Application of grifola frondosa extract in preparation of medicine for inhibiting myeloid-derived suppressor cells

Abstract

The invention discloses an application of a grifola frondosa extract in preparation of a medicine for inhibiting myeloid-derived suppressor cells. The polysaccharide extract is a grifola frondosa sporocarp polysaccharide extract, and the molecular weight range of the polysaccharide extract is 0.5 * 10 < 4 >-149 * 10 < 4 > Da. The grifola frondosa extract inhibits the spleen of a tumorigenic mouse from generating MDSCs, inhibits the proportion and quantity of MDSCs in the blood and tumor tissue of the tumorigenic mouse, and improves the content and activity of CD3 + T and CD8 + T lymphocytes, so that the immune response ability of the body is improved, and the grifola frondosa extract is not only suitable for preparing anti-tumor drugs, but also suitable for treating inflammatory-mediated tissue damage, infectious diseases and other diseases. And preparing medicines for treating MDSCs related diseases such as autoimmune diseases and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to application of Grifola frondosa extract in preparation of myeloid-derived suppressor cell inhibiting drugs. BACKGROUND

[0002] Myeloid-derived suppressor cells (MDSCs) are a group of highly heterogeneous immature myeloid cells derived from the bone marrow hematopoietic system, generated in the bone marrow and spleen and the like through myeloid generation pathway, and have significant and extensive immunosuppressive function. In pathological conditions such as tumor, chronic inflammation and infection, MDSCs abnormally proliferate and accumulate in large quantities in peripheral blood, lymphoid organs and lesion tissues, which is one of the important reasons for the impairment of immune function of the body. MDSCs are divided into granulocyte or polymorphonuclear MDSCs (PMN-MDSCs) and monocyte MDSCs (M-MDSCs). In mice, MDSCs are defined as cells expressing CD11b and Gr-1, Gr-1 contains two epitope antigens Ly6G and Ly6C, M-MDSCs are defined as cells expressing CD11b + Ly6C hi Ly6G − CD11b + Ly6C lo Ly6G + CD11b + CD33 + CD14 + CD15 - HLA - DRlo / - CD11b + CD33 + CD14 - CD15 + HLA - DR - In addition, a third type of MDSCs is found in human body, which lacks the characteristics of monocytes or granulocytes, and may be the precursor of M-MDSCs and PMN-MDSCs, but more research is needed to fully understand or confirm this potential evolutionary relationship, and this subpopulation is defined as CD11b + CD33 + CD14 - CD15 - HLA-DR- .

[0003] Studies have shown that MDSCs in a variety of types (such as breast cancer, lung cancer, colon cancer, liver cancer, prostate cancer and head and neck cancer, etc.) cancer patients or tumor-bearing mice in the peripheral blood, spleen and tumor tissue in a large number of expansion, with tumor load is positively correlated with shorter survival and anti-tumor treatment response. MDSCs have been shown to have strong immunosuppressive activity, MDSCs inhibit the body's innate immunity and acquired immunity of tumor through a variety of ways, including the expression of immune checkpoint PD-L1, CTLA-4, CD155 and other induced T cell dysfunction, consumption of T cells required amino acids, active nitrogen and active oxygen to destroy the function of T cells, inducing regulatory T cells (Treg) infiltration and proliferation, blocking NK cell cytotoxicity, preventing T lymphocyte homing and so on, forming an immunosuppressive environment, weakening immune function. In clinical animal model studies, a variety of chemotherapy drugs such as 5-fluorouracil (5-FU), gemcitabine and other significantly inhibit the number of MDSCs, and the current clinical treatment research with PD-1 or PD-L1 and other inhibitors combined with drug use. At present, the use of inhibitors targeting MDSCs alone or in combination with immune checkpoint inhibitors and chemotherapy drugs and other cancer clinical treatment is a very important scheme and strategy. As a small molecule chemotherapy drug, gemcitabine can also clear CD3 + T cells while clearing MDSCs. In patients or tumor-bearing mouse models, after clearing MDSCs, the T cells in the body are very few, and the treatment effect is generally poor, so it is of great significance and urgency to find drugs that can clear MDSCs while retaining T lymphocytes.

[0004] With further research, it was found that MDSCs also proliferate and accumulate in a large number of inflammatory sites and lymphoid organs in a variety of diseases, such as infection, autoimmune diseases and inflammatory-mediated tissue damage, etc., inhibit T cell response, and form an immunosuppressive environment. In psoriasis patients and psoriasis-like mouse models, MDSCs show aggregated expansion, which is positively correlated with the severity of the disease, but acitretin, polyethylene glycol ointment, etc., can reduce the number of MDSCs and Th17 cells in mice, and improve symptoms. Studies have found that MDSCs inhibit T cell function in a manner dependent on arginase in hepatitis B virus infection, which also provides a new direction and target for viral treatment.

[0005] Grifola frondosa is a traditional edible and medicinal fungus, and its polysaccharide components have a good research foundation in immunomodulation and anti-tumor. Previous studies have shown that Grifola frondosa polysaccharide can regulate the immune function of the body, enhance the activity of T cells and natural killer cells, and thus play an anti-tumor role. However, existing studies are mostly focused on its overall immune enhancement or anti-tumor effect, and there are limited reports on whether it can target the regulation of tumor immunosuppressive microenvironment, especially whether it can inhibit the expansion and function of MDSCs and its key subpopulation PMN-MDSCs.

[0006] Recent studies have shown that crude polysaccharide components derived from Grifola frondosa can significantly reduce the accumulation of MDSCs in tumor tissues and peripheral immune organs in tumor models such as breast cancer, especially showing a significant inhibitory effect on PMN-MDSCs, while enhancing the anti-tumor effect function of CD8⁺ T cells, thereby inhibiting tumor growth. However, as of now, there has been no systematic patent disclosure on drugs or compositions that use Grifola frondosa polysaccharide as an effective ingredient to inhibit or regulate MDSCs. SUMMARY

[0007] In view of the shortcomings and deficiencies of the prior art, the present application provides an application of Grifola frondosa extract in the preparation of a myeloid-derived suppressor cell inhibiting drug. Specifically, it relates to the use of Grifola frondosa extract as a myeloid-derived suppressor cell (MDSCs) inhibitor, which provides new substances for the manufacture of biological drugs and promotes the development of the biological pharmaceutical industry.

[0008] In order to achieve the above-mentioned purpose, the technical scheme created by the present application is as follows:

[0009] The first object of the present application is to provide an application of Grifola frondosa extract in the preparation of a myeloid-derived suppressor cell inhibiting drug, wherein the Grifola frondosa extract is a Grifola frondosa fruiting body polysaccharide extract with a molecular weight range of 0.5×10 4 -149×10 4 Da, and the Grifola frondosa extract is prepared by the following method:

[0010] 1) Grifola frondosa fruiting bodies are crushed and then extracted with pure water at a temperature of 90-110°C, and the filtrate is collected by filtration. The filter residue is added with pure water and repeatedly extracted, and the filtrates are combined and concentrated to obtain a concentrated solution;

[0011] 2) 0.5-4 volumes of anhydrous ethanol are added to the concentrated solution, and the precipitate is obtained by centrifugation after standing. The precipitate is washed and then dissolved in 65-70°C pure water, and the filtrate is obtained after removing the ethanol and filtering. The filtrate is dialyzed and then freeze-dried to obtain a freeze-dried product;

[0012] 3) dissolving the freeze-dried product, loading it on an anion exchange chromatography column, eluting it with pure water and 0.1-0.2 M NaCl solution in sequence; collecting the NaCl solution elution part, concentrating it, adding 0.5-4 times volume of anhydrous ethanol, standing, centrifuging, obtaining a precipitate; dissolving the precipitate with 65-70 °C pure water after washing, filtering after removing ethanol, obtaining a filtrate; concentrating, freeze-drying the filtrate to obtain the Grifola frondosa extract.

[0013] Preferably, the Grifola frondosa extract is used for preparing a medicament for inhibiting myeloid-derived suppressor cells and promoting CD3 + T and CD8 + T lymphocytes, and increasing the content and activity of T lymphocytes.

[0014] The Grifola frondosa fruiting body polysaccharide extract can inhibit myeloid-derived suppressor cells, increase the content and activity of CD3 + T and CD8 + T lymphocytes, and improve the immune response ability of the body.

[0015] Preferably, the Grifola frondosa extract can also down-regulate the protein expression of PD-L1 and PD-1 in tumor tissues.

[0016] Preferably, the myeloid-derived suppressor cells are myeloid-derived suppressor cells in blood, spleen and / or tumor, and the CD3 + T and CD8 + T lymphocytes are CD3+T and CD8 + T lymphocytes in blood, spleen and / or tumor.

[0017] Preferably, the Grifola frondosa extract can inhibit myeloid-derived suppressor cells by inhibiting the production of myeloid-derived suppressor cells in the body, eliminating peripheral blood myeloid-derived suppressor cells, or inhibiting the recruitment of myeloid-derived suppressor cells at the tumor site.

[0018] Preferably, the Grifola frondosa extract is used in an amount of 50-200 mg / kg / day.

[0019] Preferably, in step 1), the pulverization refers to pulverization to 20-60 mesh, in the extraction and the repeated extraction, the amount of pure water added is 1 g:15-25 mL of material liquid ratio based on the mass of the Grifola frondosa fruiting body or filter residue, the extraction or repeated extraction time is 1~3h, and the concentration is vacuum concentration to 10%~20% of the volume of the combined filtrate; in steps 2) and 3), the 0.5-4 volumes of anhydrous ethanol is 4 volumes of anhydrous ethanol, the standing refers to standing overnight at 4℃, the centrifugation refers to centrifugation at 8000 rpm for 15 min, the washing refers to washing with cold ethanol at 0~10℃, and the filtration after removing ethanol is filtered with a 0.45μm filter membrane; in step 2), the dialysis refers to 48h dialysis with a 3500Da dialysis bag.

[0020] Preferably, the Grifola frondosa extract preparation method comprises the following specific steps:

[0021] 1) The fruiting body of Grifola frondosa is pulverized to 40 mesh, 20 times the mass of the raw material is added to it to extract pure water for 2 hours, the extraction temperature is 90-110℃; the obtained extract is filtered, the filtrate is collected, the residue is added with pure water solvent in an amount of 20 times the mass, and then heated to 90-110℃ to extract for 2 hours again, the obtained extract is filtered, and then the filtrate obtained after each extraction is combined and concentrated under vacuum to obtain a concentrated solution with a volume of 15% of the combined filtrate;

[0022] 2) 0.67-4 times the volume of anhydrous ethanol of the concentrated solution is added while stirring, fully mixed, and left to stand overnight at 4℃, centrifuged at 8000 rpm for 15 min to obtain precipitate, and washed with 0~10℃ cold ethanol; the precipitate is redissolved with 65~70℃ pure water, evaporated with ethanol under vacuum at 48~52℃, centrifuged, and filtered with a 0.45μm filter membrane to obtain a filtrate; the filtrate is dialyzed with a 3500Da dialysis bag for 48 hours, vacuum concentrated again, freeze-dried to obtain a freeze-dried product;

[0023] 3) The freeze-dried product obtained in step 2) is weighed, redissolved with water according to the material ratio of 40 g:1000 mL, loaded onto an anion exchange chromatography column, eluted with pure water, 0.1 M NaCl, and 0.3 M NaCl in sequence, the 0.1M NaCl elution part is collected, and the eluate is concentrated; 4 times the volume of anhydrous ethanol of the concentrated solution is added while stirring, fully mixed, left to stand overnight at 4℃, centrifuged at 8000 rpm for 15 min to obtain precipitate, and washed with 0~10℃ cold ethanol; the precipitate is redissolved with 65~70℃ pure water, evaporated with ethanol under vacuum at 50℃, centrifuged at 8000 rpm for 15 min to take the supernatant, filtered with a 0.45μm filter membrane to obtain a filtrate; the filtrate is concentrated and freeze-dried to obtain a Grifola frondosa extract.

[0024] A second object of the present application is to provide a myeloid-derived suppressor cell inhibiting drug comprising a Grifola frondosa extract, the Grifola frondosa extract being a Grifola frondosa fruiting body polysaccharide extract having a molecular weight range of 0.5 x 10 4 - 149 x 10 4 Da, the Grifola frondosa extract containing glucose, mannose, fucose, glucosamine hydrochloride, galactose, galacturonic acid, and glucuronic acid.

[0025] Preferably, the myeloid-derived suppressor cell inhibiting drug is an anti-tumor drug, an inflammatory-mediated tissue damage treatment drug, an autoimmune disease treatment drug, or an infectious disease treatment drug.

[0026] Preferably, the myeloid-derived suppressor cell inhibiting drug is an anti-tumor drug, an inflammatory-mediated tissue damage treatment drug, an autoimmune disease treatment drug, or an infectious disease treatment drug.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] The present application first proposes the use of a Grifola frondosa extract in the preparation of a myeloid-derived suppressor cell inhibiting drug.

[0029] The present application provides the use of a Grifola frondosa extract in the preparation of a myeloid-derived suppressor cell inhibiting drug. The Grifola frondosa extract is a Grifola frondosa fruiting body polysaccharide extract having a molecular weight range of 0.5 x 10 4 - 149 x 10 4 Da. The Grifola frondosa extract inhibits the production of MDSCs in the spleen of a tumor-causing mouse, inhibits the proportion and quantity of MDSCs in the blood and tumor tissue of a tumor-causing mouse, and increases the content and activity of CD3 + T lymphocytes, thereby improving the immune response capacity of the body. The Grifola frondosa extract is not only suitable for the preparation of an anti-tumor drug, but also suitable for the preparation of a drug for MDSCs-related diseases such as inflammatory-mediated tissue damage, infectious diseases, and autoimmune diseases.

[0030] The present application develops a drug for inhibiting myeloid-derived suppressor cells and improving T cells. The results show that the Grifola frondosa extract removes the MDSC cell population in the spleen, blood, and tumor site of a tumor-causing mouse. The present application verifies the inhibition and removal of a large number of myeloid-derived suppressor cells in a breast cancer mouse in vivo by a Grifola frondosa polysaccharide extract, and increases the number of CD3 + T lymphocytes in vivo, thereby enhancing the immune response capacity of the body. Therefore, the Grifola frondosa polysaccharide extract has great prospects for use in the treatment of MDSCs-related diseases and the preparation of a drug for the treatment of MDSCs-related diseases.

[0031] The present application develops a kind of preparation based on Grifola frondosa polysaccharide, which can selectively inhibit MDSCs and improve the immunosuppressive state of tumor, and has important theoretical significance and broad application prospect for improving the effect of tumor immunotherapy, reducing the side effects of treatment and expanding its application in tumor and immune-related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 It is the molecular weight distribution characteristic map of Grifola frondosa extract GFI-2.

[0033] Figure 2 It is the ion chromatogram of 16 kinds of monosaccharide markers.

[0034] Figure 3 It is the ion chromatogram of GFI-2 monosaccharide.

[0035] Figure 4 It is the tumor change graph of model group and GFI-2 administration group mice.

[0036] Figure 5 It is the tumor tissue size comparison graph of model group and GFI-2 administration group mice.

[0037] Figure 6 It is the flow detection result graph of Grifola frondosa extract GFI-2 reducing the proportion and quantity of MDSCs in the blood of treatment group mice.

[0038] Figure 7 It is the flow detection result graph of Grifola frondosa extract GFI-2 reducing the proportion and quantity of MDSCs in the spleen of treatment group mice.

[0039] Figure 8 It is the flow detection result graph of Grifola frondosa extract GFI-2 reducing the proportion and quantity of MDSCs in the tumor tissue of treatment group mice.

[0040] Figure 9 It is the flow detection result graph of Grifola frondosa extract GFI-2 promoting the proportion and quantity of CD3 + T cells in the blood circulation of treatment group mice.

[0041] Figure 10 It is the flow detection result graph of Grifola frondosa extract GFI-2 promoting the proportion and quantity of CD3 + T cells in the peripheral lymphatic organs spleen of treatment group mice.

[0042] Figure 11 It is the flow detection result graph of Grifola frondosa extract GFI-2 promoting the proportion and quantity of CD3 + T cells in the tumor tissue of treatment group mice.

[0043] Figure 12GFI-2 promotes CD8 + Figure of flow cytometry results of the proportion and number of T cells in the blood circulation of mice in the treatment group.

[0044] Figure 13 GFI-2 promotes CD8 + Figure of flow cytometry results of the proportion and number of T cells in the peripheral lymphatic organs of mice in the treatment group.

[0045] Figure 14 Figure of immunohistochemical results of GFI-2 inhibiting the protein expression of immune checkpoints PD-1 and PD-L1 in tumor tissues. DETAILED DESCRIPTION

[0046] The following examples are further illustrations of the application and are not intended to limit the application.

[0047] Example 1: Preparation of Grifola frondosa extract and determination of polysaccharide content

[0048] 1. Preparation of Grifola frondosa extract

[0049] The fruiting bodies of Grifola frondosa were ground to 40 mesh, and then pure water was added to the ground fruiting bodies in an amount of about 20 times the mass of the raw material to extract for 2 hours. The extraction temperature was 100°C. The resulting extract was filtered, the filtrate was collected, and pure water solvent was added to the residue in an amount of about 20 times the mass, and then extracted at 100°C for another 2 hours. The resulting extract was then filtered, and the filtrate obtained after each extraction was combined and concentrated under vacuum to obtain a concentrated solution at 15% of the combined volume of the filtrate.

[0050] Extract 1: Anhydrous ethanol was added to the concentrated solution in an amount of 0.67 times the volume of the concentrated solution while stirring, mixed thoroughly, and left to stand at 4°C overnight. The precipitate was obtained by centrifugation at 8000 rpm for 15 min, and washed thoroughly with cold ethanol at 4°C. The precipitate was redissolved with pure water at 65-70°C, and the ethanol was evaporated by vacuum concentration at 50°C. The supernatant was obtained by centrifugation at 8000 rpm for 15 min, and filtered through a 0.45 μm filter membrane. The filtrate was dialyzed in a dialysis bag (3500 Da) for 48 hours, and then vacuum concentrated and lyophilized to obtain sample GFI-40, which was stored for later use.

[0051] Extract 2: To the concentrated solution, 1 times volume of absolute ethanol was added while stirring, mixed well, and left to stand at 4°C overnight. The precipitate was obtained by centrifugation at 8000 rpm for 15 min, and washed with cold ethanol at 4°C. The precipitate was redissolved with pure water at 65-70°C, and the ethanol was evaporated by vacuum concentration at 50°C. The supernatant was obtained by centrifugation at 8000 rpm for 15 min, and filtered with a 0.45 μm filter to obtain the filtrate. The filtrate was dialyzed for 48 hours with a dialysis bag (3500 Da), and vacuum concentrated and freeze-dried again to obtain sample GFI-50, which was stored for later use.

[0052] Extract 3: To the concentrated solution, 1.5 times volume of absolute ethanol was added while stirring, mixed well, and left to stand at 4°C overnight. The precipitate was obtained by centrifugation at 8000 rpm for 15 min, and washed with cold ethanol at 4°C. The precipitate was redissolved with pure water at 65-70°C, and the ethanol was evaporated by vacuum concentration at 50°C. The supernatant was obtained by centrifugation at 8000 rpm for 15 min, and filtered with a 0.45 μm filter to obtain the filtrate. The filtrate was dialyzed for 48 hours with a dialysis bag (3500 Da), and vacuum concentrated and freeze-dried again to obtain sample GFI-60, which was stored for later use.

[0053] Extract 4: To the concentrated solution, 2.33 times volume of absolute ethanol was added while stirring, mixed well, and left to stand at 4°C overnight. The precipitate was obtained by centrifugation at 8000 rpm for 15 min, and washed with cold ethanol at 4°C. The precipitate was redissolved with pure water at 65-70°C, and the ethanol was evaporated by vacuum concentration at 50°C. The supernatant was obtained by centrifugation at 8000 rpm for 15 min, and filtered with a 0.45 μm filter to obtain the filtrate. The filtrate was dialyzed for 48 hours with a dialysis bag (3500 Da), and vacuum concentrated and freeze-dried again to obtain sample GFI-70, which was stored for later use.

[0054] Extract 5: To the concentrated solution, 4 times volume of absolute ethanol was added while stirring, mixed well, and left to stand at 4°C overnight. The precipitate was obtained by centrifugation at 8000 rpm for 15 min, and washed with cold ethanol at 4°C. The precipitate was redissolved with pure water at 65-70°C, and the ethanol was evaporated by vacuum concentration at 50°C. The supernatant was obtained by centrifugation at 8000 rpm for 15 min, and filtered with a 0.45 μm filter to obtain the filtrate. The filtrate was dialyzed for 48 hours with a dialysis bag (3500 Da), and vacuum concentrated and freeze-dried again to obtain sample GFI-80, which was stored for later use.

[0055] 2. Preparation of main components of Grifola frondosa extract

[0056] GFI-80 40 g obtained in step 1 was weighed out and dissolved in 1000 mL of pure water to prepare a 40 mg / mL solution. A DEAE-Sepharose Fast Flow gel-filled column (column inner diameter: 45 mm, column length: 75 cm) was used, and first of all, a pure water equilibration was performed at 3-4 times the column volume. Subsequently, the GFI-80 solution was loaded, and elution was performed using pure water and 0.1 M NaCl solution in sequence at a flow rate of 10 mL per minute. Elution fractions were collected in sequence, and the eluate was concentrated in vacuo, with the 0.1 M NaCl component being named GFI-2. To the GFI-2 concentrate, anhydrous ethanol was added to 4 times the volume of the concentrate while stirring, and after thorough mixing, it was left to stand at 4°C overnight, and a precipitate was obtained by centrifugation at 8000 rpm for 15 min, and the precipitate was washed with cold ethanol at 4°C. The precipitate was redissolved with pure water at 65-70°C, and the ethanol was evaporated by concentration in vacuo at 50°C, and the supernatant was obtained by centrifugation at 8000 rpm for 15 min, and the filtrate was obtained by filtration through a 0.45 μm filter. The filtrate was again concentrated in vacuo and freeze-dried to obtain GFI-2 freeze-dried powder.

[0057] 3. Determination of the molecular weight distribution of Grifola frondosa polysaccharide GFI-2 (HPGPC method)

[0058] Standard: molecular weight 5000 (Sigma, 102084138), molecular weight 11600 (Sigma, 102136543), molecular weight 23800 (Sigma, 102124529), molecular weight 48600 (Sigma, 102104509), molecular weight 80900 (Sigma, 102108375), molecular weight 148000 (Sigma, 102089360), molecular weight 273000 (Sigma, 102110878), molecular weight 409800 (Sigma, 102124507), molecular weight 667800 (Sigma, 102104510).

[0059] Sample and standard preparation: 5 mg of sample GFI-2 and standard were accurately weighed out and dissolved in 0.01 M NaCl solution, and the sample was prepared as a 5 mg / mL solution, and the supernatant was obtained by centrifugation at 12000 rpm for 10 min, and then the sample was transferred to a 1.8 mL sample vial.

[0060] Mobile phase preparation: 0.58 g of chromatographic grade NaCl was accurately weighed out and dissolved in 1 L of ultrapure water, filtered through a 0.45 μm filter, and sonicated (40 kHz) for 10 min, and used as needed.

[0061] Instrument parameters: HPLC (Agilent 1260), chromatographic columns: TSKgel G5000 PWXL column (300 mm × 7.8 mm; inner diameter, 10 μm) and TSKgel G3000 PWXL column (300 mm × 7.8 mm; inner diameter, 7 μm).

[0062] Detection parameters: mobile phase: 0.01M NaCl, flow rate: 0.28 mL / min, column temperature: 35℃, injection volume: 20μL, detector: differential detector.

[0063] Experimental results are as follows Figure 1 As shown in Table 1, the GFI-2 content of the three components shows that the retention time of the main component is 45.79 min and the molecular weight is 94 × 10⁻⁶. 4 ~149×10 4 The retention time of the second component was 62.59 min, and its molecular weight was 1.2 × 10⁻⁶. 4 ~1.3×10 4 The retention time of the third component was 71.22 min, approximately 0.5 × 10⁻⁶. 4 Da.

[0064] 4. Monosaccharide composition of Grifola frondosa polysaccharide GFI-2

[0065] GFI-2 was degraded by acid hydrolysis, and its monosaccharide composition and proportion were analyzed by ion chromatography and compared with monosaccharide standards (Reference: Determination of Monosaccharide Composition in Pueraria Polysaccharides from Different Origins by Ion Chromatography, Zhao Dan et al., 2017). The results showed that the main monosaccharides of GFI-2 were glucose and mannose, and it also contained fucose, glucosamine hydrochloride, galactose, galacturonic acid, and glucuronic acid. Figure 2 and Figure 3 .

[0066] Example 2: Grifola frondosa polysaccharide GFI-2 can reduce the content of MDSCs in tumor-bearing mice and improve the tumor microenvironment.

[0067] The results of Example 1 showed that the Grifola frondosa extract inhibited myeloid-derived suppressor cells and increased CD3 levels. + CD8 + It reduces the number and activity of T cells, downregulates the protein expression of PD-L1 and PD-1 in tumor tissue, and enhances the body's immune response.

[0068] 1 Experimental Methods

[0069] 1.1 Preparation of 4T1 mouse breast cancer cells

[0070] 4T1 cells were taken out from the liquid nitrogen storage tank, and were resuscitated, activated and cultured in DMEM medium containing 1% penicillin, 1% streptomycin and 10% fetal bovine serum (FBS), under the condition of 37°C and 5% CO2 concentration in a cell incubator, until the logarithmic growth phase.

[0071] 1.2 Establishment of 4T1 subcutaneous tumor model

[0072] The mice were BALB / C female mice (5-7 weeks old, weighing 20±2g) from Guangdong Experimental Animal Center, and were adaptively fed for one week before the experiment. The logarithmically growing 4T1 cells were digested, counted, and adjusted to a cell density of 5×10 5 cells / mL, and were implanted subcutaneously in mice for 100 μL. After one week of modeling, the mice were randomly divided into two groups (control model group, GFI-2 group), with 10 mice in each group. A normal control group was also set up.

[0073] 1.3 Drug administration

[0074] The control model group was only given normal saline (0.9% sodium chloride aqueous solution). The GFI-2 group was given intraperitoneal administration at a dose of 100 mg / kg / day every other day for 4 weeks. The tumor length was recorded.

[0075] After the last administration, the mice were sacrificed the next day, and blood, spleen, tumor and other samples were taken using a heparin sodium cannula. The samples were stored according to the following experimental purposes.

[0076] 1.4 Flow cytometry detection of MDSCs and CD3 + CD8 + T cells

[0077] 1) Preparation of single-cell suspension of spleen cells

[0078] After the administration was completed, the 5 groups of tumor-bearing mice were sacrificed by decapitation, and the sacrificed BalB / C mice were sterilized in alcohol in a clean bench for 5 minutes. After removal, the spleen was taken out along the midline of the abdomen, washed in pre-cooled PBS, and transferred to a 1.5 mL centrifuge tube containing RPMI1640, and ground using a rubber grinding rod. The grinding liquid was filtered using a 70 μM cell filter, and the filter was washed with PBS, centrifuged at 350g for 7 min, and the supernatant was discarded to obtain the precipitated cells; the cells were resuspended with 2 mL of red blood cell lysis solution, blown evenly, and lysed at room temperature for 10 minutes, centrifuged at 350g for 7 min to discard the supernatant, and the precipitated cells were obtained; the cells were resuspended and washed with PBS, and centrifuged at 350g for 7 min to discard the supernatant. The cells were resuspended with PBS buffer to obtain a single-cell suspension, which was placed on ice and ready for use.

[0079] 2) Preparation of single-cell suspension of tumor tissue

[0080] After the end of administration, 5 groups of tumor-bearing mice were executed by cervical dislocation, and the sacrificed BalB / C mice were placed in an alcohol in a clean bench for 5 minutes. After taking out, cut along the midline of the abdomen, take out the tumor tissue, cut into small pieces in ice PBS, about 2-4 mm. The tissue was quickly transferred to 5 mL of tissue digestion solution (1640 medium containing w / v 0.1% collagenase type I, 0.2% dispase type I, 1% DNAse I stock), incubated at 37°C for 30 min, vortexed every 5 min (during this process, continuous observation was carried out until the tissue was dissociated). Add 10 mL FACS buffer, filter with 70 μm cell filter, and rinse the cell filter with 10-15 mL FACS buffer. Centrifugation at 350g, 5min, at 4°C, discard the supernatant (gently pour out), leave the precipitate. Add 1 mL of red blood cell lysis solution, incubate at room temperature for 3 minutes. Immediately place the sample on ice and resuspend it in 10 milliliters of FACS buffer to neutralize the red blood cell lysis buffer. Pipette blow, then filter with a 40 μm cell filter into a 50 mL test tube. Centrifugation at 350g, 5min, at 4°C. Discard the supernatant (gently pour out), resuspend with PBS buffer to obtain a single cell suspension, place on ice, ready for use.

[0081] 3) Single cell preparation of blood samples

[0082] After the end of administration, peripheral blood was collected using a blood collection tube containing sodium heparin by the method of orbital blood collection. Take 60 μL of blood sample, add 2 mL of red blood cell lysis solution (ACK), vortex well, avoid light incubation for 10 min. 350xg, 4°C centrifugation for 5 min, discard the supernatant, wash with 2 mL PBS buffer for 2 times, discard the supernatant, obtain the cell precipitate. Resuspend the cells with PBS buffer to obtain a single cell suspension, place on ice, ready for use.

[0083] 4) Flow detection

[0084] The blood, spleen and tumor single cell suspensions were centrifuged, resuspended with 100 μL PBS, and negative control and single positive staining control groups were set up, respectively, wherein the single positive staining group included MDSCs and T cell surface markers CD45, CD11b, Gr-1, CD3, CD8+. The antibodies involved in the experiment included: Anti-mouse CD45-APC-eFluor 780 (30-F11) (Thermo Fisher Scientific, 47-0451-82), Anti-mouse CD11b-APC (M1 / 70) (Thermo Fisher Scientific, 11-0031-82), Anti-mouse CD3e-FITC (145-2C11) (Thermo Fisher Scientific, 17-0112-82), Anti-mouse GR-1-PE-Cyanine7 (RB6-8C5) (Thermo Fisher Scientific, 25-5931-82), Anti-mouse CD8-PE (53-6.7) (Thermo Fisher Scientific, 12-0081-82), Anti-Mouse CD16 / CD32 (BD, 553141).

[0085] An antibody mixture for 20 samples was prepared: 2.5 μL of Anti-mouse CD45-APC-eFluor 780, 10 μL of Anti-mouse CD3e-FITC, 10 μL of Anti-mouse CD8-PE, 2.5 μL of Anti-mouse CD11b-APC and 2.5 μL of Anti-mouse GR-1-PE-Cyanine7 were diluted into 500 μL of FACS buffer to prepare the antibody mixture, which was placed on ice for standby.

[0086] The experimental steps are briefly described as follows:

[0087] (1) Resuspend 2-5 x 10 6 cells in 20 μL PBS.

[0088] (2) Add 25 μL of 7-ADD (BD, 559925) reagent, incubate on ice for 30 min, and avoid light.

[0089] (3) Add 100 μL of PBS to each tube, centrifuge at 350 g for 5 min at 4°C, and gently pour out the supernatant.

[0090] (4) Add 25 μL of Anti-Mouse CD16 / CD32 dilution buffer (2 μL diluted to 23 μL FACS buffer), incubate on ice for 30 min, protected from light.

[0091] (5) Add 25 μL of antibody mixture and incubate on ice for 30 min in the dark.

[0092] (6) Add 100 μL of PBS to each tube, centrifuge at 350 g for 5 min at 4 °C, and gently pour out the supernatant.

[0093] (7) Add 100 μL of FACS buffer, transfer to a flow cytometry tube, add 300 μL of FACS buffer, and then perform the detection.

[0094] 4) Calculation and Analysis

[0095] The above data was calculated and analyzed using Prism 6.07 and FlowJo.

[0096] 2. Experimental Results

[0097] 2.1 Grifola frondosa extract GFI-2 can inhibit tumor growth in 4T1 xenograft mouse models at dose-dependent rates.

[0098] like Figure 4 , Figure 5 As shown in Table 1, the experimental data indicate that, compared with the control group, Grifola frondosa extract GFI-2 effectively inhibited tumor growth in mice, demonstrating significant efficacy. Furthermore, this dosage range is acceptable for humans.

[0099] 2.2 Grifola frondosa extract GFI-2 reduced the proportion of MDSCs cells in the blood, spleen, and tumors of tumor-bearing mice in the treatment group.

[0100] like Figure 6 As shown, flow cytometry analysis demonstrated that, compared with the control group, Grifola frondosa extract reduced the proportion and number of MDSCs in the blood of mice in the treatment group.

[0101] like Figure 7 As shown, flow cytometry analysis demonstrated that, compared with the control model group, Grifola frondosa extract reduced the proportion and number of MDSCs in the spleen of mice in the treatment group.

[0102] like Figure 8 As shown, flow cytometry analysis demonstrated that, compared with the control model group, Grifola frondosa extract reduced the proportion and number of MDSCs in the tumor tissues of mice in the treatment group.

[0103] 2.3 Grifola frondosa extract GFI-2 increased CD3 levels in the blood, spleen, and tumor tissue of tumor-bearing mice in the treatment group. +T cell ratio

[0104] As shown in Figure 9 Figure 6, flow cytometry proved that, compared with the control model group, the Grifola extract promoted the ratio and number of CD3 + T cells in the blood circulation of the treatment group mice increased.

[0105] As shown in Figure 10 Figure 6, flow cytometry proved that, compared with the control model group, the Grifola extract promoted the ratio and number of CD3 + T cells in the peripheral lymphatic organs of the treatment group mice increased.

[0106] As shown in Figure 11 Figure 6, flow cytometry proved that, compared with the control model group, the Grifola extract promoted the ratio and number of CD3 + T cells in the peripheral lymphatic organs of the treatment group mice increased.

[0107] 2.4 Grifola extract GFI-2 increased the ratio of CD8 + T cells in the blood and spleen of the treatment group tumor mice

[0108] As shown in Figure 12 Figure 6, flow cytometry proved that, compared with the control model group, the Grifola extract promoted the ratio and number of CD3 + T cells in the blood circulation of the treatment group mice increased.

[0109] As shown in Figure 13 Figure 6, flow cytometry proved that, compared with the control model group, the Grifola extract promoted the ratio and number of CD3 + T cells in the peripheral lymphatic organs of the treatment group mice increased.

[0110] Example 3: Grifola extract GFI-2 inhibits MDSCs to enhance immune response

[0111] In order to further detect that the Grifola extract GFI-2 improves and enhances the immune response in the tumor tissue by inhibiting MDSCs, this embodiment detects the killing T cell activity and the immune suppression checkpoint PD-L1 and PD-1 of the tumor tissue.

[0112] 3.1 Preparation of sample tissue sections

[0113] Take the tumor tissue obtained in Example 2, fix it with 10% neutral formalin overnight, then embed it with paraffin, cut and patch it, and prepare the tumor tissue paraffin section.

[0114] 3.2 Tumor tissue immune cell killing T cell CD8 +T cells, MDSCs and tumor cell surface inhibitory molecules PD-L1 and CD8 + T cell-expressed PD-1 was immunohistochemically stained.

[0115] Experimental procedures:

[0116] (1) The slice was routinely dewaxed with xylene and dehydrated with gradient alcohol

[0117] The paraffin section was baked in an oven at 65-68°C for 2h, the paraffin was dissolved, and then the paraffin section was dewaxed with xylene I (soaking time 15 min), xylene II (soaking time 15 min), xylene III (soaking time 15 min).

[0118] The dewaxed section was dehydrated by soaking in 100% alcohol I for 5 min, 100% alcohol II for 5 min, 90% alcohol I for 5 min, 90% alcohol II for 5 min, 80% alcohol for 5 min, and 75% alcohol for 5 min, and then washed with pure water for 3 times, 5 min each time.

[0119] (2) Inactivation of endogenous antioxidant enzymes

[0120] The section was placed in 0.3% hydrogen peroxide freshly prepared with methanol, and left at room temperature for 15 min, and then washed with 1×PBS buffer for 3 times, 5 min each time.

[0121] (3) Antigen repair

[0122] The section was placed in 0.01 mol / L citrate buffer (make sure the liquid level can completely cover the section), heated to boiling in a microwave oven at medium-high fire for 8 min, incubated for 7 min, then adjusted to medium fire for 6 min, and then incubated for 6 min, and then removed and left to room temperature, and then the repaired section was placed in PBS buffer for washing for 3 times, 5 min each time.

[0123] (4) Serum blocking

[0124] The section was incubated with 10% normal goat serum blocking solution at 37°C for 30 min, and then poured off without washing.

[0125] (5) Incubation of primary antibody

[0126] The primary antibody (Anti-PD-L1 antibody [28-8], abcam company, ab205921; Anti-PD1 antibody [NAT105], abcam company, ab52587) was added to the section, placed in a wet box and incubated in a 37°C incubator for 1 h, and then incubated overnight at 4°C, and then washed with 1×PBS buffer for 3 times, 5 min each time.

[0127] (6) Secondary antibody incubation

[0128] Add secondary antibody (goat anti-rabbit IgG H&L (HRP), Abcam, ab205718) to the slides, incubate at room temperature for 50 min, then wash three times with 1×PBS buffer, 5 min each time.

[0129] (7) DAB color development

[0130] Prepare fresh DAB chromogenic solution, add it to the slide under a microscope, and develop the chromogenic solution in the dark. Control the degree of chromogenic development under a microscope. Then wash with 1×PBS buffer or tap water for 10 minutes to stop the chromogenic process.

[0131] (8) Restaining

[0132] Place the slides in hematoxylin staining solution for 5 minutes, rinse with pure water (do not rinse the tissue with running water to prevent detachment); then, place them in hematoxylin permeation solution staining box for 10 seconds for differentiation, rinse, and then place them in hematoxylin blue solution for 10 seconds.

[0133] (9) Routine dehydration, clearing, and sealing.

[0134] The sections were sequentially immersed in 75% alcohol (5 min), 85% alcohol (5 min), anhydrous ethanol I (5 min), anhydrous ethanol II (5 min), and xylene I (5 min) to dehydrate until they became transparent. The sections were then removed from xylene I and allowed to dry slightly before being mounted with neutral resin.

[0135] 3.3 Results

[0136] like Figure 14 As shown, the expression of MDSCs and the immunosuppressive factor programmed death ligand PD-L1 on the surface of tumor cells was significantly downregulated within tumor tissue. Furthermore, CD8-inducing enzymes were detected. + PD-1 protein expression is also downregulated in T cell apoptosis.

[0137] Table 1 Molecular weight distribution of GFI-2

[0138] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of Grifola frondosa extract for the manufacture of a drug for suppressing myeloid-derived suppressor cells, characterized in that, The Grifola frondosa extract is a Grifola frondosa fruiting body polysaccharide extract having a molecular weight range of 0.5 x 10 4 -149 x 10 4 Da, and is prepared by the following method: -149 x 10 4 Da, and is prepared by the following method: 1) Grifola frondosa fruiting body is crushed and extracted with pure water at 90-110℃, the filtrate is collected, the residue is added with pure water and extracted repeatedly, the filtrates are combined, concentrated, and a concentrated solution is obtained; 2) 0.5-4 volumes of anhydrous ethanol is added to the concentrated solution, it is allowed to stand, centrifuged, and a precipitate is obtained; The precipitate is washed, dissolved with 65-70℃ pure water, filtered after the ethanol is removed, and a filtrate is obtained; the filtrate is dialyzed and freeze-dried to obtain a freeze-dried product; 3) The freeze-dried product is dissolved and loaded onto an anion exchange chromatography column, and sequentially eluted with pure water and 0.1-0.2 M NaCl solution; The NaCl solution eluted part is collected, concentrated, added with 0.5-4 volumes of anhydrous ethanol, allowed to stand, centrifuged, and a precipitate is obtained; the precipitate is washed, dissolved with 65-70℃ pure water, filtered after the ethanol is removed, and a filtrate is obtained; the filtrate is concentrated and freeze-dried to obtain a Grifola frondosa extract.

2. Use according to claim 1, characterized in that, Use of grifola frondosa extract for preparation of myeloid-derived suppressor cell inhibition and promotion of CD3 + T and CD8 + Use in medicaments with increased content and activity of T lymphocytes.

3. Use according to claim 2, characterized in that, The Grifola frondosa extract also down-regulates the protein expression of PD-L1 and PD-1 in tumor tissues.

4. Use according to claim 1 or 2, characterized in that, The myeloid-derived suppressor cells are myeloid-derived suppressor cells in the blood, spleen and / or tumor, the CD3 + T and CD8 + T lymphocytes are CD3+T and CD8 + T lymphocytes in the blood, spleen and / or tumor.

5. Use according to claim 1 or 2, characterized in that, The Grifola frondosa extract achieves the effect of inhibiting myeloid-derived suppressor cells by inhibiting the production of myeloid-derived suppressor cells in the body, eliminating peripheral blood myeloid-derived suppressor cells, or inhibiting the recruitment of myeloid-derived suppressor cells at the tumor site.

6. Use according to claim 1 or 2, characterized in that, The dosage of the Grifola frondosa extract is 50-200 mg / kg / day.

7. The use according to claim 1, characterized in that, In step 1), the crushing refers to crushing to 20-60 mesh, in the extraction and the repeated extraction, the amount of pure water added is 1 g:15-25 mL based on the material liquid ratio, the extraction or repeated extraction time is 1-3 h, and the concentration is vacuum concentration to 10%-20% of the volume of the combined filtrate; in steps 2) and 3), the 0.5-4 volumes of anhydrous ethanol is 4 volumes of anhydrous ethanol, the standing refers to standing overnight at 4℃, the centrifugation refers to centrifugation at 8000 rpm for 15 min, the washing refers to washing with 0-10℃ cold ethanol, and the filtration after the ethanol is removed is filtered with a 0.45 μm filter; in step 2), the dialysis refers to 48 h dialysis with a 3500 Da dialysis bag.

8. The use according to claim 1, characterized in that, The preparation method of the Grifola frondosa extract comprises the following specific steps: 1) Grifola frondosa fruiting body is crushed to 40 mesh, 20 times the amount of pure water is added to the raw material for extraction for 2 hours, and the extraction temperature is 90-110℃; the obtained extract is filtered, the filtrate is collected, 20 times the amount of pure water is added to the residue, then heated to 90-110℃ for 2 hours of extraction, the obtained extract is filtered, and then the filtrates obtained after each extraction are combined and concentrated under vacuum to obtain a concentrated solution with a volume of 15% of the combined filtrate; 2) to the concentrated liquid, 0.67-4 times the volume of the concentrated liquid of anhydrous ethanol is added while stirring, and the mixture is uniformly mixed, and then is left to stand at 4 DEG C overnight, and is centrifuged at 8000 rpm for 15 min to obtain a precipitate, and the precipitate is washed with cold ethanol at 0-10 DEG C; the precipitate is redissolved with pure water at 65-70 DEG C, and is concentrated under vacuum at 48-52 DEG C to evaporate ethanol, and is centrifuged, and is filtered through a 0.45 mu m filter membrane to obtain a filtrate; the filtrate is dialyzed for 48 h using a 3500 Da dialysis bag, and is concentrated under vacuum again, and is freeze-dried to obtain a freeze-dried product; 3) the freeze-dried product obtained in step 2) is weighed, and is redissolved with water according to a material ratio of 40 g:1000 mL, and is loaded onto an anion exchange chromatography column, and is eluted with pure water, 0.1 M NaCl and 0.3 M NaCl in sequence, and the 0.1 M NaCl elution part is collected, and the eluate is concentrated; to the concentrated liquid, 4 times the volume of the concentrated liquid of anhydrous ethanol is added while stirring, and the mixture is uniformly mixed, and then is left to stand at 4 DEG C overnight, and is centrifuged at 8000 rpm for 15 min to obtain a precipitate, and the precipitate is washed with cold ethanol at 0-10 DEG C; the precipitate is redissolved with pure water at 65-70 DEG C, and is concentrated under vacuum at 50 DEG C to evaporate ethanol, and is centrifuged at 8000 rpm for 15 min to obtain a supernatant, and the supernatant is filtered through a 0.45 mu m filter membrane to obtain a filtrate; the filtrate is concentrated and freeze-dried to obtain a Grifola frondosa extract.

9. A myeloid-derived suppressor cell inhibiting drug, characterized by, The composition comprises a Grifola frondosa extract, which is a Grifola frondosa fruiting body polysaccharide extract having a molecular weight range of 0.5 x 10 4 -149 x 10 4 Da, and contains glucose, mannose, fucose, glucosamine hydrochloride, galactose, galacturonic acid, and glucuronic acid. The composition comprises a Grifola frondosa extract, which is a Grifola frondosa fruiting body polysaccharide extract having a molecular weight range of 0.5 x 10 4 -149 x 10 4 Da, and contains glucose, mannose, fucose, glucosamine hydrochloride, galactose, galacturonic acid, and glucuronic acid.

10. The medicament according to claim 9, characterized in that, The myeloid-derived suppressor cell inhibiting drug is an anti-tumor drug, an inflammatory-mediated tissue damage treatment drug, an autoimmune disease treatment drug or an infectious disease treatment drug.

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