Intervention application of compound with benzophenone mother nucleus structure to CAG cell model

Abstract

The invention relates to the technical field of biomedical research, and provides intervention application of a compound with a benzophenone mother nucleus structure to a CAG cell model, which comprises the following steps: step 1, quickly unfreezing cell seeds in a water bath kettle at 37 DEG C, dropwise adding into a sterile PBS (Phosphate Buffer Solution), uniformly mixing, centrifuging, discarding supernatant, adding into a complete culture medium, and incubating overnight; 2, carrying out passage once every 3 days according to a passage ratio of 1: 3, digesting with 0.25% of pancreatin, terminating digestion, centrifuging, discarding supernatant, adding a complete culture medium, and incubating overnight; and step 3, adding an MNNG reagent into the complete medium, culturing to a fourth generation, and detecting and verifying modeling conditions by using RT-PCR (Reverse Transcription-Polymerase Chain Reaction). The aquilaria sinensis leaves have remarkable anti-inflammatory activity, the anti-inflammatory substance basis of the aquilaria sinensis leaves may be four benzophenone compounds in an n-butyl alcohol extraction part, and the active ingredients may influence tissue cell apoptosis by regulating and controlling an RTK / P I 3K-Akt / Bc l-2 signal axis, so that the anti-CAG effect is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical research, and specifically to an intervention application of a compound having a benzophenone-type mother nucleus structure on a CAG cell model. Background Art

[0002] Chronic atrophic gastritis (CAG) is a chronic digestive system disease characterized by atrophy of gastric mucosal epithelium and glands, decreased number, thinning of gastric mucosa, thickening of mucosal basal layer, or accompanied by pyloric metaplasia and intestinal metaplasia, or mucosal epithelial metaplasia or dysplasia. The lesions are mainly located in the gastric antrum and gastric body, and often manifest as upper abdominal pain, fullness, belching, loss of appetite, or weight loss, anemia, etc., without specificity. CAG is an important starting stage in the development of intestinal gastric cancer. It is a multi-pathogenic disease and precancerous lesion. This disease is more common in middle-aged and older people, and there are obvious regional differences. According to the national CAG patient consultation data, about 7.62% of patients eventually develop gastric cancer due to CAG, and 43.23% of patients have varying degrees of mucosal epithelial dysplasia.

[0003] At present, the classic quadruple therapy is generally used as clinical symptomatic treatment for chronic gastritis caused by Hp infection. However, this therapy can only achieve clinical symptomatic treatment, and it is difficult to reverse a series of secondary symptoms such as mucosal epithelial metaplasia caused by CAG. In addition, the toxic and side effects of the drug itself cannot be ignored. Research results show that long-term use of PPIs by Hp-infected people can change the type of gastritis, from antral gastritis to corpus gastritis. This is because the pH in the stomach rises after taking PPIs, which is conducive to the displacement of Hp from the antrum to the corpus, and inflammation and atrophy of the corpus further reduce gastric acid secretion. The risk of gastric cancer in low-acid or acid-free gastritis with corpus atrophy is significantly increased. Bismuth preparations are no longer approved for production in many countries and regions in the world due to inherent safety issues such as dosage, course of treatment, and adverse reaction rate. Therefore, how to provide continuous, effective and safe treatment for CAG is still a key issue that needs to be urgently solved in the current gastroenterology clinic.

[0004] Mucosal atrophy manifested by CAG is an important starting link of intestinal gastric cancer. As the main precancerous state of gastric cancer, CAG can develop into precancerous lesions such as atypical hyperplasia of gastric mucosal epithelium and intestinal metaplasia on the basis of gastric mucosal atrophy, thereby promoting the occurrence of tissue cancer. A common feature of malignant tumors is the change of cell cycle, including dysregulation of cell hyperproliferation and cell apoptosis. In the development of CAG, there will be a significant increase in cell apoptosis, and the interaction between Fas and its ligand will cause apoptosis of gastric gland cells. Some studies have also pointed out that IL-6 plays a major role in this process, promoting the apoptosis of gastric parietal cells and causing atrophy of gastric mucosa. At the same time, Helicobacter pylori infection, as the main pathogenic factor of CAG, also affects the apoptosis of CAG tissue, making CAG more likely to transform into gastric cancer. After Helicobacter pylori infection, the level of survivin protein, a member of the apoptosis inhibitory protein family, in the gastric mucosa is significantly decreased, the cell survival rate is reduced, and cell apoptosis is caused. The PI3K / AKT signaling pathway is one of the important pathways for regulating cell proliferation and apoptosis. A large number of studies have confirmed that in the occurrence and development of CAG, PI3K is one of the important pathways mediating cell apoptosis. After activation, PI3K can produce PIP3, which acts as the second messenger in the cell to induce AKT to translocate to the cell membrane and activate. The activated AKT regulates downstream target proteins such as caspase9 and Bad through phosphorylation, thereby inhibiting cell apoptosis.

[0005] Previously, the development, research and utilization of Aquilaria sinensis resources were basically limited to the traditional medicinal parts (including resin heartwood), and other parts of Aquilaria sinensis with medicinal development potential, such as branches, leaves, seeds, etc., were completely ignored. As an evergreen tree in the Thymelaeaceae family, Aquilaria sinensis has the highest yield in leaves. Under normal circumstances, artificial cultivation of Aquilaria sinensis requires pruning of branches and leaves 2-4 times a year, which means that Aquilaria sinensis leaves can be picked at least twice a year, and its yield is very considerable. However, since the leaves are not the traditional medicinal parts of Aquilaria sinensis, the pruned branches and leaves are often discarded, resulting in serious waste of resources. In 2014, Aquilaria sinensis leaves were officially included in the catalog of new food additives, proving that Aquilaria sinensis leaves have been recognized by the industry in terms of food safety. At present, some companies have developed Aquilaria sinensis leaves into different types of health products and have put them on the market. Therefore, comprehensive development and research on the non-medicinal parts of Aucklandia odora, mainly leaves, can expand the medicinal range of Aucklandia odora. This can not only make full use of rare plant resources, but also provide a theoretical basis and research foundation for the further development of Aucklandia odora leaves and its series of related products. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides an intervention application of a compound having a benzophenone-type nucleus structure on a CAG cell model, which solves the problem of improving the accuracy and efficiency of drug research and development by optimizing cell models and drug screening technology.

[0007] To achieve the above objectives, the present invention is implemented by the following technical scheme: an intervention application of a compound having a benzophenone-like nucleus structure on a CAG cell model comprises the following steps:

[0008] Step 1: Thaw the cell seeds quickly in a 37°C water bath, add dropwise into sterile PBS buffer, mix well and centrifuge, discard the supernatant, add complete medium, and incubate overnight;

[0009] Step 2: Subculture once every 3 days at a subculture ratio of 1:3. After digestion with 0.25% trypsin, terminate digestion, centrifuge, discard supernatant, add complete medium, and incubate overnight;

[0010] Step 3: Add MNNG reagent to the complete culture medium, culture to the fourth generation, and use RT-PCR to verify the modeling conditions;

[0011] Step 4: Inoculate cells in a 96-well plate, set up sample groups with different concentration gradients, incubate for 24 hours after administration, and use the MTT method to detect the relative viability of cells;

[0012] Step 5: Inoculate cells into 6-well plates, collect cells after administration, extract RNA, reverse transcribe to synthesize cDNA, and perform real-time quantitative PCR detection;

[0013] Step 6: Inoculate cells into 6-well plates, collect cells after administration, and use Annexin V-FITC and propidium iodide staining to detect cell apoptosis rate;

[0014] Step 7, inoculating cells into 6-well plates, collecting cells after administration, and staining with propidium iodide to detect cell cycle distribution;

[0015] Step 8, inoculating cells into 6-well plates, and using EDU staining after administration to detect cell proliferation rate;

[0016] Step 9: Use GraphPad Prism software to analyze the data and use two-way analysis of variance to determine significant differences.

[0017] Preferably, the seed cells in the step 1 are sent in the form of dry ice cryopreservation, and 10 mL of sterile PBS buffer is taken, wherein the cryopreservation tube storing the seed cells is quickly thawed in a 37°C water bath, the cell suspension is added dropwise to the sterile PBS buffer, and then the mixture is slowly blown and mixed, and centrifuged at 1000 rpm for 5 min, the supernatant is discarded, 5 mL of complete culture medium is added, and the cell suspension is added to a T25 cell culture flask after being blown and mixed, and the cells are evenly spread in the culture flask by cross-shaking, and the flask is placed in an incubator and incubated overnight, wherein the culture medium is 90% h-DMEM+10% fetal bovine serum, and the conditions of the incubator are 37°C, 5% CO2 and 95% RH.

[0018] Preferably, in the step 2, after confirming the cell state, the culture medium is discarded in the clean bench, 3 mL PBS is added to wash the cells and then discarded, 1 mL 0.25% trypsin is added for 2 min, and the cells are observed under an inverted microscope. After the cells shrink and become round, 2 mL complete culture medium is added to terminate the digestion. Centrifuge for 5 min at 1000 rpm, discard the supernatant, add complete culture medium, blow and mix, and add the cell suspension to a T25 cell culture flask or a 100 mm cell culture dish. Cross-shake to evenly spread the cells in the culture flask, and place in an incubator for overnight incubation. The culture medium is 90% h-DMEM + 10% fetal bovine serum, and the conditions of the incubator are 37 ° C, 5% CO2 and 95% RH.

[0019] Preferably, the cells in the step 4 are inoculated at a density of 1E4 cells / well. 1. Cell inoculation, and incubation in an incubator overnight, and a solvent control group and 8 sample groups with concentration gradients are set. Three replicate wells are set under each concentration gradient, and the drug is administered when the cell plating rate in the 96-well plate reaches 40% to 60%. After the drug administration is completed, the 96-well plate is placed in an incubator for 24 hours and the supernatant is discarded, 0.5 mg / mL of MTT working solution is added and incubated at 37°C in the dark for 4 hours, and the supernatant is discarded after the incubation is completed, 150 μL DMSO is added to each well, and the OD value is read at 490nm. The relative cell viability is calculated according to the formula, and the conditions of the incubator are 37°C, 5% CO2 and 95% RH, and 200 μL of complete culture solution is added to each well of the solvent control group; 200 μL of complete culture solution containing the corresponding concentration of the test substance is added to each well of the sample group; the zero-adjusted well is inoculated with no cells, and only 200 μL of complete culture solution is added.

[0020] Preferably, the calculation formula for the relative cell viability is:

[0021] Relative cell viability (%) = (sample group - zero-adjusted well) / (solvent control group - zero-adjusted well) × 100.

[0022] Preferably, when the inoculated cells in step 5 to step 8 have a density of 2E5 cells / well and the plating rate in a 6-well plate reaches 40% to 50%, group administration is performed, the dosage per well is 2 mL, 3 replicate wells are set for each group, and culture is continued in an incubator for 24 hours.

[0023] Preferably, the culture medium is discarded after culturing in an incubator for 24 hours in the PCR test, and 1 mL / well PBS is used for washing twice. 1 mL of Total RNA Extractor (Trizol) is added to each well, and the cells are lysed by blowing, and then the samples are stored in a -80°C refrigerator. After the cell lysate is taken out from the -80°C refrigerator, it is placed at room temperature for 5 minutes until it melts. 200 μL of chloroform is added to each 1 mL of Total RNA Extractor, and the mixture is shaken vigorously for 15 seconds, and placed at room temperature for 5 minutes. Centrifuge at 12000 r / min and 4°C for 15 minutes, and the upper water sample is taken after centrifugation. Layer into a new 1.5mL Eppendorf tube, add 400μL isopropanol, shake gently 6-9 times, let stand at room temperature for 10min, then centrifuge at 12000r / min, 4℃ for 10min. After centrifugation, carefully discard the supernatant, add 1mL 75% ethanol, flick the bottom of the tube, wash thoroughly, suspend the precipitate, centrifuge again at 7500r / min, 4℃ for 5min, discard the supernatant, dry naturally in a fume hood for 10min, add 30-50μL water according to the amount of precipitate, bathe in 55℃-60℃ water for 10min, and detect RNA concentration with an ultramicro spectrophotometer after complete dissolution.

[0024] Preferably, in the cell apoptosis rate detection, after culturing in the incubator for 24 hours, the cell culture fluid is collected, and the cells are digested according to the cell passaging method. The digested cells are combined with the cell culture fluid and centrifuged. After centrifugation, the supernatant is carefully removed, and about 1 ml of PBS pre-cooled in an ice bath is added to wash the cells. The cells are resuspended and transferred to a 1.5 ml centrifuge tube, and the above washing process is repeated once, 50,000 to 100,000 resuspended cells are taken, centrifuged and the supernatant is discarded, and the staining solution is added to the cell pellet according to 195 μl Annexin V-FITC binding solution + 5 μl Annexin V-FITC + 10 μl propidium iodide staining solution per well, and gently blow to mix, and incubate in the dark at room temperature of 20 to 25°C for 10 to 20 minutes, then place in an ice bath, and immediately detect on the machine.

[0025] Preferably, after 24 hours of incubation in the incubator in the cell cycle detection, the cells are digested according to the cell passaging method and then centrifuged to carefully remove the supernatant, about 1 ml of ice-bath pre-cooled PBS is added to wash the cells, the cells are resuspended, and the cells are transferred to a 1.5 ml centrifuge tube, and the above washing process is repeated twice, 300 μL of pre-cooled PBS is added, and the bottom of the centrifuge tube is gently tapped to properly disperse the cells, followed by adding 700 μL of ice-bath pre-cooled anhydrous ethanol, gently pipetting to mix, and fixed at 4°C overnight, and the supernatant is carefully removed after centrifugation, about 1 ml of ice-bath pre-cooled PBS is added to wash the cells twice, and the cells are resuspended, and the staining solution is added to the cell pellet according to 0.5 mL of sample staining buffer + 25 μL of propidium iodide staining solution (20X) + 10 μL RNaseA (50X) per well, and the cells are warmed at 37°C in the dark for 30 minutes, and then immediately tested on the machine.

[0026] Preferably, after 24 hours of incubation in the incubator in the cell proliferation assay, EdU is diluted 1:500 with complete culture medium to obtain 2X EdU working solution, and an equal volume of 2X EdU working solution preheated at 37°C is added to a 6-well plate to make the final EdU concentration in the 6-well plate become 1X, and the cells are incubated for 6 hours. After the staining is completed, the cells are digested with reference to the cell passaging method, the cells are washed once with PBS, 1 mL of fixative is added, and the cells are fixed at room temperature for 15 minutes. After centrifugation, the supernatant is carefully removed, and about 1 ml of PBS is added to wash the cells 3 times. The cells are resuspended, and the dye is added to the cell pellet according to 0.43 mL Click Reaction Buffer + 20 μL CuSO4 + 1 μL Azide 5555 + 50 μL Click Additive Solution per well, incubated at 37°C in the dark for 30 minutes, centrifuged to remove the dye, the cells are washed 3 times with PBS, and 500 μL PBS is added for immediate detection.

[0027] The present invention provides an intervention application of a compound having a benzophenone-type nucleus structure on a CAG cell model, which has the following beneficial effects:

[0028] 1. The present invention utilizes Aquilaria sinensis leaves with significant anti-inflammatory activity, and its anti-inflammatory material basis may be the four benzophenone compounds in the n-butanol extraction part. The above active ingredients may affect tissue cell apoptosis by regulating the RTK / PI3K-Akt / Bcl-2 signaling axis, thereby exerting an anti-CAG effect.

[0029] 2. The 10% ethanol, 30% ethanol and 50% ethanol macroporous resin elution parts of the n-butanol extraction part of the white atractylodes leaf of the present invention have different degrees of in vitro anti-inflammatory activity within their safe concentration range, and the efficacy shows a certain concentration dependence. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1It is the technical roadmap of the present invention;

[0031] Figure 2 A schematic diagram of the experimental design in the present invention;

[0032] Figure 3 This is a schematic diagram of the morphological changes of GES-1 cells after MNNG acts on them in the present invention;

[0033] Figure 4 This is a schematic diagram of the expression of genes related to epithelial-mesenchymal transition after MNNG acts on GES-1 cells in the present invention;

[0034] Figure 5 It is a schematic diagram of the cell viability curve in the present invention;

[0035] Figure 6 Schematic diagram of the effect of 2-Oa-rhamnosyl-4,6,4'-trihydroxybenzophenone on the gene expression of GES-1 and GES-1 / MNNG cells in the present invention;

[0036] Figure 7 Schematic diagram of the effect of 2-Oa-rhamnosyl-4,6,4'-trihydroxybenzophenone on the cell cycle of GES-1 and GES-1 / MNNG in the present invention;

[0037] Figure 8 Schematic diagram of the effect of 2-Oa-rhamnosyl-4,6,4'-trihydroxybenzophenone on the proliferation (Edu) of GES-1 and GES-1 / MNNG cells in the present invention;

[0038] Fig. 9 Schematic diagram of the effect of 2-Oa-rhamnosyl-4,6,4'-trihydroxybenzophenone on apoptosis of GES-1 and GES-1 / MNNG cells in the present invention. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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 creative work are within the scope of protection of the present invention.

[0040] Example:

[0041] As one aspect of the present invention, please refer to the attached Figure 1 and attached Figure 2 The present invention provides an intervention application of a compound having a benzophenone-type core structure on a CAG cell model, comprising the following steps:

[0042] 1. Cell Culture

[0043] 1. Cell recovery: Seed cells are sent frozen on dry ice and recovered immediately in the clean bench upon arrival. Prepare 10mL sterile PBS buffer in advance. After the cryotubes are quickly thawed in a 37°C water bath, drop the cell suspension into sterile PBS and slowly blow and mix. Centrifuge at 1000rpm for 5min, discard the supernatant, add 5mL complete culture medium (90% h-DMEM + 10% fetal bovine serum), blow and mix, then add the cell suspension to a T25 cell culture flask, cross-shake to spread the cells evenly in the culture flask, and incubate overnight in an incubator (37°C, 5% CO2, 95% RH).

[0044] 2. Cell medium replacement: The next day after recovery and passage, confirm the cell status and replace the medium. After discarding the culture medium in the clean bench, add 3 mL of PBS to wash the cells and then discard, then add 5 mL of fresh complete culture medium and transfer to the incubator (37°C, 5% CO2, 95% RH) for continued culture.

[0045] 3. Cell passaging: The cell passaging interval is usually 3 days, and the passaging ratio is 1:3. After confirming the cell status, discard the culture medium in the clean bench, add 3mL PBS to wash the cells and then discard, add 1mL 0.25% trypsin to digest for 2min, observe under an inverted microscope, and add 2mL complete culture medium to terminate digestion after the cells shrink and become round. Centrifuge at 1000rpm for 5min, discard the supernatant, add complete culture medium (90% h-DMEM + 10% fetal bovine serum), blow and mix, and add the cell suspension to a T25 cell culture flask or a 100mm cell culture dish. Cross-shake to spread the cells evenly in the culture flask, and incubate overnight in an incubator (37°C, 5% CO2, 95% RH).

[0046] 4. Cell cryopreservation: The pretreatment steps are the same as cell passaging. After centrifugation, add freezing solution (1.5mL / cryotube) according to the freezing quantity. After pipetting and mixing, add the cell suspension into the cryotube, and then transfer it to a -80℃ refrigerator overnight. For long-term storage, transfer the cell lines to a liquid nitrogen tank the next day.

[0047] 5.MNNG-induced modeling: Add MNNG reagent to complete culture medium according to the experimental design, subculture to the fourth generation according to the above cell maintenance operation, collect samples for RT-PCR detection to verify the optimal MNNG modeling conditions. It was confirmed that the formal experiment used 5μM MNNG treatment for 24 hours and then subcultured to the fourth generation as the modeling condition.

[0048] Table 1 MNNG induced modeling experimental design

[0049]

[0050]

[0051] 2. Cytotoxicity Assay

[0052] 1. Cell seeding: Cells were seeded into 96-well plates at a density of 1E4 cells / well and incubated overnight in an incubator (37°C, 5% CO2, 95% RH).

[0053] 2. Experimental grouping: The experiment sets up a solvent control group and 8 sample groups with concentration gradients. Set up 3 replicate wells under each concentration gradient. Prepare working solutions of the test substances with different concentrations according to the experimental design.

[0054] 3. Administration: When the cell plating rate in the 96-well plate reaches 40% to 60%, administration is performed. Add 200 μL of complete culture medium to each well of the solvent control group; add 200 μL of complete culture medium containing the corresponding concentration of the test substance to each well of the sample group; the zero-adjusted well has no cell inoculation and only 200 μL of complete culture medium is added. After administration, place the 96-well plate in an incubator (37°C, 5% CO2, 95% RH) for incubation.

[0055] 4. Detection: After incubating the cells for 24 hours, discard the supernatant, add MTT working solution (0.5 mg / mL, freshly prepared and used), incubate at 37°C in the dark for 4 hours. After incubation, discard the supernatant, add 150 μL DMSO to each well, and read the OD value at 490 nm.

[0056] 5. Calculation of relative cell viability: The calculation formula is:

[0057] Relative cell viability (%) = (sample group - zero-adjusted well) / (solvent control group - zero-adjusted well) × 100%.

[0058] 3. Real-time quantitative PCR detection

[0059] 1. Inoculation: Inoculate cells into 6-well plates at a density of 2E5 cells / well and incubate overnight in an incubator (37°C, 5% CO2, 95% RH).

[0060] 2. Liquid preparation: According to the experimental design (Table 1, Figure 2 ) Prepare the test substance working solution;

[0061] 3. Drug administration: According to the experimental design, when the cell plating rate in the 6-well plate reaches 40% to 50%, the drug administration is carried out in groups, with a dosage of 2 mL per well and 3 replicate wells per group. The cells are cultured in an incubator (37°C, 5% CO2, 95% RH) for 24 hours.

[0062] 4. Collect cells: After culturing for 24 hours, discard the culture medium, wash twice with 1 mL / well PBS, add 1 mL Total RNA Extractor (Trizol) to each well, blow and lyse the cells, and store the samples in a -80°C refrigerator.

[0063] 5. RNA extraction: Take out the cell lysate from the -80℃ refrigerator and let it stand at room temperature for 5 minutes until it melts. Add 200μL of chloroform to each 1mL of TotalRNAExtractor, shake vigorously for 15s, and let it stand at room temperature for 5min. Centrifuge at 12000r / min, 4℃ for 15min. After centrifugation, take the upper aqueous layer to a new 1.5mL Eppendorf tube (about 400μL per sample), add 400μL of isopropanol, shake gently 7-8 times, and let it stand at room temperature for 10min. Centrifuge at 12000r / min, 4℃ for 10min. After centrifugation, carefully discard the supernatant and add 1mL of 75% ethanol (prepared with DEPC water). Flick the bottom of the tube, wash thoroughly, and suspend the precipitate. Centrifuge at 7500r / min, 4℃ for 5min. After centrifugation, discard the supernatant and dry naturally in the fume hood for 10min. Add appropriate amount of DEPC water (30-50 μL) according to the amount of precipitation, incubate in a 55℃-60℃ water bath for 10 min, and detect RNA concentration using an ultra-micro spectrophotometer after complete dissolution.

[0064] 6. Reverse transcription: Calculate the amount of cDNA required based on the detection amount, and synthesize cDNA using RNA as a template according to the RNA reverse transcription kit product instructions. After completion, the sample is stored at -20°C for future use.

[0065] 7. Real-time quantitative PCR: Take out the cDNA sample from the refrigerator, wait for it to melt, add an equal volume of sterile water to dilute, shake and mix, and centrifuge briefly. The type and quantity of reagents added to each well are based on the TBGreen Premix Ex Taq II fluorescent dye product manual. After adding the sample, the machine is tested and the results are calculated using the 2-△△CT method.

[0066] 8. Primer information:

[0067] Table 2 Primer sequence list

[0068]

[0069]

[0070] IV. Cell apoptosis detection

[0071] 1. Inoculation: Inoculate cells into 6-well plates at a density of 2E5 cells / well and incubate overnight in an incubator (37°C, 5% CO2, 95% RH).

[0072] 2. Liquid preparation: According to the experimental design ( Figure 2 )Prepare the test substance working solution.

[0073] 3. Dosage: According to Figure 2 The experimental design was that when the cell plating rate in the 6-well plate reached 40% to 50%, the cells were grouped and dosed with 2 mL per well, and 3 replicate wells were set for each group. The cells were cultured in an incubator (37° C., 5% CO2, 95% RH) for 24 hours.

[0074] 4. Collect cells: After culturing for 24 hours, collect the cell culture fluid, digest the cells according to the cell passaging method, combine the digested cells with the cell culture fluid and centrifuge. After centrifugation, carefully remove the supernatant, add about 1 ml of ice-cold PBS to wash the cells, resuspend the cells, and transfer them to a 1.5 ml centrifuge tube. Repeat the above washing process once.

[0075] 5. Take 50,000 to 100,000 resuspended cells, centrifuge and discard the supernatant, add staining solution to the cell pellet according to 195μl AnnexinV-FITC binding solution + 5μl AnnexinV-FITC + 10μl propidium iodide staining solution per well, and gently blow to mix. Incubate at room temperature (20-25℃) in the dark for 10-20 minutes, then place in an ice bath and immediately test on the machine.

[0076] 5. Cell cycle detection

[0077] 1. Inoculation: Inoculate cells into 6-well plates at a density of 2E5 cells / well and incubate overnight in an incubator (37°C, 5% CO2, 95% RH).

[0078] 2. Liquid preparation: According to the experimental design ( Figure 2 )Prepare the test substance working solution.

[0079] 3. Dosage: According to Figure 2 The experimental design was that when the cell plating rate in the 6-well plate reached 40% to 50%, the cells were grouped and dosed with 2 mL per well, and 3 replicate wells were set for each group. The cells were cultured in an incubator (37° C., 5% CO2, 95% RH) for 24 hours.

[0080] 4. Collect cells: After 24 hours of culture, digest the cells according to the cell passaging method. After centrifugation, carefully remove the supernatant, add about 1 ml of ice-cold PBS to wash the cells, resuspend the cells, and transfer them to a 1.5 ml centrifuge tube. Repeat the above washing process twice.

[0081] 5. Fix the cells: Add 300 μL of pre-cooled PBS. Gently tap the bottom of the centrifuge tube to properly disperse the cells and avoid cell clumping. Then add 700 μL of ice-cold anhydrous ethanol, gently pipette to mix, and fix overnight at 4°C.

[0082] 6. Cell staining: After centrifugation, carefully remove the supernatant, add about 1 ml of ice-cold PBS to wash the cells twice, and resuspend the cells. Add staining solution to the cell pellet according to 0.5 mL sample staining buffer + 25 μL propidium iodide staining solution (20X) + 10 μL RNaseA (50X) per well. Incubate at 37℃ in the dark for 30 minutes. Then immediately test on the machine.

[0083] VI. Cell Proliferation Detection

[0084] 1. Inoculation: Inoculate cells into 6-well plates at a density of 2E5 cells / well and incubate overnight in an incubator (37°C, 5% CO2, 95% RH).

[0085] 2. Liquid preparation: According to the experimental design ( Figure 2 )Prepare the test substance working solution.

[0086] 3. Dosage: According to Figure 2 The experimental design was that when the cell plating rate in the 6-well plate reached 40% to 50%, the cells were grouped and dosed with 2 mL per well, and 3 replicate wells were set for each group. The cells were cultured in an incubator (37° C., 5% CO2, 95% RH) for 24 hours.

[0087] 4. EDU staining: dilute EdU (10 mM) with complete culture medium at 1:500 to obtain 2X EdU working solution (20 μM). Add an equal volume of 2X EdU working solution (20 μM) preheated at 37°C to a 6-well plate to make the final EdU concentration in the 6-well plate 1X. Continue to incubate the cells for 6 hours.

[0088] 5. Collect cells: After staining, digest the cells according to the cell passaging method. Wash the cells once with PBS.

[0089] 6. Fix cells: Add 1 mL of fixative and fix at room temperature for 15 minutes.

[0090] 7. Cell staining: After centrifugation, carefully remove the supernatant, add about 1 ml of PBS to wash the cells 3 times, and resuspend the cells. Add stain to the cell pellet according to 0.43 mL Click Reaction Buffer + 20 μL CuSO4 + 1 μL Azide 5555 + 50 μL Click Additive Solution per well. Incubate at 37°C in the dark for 30 minutes. Centrifuge to remove the staining solution, wash the cells 3 times with PBS, add 500 μL PBS and immediately test on the machine.

[0091] 7. Statistical analysis of results

[0092] GraphPad Prism Program software was used for drawing, and two-way analysis of variance was used among the groups. *p<0.05 indicated a significant difference, and **p<0.01 indicated an extremely significant difference.

[0093] Model cells and drugs

[0094] The cells used in this study were human gastric mucosal cells (GES-1), purchased from Ubiquitin (Shanghai) Trading Co., Ltd. (Cat. No.: U80113).

[0095] The experimental drug is: 2-Oa-rhamnoside-4,6,4'.-trihydroxybenzophenone extracted from the leaves of Aquilaria sinensis, a plant of the Thymelaeaceae family, provided by the Department of Pharmacy of Xi'an Traditional Chinese Medicine Hospital.

[0096] Reagents and main consumables

[0097] Table 3 List of reagents and main consumables

[0098]

[0099]

[0100] Main Equipment

[0101] Table 4 List of main equipment

[0102]

[0103] As one aspect of the present invention, please refer to the attached Figure 3 - Attachment Fig. 9 The present invention provides an intervention application of a compound having a benzophenone-type nucleus structure on a CAG cell model, comprising the following experimental steps:

[0104] 1. Screening of cell MNNG modeling conditions:

[0105] After MNNG acted on GES-1 cells, the cell morphology was observed using an inverted microscope. The results showed that when the concentration of MNNG was greater than 20μM, GES-1 cells could not proliferate normally, and the cells died immediately after passage; after continuous action at 5μM and 10μM concentrations (chronic exposure), the cells had no ability to proliferate after the third generation; after 5μM and 10μM MNNG acted on GES-1 cells for 24 hours and then replaced with normal culture medium (acute exposure), the cells could stably passage to the fourth generation, which was similar to the results of previous related reports.

[0106] Reference Figure 3The results showed that the normal GES-1 cells had regular morphology and a small number of floating dead cells in the supernatant; after MNNG treatment, the number of floating dead cells in the supernatant increased and the morphological polarization became more obvious. Pictures were collected at the same time after subculture, and the cell plating rate after MNNG treatment was significantly lower than that of normal GES-1 cells, indicating that the cell proliferation rate slowed down after MNNG treatment.

[0107] See also Figure 4 Compared with normal GES-1 cells, MNNG can significantly increase the level of epithelial-mesenchymal transition (EMT) of GES-1. After 5μM MNNG acted on the cells for 24 hours, the EMT-related genes were significantly upregulated in the fourth generation; after 10μM acted on the cells for 24 hours, the gene levels showed significant changes from the second generation. Chemical factors are one of the common causes of gastric diseases, and nitrite is the most representative of them. Methylnitronitrosoguanidine (MNNG) is a synthetic nitroso compound, which is often used in experimental studies to simulate gastric mucosal cell lesions caused by the body's ingestion of nitroso compounds. Epithelial-mesenchymal transition (EMT) is an important indicator of the malignant transformation and metastatic ability of reactive cells. This experiment used different time and MNNG concentration conditions, combined with the verification of epithelial-mesenchymal transition marker genes N-cadherin, Snail, and Twist, and finally determined to use 5μM MNNG to act on GES-1 cells for 24 hours, then pass them to the fourth generation to construct a stable gastric mucosal lesion cell model for functional verification of 2-Oa-rhamnoside-4,6,4'.-trihydroxybenzophenone.

[0108] 2. Effect of 2-Oa-rhamnosyl-4,6,4'-trihydroxybenzophenone on the cytotoxicity of GES-1 and GES-1 / MNNG cells:

[0109] The samples were dosed at 8 concentrations from high to low, and cytotoxicity tests were performed on GES-1 and GES-1 / MNNG cells. The trends of cell viability changes are shown in the following table. Figure 5As shown, the cell relative viability curve was drawn with the 8 selected concentrations of 2-Oa-rhamnoside-4,6,4'.-trihydroxybenzophenone as the horizontal axis and the cell relative viability value as the vertical axis. Calculated using GraphPadPrism software, the IC50 value of 2-Oa-rhamnoside-4,6,4'.-trihydroxybenzophenone based on GES-1 normal cells was 3.624 mg / mL; the IC50 value based on GES-1 / MNNG cells was 1.300 mg / mL. Based on the MTT results, it is speculated that the toxic side effects of 2-Oa-rhamnoside-4,6,4'.-trihydroxybenzophenone on normal gastric mucosal epithelial cells are lower than those on diseased gastric mucosal cells, so in later experiments, 2-Oa-rhamnoside-4,6,4'.-trihydroxybenzophenone was added to the functional detection of GES-1 cells to further verify this hypothesis;

[0110] 3. Effects of 2-Oa-rhamnosyl-4,6,4'-trihydroxybenzophenone on gene expression in GES-1 and GES-1 / MNNG cells:

[0111] When cells were treated with 4 mg / mL 2-Oa-rhamnoside-4,6,4'.-trihydroxybenzophenone for 24 hours, compared with the GES-1 / MNNG control, the expression of PI3K, AKT, Caspase3, CYTC, PTEN, and BAX genes changed significantly (P<0.05); compared with the GES-1 control, the expression of Caspase3 and CYTC genes changed significantly (P<0.05); when cells were treated with 3 mg / mL 2-Oa-rhamnoside-4,6,4'.-trihydroxybenzophenone for 24 hours, compared with the GES-1 / MNNG control, PI3K , Caspase3, CYTC, PTEN, and BAX gene expressions showed significant changes (P<0.05). Compared with the GES-1 control, Caspase3 gene expression showed significant changes (P<0.01). When cells were treated with 2mg / mL 2-Oa-rhamnosyl-4,6,4'.-trihydroxybenzophenone for 24h, compared with the GES-1 / MNNG control, Caspase3, CYTC, and PTEN gene expressions showed significant changes (P<0.05). Compared with the GES-1 control, Caspase3 gene expression showed significant changes (P<0.05). The effect trend of 2-Oa-rhamnosyl-4,6,4'.-trihydroxybenzophenone on gene expression in GES-1 and GES-1 / MNNG cells is as follows: Figure 6 As shown;

[0112] GraphPad Prism Program software was used for drawing, and two-way ANOVA statistical analysis was performed between the groups. Compared with the GES-1-control group, the significance was indicated by *, and P-value < 0.05 was indicated by *; compared with the GES-1 / MNNG-control group, the significance was indicated by #, and P-value < 0.05 was indicated by #;

[0113] 4. Effects of 2-Oa-rhamnosyl-4,6,4'-trihydroxybenzophenone on the cell cycle of GES-1 and GES-1 / MNNG

[0114] When cells were treated with 3mg / mL and 4mg / mL 2-Oa-rhamnoside-4,6,4'.-trihydroxybenzophenone for 24h, the ratio of cells in the S phase increased significantly compared with the GES-1 / MNNG control (P<0.05); when cells were treated with 2mg / mL, 3mg / mL, and 4mg / mL 2-Oa-rhamnoside-4,6,4'.-trihydroxybenzophenone for 24h, the ratio of cells in the S phase increased significantly compared with the GES-1 control (P<0.05), indicating that 2-Oa-rhamnoside-4,6,4'.-trihydroxybenzophenone can cause cell S phase arrest and interfere with the construction of DNA molecules. Effects of 2-Oa-rhamnoside-4,6,4'.-trihydroxybenzophenone on the cell cycle of GES-1 and GES-1 / MNNG Figure 7 As shown, GraphPad Prism Program software was used for drawing, and Two-way ANOVA statistical analysis was performed between the groups. Compared with the GES-1-control group, the significance is indicated by *, and P-value < 0.05 is indicated by *; compared with the GES-1 / MNNG-control group, the significance is indicated by #, and P-value < 0.05 is indicated by #;

[0115] 5. Effects of 2-Oa-rhamnosyl-4,6,4'-trihydroxybenzophenone on the proliferation of GES-1 and GES-1 / MNNG cells:

[0116] The cells were treated with 2mg / mL, 3mg / mL, and 4mg / mL 2-Oa-rhamnosyl-4,6,4'.-trihydroxybenzophenone for 24 hours. Compared with the GES-1 and GES-1 / MNNG controls, the cell proliferation rate was significantly decreased (P<0.05), indicating that 2-Oa-rhamnosyl-4,6,4'.-trihydroxybenzophenone can inhibit cell proliferation. Combined with the cell cycle results, the decrease in proliferation rate may be caused by cell cycle arrest. Effects of 2-Oa-rhamnosyl-4,6,4'.-trihydroxybenzophenone on the proliferation of GES-1 and GES-1 / MNNG cells Figure 8 As shown, GraphPad Prism Program software was used for drawing, and Two-way ANOVA statistical analysis was performed between the groups. Compared with the GES-1-control group, the significance is indicated by *, and P-value < 0.05 is indicated by *; compared with the GES-1 / MNNG-control group, the significance is indicated by #, and P-value < 0.05 is indicated by #.

[0117] VI. Effects of 2-Oa-rhamnosyl-4,6,4'-trihydroxybenzophenone on apoptosis of GES-1 and GES-1 / MNNG cells:

[0118] When cells were treated with 2mg / mL, 3mg / mL, and 4mg / mL 2-Oa-rhamnosyl-4,6,4'.-trihydroxybenzophenone for 24h, the apoptosis rate was significantly increased compared with the GES-1 / MNNG control (P<0.05), indicating that 2-Oa-rhamnosyl-4,6,4'.-trihydroxybenzophenone can promote cell apoptosis. Effects of 2-Oa-rhamnosyl-4,6,4'.-trihydroxybenzophenone on apoptosis of GES-1 and GES-1 / MNNG cells Fig. 9 As shown, GraphPad Prism Program software was used for drawing, and Two-way ANOVA statistical analysis was performed between the groups. Compared with the GES-1-control group, the significance is indicated by *, and P-value < 0.05 is indicated by *; compared with the GES-1 / MNNG-control group, the significance is indicated by #, and P-value < 0.05 is indicated by #.

[0119] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An intervention application of a compound having a benzophenone-like core structure on a CAG cell model, characterized in that: The following steps are involved: Step 1: Thaw the cell seeds quickly in a 37°C water bath, add dropwise into sterile PBS buffer, mix well and centrifuge, discard the supernatant, add complete medium, and incubate overnight; Step 2: Subculture once every 3 days at a subculture ratio of 1:

3. After digestion with 0.25% trypsin, terminate digestion, centrifuge, discard supernatant, add complete medium, and incubate overnight; Step 3: Add MNNG reagent to the complete culture medium, culture to the fourth generation, and use RT-PCR to verify the modeling conditions; Step 4: Inoculate cells in a 96-well plate, set up sample groups with different concentration gradients, incubate for 24 hours after administration, and use the MTT method to detect the relative viability of cells; Step 5: Inoculate cells into 6-well plates, collect cells after administration, extract RNA, reverse transcribe to synthesize cDNA, and perform real-time quantitative PCR detection; Step 6: Inoculate cells into 6-well plates, collect cells after administration, and use Annexin V-FITC and propidium iodide staining to detect cell apoptosis rate; Step 7, inoculating cells into 6-well plates, collecting cells after administration, and staining with propidium iodide to detect cell cycle distribution; Step 8, inoculating cells into 6-well plates, and using EDU staining after administration to detect cell proliferation rate; Step 9: Use GraphPad Prism software to analyze the data and use two-way analysis of variance to determine significant differences.

2. The use of a compound having a benzophenone-like core structure for intervention on a CAG cell model according to claim 1, characterized in that: The seed cells in the step 1 are sent in the form of dry ice freezing, and 10 mL of sterile PBS buffer is taken, and the cryopreservation tube storing the seed cells is quickly thawed in a 37°C water bath, and the cell suspension is added dropwise to the sterile PBS buffer and then slowly blown to mix, and centrifuged at 1000 rpm for 5 minutes, the supernatant is discarded, 5 mL of complete culture medium is added, and the cell suspension is added to a T25 cell culture flask after blowing and mixing, and the cells are evenly spread in the culture flask by cross-shaking, and incubated in an incubator overnight, the culture medium is 90% h-DMEM+10% fetal bovine serum, and the conditions of the incubator are 37°C, 5% CO2 and 95% RH.

3. The use of a compound having a benzophenone-like core structure for intervention on a CAG cell model according to claim 1, characterized in that: In the step 2, after confirming the cell status, the culture medium was discarded in the clean bench, 3 mL of PBS was added to wash the cells and then discarded, 1 mL of 0.25% trypsin was added to digest for 2 min, and the cells were observed under an inverted microscope. After the cells shrank and became round, 2 mL of complete culture medium was added to terminate the digestion. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add complete culture medium, mix by pipetting, and then add the cell suspension to a T25 cell culture flask or a 100 mm cell culture dish. Cross-shake to spread the cells evenly in the culture flask, and incubate in an incubator overnight. The culture medium is 90% h-DMEM + 10% fetal bovine serum, and the conditions of the incubator are 37°C, 5% CO2 and 95% RH.

4. The use of a compound having a benzophenone-like core structure for intervention on a CAG cell model according to claim 1, characterized in that: In the step 4, the cells are inoculated at a density of 1E4 cells / well.

1. Cell inoculation, and incubated overnight in an incubator, and a solvent control group and 8 sample groups with concentration gradients are set. Three replicate wells are set under each concentration gradient, and the drug is administered when the cell plating rate in the 96-well plate reaches 40% to 60%. After the drug administration is completed, the 96-well plate is placed in an incubator for 24 hours and the supernatant is discarded, 0.5 mg / mL of MTT working solution is added and incubated at 37°C in the dark for 4 hours, and the supernatant is discarded after the incubation is completed, 150 μL DMSO is added to each well, and the OD value is read at 490nm. The relative cell viability is calculated according to the formula, and the conditions of the incubator are 37°C, 5% CO2 and 95% RH, and 200 μL of complete culture solution is added to each well of the solvent control group; 200 μL of complete culture solution containing the corresponding concentration of the test substance is added to each well of the sample group; there is no cell inoculation in the zero-adjusted well, and only 200 μL of complete culture solution is added.

5. The use of a compound having a benzophenone-like core structure for intervention on a CAG cell model according to claim 4, characterized in that: The calculation formula of the relative cell viability is: Relative cell viability (%) = (sample group - zero-adjusted well) / (solvent control group - zero-adjusted well) × 100.

6. The use of a compound having a benzophenone-like core structure for intervention on a CAG cell model according to claim 1, characterized in that: When the density of the inoculated cells in step 5 to step 8 reaches 40% to 50% in a 6-well plate at 2E5 cells / well, the cells are dosed in groups, with a dosage of 2 mL per well, and three replicate wells are set for each group. The cells are cultured in an incubator for 24 hours.

7. The use of a compound having a benzophenone-like core structure for intervention on a CAG cell model according to claim 6, characterized in that: The culture medium in the PCR test was cultured in an incubator for 24 hours, and the culture medium was discarded. The cells were washed twice with 1 mL / well PBS, and 1 mL of Total RNA Extractor (Trizol) was added to each well. After blowing and lysing the cells, the samples were collected and stored in a -80°C refrigerator. After the cell lysate was taken out from the -80°C refrigerator, it was placed at room temperature for 5 minutes until it melted. 200 μL of chloroform was added to each 1 mL of Total RNA Extractor, and the cells were shaken vigorously for 15 seconds, and the cells were placed at room temperature for 5 minutes. Centrifugation was performed at 12000 r / min and 4°C for 15 minutes. The upper water layer was collected after centrifugation to To a new 1.5mL Eppendorf tube, add 400μL isopropanol, shake gently 6 to 9 times, let stand at room temperature for 10 minutes, centrifuge at 12000r / min, 4℃ for 10 minutes, carefully discard the supernatant, add 1mL 75% ethanol, flick the bottom of the tube, wash thoroughly, suspend the precipitate, centrifuge again at 7500r / min, 4℃ for 5 minutes, discard the supernatant, dry naturally in a fume hood for 10 minutes, add 30 to 50μL water according to the amount of precipitate, bathe in 55℃ to 60℃ water for 10 minutes, and detect the RNA concentration after complete dissolution using an ultramicro spectrophotometer.

8. The use of a compound having a benzophenone-like core structure for intervention on a CAG cell model according to claim 6, characterized in that: In the cell apoptosis rate detection, after culturing in the incubator for 24 hours, the cell culture fluid is collected, and the cells are digested according to the cell passaging method. The digested cells are combined with the cell culture fluid and centrifuged. After centrifugation, the supernatant is carefully aspirated, and about 1 ml of PBS pre-cooled in an ice bath is added to wash the cells. The cells are resuspended and transferred to a 1.5 ml centrifuge tube. The above washing process is repeated once, 50,000 to 100,000 resuspended cells are taken, centrifuged and the supernatant is discarded, and the staining solution is added to the cell pellet according to 195 μl Annexin V-FITC binding solution + 5 μl Annexin V-FITC + 10 μl propidium iodide staining solution per well, and gently blow to mix, and incubate in the dark at room temperature of 20 to 25°C for 10 to 20 minutes, then put in an ice bath, and immediately detect on the machine.

9. The use of a compound having a benzophenone-like core structure for intervention on a CAG cell model according to claim 6, characterized in that: After 24 hours of incubation in the incubator in the cell cycle detection, the cells were digested according to the cell passaging method and then centrifuged, the supernatant was carefully removed, about 1 ml of ice-bath pre-cooled PBS was added to wash the cells, the cells were resuspended, and the cells were transferred to a 1.5 ml centrifuge tube, and the above washing process was repeated twice, 300 μL of pre-cooled PBS was added, and the bottom of the centrifuge tube was gently tapped to properly disperse the cells, followed by adding 700 μL of ice-bath pre-cooled anhydrous ethanol, gently blowing and mixing, and fixed at 4°C overnight, and the supernatant was carefully removed after centrifugation, and about 1 ml of ice-bath pre-cooled PBS was added to wash the cells twice, and the cells were resuspended, and the staining solution was added to the cell pellet according to 0.5 mL of sample staining buffer + 25 μL of propidium iodide staining solution (20X) + 10 μL RNaseA (50X) per well, and the cells were incubated at 37°C in the dark for 30 minutes, and then immediately tested on the machine.

10. The use of a compound having a benzophenone-like core structure for intervention on a CAG cell model according to claim 6, characterized in that: After 24 hours of incubation in the incubator in the cell proliferation assay, EdU was diluted 1:500 with complete culture medium to obtain 2X EdU working solution, and an equal volume of 2X EdU working solution preheated at 37°C was added to a 6-well plate to make the final EdU concentration in the 6-well plate 1X, and the cells were incubated for 6 hours. After the staining was completed, the cells were digested according to the cell passaging method, the cells were washed once with PBS, 1 mL of fixative was added, and the cells were fixed at room temperature for 15 minutes. After centrifugation, the supernatant was carefully removed, and about 1 ml of PBS was added to wash the cells 3 times. The cells were resuspended, and the stain was added to the cell pellet according to 0.43 mL Click Reaction Buffer + 20 μL CuSO4 + 1 μL Azide 5555 + 50 μL Click Additive Solution per well, incubated at 37°C in the dark for 30 minutes, centrifuged to remove the staining solution, the cells were washed 3 times with PBS, and 500 μL PBS was added to the machine for immediate detection.

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