produce 5-hydroxytryptamine. This process is the rate-limiting step in 5-hydroxytryptamine biosynthesis, meaning that TPH1 activity directly affects 5-hydroxytryptamine production. Therefore, in this example, Western blot analysis of TPH1 was used to indirectly reflect the 5-hydroxytryptamine content in each of the aforementioned groups, thereby verifying the cell sensitization phenotype. The concentrations of isovaleric acid and paeoniflorin used in the BON sensitized cell model were as follows (three replicates were performed in each group, and the average value was taken):

[0107] 1) The original cell line without any treatment was used as the control group

[0108] 2) Isovaleric acid 25 μM

[0109] 3) Isovaleric acid 25 μM + paeoniflorin 100 μM

[0110] 4) Isovaleric acid 25 μM + paeoniflorin 200 μM

[0111] 5) Isovaleric acid 25 μM + paeoniflorin 400 μM

[0112] The results obtained are as follows Figure 1 As shown, the experimental results showed that when the concentration of paeoniflorin was 200μM and 400μM, there was no statistical difference between it and the control group 1) (P>0.05), but there was a significant difference between it and the model group 2) (P<0.05), so paeoniflorin at concentrations of 200μM and 400μM was screened for subsequent experiments.

[0113] Example 6

[0114] An application of a BON sensitized cell model comprises the following steps:

[0115] S1. Cell culture:

[0116] BON-1 cells in the logarithmic growth phase were obtained, digested with trypsin to form a single cell suspension and counted. The inoculation density was 5 × 10 5 Cells / well were seeded in 12-well plates and cultured at 37°C in a 5% CO environment for 8 h;

[0117] S2, sensitization treatment and paeoniflorin intervention:

[0118] The plate was removed, the culture medium was removed, and the cells were sensitized with a medium containing 25 μM isovaleric acid for 48 h according to the following groupings. The supernatant was aspirated, and the cells were then added with a medium containing 200 μM or 400 μM paeoniflorin, respectively, and incubated in a 37°C, 5% CO2 incubator for 48 h.

[0119] S3. Target screening:

[0120] LiP-MS technology was used to screen the binding targets of paeoniflorin and identify the protein sequences to which paeoniflorin binds.

[0121] Based on the principle of limited enzyme digestion to find small molecule binding targets, if the abundance of a longer peptide segment of a certain sequence is detected to increase and the abundance of its corresponding shorter peptide segment decreases, it indicates that the sequence may have blocked the enzyme cleavage site on the sequence due to drug binding. In this case, the possibility of this sequence being the target sequence is relatively high, and this sequence is given priority. Based on this, target sequences such as SLC25A5, DLD, PSMB4, KRT9, RPS4X, EIF5A and TUBA1C were screened. The characteristic sequence peptides of SLC25A5, DLD, PSMB4, KRT9, RPS4X, EIF5A and TUBA1C are shown in the attached figure. Figures 2 to 8 shown.

[0122] In summary, this application uses isovaleric acid-sensitized BON cells as a BON sensitized cell model to participate in the target screening of paeoniflorin intervention in BON cell sensitization, which has the advantages of simple operation, high sensitivity and high specificity. It can also be extended to the target screening and mechanism of action research of other drugs, and has broad application prospects.

Claims

1. A method for preparing a BON-sensitized cell model, characterized in that: BON-1 cells were sensitized with isovaleric acid.

2. The method for preparing a BON-sensitized cell model according to claim 1, wherein: The following steps are involved: S1. Cell culture: BON-1 cell line was used and cultured in culture medium; S2. Sensitization treatment: BON-1 cells were sensitized with isovaleric acid to obtain a BON-sensitized cell model.

3. The method for preparing a BON-sensitized cell model according to claim 2, wherein: The step S2 specifically comprises taking out the well plate, removing the culture medium, adding a culture medium containing isovaleric acid, and performing sensitization in a 37° C., 5% CO 2 incubator.

4. The method for preparing a BON-sensitized cell model according to claim 3, wherein: The concentration of isovaleric acid in the culture medium containing isovaleric acid is 10 μM to 200 μM.

5. The method for preparing a BON-sensitized cell model according to any one of claims 2 to 4, characterized in that: The sensitization time in step S2 is 18 hours to 72 hours.

6. A BON-sensitized cell model prepared by the method for preparing a BON-sensitized cell model according to any one of claims 1 to 5.

7. A method for preparing a BON-sensitized cell model according to any one of claims 1 to 5 or use of the BON-sensitized cell model according to claim 6 in screening targets for paeoniflorin-mediated BON cell sensitization, characterized in that: The BON-sensitized cell model according to claim 6 is intervened by using paeoniflorin, and the small molecule target of paeoniflorin is screened by a chemical proteomics method using limited enzyme digestion.

8. The use according to claim 7, comprising the following steps: S1. Cell culture: BON-1 cell line was used and cultured in medium; S2, sensitization treatment and paeoniflorin intervention: BON-1 cells were treated with isovaleric acid and paeoniflorin to obtain BON-sensitized cells induced by paeoniflorin. S3. Target screening: LiP-MS technology was used to screen the binding targets of paeoniflorin and identify the protein sequences to which paeoniflorin binds.

9. The use according to claim 8, characterized in that The step S2 specifically comprises: taking out the well plate, removing the culture medium, adding a culture medium containing isovaleric acid to sensitize the cells, aspirating the supernatant, then adding a culture medium containing paeoniflorin, and incubating in a 37° C., 5% CO 2 incubator.

10. The use according to claim 9, characterized in that The concentration of isovaleric acid in the culture medium containing isovaleric acid in step S2 is 0 to 1000 μM, the concentration of paeoniflorin in the culture medium containing paeoniflorin is 50 μM to 2000 μM, the sensitization time is 18 h to 72 h, and the incubation time is 18 h to 72 h.

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