Traditional Chinese medicine composition for improving tumor immune microenvironment and inhibiting gastric cancer as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention provides a traditional Chinese medicine composition for improving tumor immune microenvironment and inhibiting gastric cancer. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 10-20 parts of radix astragali preparata, 10-20 parts of radix codonopsis, 5-15 parts of fried rhizoma atractylodis macrocephalae, 5-15 parts of fried radix paeoniae alba, 5-15 parts of radix angelicae sinensis, 5-15 parts of poria cocos, 5-15 parts of rhizoma pinellinae praeparata, 4-8 parts of pericarpium citri reticulatae, 3-8 parts of radix aucklandiae, 1-5 parts of fructus amomi, 5-15 parts of rhizoma sparganii and 5-15 parts of rhizoma curcumae. The traditional Chinese medicine composition is prepared from, by weight, 20-40 parts of salvia chinensis, 20-40 parts of oldenlandia diffusa and 3-8 parts of honey-fried licorice roots, and can regulate polarization of tumor-related macrophages and improve the tumor immune microenvironment by regulating the activity of protein phosphatase 2A and mediating a PI3K / AKT / NF-kappa B pathway, thereby inhibiting proliferation and migration of gastric cancer cells, promoting apoptosis of the gastric cancer cells and reversing the epithelial-mesenchymal transition process. In-vivo verification safety is good, a new direction and thought are provided for treatment of gastric cancer, and a preparation method and application of the compound are disclosed.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for improving tumor immune microenvironment to inhibit gastric cancer and a preparation method and application thereof. BACKGROUND

[0002] Gastric cancer is a malignant tumor occurring in gastric mucosal epithelial cells. Although the application of new drugs such as targeted therapy and immunotherapy enables the existing comprehensive therapy to prolong the survival period of some gastric cancer patients to more than 1 year, the recurrence and metastasis rate of gastric cancer is still at a high level. At the same time, the multiple system adverse reactions and sequelae caused by radiotherapy, chemotherapy and surgical treatment seriously damage the quality of life of patients. To break through this treatment dilemma, it is urgent to explore more optimized treatment strategies for gastric cancer.

[0003] In recent years, with the development of immunology, more and more studies have shown that tumor progression is related to tumor immune microenvironment. The tumor immune microenvironment is a complex network of interactions established by stromal cells and immune cells along the extracellular matrix, in which immune cells, stromal cells and cytokines can interact with tumor cells, affecting tumor occurrence, development and immunotherapy response. Tumor-associated macrophages, as the most abundant immune cells infiltrating the tumor microenvironment, have high plasticity: classical activated M1 macrophages (pro-inflammatory, anti-tumor) and alternative activated M2 macrophages (immune suppression, pro-tumor) regulate tumor progression and treatment response through dynamic polarization conversion. In the gastric cancer microenvironment, tumor-associated macrophages promote gastric cancer cell proliferation, invasion and migration by secreting various cytokines, and play a key regulatory role in the immune regulation, metabolism and drug resistance of gastric cancer cells. It is worth noting that the transformation of macrophages from M1 to M2 phenotype is a key feature of malignant cancer progression. Therefore, targeting TAM polarization is expected to become a highly potential treatment strategy for gastric cancer.

[0004] The unique advantages of traditional Chinese medicine in improving the "holism, balance, multi-system and multi-target" of tumor microenvironment are attracting widespread attention from researchers, and its ability to synergistically regulate multiple signaling pathways provides a new idea for the comprehensive intervention of gastric cancer. Combining tumor immune microenvironment with traditional Chinese medicine can reveal the mechanism of TCM syndrome differentiation and treatment of gastric cancer from a new microscopic perspective. Studying the effect of traditional Chinese medicine compound on the polarization of tumor-associated macrophages and combining the holistic concept of traditional Chinese medicine with the concept of modern precision medicine also opens up a new way for the prevention and treatment of gastric cancer. SUMMARY

[0005] Invention purposes: The purpose of the present application is to improve the tumor immune microenvironment to inhibit gastric cancer, and provide a traditional Chinese medicine composition and its preparation method and application. The composition is based on the TCM principle of "tonifying qi and invigorating the spleen, removing blood stasis and detoxifying", and through the unique combination of monarch, minister, assistant and messenger, it can not only directly inhibit tumor cells, but also can enhance the activity of protein phosphatase 2A (PP2A) enzyme, and then regulate the PI3K / AKT / NF-kB signaling pathway, reprogram the polarization of tumor-associated macrophages (TAMs), and improve the immune suppressive microenvironment.

[0006] Technical scheme: The purpose of the present application is realized by the following scheme:

[0007] A traditional Chinese medicine composition for improving tumor immune microenvironment to inhibit gastric cancer is prepared from the following raw materials in the following proportions by weight: 10-20 parts by weight of fried astragalus, 10-20 parts by weight of dangshen, 5-15 parts by weight of fried atractylodes, 5-15 parts by weight of fried white peony root, 5-15 parts by weight of angelica, 5-15 parts by weight of poria, 5-15 parts by weight of fried pinellia, 4-8 parts by weight of dried tangerine or orange peel, 3-8 parts by weight of saussurea, 1-5 parts by weight of amomum villosum, 5-15 parts by weight of sparganium, 5-15 parts by weight of curcuma, 20-40 parts by weight of semen astragali complanati, 20-40 parts by weight of oldenlandia, and 3-8 parts by weight of fried licorice root.

[0008] The traditional Chinese medicine composition for improving tumor immune microenvironment to inhibit gastric cancer is prepared from the following raw materials in the following proportions by weight: 15 parts by weight of fried astragalus, 15 parts by weight of dangshen, 10 parts by weight of fried atractylodes, 10 parts by weight of fried white peony root, 10 parts by weight of angelica, 10 parts by weight of poria, 10 parts by weight of fried pinellia, 6 parts by weight of dried tangerine or orange peel, 6 parts by weight of saussurea, 3 parts by weight of amomum villosum, 10 parts by weight of sparganium, 10 parts by weight of curcuma, 30 parts by weight of semen astragali complanati, 30 parts by weight of oldenlandia, and 5 parts by weight of fried licorice root.

[0009] The traditional Chinese medicine composition for improving tumor immune microenvironment to inhibit gastric cancer is prepared by the following method: taking traditional Chinese medicine raw materials by weight fraction, soaking in water for 30-60 minutes, decocting and extracting twice, adding water in the first time at 10-12 times the weight of the medicine, decocting for 20-25 minutes, after decocting, taking out the decoction, filtering to obtain the filtrate. The residue is added with 8-10 times the weight of water, and decocted for the second time for 15-20 minutes, filtered, and the filtrate is taken. The two decoctions are combined, concentrated, and prepared into dry extract, and then added with excipients to prepare one of tablets, capsules and granules.

[0010] The traditional Chinese medicine composition for improving tumor immune microenvironment to inhibit gastric cancer, the preparation method of the traditional Chinese medicine composition is as follows: taking the traditional Chinese medicine composition by weight fraction, soaking in water for 30-60 minutes, decocting and extracting twice, filtering through double-layer gauze, combining the two filtrates, concentrating the filtrate to 1.8g / ml of the concentration of crude drug, and preparing the filtered medicinal liquid into freeze-dried powder by using a freeze dryer, sealing and storing in a-20°C refrigerator for standby.

[0011] The traditional Chinese medicine composition for improving tumor immune microenvironment to inhibit gastric cancer, the preparation method of the traditional Chinese medicine composition is as follows: taking the traditional Chinese medicine composition by weight fraction, soaking in water for 30-60 minutes, decocting and extracting twice, filtering the filtrate after the first decocting and extracting, adding 8-10 times of water by weight of the medicine to the residue, performing second decocting, filtering the filtrate, combining the two filtrates, and concentrating to prepare one of a decoction and a mixture.

[0012] The application of the traditional Chinese medicine composition for improving tumor immune microenvironment to inhibit gastric cancer in the preparation of a gastric cancer treatment.

[0013] The application of the traditional Chinese medicine composition for improving tumor immune microenvironment to inhibit gastric cancer in the preparation of a gastric cancer treatment, wherein the traditional Chinese medicine composition regulates the activity of PP2A enzyme, mediates the PI3K / AKT / NF-κB pathway to regulate the polarization of tumor-associated macrophages.

[0014] The application of the traditional Chinese medicine composition for improving tumor immune microenvironment to inhibit gastric cancer in the preparation of a gastric cancer treatment, wherein the traditional Chinese medicine composition regulates the activity of PP2A enzyme, mediates the PI3K / AKT / NF-κB pathway to regulate the polarization of tumor-associated macrophages.

[0015] Beneficial effects

[0016] There is no "gastric cancer" disease name in the ancient Chinese medical books, but according to its clinical manifestations, the relevant discussion of gastric cancer can be found in the disease category of "stomach counterflow", "stomach counterflow", "dysphagia", "accumulation", "stomach pain" and the like. The pathogenesis of gastric cancer involves deficiency of vital qi, external evil of six evils, improper diet, emotional failure, excessive worry and the like, and pathological products such as "phlegm", "stasis" and "toxicity" are involved, involving multiple visceral dysfunction such as liver and kidney, and deficiency of spleen is the root and stasis and toxicity are the cause, which is generally deficiency in root and excess in branch. Deficiency of spleen is the root of the pathogenesis of gastric cancer, so it can be treated from the spleen, and "tonifying qi and invigorating spleen, removing blood stasis and resolving toxicity" can be the treatment principle. The traditional Chinese medicine composition for regulating immune microenvironment of gastric cancer provided by the present application is based on the pathogenesis theory of "spleen deficiency and stasis and toxicity", and the composition is composed of fried astragalus, radix codonopsis, fried atractylodes, fried white peony root, angelica, poria cocos, prepared pinellia, dried tangerine or orange peel, costus, amomum villosum, rhizoma sparganii, curcuma zedoary, stone see through, white flower snake tongue grass and licorice. The innovation lies in the construction of a triple synergistic traditional Chinese medicine treatment system of "tonifying qi and invigorating spleen (treating the root), removing blood stasis and resolving mass (treating the branch), and clearing heat and resolving toxicity (anti-cancer)". In the formula, fried astragalus and radix codonopsis are the monarch, focusing on invigorating the spleen and supporting the root of acquired constitution; fried atractylodes, poria cocos, dried tangerine or orange peel and prepared pinellia are the ministers, drying dampness and invigorating the spleen, regulating qi and harmonizing the stomach, and assisting the monarch in strengthening the middle jiao transportation and transformation; rhizoma sparganii and curcuma zedoary are used to break blood and disperse qi, remove blood stasis and resolve mass to treat the branch, and at the same time, stone see through and white flower snake tongue grass are combined to clear heat and resolve toxicity, and modern pharmacological studies have confirmed their direct anti-tumor activity, and they can directly reach the lesion; angelica and fried white peony root nourish blood and promote blood circulation to relieve pain; and licorice is used to harmonize all medicines. The whole formula follows the principle of "supporting the healthy qi and removing the evil qi without damaging the healthy qi", the combination of monarch, minister and assistant is strict, and it forms the overall treatment advantage of multiple pathways and multiple targets according to the core pathogenesis of "spleen deficiency as the root and stasis and toxicity mutual knotting" of gastric cancer. The compatibility principle and drug strength are essentially different from those of ordinary prescriptions for treating gastric diseases: the traditional Chinese medicine composition in the present application creatively adds a large amount of blood-breaking and heat-clearing and toxicity-resolving anticancer drugs on the basis of invigorating the spleen and tonifying qi, and the treatment goal and drug thinking are fundamentally different from those of ordinary gastric disease prescriptions; there is a delicate synergy and restraint relationship between the blood-breaking drugs such as rhizoma sparganii and curcuma zedoary and the spleen and stomach, but a large amount of spleen-invigorating and qi-tonifying drugs and licorice in the present application can effectively control their side effects, so that the whole formula can be safely and long-term used for gastric cancer patients.

[0017] The inventors combine traditional Chinese medicine theory and cell biology for the first time to find and verify that the traditional Chinese medicine composition provided by the application can specifically enhance the activity of PP2A enzyme, and then regulate the PI3K / AKT / NF-κB signaling pathway, so as to reprogram TAMs, and make them polarize from the tumor-promoting M2 phenotype to the anti-tumor M1 phenotype. This remodeling of the microenvironment from “immune suppression” to “immune activation” embodies the modern treatment concept of precise immune regulation. Further rescue experiments have confirmed that the activation of PP2A enzyme is the key and indispensable link for the compound to play a role, highlighting the specificity and uniqueness of its mechanism of action, which is a new synergistic effect that cannot be achieved by any single component or known simple combination. The composition provided by the application can not only directly inhibit the proliferation and migration of gastric cancer cells (MKN-45), but also can synergistically promote tumor cell apoptosis and reverse the process of epithelial-mesenchymal transition (EMT) by improving the root cause of the tumor immune microenvironment. In vivo and in vitro experiments have confirmed that the composition significantly inhibits tumor growth while having good safety and no obvious toxic side effects.

[0018] Existing literature reports that most of the drugs such as Huangqi and Dangshen can generally enhance immune cells such as T cells and NK cells, or non-specifically regulate cytokines. However, the present application confirms that the specific compound specifically targets PP2A enzyme activity to induce phenotypic polarization of tumor-associated macrophages (TAMs), and reverses them from the tumor-promoting M2 type to the anti-tumor M1 type. The target cells and depth of action of this effect are fundamentally different from known prescriptions. The present application combines traditional Chinese medicine holism with modern tumor immunology to provide a new treatment strategy and drug selection with independent intellectual property rights for the treatment of gastric cancer, and its effect is far beyond the simple addition of each single herb or known prescription, and unexpected technical effects have been achieved. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a diagram showing that TAMs model in vitro presents a tumor-promoting M2 phenotype and is related to PP2A enzyme activity. Among them Figure 1 A is a diagram of the cell morphology of THP-1, M0 macrophages, and TAM cells under an inverted phase contrast microscope. Figure 1 B-1C is a diagram of the protein expression levels of iNOS, Arg-1, TNF-α, and IL-10 in the three groups of cells. Figure 1 D-1F is a diagram of the proportion of M1 / M2 phenotype macrophages in the three groups detected by flow cytometry. Figure 1 G is a diagram of the PP2A enzyme activity of the three groups of cells.

[0020] Figure 2 is a diagram showing that the traditional Chinese medicine composition provided by the application inhibits the progression of gastric cancer by regulating TAMs in vitro. Among themFigure 2 A is a macrophage cell viability chart after treating with traditional Chinese medicine composition for 24 hours and 48 hours in cell viability assay, Figure 2 B is a MKN-45 cell viability chart co-cultured with TAMs. Figure 2 C-2D is a cell scratch test chart for detecting MKN-45 cell migration. Figure 2 E-2F is a chart of E-cadherin, N-cadherin, Bax, Bcl-2 protein expression levels of MKN-45 gastric cancer cells in each group.

[0021] Figure 3 The traditional Chinese medicine composition provided by the present application enhances PP2A enzyme activity, inhibits PI3K / AKT axis, and regulates NF-kB signal, and reprograms TAMs polarization chart. Figure 3 A-3B is a chart of iNOS, Arg-1, TNF-α, IL-10 protein expression levels of macrophages in each group. Figure 3 C-3D is a chart of p-PI3K / PI3K, p-AKT / AKT, p-NF-κB, NF-κB, PP2Ac protein expression levels of macrophages in each group. Figure 3 E-3G is a chart of M1 / M2 cell ratio of macrophages in each group analyzed by flow cytometry. Figure 3 H is a chart of PP2A enzyme activity of macrophages in each group determined by PP2A enzyme activity.

[0022] Figure 4 The traditional Chinese medicine composition provided by the present application inhibits the growth of gastric cancer in a gastric cancer xenograft tumor model chart. Figure 4 A is a schematic diagram of in vivo experiment. Figure 4 B is a representative diagram of tumors of mice treated with traditional Chinese medicine composition (11.7 g / kg and 23.4 g / kg), 5-Fu (25 mg / kg) or control. Figure 4 C is a chart of tumor volume in each group. Figure 4 D is the change of body weight of mice during the whole experiment, which is measured every 3 days. Figure 4 E is a chart of tumor weight in each group.

[0023] Figure 5 The traditional Chinese medicine composition provided by the present application inhibits the growth of gastric cancer by regulating TAMs polarization in vivo chart. Figure 5 A-5B is a chart of E-cadherin, N-cadherin, Bax, Bcl-2, PP2Ac protein expression levels in tumor samples in each group. Figure 5 C is a chart of PP2A enzyme activity in tumor samples in each group. Figure 5 D-5E is a chart of M2 macrophage ratio in tumor tissue detected by flow cytometry. Figure 5F is a representative image of DAPI, F4 / 80, and CD86 immunofluorescence staining in a tumor tissue section. Figure 5 G is a representative image of DAPI, F4 / 80, and CD206 immunofluorescence staining in tumor tissue sections. Figure 5 H represents representative images of H&E staining and Ki-67, F4 / 80, and CD206 immunohistochemical staining in each group of tumor tissue sections. Figure 5 Image I is a representative HE staining image of mouse liver, spleen, and kidney.

[0024] Figure 6 This diagram shows that the rescue experiment confirms that PP2A enzyme activation is a key step in YJD reprogramming TAMs. Figure 6 A-6B is a graph showing the protein expression levels of iNOS, Arg-1, TNF-α, and IL-10 in macrophages of each group. Figure 6 C-6D is a graph showing the expression levels of p-PI3K / PI3K, p-AKT / AKT, p-NF-κB, NF-κB, and PP2Ac proteins in macrophages of each group. Figure 6 E-6G is a flow cytometry analysis of the M1 / M2 ratio of macrophages in each group. Figure 6 H represents the PP2A enzyme activity assay, which measures the PP2A enzyme activity of macrophages in each group. Detailed Implementation

[0025] The following examples further illustrate the above-mentioned content of the present invention in detail, but should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention.

[0026] Example 1: Take 150g of roasted Astragalus membranaceus, 150g of Codonopsis pilosula, 100g of stir-fried Atractylodes macrocephala, 100g of stir-fried Paeonia lactiflora, 100g of Angelica sinensis, 100g of Poria cocos, 100g of processed Pinellia ternata, 60g of Citrus reticulata peel, 60g of Aucklandia lappa, 30g of Amomum villosum, 100g of Sparganium stoloniferum, 100g of Curcuma zedoaria, 300g of Hedyotis diffusa, 300g of roasted Glycyrrhiza uralensis. Weigh the above-mentioned Chinese herbal ingredients, soak them in water for 60 minutes, and decoct twice. For the first decoction, add 12 times the weight of the herbs and decoct for 20 minutes. After decoction, remove the decoction, filter and collect the filtrate. Add 10 times the weight of the herbs to the dregs and decoct again for 15 minutes. Filter and collect the filtrate. Combine the two decoctions, concentrate and prepare a compound.

[0027] Example 2: Take the 150g of fried Astragalus, 150g of Codonopsis, 100g of fried Atractylodes, 100g of fried White Peony Root, 100g of Angelica, 100g of Poria, 100g of fried Pinellia, 60g of dried tangerine or orange peel, 60g of costus root, 30g of amomum villosum, 100g of rhizoma sparganii, 100g of curcuma zedoary, 300g of Radix Scrophulariae, 300g of Herba Belamcanda, 50g of fried licorice. Take the above-mentioned traditional Chinese medicine composition, soak in water for 30 minutes, decoct and extract twice, filter with double-layer gauze, combine the two filtrates, take the filtrate, concentrate to 1.8g / ml of crude drug concentration, use a freeze dryer to prepare the filtered liquid into a freeze-dried powder, seal it in a-20°C refrigerator.

[0028] Example 3: Take the 150g of fried Astragalus, 150g of Codonopsis, 100g of fried Atractylodes, 100g of fried White Peony Root, 100g of Angelica, 100g of Poria, 100g of fried Pinellia, 60g of dried tangerine or orange peel, 60g of costus root, 30g of amomum villosum, 100g of rhizoma sparganii, 100g of curcuma zedoary, 300g of Radix Scrophulariae, 300g of Herba Belamcanda, 50g of fried licorice. Take the above-mentioned traditional Chinese medicine composition, soak in water for 30 minutes, decoct and extract twice, the first time add water to 12 times the weight of the medicine, decoct for 20 minutes, after decoction, take out the decoction, filter the filtrate. Add 10 times the weight of water to the dregs and decoct for the second time, filter, take the filtrate, combine the two decoctions, concentrate, add starch, and prepare granules.

[0029] Example 4: Take the 150g of fried Astragalus, 150g of Codonopsis, 100g of fried Atractylodes, 100g of fried White Peony Root, 100g of Angelica, 100g of Poria, 100g of fried Pinellia, 60g of dried tangerine or orange peel, 60g of costus root, 30g of amomum villosum, 100g of rhizoma sparganii, 100g of curcuma zedoary, 300g of Radix Scrophulariae, 300g of Herba Belamcanda, 50g of fried licorice. Take the above-mentioned traditional Chinese medicine composition, soak in water for 60 minutes, decoct and extract twice, the first time add water to 10 times the weight of the medicine, decoct for 25 minutes, after decoction, take out the decoction, filter the filtrate. Add 8 times the weight of water to the dregs and decoct for the second time, filter, take the filtrate, combine the two decoctions, concentrate, add dextrin, and prepare capsules.

[0030] Take the freeze-dried powder in the typical prescription of Example 2, dissolve it in complete culture medium, and filter sterilize with a sterile filter. Conduct cell experiments and animal experiments (Examples 5-9) on the traditional Chinese medicine composition, and the other implementation effects are equivalent, as follows:

[0031] Example 5: Construct an in vitro tumor cell-macrophage co-culture system and verify it.

[0032] Experimental grouping: THP-1 cell group (THP-1 group), M0 macrophage group (Control group), M0 macrophage and MKN-45 cell co-culture group (Co-culture group)

[0033] Experimental methods:

[0034] (1) To construct the in vitro tumor cell-macrophage co-culture system, MKN-45 cells were seeded into the upper chamber of a 0.4 pm Transwell chamber (5 x 10 5 cells / well) for 24 hours, adhering to the wall; THP-1 cells were seeded into the bottom of a 6-well plate (1 x 10 6 cells / well) and induced to differentiate into M0 macrophages by exposure to 50 ng / ml phorbol ester for 24 hours. The chamber containing MKN-45 cells was placed directly on top of the six-well plate containing M0 macrophages, and the two cells were co-incubated in serum-free medium for 24 hours. According to the experimental design, the positions of the two cells in the co-culture system were reversed, and the co-culture supernatant containing the released factors was collected for Example 6.

[0035] (2) The cell morphology of THP-1, M0 macrophages, and TAM cells was observed by inverted phase contrast microscopy.

[0036] (3) Western Blot detection: Cell proteins were extracted, and after SDS-PAGE separation of the proteins, the samples were transferred to a methanol-activated PVDF membrane. The membrane was blocked with 5% BSA for 1.5 hours, and then taken out and placed in iNOS, Arg-1, TNF-a, IL-10, PP2Ac, β-actin, and primary antibodies, respectively, and incubated at 4°C overnight. The next day, the membrane was rinsed with TBST three times, and then placed in the corresponding secondary antibody (1:10,000) and incubated at room temperature for 1 hour. The membrane was developed using enhanced chemiluminescence reagent, and the results were analyzed using Image J image analysis software to measure the gray value of the bands and compare the differences between groups.

[0037] (4) Flow cytometry detection: The cells were collected and resuspended with PBS to prepare a 1 ml single-cell suspension. The cells were stained with CD86 and CD206 antibodies at room temperature for 30 minutes in the dark. The cells were washed twice and resuspended in staining buffer. The labeled cells were analyzed using a FACSCelesta flow cytometer.

[0038] (5) PP2A enzyme activity detection: According to the instructions of the serine / threonine phosphatase assay system, the PP2A activity of the cells was determined by molybdenum blue colorimetry. The supernatant was treated with an ion chromatography column to remove endogenous phosphate ions before determining the PP2A activity. A phosphate standard curve was prepared, and the protein was quantified. After the reaction was stopped, the absorbance at 630 nm was measured.

[0039] The untreated THP-1 cells were in a round and transparent suspended growth state. After being stimulated by phorbol ester for 24 hours, the cells were induced to be M0 macrophages, which were transformed into gray and opaque spindle-shaped adherent cells under a microscope, consistent with the typical morphological characteristics of macrophages. After further co-culturing the M0 macrophages with MKN-45 cells for 24 hours, the cells were differentiated into TAMs, which showed spindle-shaped adherent growth characteristics with long pseudopods extending under a microscope, and showed an aggregated or colony-like growth pattern. Figure 1 A). To verify the molecular characteristics of cell phenotype transformation, the expression characteristics of macrophage markers in the three kinds of cells were detected by Western blot experiment and flow cytometry. The results showed that, compared with the undifferentiated macrophages (Control group), the expression of M2 type macrophage related proteins Arg-1 and IL-10 in TAMs (Co-culture group) was significantly up-regulated, and the expression of M1 type macrophage related proteins iNOS and TNF-α was significantly down-regulated Figure 1 B-1C). Compared with the Control group, the proportion of CD86+ cells in the Co-culture group was significantly reduced, and the proportion of CD206+ cells was significantly increased Figure 1 D-1F). The above results confirmed that the TAMs model used in the subsequent experiments in the present study was successfully established. In addition, we found that the expression level of PP2Ac protein in the Co-culture group did not have a significant difference compared with the Control group Figure 1 B-1C). However, the PP2A enzyme activity in the Co-culture group was significantly lower than that in the Control group. This result suggested that PP2A might be involved in the polarization process of TAMs in the gastric cancer microenvironment by regulating enzyme activity Figure 1 G).

[0040] Example 6: Verification of the inhibition of gastric cancer progression by regulating the polarization of tumor-associated macrophages by the traditional Chinese medicine composition through in vitro experiments

[0041] Experimental grouping: MKN-45 cell group (Control group), MKN-45 cells co-cultured with M0 macrophages group (Co-culture group), low-dose intervention co-culture group of the traditional Chinese medicine composition prepared in Example 2 of the present application (designated as YJD-L group), and high-dose intervention co-culture group of the traditional Chinese medicine composition prepared in Example 2 of the present application (designated as YJD-H group)

[0042] Experimental method:

[0043] (1) Cell viability determination: the cell viability was evaluated using a Cell Counting Kit-8 reagent. THP-1 cells (5 x 10 3Cells (cells / well) were seeded into 96-well plates and induced to adhere to the plates with phorbol ester. Then, different concentrations of traditional Chinese medicine composition were used for intervention. After culturing for 24 hours and 48 hours, cell supernatant was collected, and 10 μL of CCK-8 reagent was added to each well. The cells were incubated at 37°C in the dark for 1 hour, and the OD value of each well was measured at 450 nm using an ELISA reader.

[0044] (2) Cell scratch assay: MKN-45 cells (5×10⁻⁶) were scratched. 5 Cells (per well) were seeded into 6-well plates. When the cells adhered and reached 80% confluence, a straight line was drawn on the bottom of the plate using a 10 μL pipette tip, followed by rinsing three times with PBS. Images were taken under an inverted phase-contrast microscope, and the area of ​​the scratched region was measured. The supernatant collected from co-culture under different intervention conditions was added to the wells, and images were taken again at 24 and 48 hours. Changes in the scratched region area were measured using ImageJ software.

[0045] (3) Western Blot detection: Cell proteins were extracted, and after protein separation by SDS-PAGE, the samples were transferred to a methanol-preactivated PVDF membrane. The membrane was blocked with 5% BSA for 1.5 h. The blocked membrane was then placed in primary antibodies Bax, Bcl-2, E-cadherin, N-cadherin, and β-actin, respectively, and incubated overnight at 4°C. The next day, the membrane was washed three times with TBST and then incubated with the corresponding secondary antibody (1:10000) at room temperature for 1 h. The membrane was developed using an enhanced chemiluminescence reagent, and the results were analyzed using ImageJ image analysis software. The gray values ​​of the bands were measured, and the differences between the groups were compared.

[0046] Experimental results: Within the low concentration range of 0-2 mg / ml, the herbal composition of this invention did not significantly inhibit the growth of macrophages. Figure 2 A); when MKN-45 cells were treated with macrophage supernatant containing a traditional Chinese medicine composition, the traditional Chinese medicine composition inhibited the proliferation of MKN-45 cells in a dose- and time-dependent manner. Figure 2 B). The above results indicate that low concentrations of the traditional Chinese medicine composition (0-2 mg / ml) can inhibit the proliferation of gastric cancer cells through macrophage-mediated antitumor effects, without significantly inhibiting macrophages themselves. Based on this, subsequent experiments used concentrations of 1 mg / ml (traditional Chinese medicine composition, low-dose group, YJD-L) and 2 mg / ml (traditional Chinese medicine composition, high-dose group, YJD-H) for further investigation. Figure 2 As shown in C-2D, compared with the Co-culture group, the migration ability of MKN-45 cells was significantly reduced after intervention with the traditional Chinese medicine composition, and the effect was more obvious in the YJD-H group. Figure 2E-2F showed that compared with the Co-culture group, the expression of pro-apoptotic protein Bax and epithelial phenotype marker protein E-cadherin in the YJD-H group was significantly up-regulated, and the expression of anti-apoptotic protein Bcl-2 and mesenchymal phenotype marker protein N-cadherin was significantly down-regulated. The above results confirmed at the protein level that the traditional Chinese medicine composition can effectively reverse the EMT process of human gastric cancer cells MKN-45 and promote its apoptosis.

[0047] Example 7: Through in vitro experiments, it is verified that the traditional Chinese medicine composition prepared in Example 2 of the application enhances the PP2A enzyme activity, inhibits the PI3K / AKT axis, and regulates the NF-kB signal, reprograms TAMs polarization

[0048] Experimental grouping: M0 macrophage group (Control group), M0 macrophage and MKN-45 cell co-culture group (Co-culture group), low-dose intervention co-culture group of the traditional Chinese medicine composition prepared in Example 2 of the application (code: YJD-L group), and high-dose intervention co-culture group of the traditional Chinese medicine composition prepared in Example 2 of the application (code: YJD-H group)

[0049] Experimental method:

[0050] (1) Western Blot detection: After extracting the cell protein, separating the protein by SDS-PAGE, and transferring it to a PVDF membrane pre-activated with methanol, the membrane was blocked with 5% BSA for 1.5 h. The blocked membrane was taken out and placed in iNOS, Arg-1, TNF-α, IL-10, β-actin, PI3K, p-PI3K, AKT, p-AKT, NF-κB, p-NF-κB, and PP2Ac primary antibodies, and incubated at 4°C overnight. The next day, it was rinsed with TBST for 3 times, and then placed in the corresponding secondary antibody (1:10000) for incubation at room temperature for 1 h. The membrane was developed using enhanced chemiluminescence reagent, and the results were analyzed using Image J image analysis software to measure the gray value of the band and compare the differences between groups.

[0051] (2) Flow cytometry detection: The detection method is the same as in Example 5.

[0052] (3) PP2A enzyme activity detection: The detection method is the same as in Example 5.

[0053] Experimental results: There was no significant difference in the expression level of PP2Ac protein between groups Figure 3 C- Figure 3 D); compared with the Co-culture group, the PP2A enzyme activity of the traditional Chinese medicine composition group was significantly increased, and the high-dose group had a more significant effect Figure 3H). The ratio of p-PI3K / PI3K and p-AKT / AKT phosphorylation levels in the YJD-H group was significantly reduced, and the p-NF-κB / NF-κB was significantly increased Figure 3 C-3D). Compared with the Co-culture group, the M1 macrophage-related protein iNOS and TNF-α levels in the traditional Chinese medicine composition group prepared in Example 2 of the application were significantly up-regulated Figure 3 A-3B), and the proportion of M1 type surface marker CD86+ cells was significantly increased Figure 3 E-3F); the M2 type macrophage-related proteins Arg-1 and IL-10 in the high-dose group were significantly down-regulated Figure 3 A-3B), and the proportion of M2 type surface marker CD206+ cells was significantly reduced Figure 3 E、 Figure 3 G). The above results suggest that the traditional Chinese medicine composition of the application may activate PP2A enzyme activity, regulate the PI3K / AKT / NF-κB signaling pathway, promote the polarization of macrophages to M1 type, and inhibit the polarization of M2 type.

[0054] Example 8: Construction of a nude mouse gastric cancer xenograft model to verify that the traditional Chinese medicine composition inhibits the growth of gastric cancer by regulating the polarization of tumor-associated macrophages

[0055] Experimental method:

[0056] (1) Animal experiment: The animal ethics and research procedures were approved by the Experimental Animal Ethics Committee of the Affiliated Hospital of Nanjing University of Chinese Medicine (Approval No.: 2025DW-007-01, Approval Date: January 14, 2025). BALB / c nude mice (male, SPF level, 6-8 weeks old, 18-22 grams) were purchased from Sibeifu Biotechnology Co., Ltd. The mice were fed in a specific pathogen-free environment and regularly irradiated with ultraviolet rays indoors. Each mouse was inoculated with MKN-45 cells (5 x 10 6 cells per mouse) in the right armpit to establish a xenotransplantation tumor model. After 7 days, when the tumor volume reached 100 mm 3 , it was an indication of the successful establishment of the xenotransplantation mouse model. The mice were randomly divided into four groups: control group (normal saline, 0.2 ml / 10 g, gavage), 5-FU group (5-FU, 25 mg / kg, intraperitoneal injection), YJD-L group (traditional Chinese medicine composition prepared in Example 2 of the application, 11.7 g / kg, 0.2 ml / 10 g, gavage), YJD-H group (traditional Chinese medicine composition prepared in Example 2 of the application, 23.4 g / kg, 0.2 ml / 10 g, gavage). All mice were treated with drugs continuously for 14 days (5-Fu was injected intraperitoneally every other day). The tumor volume and body weight were evaluated every 3 days, and finally the animals were euthanized, and the spleen, liver, kidney, and tumor tissue were obtained for further analysis in subsequent experiments.

[0057] (2) Immunofluorescence staining: for analyzing macrophage phenotype in tumor-bearing mice. Tumor tissues were fixed in 4% paraformaldehyde, dehydrated, embedded and sectioned. Slides were incubated with primary antibodies (F4 / 80, CD206, CD86 antibodies) at 4°C overnight, the next day slides were incubated with fluorescent secondary antibodies and DAPI, representative images were captured using a fluorescence microscope (Nikon, Japan). Co-localization analysis was performed using SlideViewer software.

[0058] (3) Hematoxylin-eosin (HE) staining: Spleen, liver, kidney and tumor tissues of mice in each group were collected, fixed in 4% paraformaldehyde, dehydrated, embedded and sectioned. Paraffin sections were deparaffinated and dehydrated. Cell nuclei were stained with hematoxylin, followed by eosin staining. Image acquisition was performed using a Virtual Slide System (Olympus, Japan). Analysis was performed using SlideViewer software.

[0059] (4) Immunohistochemistry (IHC) staining: paraffin section preparation followed the same protocol as HE staining. After antigen retrieval, the sections were blocked, incubated with primary antibodies (Ki-67, F4 / 80, CD206 antibodies) at 4°C overnight, and incubated with conjugated secondary antibodies for 30 minutes. Sections were developed with diamino benzidine substrate and counterstained with hematoxylin. Analysis was performed using SlideViewer software.

[0060] (5) Flow cytometry detection: animal tumor tissues were cut into small pieces, digested with DNase and collagenase type IV in RPMI1640 culture medium, and then the tumor samples were filtered with a 200-mesh screen. CD45, CD11b, F4 / 80, CD206 antibodies were used to stain the cells at room temperature for 30 minutes in the dark, and the labeled cells were analyzed using a FACSCelest flow cytometer. The above results were analyzed using FlowJo software.

[0061] (6) Western Blot detection: tumor tissue proteins were extracted, and after SDS-PAGE separation of the proteins, the samples were transferred to a PVDF membrane pre-activated with methanol. The membrane was blocked with 5% BSA for 1.5 hours, and then removed and placed in Bax, Bcl-2, E-cadherin, N-cadherin, PP2Ac, β-actin primary antibodies at 4°C overnight. The next day, the membrane was washed with TBST three times, and then placed in the corresponding secondary antibody (1:10000) at room temperature for 1 hour. The membrane was developed using enhanced chemiluminescence reagent, and the results were analyzed using Image J image analysis software to measure the gray value of the bands and compare the differences between groups.

[0062] (7) PP2A enzyme activity detection: according to the instructions of the serine / threonine phosphatase assay system, the PP2A activity of the tissue was detected by molybdenum blue colorimetry. The tissue lysate was prepared according to the instructions, and the supernatant was treated with an ion chromatography column to remove endogenous phosphate ions before the PP2A activity was detected. The phosphate standard curve was prepared, the protein was quantified, and the absorbance at 630 nm was measured after the reaction was stopped.

[0063] Experimental results: four treatment schemes were verified by constructing a gastric cancer xenograft model Figure 4 A), the drug concentrations were selected as human equivalent traditional Chinese medicine composition (23.4 g / kg) and half equivalent traditional Chinese medicine composition (11.7 g / kg) as the high-dose group (YJD-H) and the low-dose group (YJD-L). Compared with the Control group, the 5-Fu group and the traditional Chinese medicine composition group can significantly reduce the tumor weight and tumor volume Figure 4 B-4C、 Figure 4 E). Compared with the Control group, the tumor cells of the 5-Fu group and the traditional Chinese medicine composition group appeared vacuole-like deformation, the cytoplasm was pinkly dyed, the cell membrane boundary was blurred, and obvious karyopyknosis was observed, and the distribution range of the tumor necrosis area of the high-dose traditional Chinese medicine composition group was larger Figure 5 H). The expression level of Ki-67 of the 5-Fu group and the traditional Chinese medicine composition group was reduced, and the improvement of the tumor histopathology of the high-dose traditional Chinese medicine composition group was more significant Figure 5 H). The traditional Chinese medicine composition group effectively slowed down the weight loss trend of the tumor-bearing mice Figure 4 D). The HE staining observation of the mouse spleen, liver and kidney showed no abnormal pathological changes Figure 5 I), indicating that the traditional Chinese medicine composition has good in-vivo drug safety.

[0064] Compared with the Control group, the expression of Bax and E-cadherin in the high-dose traditional Chinese medicine composition group was significantly up-regulated, and the expression of Bcl-2 and N-cadherin was significantly down-regulated, suggesting that the traditional Chinese medicine composition can inhibit the EMT process of gastric cancer cells and promote their apoptosis in vivo Figure 5 A-5B). There was still no significant difference in the expression of PP2Ac protein among the groups, but compared with the Control group, the PP2A enzyme activity of the traditional Chinese medicine composition group was significantly increased Figure 5 A-5C). The proportion of M2 type macrophages in the traditional Chinese medicine composition group was significantly decreased Figure 5 D-5E). Immunofluorescence staining results showed that compared with the Control group, the number of F4 / 80+CD86+ cells in the traditional Chinese medicine composition group increased, and the number of F4 / 80+CD206+ cells decreased, and the change of the high-dose traditional Chinese medicine composition group was more significant. The immunohistochemical results further confirmed that the traditional Chinese medicine composition group can inhibit the expression of F4 / 80 and CD206 in the tumor tissueFigure 5 H). The above results verify in vivo that the traditional Chinese medicine composition can reduce M2 type macrophages in the gastric cancer tumor microenvironment by activating PP2A enzyme activity, and inhibit the progression of gastric cancer.

[0065] Example 9: PP2A enzyme activation is a key link in reprogramming TAMs polarization by YJD

[0066] Experimental grouping: M0 macrophage and MKN-45 cell co-culture group (Co-culture group), high-dose traditional Chinese medicine composition prepared in Example 2 of the application intervention co-culture group (code: YJD-H group), PP2A inhibitor intervention co-culture group (LB-100 group), PP2A inhibitor and high-dose traditional Chinese medicine composition prepared in Example 2 of the application intervention co-culture group (YJD-H+LB-100 group)

[0067] Experimental method: the detection method is the same as that in Example 7.

[0068] Experimental results: In order to further verify the core position of PP2A enzyme activation in the traditional Chinese medicine composition regulating TAMs polarization against gastric cancer, we used LB-100 to specifically inhibit PP2A activity, and the results showed that after the inhibition of PP2A activity, the regulation effect of the traditional Chinese medicine composition on the PI3K / AKT / NF-κB signaling pathway in TAMs was significantly weakened. Specifically, compared with the YJD-H group, the expression of p-PI3K / PI3K and p-AKT / AKT in the YJD-H+LB-100 group was significantly increased, the expression of p-NF-κB / NF-κB was significantly decreased, and there was no obvious difference in the expression of PP2Ac protein (Fig. 6A-6C). Figure 6 C-6D), while the PP2A activity of the YJD-H+LB-100 group was significantly reduced (Fig. 6D). Figure 6 H). LB-100 treatment can weaken the reprogramming effect of the traditional Chinese medicine composition on macrophages: compared with the YJD-H group, the expression of iNOS and TNF-α in the YJD-H+LB-100 group was significantly down-regulated, the expression of Arg-1 and IL-10 was significantly up-regulated (Fig. 6E-6F), the proportion of CD86+ cells decreased, and the proportion of CD206+ cells increased (Fig. 6G). Figure 6 A-6B), CD86+ cell proportion decreased, and CD206+ cell proportion increased (Fig. 6G). Figure 6 E-6G). The above results confirm that the activation of PP2A enzyme is a key functional link in the regulation of TAMs polarization phenotype conversion by the traditional Chinese medicine composition. When the PP2A activity is inhibited, the reprogramming effect of the traditional Chinese medicine composition on macrophages and the regulation effect on the signaling pathway are significantly reversed.

[0069] From the above experiments, it can be seen that the traditional Chinese medicine composition provided by the application can improve the tumor immune microenvironment by regulating TAMs polarization to exert an anti-gastric cancer effect, and also exhibits good safety and significant effectiveness, which embodies the unique effect of the traditional Chinese medicine composition.

[0070] The above description is only the preferred embodiment of the present application, it should be pointed out that for the ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, also should be considered as the protection scope of the present application.

Claims

1. A traditional Chinese medicine composition for improving the tumor immune microenvironment to inhibit gastric cancer, characterized in that, The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 10-20 parts by weight of fried Astragalus, 10-20 parts by weight of Codonopsis, 5-15 parts by weight of fried Atractylodes, 5-15 parts by weight of fried White Peony Root, 5-15 parts by weight of Angelica, 5-15 parts by weight of Poria, 5-15 parts by weight of fried Pinellia, 4-8 parts by weight of dried tangerine or orange peel, 3-8 parts by weight of Saussurea, 1-5 parts by weight of Amomum villosum, 5-15 parts by weight of Sparganium, 5-15 parts by weight of Curcuma, 20-40 parts by weight of Oldenlandia, 20-40 parts by weight of White Flower Snake Weed, and 3-8 parts by weight of fried Licorice.

2. The traditional Chinese medicine composition for improving the tumor immune microenvironment to inhibit gastric cancer according to claim 1, characterized in that, The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 15 parts by weight of fried Astragalus, 15 parts by weight of Codonopsis, 10 parts by weight of fried Atractylodes, 10 parts by weight of fried White Peony Root, 10 parts by weight of Angelica, 10 parts by weight of Poria, 10 parts by weight of fried Pinellia, 6 parts by weight of dried tangerine or orange peel, 6 parts by weight of Saussurea, 3 parts by weight of Amomum villosum, 10 parts by weight of Sparganium, 10 parts by weight of Curcuma, 30 parts by weight of Oldenlandia, 30 parts by weight of White Flower Snake Weed, and 5 parts by weight of fried Licorice. 3.The traditional Chinese medicine composition for improving tumor immune microenvironment to inhibit gastric cancer according to claim 1, characterized in that, The preparation method of the traditional Chinese medicine composition is as follows: the traditional Chinese medicine raw materials are taken in parts by weight, soaked in water for 30-60 minutes, extracted by decoction twice, the first time with 10-12 times the weight of the medicine, decocted for 20-25 minutes, after the decoction is finished, the decoction is taken out, filtered to obtain the filtrate. The residue is added with 8-10 times the weight of the medicine, extracted by decoction again, decocted for 15-20 minutes, filtered to obtain the filtrate, the two filtrates are combined, concentrated, prepared into dry extract, added with excipients, and prepared into one of tablets, capsules and granules. 4.The traditional Chinese medicine composition for improving tumor immune microenvironment to inhibit gastric cancer according to claim 1, characterized in that, The preparation method of the traditional Chinese medicine composition is as follows: the traditional Chinese medicine composition is taken in parts by weight, soaked in water for 30-60 minutes, extracted by decoction twice, filtered with double-layer gauze, the two filtrates are combined, the filtrate is concentrated to a concentration of 1.8 g / ml of crude drug, and the filtered liquid is prepared into freeze-dried powder by using a freeze-drying machine, sealed and stored in a-20°C refrigerator.

5. The traditional Chinese medicine composition for improving the tumor immune microenvironment to inhibit gastric cancer according to claim 1, characterized in that, The preparation method of the traditional Chinese medicine composition is as follows: the traditional Chinese medicine composition is taken in parts by weight, soaked in water for 30-60 minutes, extracted by decoction twice, the first time with 10-12 times the weight of the medicine, decocted for 20-25 minutes, after the decoction is finished, the decoction is taken out, filtered to obtain the filtrate. The residue is added with 8-10 times the weight of the medicine, extracted by decoction again, decocted for 15-20 minutes, filtered to obtain the filtrate, the two filtrates are combined, concentrated, prepared into one of decoction and mixture.

6. The application of the traditional Chinese medicine composition for improving tumor immune microenvironment to inhibit gastric cancer in the preparation of a drug for treating gastric cancer according to claim 1.

7. The application of the traditional Chinese medicine composition for improving tumor immune microenvironment to inhibit gastric cancer in the preparation of a drug for treating gastric cancer according to claim 1. Application, characterized in that The traditional Chinese medicine composition regulates the activity of protein phosphatase 2A, mediates the PI3K / AKT / NF-κB pathway, and regulates the polarization of tumor-associated macrophages. 8.The application of the traditional Chinese medicine composition for improving tumor immune microenvironment to inhibit gastric cancer according to claim 1, wherein, The traditional Chinese medicine composition regulates the immune microenvironment of gastric cancer, inhibits the proliferation and migration of gastric cancer cells, promotes the apoptosis of the cells, and reverses the process of epithelial-mesenchymal transition.

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