Application of polysaccharide monomer separated from schisandra chinensis in preparation of PD-L1 + TAMs activator

Abstract

The invention discloses an application of a polysaccharide monomer separated from schisandra chinensis in preparation of a PD-L1 + TAMs activator. A large number of experiments show that the polysaccharide monomer schisanan B separated from schisandra chinensis can effectively activate PD-L1 + TAMs cell subsets, so that the phagocytosis of TAMs is enhanced, and the proliferation of tumor cells is inhibited; therefore, it is determined that the polysaccharide monomer schisanan B separated from schisandra chinensis can serve as a natural PD-L1 + TAMs cell subset activator to be used for treating cancers such as non-small cell lung cancer, intestinal cancer, melanoma, urothelial carcinoma or liver cancer, and the polysaccharide monomer schisanan B has the advantages of being free of toxic and side effects, low in price, wide in adaptive patient population and the like.

Description

Technical Field

[0001] This invention relates to novel pharmacological uses of active ingredients isolated from traditional Chinese medicinal materials, particularly to the use of the polysaccharide monomer schisanan B isolated from Schisandra chinensis in the preparation of PD-L1. + Novel pharmacological applications, such as TAMs subgroup activators, belong to the field of novel pharmacological applications of the polysaccharide monomer schisanan B isolated from Schisandra chinensis. Background Technology

[0002] Cancer refers to malignant tumors originating from epithelial tissue and is the most common type of malignant tumor. The blood of cancer patients is often acidic. Generally, the term "cancer" is used to refer to all malignant tumors. Cancer is characterized by abnormal cell differentiation and proliferation, uncontrolled growth, invasiveness, and metastasis. Its occurrence is a complex, multi-factorial, and multi-step process. Therefore, studying the current state of drug treatment for this disease and effectively preventing and treating it is of great practical significance.

[0003] In recent years, scientific research on cancer treatment has made many significant breakthroughs, including the discovery of immune checkpoints and the discovery of immunotherapy, which utilizes the inhibition of immune checkpoints to enable the human immune system to function normally. Immunotherapy has made significant progress in the field of cancer, with PD-L1, as an important immune checkpoint protein, attracting much attention. Programmed cell death ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274), is expressed on both immune cells and tumor cells. TAMs are the main cells in human tumors, besides cancer cells, that express PD-L1.

[0004] In traditional understanding, PD-L1 + TAMs (tumor cytotoxic cells) are believed to play an immunosuppressive role in the tumor immune microenvironment. TAMs inhibit the cytotoxic activity of T lymphocytes and NK cells by releasing immunomodulatory factors such as PGE2, IL-10, and TGF-β. After secreting IL-10 and TGF-β, TAMs induce tregs to suppress the activity of effector T lymphocytes. Furthermore, TAMs participate in the transformation of Th cells into tregs, which further suppress the immune response in an antigen-specific manner. In summary, as the most abundant cell population in tumor infiltrating cells, they participate in numerous immunosuppressive pathways; however, researchers often overlook their phagocytic capacity. Studies by Wang Lei et al. have shown that PD-L1… + TAMs exhibit more mature phagocytosis and further activated cytotoxicity. Furthermore, they not only possess immunostimulatory effects but also have effects on CD8...+ T cells also exhibit spatial preference. This evidence suggests that PD-L1 + TAMs subgroup activators are of great research value. On the other hand, as an emerging treatment option, the limited variety and scarcity of current treatment drugs is a significant drawback.

[0005] Schisandra chinensis (Turcz.) Baill., belonging to the Schisandraceae family, is commonly found in the dried, ripe fruit of the Schisandra chinensis plant, and is commonly known as "Northern Schisandra." It possesses astringent, qi-tonifying, fluid-generating, kidney-tonifying, and heart-calming effects, and is recorded in numerous ancient books and pharmacopoeias. Northern Schisandra is cold-resistant and mainly produced in the three northeastern provinces of China, and is one of the most precious authentic medicinal materials from Heilongjiang Province.

[0006] To date, no literature has demonstrated that Schisandra chinensis polysaccharides induce PD-L1. + TAMs can thus inhibit and treat tumors. Therefore, there is an urgent need to develop a PD-L1 inhibitor that is suitable for most clinical patients and has low production costs. + TAMs activators, thereby fundamentally breaking the current PD-L1 + To address the shortage of TAMs activators and improve their clinical efficacy in cancer patients. Summary of the Invention

[0007] One objective of this invention is to provide the polysaccharide monomer schisanan B isolated from Schisandra chinensis for the preparation of tumor-associated macrophage PD-L1 cells. + Positive expression subset (PD-L1) + Uses of TAMs (Tactic Acids) activators;

[0008] The second objective of this invention is to use the polysaccharide monomer schisanan B isolated from Schisandra chinensis in the preparation of antitumor drugs that activate tumor-associated macrophages to express PD-L1 protein and / or transform into the M1 type.

[0009] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0010] The polysaccharide monomer schisanan B isolated from Schisandra chinensis in this invention is the polysaccharide monomer WWZ-2 isolated from Schisandra chinensis as disclosed in Chinese invention patent application No. CN116284466A; the polysaccharide monomer schisanan B is also the monomeric polysaccharide SCP-2 described in the article published in the journal Carbohydrate Polymers (2024 Dec 15; 346:122644. doi:10.1016 / j.carbpol.2024.122644.); the polysaccharide monomer schisanan B (i.e., polysaccharide monomer WWZ-2 or SCP-2) mainly contains glucose, galacturonic acid and trace amounts of arabinose, rhamnose and galactose; wherein, the theoretical molar ratio of glucose to galacturonic acid is 25:3.

[0011] More preferably, the polysaccharide monomer schisanan B sugar residue backbone is 1,4-Glcp, and the side chains have T-Glcp, 1,3,4-Glcp, 1,4,6-Glcp, 1,3,4,6-Glcp, 1,4-GalAMep, and T-GalAMep linkages, with the theoretical sugar residue linkage ratio being: 1,4-Glcp: T-Glcp: 1,3,4-Glcp: 1,4,6-Glcp: 1,3,4,6-Glcp: 1,4-GalAMep: T-GalAMep = 63:19:9:6:3:10:2;

[0012] More preferably, the polysaccharide monomer Schisanan B is a white flocculent substance that is extremely soluble in water; its absolute molecular weight Mw = 106.0 kDa and polydispersity index is 2.28.

[0013] More preferably, the main chain of the polysaccharide monomer Schisanan B is composed of 1,4-linked dextran with branch points at positions 3, 6, and 3,6, respectively. The side chains are composed of 1,4-glucan chains of different lengths with multiple branch points formed by 4-26 polymers and 1,4-galacturonic acid polysaccharides of different lengths formed by 2-8 polymers, which are composed of methyl esters linked at position 6.

[0014] The most preferred chemical structural formula of the polysaccharide monomer Schisanan B is as follows:

[0015]

[0016] To determine the pharmacological activity of Schisandra chinensis monomeric polysaccharide schisanan B, this invention constructed a lung cancer xenograft model and treated solid tumors in C57BL / 6 mice with oral administration of Schisandra chinensis monomeric polysaccharide schisanan B. Experimental results showed that a dose of 200 mg / kg of Schisandra chinensis monomeric polysaccharide significantly inhibited tumor proliferation in mice, with a significantly better effect than the positive control drug: tumor morphology changed significantly, and tumor volume gradually decreased or even disappeared. Measurements of mouse body weight and serum biochemical indicators confirmed that Schisandra chinensis monomeric polysaccharide at a dose of 200 mg / kg had no significant toxic side effects in mice.

[0017] To determine the potential mechanism of Schisandra chinensis monomeric polysaccharide schisanan B in treating tumors, the present invention performed mass spectrometry flow cytometry analysis on the aforementioned xenografts. The results showed that the TAMs cell population differed most significantly before and after treatment, followed by CD8. + T cells; among them, TAMs transformed into M1 type after treatment with Schisandra chinensis monomeric polysaccharide schisanan B, CD8 + Exhausted T cells in the T cell population increased significantly after treatment; further analysis revealed a significant increase in PD-L1 protein expression in M1-type TAMs, and a significant increase in the expression of MHC-I and MHC-II molecules in all subtypes of TAMs, demonstrating the presence of PD-L1. + TAMs cell subsets also possess antigen presentation and phagocytic capabilities. This evidence points to PD-L1 as a key factor in the treatment of tumors with the Schisandra chinensis monomeric polysaccharide schisanan B. + TAMs cell subsets.

[0018] To verify the results of mass cytometry, this invention used flow cytometry to detect mouse xenografts before and after treatment. The results showed that PD-L1 expression increased and shifted towards M1 polarization in TAMs of mouse xenografts treated with Schisandra chinensis monomeric polysaccharide schisanan B. Additionally, PD-L1... + Increased expression of pro-inflammatory factors such as TNF-α and INF-γ in TAMs subsets. Simultaneously, toxic CD8+ expression was observed in xenografts of treated mice. + The T cell population increased; this demonstrates the reliability of the mass cytometry results. However, determining PD-L1... + The TAMs subgroup is an important subgroup that kills tumors and still needs further identification.

[0019] To verify whether the monomeric polysaccharide schisanan B isolated from Schisandra chinensis promotes PD-L1 protein expression and function in macrophages, specifically whether Schisandra chinensis galacturonic acid glucan can act as a PD-L1 inhibitor... +Using TAMs subset activators, this invention first employed the CCK8 assay to detect the cytotoxicity of Schisandra chinensis monomeric polysaccharide in mouse mononuclear macrophage leukemia cells RAW264.7. The results showed that the monomeric polysaccharide had a certain inhibitory effect on RAW264.7 cells, indicating that the Schisandra chinensis monomeric polysaccharide possesses certain cytotoxicity. This invention selected a safe dose with a cell death rate below 20%, namely 400 μg / mL. Subsequently, the expression level of PD-L1 in RAW264.7 cells was further detected by Western blotting. It was found that at the safe dose with a cell death rate below 20%, the PD-L1 content in RAW264.7 cells significantly increased. This demonstrates that the PD-L1 content of the Schisandra chinensis monomeric polysaccharide Schisandra B is significantly increased. + The activation of TAMs subsets was dose-dependent. Further studies revealed that the upregulation of PD-L1 expression in RAW264.7 by the schisandra monomer polysaccharide schisanan B was both concentration-dependent and time-dependent, with the most significant upregulation of PD-L1 at a administered concentration of 400 μg / mL and an administration time of 24 hours.

[0020] To verify the cytotoxic effect of Schisandra chinensis polysaccharide schisanan B on tumor cells after acting on immune cells, this invention stimulated RAW264.7 cells and Jurkat T cells with Schisandra chinensis polysaccharide, respectively. The polysaccharide-stimulated cells were then co-cultured with LLC mouse lung cancer cells. Crystal violet staining revealed that RAW264.7 cells stimulated with Schisandra chinensis polysaccharide schisanan B exhibited stronger cytotoxicity against LLC cells than Jurkat T cells stimulated under the same conditions. Subsequently, RAW264.7 cells stimulated with Schisandra chinensis polysaccharide schisanan B and Jurkat T cells were simultaneously used on LLC cells. The results showed that the LLC cells exhibited more severe cell death after co-stimulation. Flow cytometry was then used to detect the degree of apoptosis in the co-cultured LLC cells, and the results were consistent with the crystal violet staining experiment: the LLC cells co-cultured with RAW264.7 cells stimulated with Schisandra chinensis polysaccharide showed more severe apoptosis. This suggests that Schisandra chinensis polysaccharide schisanan B may activate PD-L1. + TAMs cell subsets were transformed into the M1 type, thereby killing tumor cells. Simultaneously, PD-L1... + TAMs cell subsets may also be associated with the activation of cytotoxic T cells.

[0021] To further verify the activation of PD-L1 by Schisanan B. +The phagocytic capacity of the TAMs subset was first examined under a microscope. Different concentrations of schisanan B activated RAW264.7 cells to phagocytose neutral red. The results showed that the amount of neutral red phagocytosed by RAW264.7 cells increased in a dose-dependent manner after schisanan B stimulation, demonstrating that schisanan B activates PD-L1. + After TAMs subsets were activated, their phagocytic capacity was enhanced in a dose-dependent manner. Subsequently, flow cytometry was used to detect the expression of CD11b, a phagocytic capacity indicator protein, in RAW264.7 cells after co-culturing with different concentrations of schisanan B-activated RAW264.7 cells and LLC tumor cells. The results showed that the abundance of CD11b protein expressed in RAW264.7 cells increased with increasing schisanan B concentration. Simultaneously, the number of LLC cells gradually decreased, similar to the co-culture results. All this evidence suggests that schisanan B can activate PD-L1. + The phagocytic capacity of TAMs subpopulations; finally, to reverse-engineer the results, Schisandra chinensis monomeric polysaccharide schisanan B activates PD-L1. + TAMs kill tumors not through autotoxicity, but through the establishment of a xenograft model in BABL / c Nude immunodeficient mice. The results showed that at the same dosage as in C57BL / 6 mice, there was no significant inhibition of tumor volume in immunodeficient mice: tumor morphology and volume remained unchanged. Mouse weight was also assessed. The results demonstrated that Schisandra chinensis monomeric polysaccharide at a dose of 200 mg / kg had no significant toxic side effects on immunodeficient mice. This indirectly verifies that the Schisandra chinensis monomeric polysaccharide schisanan B kills tumors by activating PD-L1. + TAMs cell subsets kill tumors not because of drug toxicity.

[0022] Therefore, this invention provides a method for preparing PD-L1 using the polysaccharide monomer schisanan B isolated from Schisandra chinensis. + Uses of TAMs subgroup activators.

[0023] Schisandan B, a polysaccharide monomer isolated from Schisandra chinensis, can be formulated into a clinically suitable pharmaceutical preparation by combining it with a formulation-acceptable carrier or excipient and following conventional formulation methods in the art. This preparation may be an oral formulation, an injectable formulation, a sustained-release formulation, or a controlled-release formulation. The oral formulation may be a pill, tablet, granule, powder, or capsule.

[0024] The carrier refers to a carrier or excipient commonly used in the pharmaceutical field, such as diluents, disintegrants, lubricants, excipients, binders, flow aids, fillers, or surfactants; in addition, other excipients such as flavoring agents and sweeteners may be added to the composition.

[0025] The diluent can be one or more ingredients that increase the weight and volume of the tablet; commonly used diluents include lactose, starch, pregelatinized starch, microcrystalline cellulose, sorbitol, mannitol, and inorganic calcium salts. Among these, lactose, starch, and microcrystalline cellulose are the most commonly used.

[0026] The disintegrant can be one or a mixture of several of the following: crospovidone (2-6% by weight), crospovidone sodium carboxymethyl cellulose (2-6% by weight), alginate (2-5% by weight), and microcrystalline cellulose (5-15% by weight). Crospovidone (2-7% by weight) and crospovidone sodium carboxymethyl cellulose (2-6% by weight) are preferred. The optimal concentration is crospovidone (2-6% by weight).

[0027] The lubricant includes one or a mixture of several of the following: stearic acid, sodium stearate, magnesium stearate, calcium stearate, polyethylene glycol, talc, and hydrogenated vegetable oil. Magnesium stearate is the most suitable. The dosage range (by weight) of the lubricant is 0.10-1%, with a typical dosage of 0.25-0.75% and an optimal dosage of 0.5-0.7%.

[0028] The binder may be one or more components that are beneficial for granulation. It may be starch paste (10-30%, by weight of the binder), hydroxypropyl methylcellulose (2-5%, by weight of the binder), polyvinylpyrrolidone (2-20%, by weight of the binder), preferably an aqueous solution of polyvinylpyrrolidone in ethanol, and most preferably a 50% aqueous solution of polyvinylpyrrolidone in ethanol.

[0029] The flow aid can be one or a mixture of several of the following: micronized silica gel, talc, and magnesium trisilicate.

[0030] The surfactant can be one or more components that can improve wettability and increase drug dissolution, commonly sodium dodecyl sulfate (common range is 0.2-6% by weight).

[0031] This invention, through extensive experiments, has discovered that the polysaccharide monomer schisanan B isolated from Schisandra chinensis can effectively activate PD-L1. + TAM cell subsets and their transformation to the M1 type enhance TAM phagocytosis, thereby inhibiting tumor cell proliferation. Schisanan B can serve as a natural PD-L1 inhibitor. +TAMs cell subset activators are used to treat cancers such as non-small cell lung cancer, colorectal cancer, melanoma, urothelial carcinoma, or hepatocellular carcinoma, complementing PD-L1 inhibitors. + This fills a gap in TAMs cell subset activators and has broad application value. Attached Figure Description

[0032] Figure 1 This document presents the results of a mass spectrometry flow cytometry assay and a verification experiment on the antitumor effect and biosafety of Schisanan B on tumor-bearing, immunocompetent C57BL / 6 mice; (A) experimental design of mouse xenografts; (B) morphology and size of mouse xenografts; (C) statistical analysis of mouse xenograft volume; (D) analysis of mouse xenograft weight; (E) mass spectrometry flow cytometry assay; (F) dimensionality reduction analysis of the tumor microenvironment before and after treatment in tumor-bearing mice; (G) heatmap analysis of checkpoint proteins in the tumor microenvironment before and after treatment in tumor-bearing mice; (H) changes in TAMs subsets before treatment in tumor-bearing mice; (I) changes in tumor-associated CD8-positive T cell subsets before and after treatment in tumor-bearing mice; and (J) changes in PD-L1 cells before and after treatment in tumor-bearing mice. + Changes in TAMs cell subsets; (K) Changes in toxic CD8-positive T cell subsets in tumor-bearing mice before and after treatment; Statistical analysis was performed on the experimental results, where *P<0.05; **P<0.01; ***P<0.001.

[0033] Figure 2 To detect changes in various cell subpopulations and inflammatory factors in tumor tissues before and after Schisanan B treatment using flow cytometry; (A) Polarization trend of TAMs subpopulations in tumor-bearing mice before and after treatment; (B) Changes in PD-L1 protein expression in the M2 type TAMs subpopulation in tumor-bearing mice before and after treatment; (C) Changes in PD-L1 protein expression in the M1 type TAMs subpopulation in tumor-bearing mice before and after treatment; (D) Changes in PD-L1 protein expression in the M1 type TAMs subpopulation in tumor-bearing mice before and after treatment. + Changes in TNF-α protein expression in TAMs subsets; (E) M1 PD-L1 in tumor-bearing mice before and after treatment. + Changes in INF-γ protein expression levels in TAMs subsets; (F) CD8 expression in tumor-bearing mice before and after treatment. + GAMZB + Changes in IL-17 protein expression in T Cell subsets; statistical analysis of the experimental results showed that *P<0.05; **P<0.01; ***P<0.001.

[0034] Figure 3 To verify in vitro that schisanan B activates PD-L1 +The results of the TAMs subset were verified to determine the tumor cell killing ability and activation of cytotoxic T cells; (A) The effect of different concentrations of Schisandra chinensis galacturonic acid dextran (schisanan B) on the proliferation of mouse mononuclear macrophage leukemia cells RAW264.7 after 24 h using the CCK8 assay; (B) The effect of different concentrations of Schisandra chinensis galacturonic acid dextran (schisanan B) on the PD-L1 expression level of RAW264.7 cells; (C) The effect of different time points of Schisandra chinensis galacturonic acid dextran (schisanan B) on the PD-L1 expression level of RAW264.7 cells; (D) The crystal violet staining results of different concentrations of Schisandra chinensis galacturonic acid dextran (schisanan B) on RAW264.7 cells, Jurkat-T cells and LLC co-cultured, and different concentrations of Schisandra chinensis galacturonic acid dextran (schisanan B) co-cultured with RAW264.7 cells, Jurkat-T cells and LLC cells; (E) The results of different concentrations of Schisandra chinensis galacturonic acid dextran (schisanan B) on the proliferation of mouse mononuclear macrophage leukemia cells RAW264.7 cells after 24 h; (B) The effect of different concentrations of Schisandra chinensis galacturonic acid dextran (schisanan B) on the PD-L1 expression level of RAW264.7 cells; (C) The effect of different time points of Schisandra chinensis galacturonic acid dextran (schisanan B) on the PD-L1 expression level of RAW264.7 cells; (D) The crystal violet staining results of different concentrations of Schisandra chinensis galacturonic acid de The changes in different cell subpopulations after B was applied to RAW264.7 cells and Jurkat-T cells and co-cultured with LLC cells were detected by flow cytometry. The experimental results were statistically analyzed, where *P<0.05; **P<0.01; ***P<0.001.

[0035] Figure 4 To verify in vitro that schisanan B activates PD-L1 + The results of verification of the phagocytic capacity of the TAMs subpopulation; (A) Microscopic images of different concentrations of schisanan B activating PD-L1 + (A) The ability of TAMs cell subsets to phagocytose neutral red; (B) The effect of different concentrations of schisanan B on the expression level of CD11b protein in RAW264.7 cells after co-culturing with LLC cells; The experimental results were statistically analyzed, where *P<0.05; **P<0.01; ***P<0.001.

[0036] Figure 5 The results of the experimental verification of the antitumor effect and biosafety of schisanan B on tumor-bearing immunodeficient mice are as follows: (A) Experimental design of mouse xenografts; (B) Morphology and size of mouse xenografts; (C) Analysis of mouse xenograft volume; (D) Analysis of mouse body weight changes. The experimental results were statistically analyzed, where *P<0.05; **P<0.01; ***P<0.001. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0038] Experimental Example 1: Schisandra chinensis polysaccharide monomer Schisandan B in activating tumor-associated macrophages PD-L1 + Experiments on positive expression subsets or activation of tumor-associated macrophages to express PD-L1 protein

[0039] (1) In vivo pharmacodynamic experiments on the antitumor effect of Schisanan B

[0040] To investigate the in vivo antitumor effect of schisanan B (schisanan B is the polysaccharide monomer WWZ-2 isolated from Schisandra chinensis as disclosed in Chinese invention patent application CN116284466A), an in vivo lung cancer xenograft model was established in immunocompetent mice (see...). Figure 1 A). Female c57BL / C mice (20±2g) were housed in a controlled environment with ample water and food (25±2℃). After 7 days of acclimatization, 35 mice were randomly divided into a model group, a positive control group (BMS-1; 0.1mg / kg), and a Schisanan B group (50, 100, 200mg / kg). A lung cancer xenograft model was established in all experimental mice, using 1×10⁻⁶... 7 Each dose was administered subcutaneously via injection of a suspension of non-small lung cancer cells from LLC mice. Two days after modeling, administration began, with mice orally receiving either Schisanan B (50, 100, or 200 mg / kg) or BMS-1 ​​(0.1 mg / kg) once daily, according to their assigned groups. Simultaneously, the model group mice received an equal volume of physiological saline. Daily data on body weight and tumor volume were collected for dynamic analysis.

[0041] Experimental results showed that tumor growth was effectively inhibited in mouse models treated with high, medium, and low doses of Schisanan B (200 / 100 / 50 mg / kg). Figure 1 B). Specifically, with the accumulation of administration time, the overall tumor volume growth in mice in the schisanan B-treated group was slow, and schisanan B showed good anti-tumor activity (see B). Figure 1 (C, 1D). The experimental results indicate that Schisanan B has certain anti-tumor effects.

[0042] (2) Mass spectroscopy flow cytometry detection of mouse xenografts

[0043] To determine the antitumor mechanism of schisanan B, the tumor microenvironment in mice was analyzed using mass cytometry (see [link]). Figure 1 E). Five fresh tumor tissues from the Schisanan B group (200 mg / kg) and the model group collected in (1) above were randomly selected and stored in tissue preservation solution for 48 hours. Then, the following experimental steps were performed: 1. The tissues were removed from the tissue preservation solution and washed with culture medium; 2. The tissues were chopped and digested with appropriate enzymes; 3. The digested suspension was filtered; 4. The cell pellet was collected after centrifugation, diluted according to the amount of pellet, and resuspended and counted with staining solution; 5. The cell suspension obtained after pretreatment was used to block the cell population with flow cytometry blocking solution at room temperature for 4 hours. After blocking, the cells were washed twice with PBS and then stained with antibody and incubated at room temperature in the dark for 1 hour. After staining, the cells were washed twice with PBS and then the samples were detected by flow cytometry (Attune NxT, Thermo Fisher Scientific, Waltham, USA) and analyzed using FlowJo (v10.6.2, Tree Star Co., Ltd, USA).

[0044] Experimental results showed that the tumor microenvironment of xenografts in mice changed significantly before and after treatment with Schisanan B (see...). Figure 1 F). Furthermore, significant changes were observed in key protein markers such as MHC-I, CD44, PD-L1, and CD11b (see [link]). Figure 1 G).

[0045] Further data mining revealed that the M1 subgroup in TAMs is related to exhausted CD8. + The increase in T cells was more pronounced (see...) Figure 1 H, 1I). Furthermore, the M1 type TAMs subset shows increased PD-L1 protein expression and depletion of CD8. + T-cell cytotoxic CD8 + Increased expression of the T-cell marker protein Siglec-F (see...) Figure 1 J, 1K). But PD-L1 + TAMs cell subsets and toxic CD8 + Whether T cell subsets are key indicators of Schisanan B's anti-tumor activity still needs further verification.

[0046] (3) Verification of mass cytometry results by cell flow cytometry

[0047] To further validate the results of mass cytometry, cell flow cytometry was used to analyze PD-L1 after schisanan B treatment. +TAMs cell subsets and toxic CD8 + Changes in T cell subsets and the cytokines they produce were detected. First, tumor tissue was digested into single cells using trypsin. Then, following the instructions of each manufacturer, the digested cells were stained with various fluorescent flow cytometry antibodies, such as CD45, CD3, CD8, F4 / 80, PD-L1, and TNF-α. After staining, flow cytometry was used for detection, and cell gating was performed based on antibody classification relationships.

[0048] Experimental results showed that the polarization direction of TAMs treated with schisanan B gradually shifted towards the M1 type, and the ratio of M1 type TAMs to M2 type TAMs increased with increasing schisanan B concentration (see...). Figure 2 A). Further investigation revealed that with increasing schisanan B concentration, PD-L1 expression in M2-type TAMs gradually decreased, while PD-L1 expression in M1-type TAMs gradually increased (see A). Figure 2 (B, 2C). This proves that Schisanan B can reduce PD-L1 + The TAMs cell subset transformed into the M1 type. Subsequently, to verify the PD-L1 activity of the M1 type... + To determine whether TAMs cell subsets possess activity, the expression of TNF-α and IFN-γ cytokines within these subsets was analyzed (see...). Figure 2 (D, 2E). The results indicate that schisanan B can not only promote PD-L1 + TAMs cell subsets transform into M1 type, and can also induce PD-L1 + Activation of TAMs cell subsets.

[0049] On the other hand, this experiment investigated the toxicity of CD8. + The activation level of T cell subsets was detected, and the number of cells co-expressing granulamycin B (GRAMB) and IL-17 increased in a dose-dependent manner (see [link to study]). Figure 2 F). This indicates that schisanan B is toxic to CD8. + T cell subsets have a certain activating effect. In summary, PD-L1... + TAMs cell subsets and toxic CD8 + T cell subsets are likely a key indicator of schisanan B's anti-tumor activity.

[0050] (4) In vitro cytotoxicity detection

[0051] To further determine whether Schisanan B can activate PD-L1 +TAMs cell subsets were validated using in vitro assays. First, the cytotoxicity of schisanan B against RAW264.7 cells was assessed using the CCK8 assay. RAW264.7 cells were cultured at a concentration of 1 × 10⁻⁶ cells / cells. 4 The cells were seeded at a concentration of [value missing] mL in 96-well cell culture plates and incubated overnight. Schisanan B concentration gradients were prepared using culture medium, and after 24 hours of incubation, CCK8 solution was added to the 96-well cell culture plates. OD values ​​were measured at 450 nm using a microplate reader.

[0052] Experimental results showed that as the concentration gradient of schisanan B increased, it had a certain inhibitory effect on the proliferation of RAW264.7 cells (see...). Figure 3 A) Schisanan B exhibited significant cytotoxicity against RAW264.7 cells. This indicates that schisanan B itself has a certain degree of cytotoxicity. Subsequent experiments will use a safe dose, i.e., a drug concentration with a cell inhibition rate below 20%, for schisanan B as a PD-L1 inhibitor. + This study investigates the role and effects of TAMs cell subset activators.

[0053] (5) Study on the ability of Schisanan B to increase PD-L1 expression in RAW264.7 cells

[0054] To further determine in vitro whether schisanan B has an activating effect on the expression and function of PD-L1 on macrophages, i.e., whether schisanan B can act as an activator of PD-L1... + TAMs cell subset activator was used to detect PD-L1 protein expression in RAW264.7 mononuclear macrophage leukemia cells after treatment with Schisanan B by Western blotting.

[0055] RAW264.7 cells treated with different concentrations of schisanan B for 24 h were centrifuged, and the precipitate was collected. Total protein was extracted by RIPA lysis, and the total protein concentration was determined using a BCA protein concentration assay kit. After protein denaturation, SDS-PAGE was performed. Following transfer to a membrane, the cells were blocked and subjected to an immunoassay, followed by development using ECL chemiluminescence buffer. The results showed that schisanan B increased PD-L1 expression in RAW264.7 cells, with the expression level reaching its maximum at a schisanan B concentration of 400 μg / mL, and this phenomenon was dose-dependent (see [link to relevant documentation]). Figure 3 B).

[0056] RAW264.7 cells were further treated with schisanan B at the optimal concentration of 400 μg / mL for different time periods, and the protein expression was detected by Western blotting using the method described above. The results showed that PD-L1 protein expression in RAW264.7 cells increased with increasing schisanan B treatment time, reaching its maximum at 24 h (see [link to treatment details]). Figure 3 C). In summary, schisanan B has an upregulating effect on PD-L1 protein in macrophages.

[0057] (6) Schisandra chinensis monomeric polysaccharide schisanan B-mediated killing effect of RAW264.7 and Jurkat-T cells on lung cancer cells

[0058] To further investigate the antitumor effect of schisanan B in the co-culture of macrophages and T cells with tumor cells, lung cancer cells LLC were seeded in 6-well culture plates and co-cultured with RAW264.7 or Jurkat T cells stimulated with schisanan B for 24 h for 24 h. After fixation with 4% paraformaldehyde, crystal violet staining was performed, and tumor survival was observed under a microscope. The killing effect was specifically reflected in the difference in staining depth among different dose concentrations of schisanan B.

[0059] The experimental results showed that both Jurkat T and RAW264.7 cells treated with Schisanan B exhibited cytotoxic activity, and at the same concentration, RAW264.7 cells showed superior cytotoxic activity compared to Jurkat T cells (see...). Figure 3 C). RAW264.7 cells and Jurkat T cells, stimulated with Schisanan B for 24 hours, were further co-cultured with LLC cells for 24 hours, using the same treatment method as above. The results showed that the proportion of LLC cells that died when co-cultured with both types of immune cells was significantly higher than the proportion of LLC cells that died when co-cultured with any single type of immune cell (see [link to article]). Figure 3 C). This demonstrates that Jurkat T and RAW264.7 cells treated with Schisanan B do not simply perform their individual functions, but rather exhibit a certain synergistic effect, with macrophages playing a greater role in this process.

[0060] Furthermore, RAW264.7 and Jurkat T cells co-cultured with LLC cells were analyzed by flow cytometry. The results showed that Schisanan B could still induce macrophage polarization towards the M1 type in vitro, and the PD-L1 protein in M1 macrophages still showed an increasing trend. Furthermore, PD-L1... + Macrophages also showed an activated state (see...) Figure 3D). All of this data indicates that PD-L1 + Macrophage subsets can not only kill tumor cells but also synergize with T cells to kill tumor cells. In summary, PD-L1... + Activation of TAMs cell subsets plays a crucial role in the antitumor effect of schisanan B.

[0061] (7) Detection of Schisanan B-mediated enhanced phagocytic capacity of RAW264.7

[0062] Based on the co-culture results described in (6), it is believed that Schisanan B activates PD-L1. + TAMs cell subsets exhibit enhanced phagocytic capacity after activation. Therefore, RAW264.7 cells were seeded in 96-well plates and stimulated with different concentrations of Schisanan B for 24 hours after cell attachment. After stimulation, 20 μL of neutral red was added to each well for 4 hours. Results showed that activated macrophages exhibited enhanced phagocytic capacity, and the neutral red content in the cells increased with increasing Schisanan B concentration (see...). Figure 4 A).

[0063] Furthermore, in order to determine PD-L1 + The ability of TAMs to phagocytose tumor cells was assessed, as described in (6). A co-culture model of RAW264.7 cells stimulated with different concentrations of Schisanan B and LLC cells was constructed. After 24 hours of co-culture, the abundance of CD11b protein in RAW264.7 cells was examined. The results showed that the abundance of CD11b protein expressed by macrophages increased with increasing concentration after co-culture, while the number of non-macrophages (i.e., LLC tumor cells) gradually decreased (see [reference]). Figure 4 B). This is consistent with the co-culture results described in (6). The above experiments demonstrate that Schisanan B activates PD-L1. + The phagocytic capacity of a TAMs cell subset is enhanced upon immersion in the TAMs cell subset.

[0064] (8) Effects of Schisanan B on tumors in organisms with immune system deficiencies

[0065] To illustrate whether schisanan B works by activating PD-L1 in vivo... + The macrophage subset enhances its killing ability, thereby playing a therapeutic role in cancer treatment. This experiment established an in vivo lung cancer xenograft model in immunodeficient mice (see...). Figure 5A). Female BLAB / C Nude mice (20±2g) were housed in a rearing room with ample water and food (25±2℃). After 7 days of acclimatization, 14 mice were randomly divided into a model group and a Schisanan B group (200mg / kg). A lung cancer xenograft model was established in all experimental mice, using 1×10⁻⁶ mice. 7 Each mouse was injected subcutaneously with a suspension of non-small lung cancer cells from LLC mice. Starting 7 days after modeling, mice in the Schisanan B group were orally administered Schisanan B (200 mg / kg) once daily, while simultaneously receiving an equal volume of saline from the model group. Daily data on body weight and tumor volume were collected for dynamic analysis.

[0066] Experimental results showed that in nude mice treated with schisanan B at a dose of 200 / 100 mg / kg, the tumor size was almost identical to that in the model group (see...). Figure 5 B). Specifically, with the accumulation of administration time, the tumor volume in the schisanan B-treated mice did not show a decreasing trend, and schisanan B at this dose had no significant effect in immunodeficient mice (see [link to article]). Figure 5 C, 5D).

Claims

1. The polysaccharide monomer Schisanan B isolated from Schisandra chinensis was used in the preparation of tumor-associated macrophage PD-L1 cells. + Uses of positive expression subset activators.

2. The use of the polysaccharide monomer schisanan B isolated from Schisandra chinensis in the preparation of antitumor drugs that activate tumor-associated macrophages to express PD-L1 protein.

3. Use of the polysaccharide monomer schisanan B isolated from Schisandra chinensis in the preparation of antitumor drugs that activate the transformation of tumor-associated macrophages to the M1 type.

4. The use according to claim 2 or 3, characterized in that, The tumors mentioned include non-small cell lung cancer, colorectal cancer, melanoma, urothelial carcinoma, or hepatocellular carcinoma.

5. The use according to any one of claims 1-4, characterized in that, The polysaccharide monomer schisanan B isolated from Schisandra chinensis is prepared into a clinically suitable pharmaceutical formulation by means of a pharmaceutically acceptable carrier or excipient using conventional formulation methods.

6. The use according to claim 5, characterized in that, The pharmaceutical preparations mentioned include oral preparations, injectable preparations, sustained-release preparations, or controlled-release preparations.

7. The use according to claim 6, characterized in that, The oral preparations mentioned are pills, tablets, granules, powders, or capsules.

8. The use according to any one of claims 1-3, characterized in that, The polysaccharide monomer Schisanan B mainly contains glucose, galacturonic acid, and trace amounts of arabinose, rhamnose, and galactose; wherein the theoretical molar ratio of glucose to galacturonic acid is 25:

3.

9. The use according to claim 8, characterized in that, The polysaccharide monomer schisanan B has a 1,4-Glcp backbone and side chains with T-Glcp, 1,3,4-Glcp, 1,4,6-Glcp, 1,3,4,6-Glcp, 1,4-GalAMep, and T-GalAMep linkages. According to the theoretical sugar residue linkage ratio: 1,4-Glcp: T-Glcp: 1,3,4-Glcp: 1,4,6-Glcp: 1,3,4,6-Glcp: 1,4-GalAMep: T-GalAMep = 63:19:9:6:3:10:2; Preferably, the polysaccharide monomer schisanan B is a white flocculent substance that is highly soluble in water; its absolute molecular weight Mw = 106.0 kDa and polydispersity index is 2.

28.

10. The use according to claim 9, characterized in that, The main chain of the polysaccharide monomer Schisanan B is composed of 1,4-linked dextran with branch points at positions 3, 6 and 3,6 respectively. The side chains are composed of 1,4-glucan chains of different lengths with multiple branch points formed by 4-26 polymers and 1,4-galacturonic acid polysaccharides of different lengths formed by 2-8 polymers, which are composed of methyl esters linked at position 6. Preferably, the chemical structural formula of the polysaccharide monomer Schisanan B is as follows:

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