Babaodan for inhibiting PD-L1 mediated immune escape of gastric squamous cell carcinoma or gastric adenocarcinoma by blocking PI3K / AKT signal channel

Abstract

The invention discloses an eight-treasure pill for inhibiting PD-L1 mediated immune escape of gastric squamous cell carcinoma or gastric adenocarcinoma by blocking a PI3K / AKT signal channel. Specifically, the invention provides application of the Babaodan in preparation of a medicine for preventing or treating gastric squamous cell carcinoma or gastric adenocarcinoma and application of the Babaodan in preparation of a medicine for inhibiting immune escape of PD-L1 mediated gastric squamous cell carcinoma or gastric adenocarcinoma.

Description

Technical Field

[0001] The present invention belongs to medical technology, in particular to the field of new drug indications, and specifically relates to the use of Babaodan in preparing a drug for preventing or treating gastric squamous cell carcinoma or gastric adenocarcinoma. Background Art

[0002] Babao Dan (BBD) is a precious Chinese patent medicine in my country with more than 400 years of clinical application experience. Its main ingredients include the following 6 currently disclosed precious Chinese medicines, namely: natural bezoar, snake gall, antelope horn, pearl, Panax notoginseng and natural musk. Modern pharmacological studies have shown that the main ingredients of Babao Dan, bezoar, Panax notoginseng, musk and pearl, all have immunomodulatory effects. Babao Dan has a significant effect on reducing the proportion of myeloid suppressor cells, and can play an anti-tumor and improve tumor immunity. Gastric cancer is one of the malignant tumors with high incidence, poor prognosis and great harm in the world. The main treatments for gastric cancer include surgery, radiotherapy, chemotherapy, targeted therapy, and comprehensive treatment. However, since the specific signs of gastric cancer in the early stage are not obvious, most patients are already in the middle and late stages when they are discovered. The main clinical treatment methods are surgery and chemotherapy. Although conventional treatment can improve the 5-year survival rate, the recurrence rate is still very high. Targeted therapy is an increasingly mature treatment method in tumor treatment and can be used for multi-stage gastric cancer treatment. Among them, immune sentinel monoclonal antibodies targeting PD-1 / PD-L1, an important target of tumor immune escape, have been used clinically. However, due to the poor clinical effect of single-drug antibodies, it is particularly important to design and discover combination drugs and complementary treatments. Babaodan can inhibit the activation of the PI3K / AKT / mTOR signaling pathway, thereby inhibiting gastric cancer cell proliferation and promoting cell apoptosis and autophagy; and Babaodan can inhibit the formation of new blood vessels and lymphatic vessels in gastric cancer; it also has the effect of reversing the multidrug resistance of gastric cancer resistant cells, but it is not clear whether Babaodan can inhibit tumor immune escape by regulating PD-1 / PD-L1 to inhibit gastric cancer. Therefore, it is necessary to explore the pharmacodynamics of BBD affecting gastric cancer and clarify its mechanism of action through in vivo and in vitro experiments.

[0003] Pharmacological studies have found that Babaodan has strong anti-inflammatory, analgesic, liver and gallbladder protection effects. It is clinically used for various damp-heat syndromes, including jaundice and gastrointestinal symptoms caused by damp-heat, as well as liver protection. However, there are no reports on the use of Babaodan in the preparation of drugs for the prevention or treatment of gastric squamous cell carcinoma or gastric adenocarcinoma. Summary of the invention

[0004] The purpose of the present invention is to provide a new pharmaceutical use of Babaodan.

[0005] In one aspect, the present invention provides the use of Babaodan in preparing a medicament for preventing or treating gastric squamous cell carcinoma or gastric adenocarcinoma.

[0006] On the other hand, the present invention provides the use of Babaodan in preparing a medicament for inhibiting PD-L1-mediated immune escape of gastric squamous cell carcinoma or gastric adenocarcinoma.

[0007] In the present invention, Babaodan inhibits PD-L1-mediated immune escape of gastric squamous cell carcinoma or gastric adenocarcinoma by blocking the PI3K / AKT signaling pathway.

[0008] The Babaodan mentioned above is the Babaodan capsule produced by Xiamen Traditional Chinese Medicine Factory Co., Ltd., with the national medicine approval number Z10940006. Its main ingredients include bezoar, snake gall, antelope horn, pearl powder, Panax notoginseng, musk, etc.

[0009] In some embodiments, the drug may be in the form of a solid, semisolid, or liquid.

[0010] In some embodiments, the drug preparation can be an aqueous solution, a non-aqueous solution, a suspension, a lozenge, a capsule, a tablet, a granule, a pill or a powder.

[0011] In some embodiments, the medicament may include one or more pharmaceutically acceptable excipients.

[0012] In some embodiments, the pharmaceutically acceptable excipient may include an excipient, a filler, or a diluent.

[0013] In some embodiments, based on mass percentage, the drug may include 0.01-99.99% of Babaodan and 99.99-0.01% of pharmaceutically acceptable excipients.

[0014] In some embodiments, the drug can be administered orally or by injection.

[0015] Beneficial effects:

[0016] The preclinical validation of the present invention has definite and significant efficacy. Pharmacological experiments have found that Babaodan inhibits PD-L1-mediated immune escape of gastric squamous cell carcinoma or gastric adenocarcinoma by blocking the PI3K / AKT signaling pathway, with significant effects. Therefore, Babaodan can be used to prevent or treat gastric squamous cell carcinoma or gastric adenocarcinoma, and has good development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The results of the gastric cancer cell line screening process are shown. Among them: (A, B) The expression of PD-L1 in BGC-823, AGS, MGC80-3, SGC-7901, and SGC7901 / DDP cells was determined by Western blot analysis, with GAPDH as an internal reference; (C, D) Cell viability was analyzed using CCK8 assay; (E) AGS and MGC80-3 cells were incubated with BDD for 24 hours and observed under a microscope. Compared with the control group (0 mg / mL), **p<0.01.

[0018] Figure 2 The effect of BBD on the expression of PD-L1 in AGS and MGC80-3 cells is shown. Among them: (AD) The fluorescence expression intensity of PD-L1 in AGS and MGC80-3 cells after BDD culture was detected by flow cytometry; (EH) The expression of PD-L1 in AGS cells and MGC80-3 cells after BBD culture was detected by Western blot. Compared with the control group (0 mg / mL), *p<0.05 and **p<0.01.

[0019] Figure 3 The effect of BBD on the PI3K / AKT signaling pathway in AGS cells and MGC80-3 cells is shown. Among them: (AD) Western blot was used to detect the expression of PI3K, AKT and p-AKT after incubation with BBD; (E) Cell morphology was observed under a microscope after incubation with LY294002 or BBD; (FM) Western blot was used to detect the expression of PI3K, AKT, p-AKT and PD-L1 in AGS cells and MGC80-3 cells after incubation with LY294002 and BBD. Compared with the control group, *p<0.05 and **p<0.01.

[0020] Figure 4 The effect of BBD on the PI3K / AKT signaling pathway of AGS cells and MGC80-3 cells is shown. Among them: (A) After incubation with 740Y-P or BBD, the cell morphology was observed under a microscope; (BI) After incubation with 740Y-P or BBD, the expression of PI3K, AKT, p-AKT, and PD-L1 in AGS cells and MGC80-3 cells was detected by Western blotting. Compared with the control group, *p<0.05 and **p<0.01; compared with the 740Y-P group, ##p<0.01; compared with the BBD group, $p<0.05 and $$p<0.01.

[0021] Figure 5 The effect of BBD on mouse MFC transplanted tumors is shown. Among them: (A, B) BBD reduced tumor volume and weight, compared with the model group, **p<0.01.

[0022] Figure 6 The effect of BBD on PD-1 and PD-L1 in tumor tissues is shown. Among them: (A, B) IHC method was used to detect the expression of PD-L1 in tumor tissues; (C, D) Western blotting method was used to detect the expression of PD-L1 in tumor tissues; (E) qRT-PCR was used to detect the expression level of PD-L1 mRNA in tumor tissues; (F, G) Flow cytometry mean fluorescence intensity of PD-L1 expression in tumor tissues; (H, I) Flow cytometry detection of PD-1 in CD8+T cells; (J, K) Bio-plex method was used to detect the expression of IFN-γ and TNF-α in mouse blood. Compared with the model group, *p<0.05 and **p<0.01. DETAILED DESCRIPTION

[0023] Hereinafter, the present invention will be described in detail by way of examples. However, the examples provided herein are only for illustrative purposes and are not intended to limit the present invention.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0026] Example

[0027] 1 Materials and Methods

[0028] 1.1 Reagents

[0029] RPMI 1640 medium (MA0215-2), trypsin (MA0233-1), PBS (MA0015-1), ultrasensitive ECL luminescent reagent (MA0186) and CCK8 cell proliferation detection kit (MA0218) were purchased from Dalian Meilun Biotechnology Co., Ltd.; Ham's F-12 (CM-0022) was purchased from Wuhan Pronosai Life Science Co., Ltd.; penicillin-streptomycin mixture (KGY0072), Trypan Blue cell counting solution (KGY015) and cell freezing preservation solution (KGY0063) were purchased from Jiangsu Keygen Biotechnology Co., Ltd.; Fetal bovine serum (SA101.02) was purchased from Beijing Sai Aomei Cell Technology Co., Ltd.; AKT antibody (4685S) and p-AKT antibody (4060S) were purchased from Cell Signaling Technology Co., Ltd., USA Technology Company; PI3K antibody (60225-I-Ig), PD-L1 antibody (17952-1-AP), HRP-conjugated Goat Anti-Mouse IgG (H+L) (SA00001-1), HRP-conjugated Goat Anti-Rabbit IgG (H+L) (SA00001-2) and GAPDH antibody (60004-1-lg) were purchased from Wuhan Sanying Biotechnology Co., Ltd.; p-PI3K antibody (AB 182651) was purchased from Abcam, USA; LY294002 (A8250) was purchased from APExBIO, USA; 740Y-P (HY-P0175) was purchased from Med Chem Express; BCA protein quantification kit (23227), Trizol RNA extraction kit and TaKaRa RNA PCR kit (RR047A) were purchased from TaKaRa, Japan; SYBR Green Master Mix (4312704) was purchased from Thermo Fisher Scientific, USA; SDS-PAGE Gel Kit (CW0022M) was purchased from Beijing Kangwei Biotechnology Co., Ltd.; APC-Cy7-CD45 (557659), FITC-CD3 (553061), AF700-CD8 (557959), BV421-PD-1 (562584) and BV786-PD-L1 (741014) were purchased from BD Company, USA; Bio-Plex Pro Rat Cytokine 23-plex was purchased from Bio-Rad Company, USA; Babaodan (BBD) is Babaodan capsule produced by Xiamen Traditional Chinese Medicine Factory Co., Ltd., with national medicine standard number Z10940006.

[0030] 1.2 Preparation of BBD

[0031] Babaodan capsules were stored in a refrigerator at 4°C, and the powder of Babaodan capsule contents was dissolved with normal saline (R22173; Yuanye Biotechnology; China) for in vivo experiments or with PBS for in vitro experiments before use.

[0032] 1.3 Cell culture

[0033] MFC cell line, BGC-823 cell line, SGC-7901 cell line, SGC7901 / DDP cell line, and MGC80-3 cell line were purchased from Wuhan Punosai Biotechnology Co., Ltd. The cells were cultured in RPMI1640 medium containing 10% FBS and 1% penicillin / streptomycin and grown in a humidified atmosphere of 5% CO2 at 37°C. Human gastric adenocarcinoma AGS cell line was purchased from Wuhan Presella Biotechnology Co., Ltd. The cells were cultured in Ham's F-12 containing 10% FBS and 1% penicillin / streptomycin and grown in an incubator of 5% CO2 at 37°C.

[0034] 1.4 Cell viability assay

[0035] Cell viability was detected using the CCK8 cell proliferation detection kit according to the manufacturer's instructions. Based on the results of the cell viability assay, AGS and MGC80-3 gastric cancer cells were selected as experimental research subjects for subsequent related drug interventions. Human AGS and MGC80-3 gastric cancer cells were seeded in 96-well plates and treated with different concentrations of BBD for 24 hours. 10 μL of CCK8 reagent was added to each well, further incubated for 2 hours, and finally the absorbance was measured at 450 nm.

[0036] 1.5 Mouse homograft studies

[0037] Twenty 6-week-old male 615 mice (Shanghai SLAC Laboratory Animal Co., Ltd.) were used for adaptive feeding in an SPF-controlled environment by maintaining a 12-h light / dark cycle with free access to food and water. 6 After mixing MFC cells with an equal volume of saline, the cells were injected subcutaneously into the right abdomen of each mouse. 3At 14:00 p.m., mice were randomly divided into a model group and a BBD group (n = 5 per group). The model group was given normal saline by gavage, and the BBD group was given 250 mg / kg BBD for 6 consecutive days a week for a total of 13 days. Body weight and tumor size were measured every other day using an electronic balance and caliper. The tumor volume was calculated as follows: major diameter (L) × (minor diameter) 2 (W2) × π / 6. After 13 days of treatment, the tumors of the mice were collected for subsequent analysis. The experimental procedures and animal care were carried out in strict accordance with international ethical guidelines, and the Guiding Opinions on the Care and Use of Laboratory Animals (Guiding Opinions) issued by the Ministry of Science and Technology of the People's Republic of China have been approved by the Institutional Animal Care and Use Committee of Fujian University of Traditional Chinese Medicine (FjTCM IACUC 2020017).

[0038] 1.6 Flow cytometry

[0039] Reagents for preparing single-cell suspensions were purchased from Sigma-Aldrich, USA. The tumor tissue was added to the lysis and digestion solution and placed in a dual-channel grinder. After grinding, the supernatant was extracted to prepare a single-cell suspension of tumor tissue for flow cytometry. After mincing, it was digested with type IV collagenase, hyaluronidase, and DNase I at 37°C for 1 hour. Single-cell suspensions were obtained by density gradient centrifugation with 40% percoll and 80% percoll. The single-cell suspension was stained with the following antibodies: BV786-PD-L1, APCCY7-CD45, FITC-CD3, AF700-CD8, BV421-PD-1. In addition, the stained cells were detected using a BD flow cytometer (BD, USA) and analyzed using FlowJo software (BD, USA).

[0040] 1.7 Cell morphology observation

[0041] MGC80-3 and AGS cells in the logarithmic growth phase were plated in a 6-well plate (2 mL / well) at 3.0 × 10 5 They were seeded at a density of 10 cells / mL, 3 wells per group, and cultured in a cell culture incubator (37°C, 5% CO2). After intervention with corresponding drugs and reagents (BBD, PI3K agonist 740Y-P or PI3K inhibitor LY94002) in the logarithmic growth phase, the growth status of the cells was observed under an inverted microscope and photographed (100×, 200×).

[0042] 1.8 Immunohistochemistry (IHC) analysis

[0043] Paraffin-embedded gastric cancer tumor tissues were dehydrated and dewaxed, and the antigen retrieval solution was heated to boiling point in a microwave oven for antigen retrieval. After washing with PBS, the sample slides were incubated in 3% H2O2 at room temperature for 10 minutes and then incubated with PD-L1 (1:500) at 4°C overnight. The next day, the sections were incubated with the secondary antibody in an oven at 37°C for 1 hour and then stained with 3,3-diaminobenzidine. The positive rate was used for semi-quantitative analysis. The acquired images were analyzed using the image J (1.46r) image analysis system.

[0044] 1.9 Preparation of total protein samples from tumor tissue

[0045] Take the tumor tissue out of the -80℃ ultra-low temperature freezer, weigh 30-80mg of tumor tissue and transfer it to a 2mL centrifuge tube, and add grinding steel beads. Add protein lysis buffer (1mL for every 100mg) to the centrifuge tube of the tissue sample. Precool the tray bottom of the tissue grinder in advance, and then operate according to the instrument instructions, set the frequency to 70HZ, the grinding time to 1min, and grind 3 times. Centrifuge (14000rpm, 20min, 4℃), collect the supernatant into a 1.5mL centrifuge tube, and determine the protein sample concentration according to the kit instructions. Use an enzyme marker to measure the OD value at 562nm, draw a protein concentration standard curve based on the corresponding OD value, and add an appropriate amount of protein lysis buffer to the sample according to the measured protein concentration results to keep the concentration of all protein samples consistent. Finally, add 1 / 4 of the corresponding volume of loading buffer to each tube of protein, heat denature in a 100℃ metal bath for 8min, divide it into 1.5mL centrifuge tubes and store it in a -80℃ ultra-low temperature refrigerator to avoid repeated freezing and thawing.

[0046] 1.10 Preparation of total cell protein samples

[0047] The cells were inoculated with 5×10 5 / well inoculated into the well plate, wait for the cells to adhere to the wall, or perform appropriate intervention, digest the cells after the intervention time is over and collect them into centrifuge tubes by centrifugation. Add protein lysis buffer (1 mL for every 100 mg) to the centrifuge tube of the sample. Centrifuge (14000rpm, 20min, 4℃), collect the supernatant into a 1.5mL centrifuge tube, and measure the protein sample concentration according to the kit instructions. Use an enzyme marker to measure the OD value at 562nm, draw a protein concentration standard curve according to the corresponding OD value, and add an appropriate amount of protein lysis buffer to the sample according to the measured protein concentration results to keep the concentration of all protein samples consistent. Finally, add 1 / 4 of the corresponding volume of loading buffer to each tube of protein, heat denature in a 100℃ metal bath for 8min, divide into 1.5mL centrifuge tubes and store in a -80℃ ultra-low temperature refrigerator to avoid repeated freezing and thawing.

[0048] 1.11 Western Blot (WB) analysis

[0049] Tumor tissue or cell protein samples were transferred to PVDF membranes after electrophoresis. The PVDF membranes were blocked with 5% skim milk powder for 1 h, and then primary antibodies were added overnight at 4°C. The next day, appropriate secondary antibodies were incubated with these membranes for 1 h at room temperature. GAPDH was used as an internal reference, and the immunoreactive protein signals were detected with ultrasensitive ECL luminescent reagents, and the density of the gel bands was analyzed using the Bio-Image Analysis System (Bio-Red, Hercules, CA, USA).

[0050] 1.12 Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis

[0051] qRT-PCR was used to detect the expression of PD-L1 in gastric cancer tumor tissues. Total RNA was extracted with RNAiso Plus, and its purity was determined. In addition, cDNA was generated using a cDNA reverse transcription kit, and qRT-PCR was performed using SYBRTM Select MasterMix. The primer sets used in this study are listed in Table 1.

[0052] Table 1 List of primer sequences used to amplify PD-L1 and GAPDH genes.

[0053]

[0054] 1.13 Multiple Particle Detection

[0055] Serum was analyzed using the Bio-Plex Pro Rat Cytokine 23-plex according to the manufacturer's instructions to determine the levels of inflammatory mediators (tumor necrosis factor-α, interferon-α, and leukemia) using a Bio-Rad Bio-Plex 200 analyzer.

[0056] Statistical analysis

[0057] The data in this study were plotted using Prism GrapPad 8.0 and analyzed using SPSS23.0 statistical software. All data were tested for normal distribution and variance homogeneity, which were consistent with normal distribution. The t-test (Student-t test analysis) was used between two groups, and one-way analysis of variance was used between multiple groups of data. If it did not conform to the normal distribution, the rank sum test was used. The parameter values ​​were expressed as (mean ± standard deviation), and the difference was statistically significant when p < 0.05.

[0058] 2. Experimental results

[0059] 2.1 Effect of BBD on the viability of gastric cancer cells (For details of CCK8 assay, microscopic observation and western blot, see Examples, Materials and Methods 1.4, 1.7 and 1.11)

[0060] First, gastric cancer cells with high expression of PD-L1 were screened by Western blotting ( Figure 1 The results showed that PD-L1 protein was highly expressed in AGS and MGC80-3, so these two gastric cancer cells were selected as the experimental research objects for subsequent related drug intervention.

[0061] Next, the CCK8 method was used to examine the effects of different concentrations of BBD on the viability of gastric cancer cells AGS and MGC80-3 ( Figure 1 In C and D), the groups were set to 0 mg / ml, 0.25 mg / ml, 0.5 mg / ml, and 1.0 mg / ml, with 0 mg / ml as the control group, which was used to compare the experimental results without BBD intervention. It was found that BBD inhibited the activity of gastric cancer cells AGS and MGC80-3 in a concentration-dependent manner to a certain extent.

[0062] At the same time, the effects of different concentrations of BBD on the morphological changes of AGS and MGC80-3 were observed under an inverted microscope. Figure 1 Middle E). The results showed that different concentrations of BBD could reduce the number of gastric cancer cells. As the concentration of BBD increased, the inhibitory effect of BBD on AGS and MGC80-3 became more obvious, and some cells began to shrink and become round and bright, indicating that BBD had a good inhibitory effect on AGS and MGC80-3.

[0063] 2.2 BBD can inhibit the expression of PD-L1 in gastric cancer cells (for flow cytometry and protein immunoblotting, see the examples, materials and methods 1.6, 1.11)

[0064] The effects of different concentrations of BBD on the expression of PD-L1 in AGS and MGC80-3 were studied, and the groups were set as 0 mg / ml, 0.25 mg / ml, 0.5 mg / ml, and 1.0 mg / ml, among which 0 mg / ml was the control group for comparing the experimental results without BBD intervention.

[0065] Flow cytometry results showed that the mean fluorescence intensity (MFI) of PD-L1 decreased after 0.25 mg / mL BBD treatment, but not significantly, while the 1.0 mg / mL BBD group significantly decreased ( Figure 2 The results showed that BBD could inhibit the expression of PD-L1 in AGS and MGC80-3 cells in a concentration-dependent manner.

[0066] Western Blot was used to detect the effect of different concentrations of BBD on the expression of PD-L1 in two gastric cancer cells. Figure 2 The results showed that BBD could effectively inhibit the translation level of PD-L in AGS and MGC-803.

[0067] 2.3 Effect of BBD on the expression of proteins related to the PI3K / AKT pathway in gastric cancer cells (microscope observation and western blot method are detailed in the examples, materials and methods 1.7 and 1.11)

[0068] Given that many studies have shown that the expression of PD-L1 in tumor cells is mainly controlled by the activation of the PI3K / AKT pathway, the expression of PI3K / AKT pathway-related proteins in colorectal cancer cells after intervention with different concentrations of BBD was subsequently examined. The groups were set to 0 mg / ml, 0.25 mg / ml, 0.5 mg / ml, and 1.0 mg / ml, with 0 mg / ml as the control group, which was used to compare the experimental results without BBD intervention. The levels of PI3K total protein and p-AKT were significantly downregulated in AGS and MGC80-3 ( Figure 3 In the AD, the inventors hypothesized that BBD could block PI3K / AKT signaling by downregulating PI3K and inhibiting AKT activation, and the expression of PD-L1 in gastric cancer cells was further reduced, thus blocking the immune escape of tumor cells.

[0069] Next, the inventors added PI3K inhibitors and agonists to further verify this hypothesis, and set the groups as control group (the control group was not intervened and was used to compare the results of other intervention experiments), LY294002 group (intervention with LY294002), BBD group (BBD 0.5mg / ml intervention), BBD+LY294002 group (BBD and LY294002 were used in combination to verify that BBD regulates PD-L1 through the PI3K / AKT pathway). LY294002 is a common PI3K inhibitor.

[0070] First, the effects of LY294002 and BBD on the morphological changes of gastric cancer cells AGS and MGC80-3 were observed under an inverted microscope. After BBD intervention, the number of the two gastric cancer cells decreased, and some cells appeared shrunk, rounded, and brightened. Compared with the use of LY294002 and BBD alone, the trend of cell changes in the combined drug group was roughly the same ( Figure 3 Middle E).

[0071] Next, the effects of LY294002 and BBD on PI3K / AKT / PD-L1 related proteins in gastric cancer cells AGS and MGC80-3 were examined. The inventors found that BBD and LY294002 alone or in combination reduced the levels of PI3K total protein, AKT phosphorylation and PD-L1 protein expression in both cells ( Figure 3 It is noteworthy that BBD and LY294002 alone showed almost the same inhibitory effect on related proteins.

[0072] In order to further verify the inventor's hypothesis, the inventor used the PI3K agonist, 740y-p. The groups were set as the control group (the control group did not intervene and was used to compare the results of other intervention experiments), the 740y-p group (intervention with 740y-p), the BBD group (BBD 0.5mg / ml intervention), and the BBD+740y-p group (BBD and 740y-p were used in combination to verify that BBD regulates PD-L1 through the PI3K / AKT pathway). The results showed that the number of two gastric cancer cells in the 740y-p group was higher than that in the control group, while the number of two gastric cancer cells in the BBD group was lower than that in the control group. Compared with the control group and the PI3K agonist (740y-p) group, the number of gastric cancer cells in the combination drug (BBD+740y-p) group was between the two groups, some cells shrank, became round and bright, and the number of gastric cancer cells increased slightly ( Figure 4 (A).

[0073] Next, the effects of 740y-p and BBD on PI3K / AKT / PD-L1-related proteins in gastric cancer cells AGS and MGC80-3 were detected. The results showed that the PI3K agonist 740y-p activated the PI3K / AKT pathway, upregulated PI3K expression, activated p-AKT, and upregulated PD-L1 expression, while BBD downregulated PI3K expression and inhibited AKT activation, thereby blocking the PI3K / AKT signaling pathway, further reducing PD-L1 expression in gastric cancer cells, blocking tumor cell immune escape, and partially offsetting activation and inhibition after combined treatment ( Figure 4 BI).

[0074] 2.4 Effects of BBD on tumor-bearing mice (for details of animal transplant tumor model construction, flow cytometry, immunohistochemistry (IHC), protein immunoblotting, RT-qPCR and multiple microparticle detection methods, see Examples, Materials and Methods 1.5, 1.6, 1.8, 1.11, 1.12 and 1.13)

[0075] In order to verify the effect of BBD in vivo, tumor inhibition and immunotherapy-related indicators were tested by using BBD to intervene in mice with gastric cancer subcutaneous allogeneic tumors to further verify the inhibitory effect of BBD on gastric cancer. Because MFC cells are derived from the tumor tissue of MFC cells, 615 mice are mainly used for immune-related research.

[0076] The results showed that the tumor volume and weight of the BBD group were significantly smaller than those of the model group ( Figure 5 ). The expression of PD-L1 in tumors was detected by IHC, Western Blot, flow cytometry and RT-qPCR ( Figure 6 AG), indicating that BBD can inhibit the expression of PD-L1 in the transcription and translation process. In addition, the expression of PD-1 on CD8+ T cells in the tumor microenvironment was also reduced ( Figure 6 H, I). TNF-α and IFN-γ cytokines in the peripheral blood of 615 mice were detected by multiple microparticle detection (Bio-Plex). Compared with the model group, TNF-α and IFN-γ cytokines in the BBD group increased ( Figure 6 (in J, K).

[0077] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be thought of by any technician familiar with the field within the technical scope disclosed by the present invention without creative work should be covered within the protection scope of the present invention.

Claims

1. Use of Babaodan in preparing a medicine for preventing or treating gastric squamous cell carcinoma or gastric adenocarcinoma.

2. Use of Babaodan in the preparation of a drug for inhibiting PD-L1-mediated immune escape of gastric squamous cell carcinoma or gastric adenocarcinoma.

3. The use according to claim 2, wherein Babaodan inhibits PD-L1-mediated immune escape of gastric squamous cell carcinoma or gastric adenocarcinoma by blocking the PI3K / AKT signaling pathway.

4. The use according to any one of claims 1 to 3, wherein The drug is in the form of solid, semisolid or liquid.

5. The use according to claim 4, wherein The drug preparation is an aqueous solution, a non-aqueous solution, a suspension, a lozenge, a capsule, a tablet, a granule, a pill or a powder.

6. The use according to claim 1, wherein The medicament includes one or more pharmaceutically acceptable excipients.

7. The use according to claim 6, wherein The pharmaceutically acceptable excipients include excipients, fillers or diluents.

8. The use according to claim 6 or 7, wherein Based on mass percentage, the medicine includes 0.01-99.99% of Babaodan and 99.99-0.01% of pharmaceutically acceptable excipients.

9. The use according to claim 1, wherein The administration route of the drug is oral administration or injection administration.

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