Application of ganoderenic acid B in promoting proliferation and maturation of dendritic cells

Abstract

The invention discloses application of ganoderenic acid B in promoting proliferation and maturation of dendritic cells. Ganodeenoic acid B is lanostane type triterpene separated from ganoderma lucidum, and can promote proliferation and maturation of dendritic cells in a concentration range of 5-50 [mu] M. The ganoderenic acid B has a good prospect of enhancing the anti-tumor immune response effect of the DCs by promoting proliferation and maturation of the DCs and enhancing the antigen uptake and presentation capacity of the DCs.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to the application of ganoderic acid B in promoting the proliferation and maturation of dendritic cells. Background Art

[0002] Dendritic cells (DCs) are the most powerful professional antigen presenting cells (APCs) in the body. They can efficiently absorb, process and present antigens. Mature DCs can effectively activate naive T cells, promote their activation and proliferation, and induce the formation of specific cytotoxic T lymphocytes (CTLs). The activation and maturation of DCs require the upregulation of a series of molecules that play a key role in co-stimulation and antigen presentation, such as CD80, CD86, CD83 and HLA-DR, as well as the release of cytokines that affect the type and intensity of immune responses. In vitro loading of mature DCs with tumor-associated antigens can induce the formation of CTLs with specific anti-tumor immune responses, thereby promoting anti-tumor immune responses.

[0003] Lingzhi (scientific name: Ganoderma lucidum (Curtis) P. Karst.) is a fungus of the Ganoderma family and the genus Ganoderma. Most of the fruiting bodies of Lingzhi are annual, and a few are perennial. It is a precious traditional Chinese medicinal material for strengthening the body and nourishing the body. It is reported that Lingzhi has unique effects in regulating immune function and inhibiting tumor growth.

[0004] Ganoderenic acid B (GAB) is a lanostane-type triterpenoid isolated from Ganoderma lucidum. Previous studies have reported that GAB can effectively reverse the multidrug resistance of the ABCB1-mediated hepatocellular carcinoma resistant cell line HepG2 / ADM to doxorubicin, vincristine, and paclitaxel. However, to date, there have been no reports on its application in promoting the proliferation and maturation of immune-related dendritic cells. Summary of the invention

[0005] The purpose of the present invention is to provide the use of ganoderic acid B in promoting the proliferation and maturation of dendritic cells. Ganoderic acid B can be used as an immune activator to improve immune activity by promoting the proliferation and maturation of dendritic cells.

[0006] To achieve the above object, the present invention adopts the following technical solution: A ganoderma lucidum extract having the activity of promoting the proliferation and maturation of dendritic cells; the ganoderma lucidum extract is ganoderic acid B, and its structural formula is: .

[0007] Furthermore, the concentration of the Ganoderma lucidum extract in promoting the proliferation and maturation of dendritic cells is 5 to 50 μM.

[0008] The above-mentioned Ganoderma lucidum extract is used to promote the proliferation and maturation of dendritic cells for non-disease diagnosis purposes.

[0009] The application of the ganoderma lucidum extract in the preparation of products for promoting the proliferation and maturation of dendritic cells.

[0010] Application of the ganoderma lucidum extract in the preparation of an immune activator.

[0011] The beneficial effects of the present invention are as follows: The results of the present invention reveal that GAB enhances the anti-tumor immune response of DCs by promoting the proliferation and maturation of DCs and enhancing the antigen uptake and presentation ability of DCs. In anti-tumor immunotherapy, enhancing the function of DCs is an important research direction. GAB can promote the proliferation and maturation of DCs, as well as enhance their antigen uptake and presentation ability, so it has a good application prospect in improving the body's immune surveillance and clearance ability of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 :The optimal concentration of GAB in promoting dendritic cell maturation. (A) Dendritic cells were treated with different concentration gradients of GAB, and the peak overlay of FITC-dextran uptake was detected by flow cytometry; (B) Quantitative statistics (A) Median fluorescence of FITC-dextran in dendritic cells of each group. n = 3, ** p<0.01, *** p<0.001 vs. Control.

[0013] Figure 2 :The effect of GAB on the endocytic capacity of dendritic cells in vitro. (A) Dendritic cells treated with GAB, Lysate, control and GAB+Lysate, flow cytometry detection of FITC-dextran uptake peak overlay; (B) Quantitative statistics (A) Median fluorescence of FITC-dextran in dendritic cells of each group. n = 3, ** p < 0.01 vs. Control.

[0014] Figure 3 :The results of GAB's effect on the maturation of dendritic cells in vitro.

[0015] Figure 4 :The results of GAB's effect on the NO secretion ability of dendritic cells in vitro. (A)NaNO 2Concentration detection standard curve; (B) Statistical analysis of NO concentration in dendritic cells of each group. n = 3, * p<0.05, *** p<0.001 vs. Control.

[0016] Figure 5 :The effect of GAB on the acid phosphatase activity in dendritic cells in vitro. (A) Standard curve of acid phosphatase activity detection; (B) Statistical analysis of acid phosphatase activity in dendritic cells of each group. n = 3, ** p<0.01, *** p<0.001 vs. Control.

[0017] Figure 6 :The results of GAB's effect on the proliferation and maturation of dendritic cells in mouse peripheral blood.

[0018] Figure 7 :The effect of GAB on the proliferation and maturation of dendritic cells in mouse spleen cells. DETAILED DESCRIPTION

[0019] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0020] The experimental materials involved in the following embodiments are as follows: Ganoderic acid B was purchased from Wuhan Tianzhi Biotechnology Co., Ltd., with item number CFN92991, batch number: CFS202301, and purity ≥98%; Ganoderic acid B was weighed and dissolved in DMSO to prepare a 100 mM Ganoderic acid B mother solution, and DCs complete medium was diluted to the corresponding concentration for use in the following Examples 1-5. Ganoderic acid B was weighed and dissolved in DMSO to prepare a 100 mg / mL Ganoderic acid B solution, and the required volume of Ganoderic acid B solution of the corresponding concentration was taken according to the group, and then PBS was used to make up the total volume to 200 μL for use in Example 6.

[0021] Ficoll Paque PREMIUM was purchased from cytiva, product number 17544202; X-VIVO 15 Serum-free cell culture medium was purchased from Lonza, catalog number 04-418Q; IL-4 was purchased from Sino Biological, catalog number GMP-11846-HNAE-L; GM-CSF was purchased from Sino Biological, catalog number GMP-10015-HNAH; trypsin was purchased from HyClone, catalog number SH30042.02; human ovarian cancer cell A2780 was purchased from Keygen, catalog number KGG3271-1; Griess Reagent kit was purchased from Beyotime, catalog number S0021S; acid phosphatase kit was purchased from Beyotime, catalog number P0326; CD11c-FITC, CD80-APC and CD86-PE were all purchased from BioLegend, catalog numbers 117306, 104714 and 105008, respectively; red blood cell lysis buffer was purchased from Solarbio, catalog number R1010; IP cell lysis buffer was purchased from Beyotime, catalog number: P0013; IP cell lysis buffer (without inhibitor) was purchased from Beyotime, catalog number: P0013J.

[0022] Example 1 Optimal concentration of GAB for promoting phenotypic maturation of dendritic cells Draw 10 mL of fresh human blood, centrifuge at 2000 rpm for 5 min, discard the supernatant, add an equal volume of phosphate buffered saline (PBS, pH7-7.4) to the lower layer of blood cells, mix well and transfer to a new 15 mL centrifuge tube. Take 4 mL of Ficoll PaquePREMIUM solution and slowly add it to the 15 mL centrifuge tube along the tube wall, centrifuge at 2000 rpm for 20 min, and adjust the centrifuge acceleration to 8 and the deceleration acceleration to 0. After centrifugation, it is divided into three layers. The peripheral blood mononuclear cells (PBMC) (containing DCs) in the middle white film layer are drawn into another new 15 mL centrifuge tube, washed twice with 10 mL PBS, centrifuged at 1000 rpm for 10 min each time, discard the supernatant, and collect the cell pellet. Take an appropriate amount of DCs complete medium to resuspend the above DCs cell pellet, take 1 mL of a concentration of 2×10 4 The cell suspension of 100 cells / mL was inoculated into a 6-well plate. Incubate at 37°C and 5% CO 2 After culturing in a constant temperature incubator for 2-3 hours, gently blow and remove the non-adherent cells, then add 2 mL of DCs complete medium, and then add DCs complete medium every 2-3 days. DCs complete medium: X-VIVO 15 serum-free cell culture medium + IL-4 with a final concentration of 75 ng / mL + GM-CSF with a final concentration of 60 ng / mL.

[0023] 37°C, 5% CO 2 After 4 days of culture in a constant temperature incubator, DCs were digested with 0.25% trypsin for 90 seconds and 1 mL of the solution was taken at a concentration of 2×10 4 Cell suspensions of 10 cells / mL were inoculated into 6-well plates, and DCs were divided into control group, 5 µM GAB group, 10 µM GAB group and 50 µM GAB group. The control group was incubated for 48 hours without treatment; the 5 µM GAB group was induced by adding GAB with a final concentration of 5 µM to DCs for 48 hours; the 10 µM GAB group was induced by adding GAB with a final concentration of 10 µM to DCs for 48 hours; the 50 µM GAB group was induced by adding GAB with a final concentration of 50 µM to DCs for 48 hours; After adding 0.25% trypsin for 90 seconds, the cells were centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, the cell pellet was collected, FITC-dextran of the corresponding final concentration was added and incubated at 37°C for 1 hour, and the cells were washed twice with PBS precooled at 4°C for 3 hours. Finally, 300 μL PBS was added to resuspend the cells and transferred to a flow tube for analysis by flow cytometry.

[0024] Immature DCs have strong antigen capture and endocytosis capabilities. After maturation, their endocytosis capabilities decrease, while their antigen capture capabilities increase. Figure 1 As shown in Figure 2, the flow cytometry results showed that 50 µM GAB had the most significant effect on reducing the endocytic ability of DCs (e.g. Figure 1 B).

[0025] Example 2 Effect of GAB on the Endocytosis Ability of Dendritic Cells Tumor cell lysate can induce maturation and activation of dendritic cells. Preparation of tumor cell lysate: Human ovarian cancer cell A2780 was cultured in RPMI1640 medium at 37°C and 5% CO 2 When the cell adhesion rate reached 80-90% in a constant temperature incubator, the cells were digested with 0.25% trypsin for 90 seconds and centrifuged at 1000 rpm for 2 minutes. The supernatant was discarded and the cell pellet was collected. The cells were washed with PBS precooled at 4°C for 3 hours at 10 °C for 10 minutes. 7 / mL was diluted to obtain a cell suspension. The cell suspension was frozen at -80℃ for 15 min, then thawed in a 37℃ water bath for 15 min, and repeated freezing and thawing for 3-5 times to fully lyse the cells to obtain Lysate. 1 µL of Lysate was taken to detect the protein concentration in Nanodrop.

[0026] DCs were obtained by the same extraction method as in Example 1, and 1 mL of the mixture was taken at a concentration of 2×10 4The cell suspension of 50 μg / mL was inoculated into 6-well plates, and DCs were divided into control group, GAB group, Lysate group, GAB+Lysate group and control group. The control group was incubated for 48 hours without treatment; the GAB group was induced by adding GAB with a final concentration of 50 μM to DCs for 48 hours; the Lysate group was induced by adding Lysate with a final concentration of 50 μg / mL to DCs for 48 hours; the GAB+Lysate group was induced by adding GAB with a final concentration of 50 μM and Lysate with a final concentration of 50μg / mL to DCs for 48 hours.

[0027] After adding 0.25% trypsin for 90 seconds, centrifuge at 2000 rpm for 5 minutes, discard the supernatant, collect the cell pellet, add the corresponding final concentration of FITC-dextran and incubate at 37 °C for 1 hour, wash twice with PBS precooled at 4 °C for 3 hours, and finally add 300 μL PBS to resuspend the cells and transfer them to a flow tube for analysis by flow cytometry. Figure 2 The results showed that both GAB and Lysate could reduce the endocytic ability of DCs, and the GAB+Lysate group had the most significant effect on reducing the endocytic ability of DCs (e.g. Figure 2 B).

[0028] Example 3 Effect of GAB on Phenotypic Maturation of Dendritic Cells DCs were obtained by the same extraction method as in Example 1, and 1 mL of the mixture was taken at a concentration of 2×10 4 The cell suspension of 50 μg / mL was inoculated into 6-well plates, and DCs were divided into control group, GAB group, Lysate group and GAB+Lysate group. The control group was incubated for 48 hours without treatment; the GAB group was induced by adding GAB with a final concentration of 50 μM to DCs for 48 hours; the Lysate group was induced by adding Lysate with a final concentration of 50 μg / mL to DCs for 48 hours; the GAB+Lysate group was induced by adding GAB with a final concentration of 50 μM and Lysate with a final concentration of 50μg / mL to DCs for 48 hours.

[0029] After 90s of 0.25% trypsin digestion, centrifuge at 2000 rpm for 5 min, discard the supernatant, collect the cell pellet, and then wash twice with PBS. Add 1.5 μL of anti-CD80-FITC, 1.5 μL of anti-CD83-PE antibody and 50 μL of PBS to each sample tube for labeling. After incubation at room temperature in the dark for 30 min, centrifuge at 2000 rpm for 5 min, discard the antibody mixture, wash twice with 0.5mL PBS, and finally add 300 μL PBS to resuspend the cells and transfer them to the flow tube, and then analyze them on the machine.

[0030] Flow cytometry was used to analyze the effect of GAB on the expression of co-stimulatory molecules and antigen-presenting molecules CD80 and CD83 associated with DCs maturation. Figure 3 As shown in the figure, compared with the control group, the expression of CD80 and CD83 in the GAB and Lysate groups was increased, and the increase in the expression of CD80 and CD83 was more significant under the combined action of GAB and Lysate, indicating that GAB has the effect of inducing DCs maturation.

[0031] Example 4 Effect of GAB on NO secretion ability of dendritic cells After obtaining DCs by the same extraction method as in Example 1, 1 mL of the solution with a concentration of 2×10 4 A cell suspension of 100 μL / mL was inoculated into a 6-well plate, and a control group, a GAB group, a Lysate group, and a GAB+Lysate group were set up. The concentration and action time were the same as in Example 2. After 90 seconds of 0.25% trypsin digestion, centrifugation was performed at 2000 rpm for 5 minutes, the supernatant was discarded, the cell pellet was collected, and the cell pellet was washed twice with PBS. Add 100 μL IP cell lysis buffer and lyse on ice for 30-40 minutes. During the lysis process, flick the cells at intervals to allow the cells to be fully lysed, and finally 4 groups of samples for determining the NO concentration were obtained. X-VIVO 15 culture medium was used to dilute the standard 1M NaNO 2 (Purchased from Biyuntian, catalog number: S0021S), the concentration gradient of the standard is 0, 1, 2, 5, 10, 20, 40, 60 μmol / L. Add 50 μL of sample or standard with different concentration gradient dilutions to each well of the 96-well plate, and then add 50 μL of Griess Reagent Ⅰ and 50 μL of Griess Reagent Ⅱ. Measure the absorbance at 540 nm, and calculate the NaNO in the sample according to the standard curve. 2 concentration, and finally NO 2 - The concentration was converted to NO concentration. GraphPad Prism 8.0 was used for plotting and analysis ( Figure 4 A).

[0032] The production and secretion of NO is another important indicator of DCs maturation. Figure 4 As shown in B, compared with the control group, the NO production of DCs increased after treatment with 50 μM GAB, further indicating that GAB can effectively induce the maturation of DCs. The NO production of DCs increased significantly under the combined action of GAB and Lysate.

[0033] Example 5 Effect of GAB on Acid Phosphatase Activity in Dendritic Cells DCs were obtained by the same extraction method as in Example 1, and 1 mL of the mixture was taken at a concentration of 2×10 4 A cell suspension of 100 μL / mL was inoculated into a 6-well plate, and a control group, a GAB group, a Lysate group, and a GAB+Lysate group were set up, and the action concentration and action time were the same as in Example 2. After trypsin treatment, the cells were collected by centrifugation at 2000 rpm for 5 min, the supernatant was removed, 100 μL IP cell lysis buffer (without inhibitor) was added to lyse the DCs, and the supernatant was taken as a sample by centrifugation at 5000 rpm for 5 min.

[0034] Take out the acid phosphatase kit and thaw it at room temperature. The specific steps are as follows: (1) Prepare the chromogenic substrate solution: Take a tube of chromogenic substrate, centrifuge it (to allow the powder to settle to the bottom to reduce concentration error), dissolve it in 2.5 mL of detection buffer, mix thoroughly, and place on ice for later use (the freshly prepared chromogenic substrate solution must be used within 6 hours).

[0035] (2) Prepare standard working solution: Take 10 μL of p-nitrophenol (PNP) solution (10 mM) and dilute it to a final concentration of 0.5 mM by adding detection buffer. Take 0, 4, 8, 16, 24, 32 and 40 μL of standard working solution respectively, and then add detection buffer to a total volume of 80 μL. Measure the absorbance at 405 nm to establish a standard curve.

[0036] (3) Set up blank control wells (40 μL detection buffer and 40 μL chromogenic substrate solution) and sample wells. Add 40 μL sample and 40 μL chromogenic substrate solution to each well of the 96-well plate. If the acid phosphatase activity in the sample is too high, reduce the sample volume or dilute it appropriately before measuring. Set up three replicate wells for each group. Mix by gently blowing with a pipette tip, or by using a shaker. Incubate at 37°C for 5-10 min. If the acid phosphatase activity in the sample to be tested is low, the incubation time can be appropriately extended to 30 min. Add 160 μL of reaction stop solution to each well to terminate the reaction. The acid phosphatase activity in the sample can be calculated by measuring the absorbance at 405 nm ( Figure 5 A).

[0037] Intracellular acid phosphatase (ACP) activity represents the ability of DCs to digest antigens. When the antigen presentation process is completed, DCs tend to mature and their ACP activity decreases. Compared with the control group, the ACP activity in DCs treated with 50 µM GAB decreased significantly. In particular, after GAB and Lysate co-stimulated DCs, their ACP activity was further reduced compared with GAB alone. The experimental results show that GAB has the effect of promoting the maturation of DCs (see Figure 5 B).

[0038] Example 6 Effects of GAB on the proliferation and maturation of dendritic cells in mice Female Balb / c mice aged 4-6 weeks (purchased from Wushi Experimental Animal Trading Co., Ltd.) were divided into control group, positive group, 20 mg / kg GAB group and 40 mg / kg GAB group. The total volume of drug administration for all groups was 200 μL, the control group was injected with 200 μL PBS, the positive group was administered with 80 mg / kg levamisole hydrochloride (solvent PBS), the 20 mg / kg GAB group was administered with 20 mg / kg GAB, and the 40 mg / kg GAB group was administered with 40 mg / kg GAB; the weight of mice in each group was weighed and recorded before administration, and the drug was administered according to the weight of the mice. According to the above drug concentration, intraperitoneal injection was performed every 3 days for a total of 14 days. After the end, the weight of the mice was measured, and blood was collected from the eyeballs and collected into anticoagulant tubes. Prepare a 10 mL centrifuge tube and mark it, transfer the blood to a new centrifuge tube, centrifuge at 2000 rpm for 5 min, discard the upper serum, collect the lower blood cells, and then add PBS equal to the volume of blood cells to mix. Add 4 mL of Ficoll PaquePREMIUM to a new 10 mL centrifuge tube, then use a Pasteur pipette to absorb the diluted blood cells and add them slowly against the tube wall, centrifuge at 2000 rpm for 20 minutes, and adjust the centrifuge acceleration to 8 and the deceleration to 0. After centrifugation, the layers are clearly separated. After absorbing the middle white film layer, wash it 3 times with 2 mL of PBS, and label it with flow cytometry antibodies (add 1.5 μL LCD11c-FITC, 1.5 μL LCD80-APC, 1.5 μL LCD86-PE and 50 μL PBS to each sample tube). After incubating at room temperature for 30 minutes in the dark, centrifuge at 2000 rpm for 5 minutes, collect the cell pellet, wash it twice with PBS, add 300 μL PBS to resuspend the cells and transfer them to the flow tube, and then analyze it on the machine ( Figure 6 ).

[0039] After blood collection, mice were killed by dislocating the neck. After disinfection, the abdominal cavity was cut open aseptically to remove the spleen of the mice. According to the groups (control group, positive group, 20 mg / kg GAB group and 40 mg / kg GAB group), they were placed in a small dish with 2 mL of normal saline, and the spleen was blown with a 1 mL syringe. After that, the blown spleen cells were collected into centrifuge tubes, centrifuged at 2000 rpm for 5 min, the supernatant was removed, the cell pellet was collected, 2 mL of red blood cell lysis buffer was added to each tube for lysis and standing for 2 min, 2-3 times the volume of red blood cell lysis buffer was added to terminate the reaction, centrifuged at 2000 rpm for 5 min, the supernatant was removed, the cell pellet was collected, and then the spleen cells were obtained after washing with PBS twice. Then the spleen cells were divided into tubes for flow cytometry antibodies (1.5 μL CD11c-FITC, 1.5 μL CD80-APC, 1.5 μL CD86-PE and 50 μL PBS were added to each sample tube). After incubation for 30 min in the dark, the cells were centrifuged at 2000 rpm for 5 min, washed twice with PBS, resuspended in 300 µL PBS and transferred to a flow cytometer for subsequent analysis ( Figure 7 ).

[0040] CD11c, CD80 and CD86 are surface markers of DCs. The expression of CD11c can be used to measure the proliferation of DCs, and the expression of CD80 and CD86 can be used to measure the maturity of DCs. Figure 6 A and Figure 7 As shown in A, GAB can promote the proliferation of peripheral blood DCs in vivo, although the promotion effect is slightly lower than that of the positive group. Figure 6 B and Figure 7 B) The maturity of peripheral blood DCs and spleen-derived DCs was higher than that of the control group, although slightly lower than that of the positive group. The experimental results showed that GAB can also promote the proliferation and maturation of DCs in vivo.

[0041] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A ganoderma lucidum extract, characterized in that: The ganoderma lucidum extract has the activity of promoting the proliferation and maturation of dendritic cells; the ganoderma lucidum extract is ganoderic acid B, and its structural formula is: 。 2. The Ganoderma lucidum extract according to claim 1, characterized in that: The concentration of the ganoderma lucidum extract in promoting the proliferation and maturation of dendritic cells is 5 to 50 μM.

3. Use of the Ganoderma lucidum extract as claimed in claim 1 in promoting the proliferation and maturation of dendritic cells for non-disease diagnosis purposes.

4. Use of the Ganoderma lucidum extract according to claim 1 in the preparation of a product for promoting the proliferation and maturation of dendritic cells.

5. Use of the Ganoderma lucidum extract as claimed in claim 1 in the preparation of an immune activator.

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