Method and index for in-vitro prejudgment of in-vivo immune regulation relationship of algal polysaccharides

Abstract

The invention discloses a method and an index for in-vitro prejudgment of an in-vivo immunoregulation relationship of algal polysaccharides, and researches show that algal polysaccharides CRVP can regulate interaction between macrophages and liver cancer cells. The CRVP promotes macrophages to be polarized into M1 type, plays a proinflammatory role, further affects the migration ability of the liver cancer cells, promotes the liver cancer cells to generate differential metabolites GABA, and induces apoptosis of the liver cancer cells at the same time. In addition, CRVP can also act on liver cancer cells, further influence the proliferation and phagocytic ability of macrophages, and induce the macrophages to be polarized into M1 type.

Description

Technical Field

[0001] The present invention relates to the technical field of polysaccharides, and in particular to a method and index for predicting the in vivo immune regulation relationship of seaweed polysaccharides in vitro. Background Art

[0002] Polysaccharides can induce apoptosis and cause tumor cell death, thereby exerting direct anti-tumor activity. Many active polysaccharides derived from microorganisms can induce apoptosis of tumor cells through endogenous pathways dependent on cysteine ​​proteases, such as: proteoglycans and polysaccharide fragment D from Grifola frondosa induce apoptosis of gastric cancer SCG-7901 and breast cancer MCF-7 cells; proteoglycan PSP from Trametes versicolor induces apoptosis of leukemia HL-60 cells; -glucan from Pleurotus ostreatus induces apoptosis of colon cancer HT-29 cells; polysaccharides from Cordyceps sinensis induce apoptosis of liver cancer HepG2 and neuroblastoma SK-N-SH cells. In addition, some active polysaccharides derived from microorganisms can induce apoptosis of tumor cells through exogenous pathways dependent on cysteine ​​proteases, such as: polysaccharides from Fucus vesiculosus induce apoptosis of colon cancer HT-29, HCT116, HCT-15, Lewis lung cancer cells and mouse melanoma B16 cells. The current understanding of the anti-tumor immunomodulatory effects of polysaccharides can be summarized as follows: (1) Promoting the release of cytokines by immune cells; (2) Enhancing the ability of immune cells to phagocytize and destroy cancer cells; and (3) Enhancing the production of chemical messengers such as nitric oxide.

[0003] The tumor microenvironment is a complex microecological system that develops around tumor cells and is an important place for the occurrence, progression and metastasis of tumors. The tumor immune microenvironment is mainly composed of cellular components such as tumor associated macrophages (TAMs), tumor associated neutrophils, tumor infiltrating lymphocytes, and non-cellular components such as extracellular matrix and cytokines. The immune microenvironment plays an important role in tumor progression, immune escape and treatment resistance. TAMs are an important type of immune cells in the tumor microenvironment. Some cytokines in the tumor microenvironment cause undifferentiated macrophages (M0) to have two polarized phenotypes, classical activation (M1) and alternative activation (M2), in different environments. The former can kill tumor cells, microorganisms and promote inflammation, while the latter has the effects of promoting tumor growth and inhibiting inflammation.

[0004] GABA is a major neurotransmitter widely present in the central nervous system (CNS), responsible for transmitting signals between neurons. Generally speaking, GABA is synthesized by neuronal cells. In neuronal cells, glutamate is converted into GABA by glutamate decarboxylase (GAD).

[0005] Caulerparacemosa varpeltata belongs to the genus Caulerparacemosa and is a tropical and subtropical green algae. Some literature mentions that Caulerparacemosa varpeltata polysaccharides have immunomodulatory and anti-tumor effects. Our laboratory has previously demonstrated that seaweed polysaccharides can activate the immune response of RAW264.7 cells. However, whether seaweed polysaccharides can affect the proliferation and apoptosis of liver cancer cells by regulating the immune response of macrophages, and whether liver cancer cells change the state of the tumor immune microenvironment by expressing some signaling molecules as the tumor progresses, thereby regulating the polarization direction of macrophages, in order to provide a certain experimental basis and theoretical basis for the study of seaweed polysaccharides in vivo through immunomodulatory anti-tumor effects. This study also focused on the effect of GABA on TAMs. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for predicting the immune regulation relationship of seaweed polysaccharides in vivo in vitro, the method comprising:

[0007] Step 1: Preparation of seaweed polysaccharides,

[0008] Step 2: Obtain the immune regulation index 1 of seaweed polysaccharides on macrophages, analyze the direct effect of seaweed polysaccharides on macrophages to induce macrophage M1 polarization, and produce the metabolic difference product asparagine Asn;

[0009] Obtain the immune regulation index 2 of seaweed polysaccharides on liver cancer cells, and analyze the direct effect of seaweed polysaccharides on liver cancer cells to limit the migration of liver cancer cells;

[0010] Obtain the immune regulation index 3 of macrophage conditioned medium RAW-CM on liver cancer cells, analyze the effect of mediators produced by macrophages activated by seaweed polysaccharides on the apoptosis of liver cancer cells, and produce differential metabolites γ-aminobutyric acid GABA;

[0011] Obtain the macrophage immunoregulatory index 4 of liver cancer cell conditioned medium Hepa-CM, and analyze the mediator produced by liver cancer cells activated by seaweed polysaccharides to induce macrophage M1 polarization;

[0012] Step 3: Analyze and compare the relationships among immunoregulatory index 1, immunoregulatory index 2, immunoregulatory index 3 and immunoregulatory index 4, derive the immunoregulatory relationship among seaweed polysaccharides, macrophages and liver cancer cells, and predict the immunoregulatory relationship of seaweed polysaccharides in vivo.

[0013] Furthermore, the seaweed polysaccharide is made from C. racemosa var peltata, which is extracted with water, filtered, concentrated, the supernatant is taken, precipitated with alcohol, protein is removed, dialyzed in a dialysis bag with a molecular weight cutoff of 8KDa, and freeze-dried to obtain the seaweed polysaccharide.

[0014] The preparation steps of the seaweed polysaccharide are as follows:

[0015] S1. Mixed material extraction: Take naturally dried Pteris racemosus shield leaf variant as raw material, crush and mix evenly with distilled water at a solid-liquid ratio of 1:30 (g / mL), stir evenly, and extract at 60-85°C for 2-3 hours, repeat the extraction twice to obtain an extract solution;

[0016] S2. Filtration and concentration: The extract obtained in step S1 was filtered through gauze, and the filtrate was rotary evaporated at 65°C to 5% of the original volume. The concentrate was centrifuged at 12000 rpm / min for 10 min, and the supernatant was collected;

[0017] S3. Alcohol precipitation: anhydrous ethanol was added to the supernatant obtained in step S2 to a final ethanol concentration of 75%, and then placed in a refrigerator at 4°C overnight for alcohol precipitation, and the precipitate was collected by centrifugation;

[0018] S4. Removal of free protein: add water to the precipitate to redissolve it, then add ammonium sulfate until saturated, then add tert-butyl alcohol, stir thoroughly on a magnetic stirrer, then centrifuge, collect the lower layer of liquid, and repeat the protein removal operation until the middle layer disappears;

[0019] S5. Dialysis freeze-drying: The precipitate obtained by alcohol precipitation is re-dissolved in water and transferred to a dialysis bag with a molecular weight cut-off of 8KDa for dialysis. The retentate is taken out and placed in a vacuum freeze dryer for freeze-drying to obtain a seaweed polysaccharide sample.

[0020] The seaweed polysaccharide in this study was isolated and purified by our laboratory and named CRVP. The mouse macrophage cell line RAW264.7 and the liver cancer cell line Hepal-6 were preserved by our laboratory.

[0021] Seaweed polysaccharides are derived from C. racemosa varpeltata, and the voucher specimen (No.: 20170408) is preserved in the Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University.

[0022] The seaweed polysaccharide is extracted by water extraction and alcohol precipitation, which is a commonly used separation and purification step. Our laboratory also determined that the total sugar content of CRVP is 66%.

[0023] At present, there are a large number of literature reports on the anti-tumor activity of polysaccharides, but there is no report that the polysaccharide of Pteris racemosus var. peltolacca can regulate the interaction between the immune system and tumors. The present invention uses Pteris racemosus var. peltolacca as a raw material to extract polysaccharides, which are used as a new regulator for activating the body's immune system to exert anti-tumor effects, in order to provide a certain experimental basis and theoretical basis for the study of seaweed polysaccharides in the body through immune regulation to fight tumors.

[0024] Furthermore, the immune regulation index 1 includes: seaweed polysaccharide on the proliferation activity of macrophages, NO release, phagocytic ability, secreted cytokine amount, M1 marker transcription level and cytokine transcription level, and differential metabolite asparagine Asn index;

[0025] The immunomodulatory index 2 includes: the proliferation activity of seaweed polysaccharides on liver cancer cells, cell migration ability and the amount of secreted cytokines;

[0026] The immunomodulatory index 3 includes: the proliferation activity of macrophage conditioned medium RAW-CM on liver cancer cells, cell migration ability, the amount of secreted cytokines, the differential metabolite γ-aminobutyric acid GABA, the effect of apoptosis inhibitor Z-VAD-FMK on liver cancer cells, the expression of apoptosis proteins, and the index of Annexin V-FITC / PI double staining analysis;

[0027] The immunoregulatory index 4 includes: indicators of the proliferation activity of liver cancer cell conditioned medium Hepa-CM on macrophages, NO release, phagocytic ability, secreted cytokine amount, and M1 marker transcription level and cytokine transcription level.

[0028] Furthermore, the immune regulation index 1 is upregulated, and seaweed polysaccharides directly act on macrophages, induce macrophage M1 polarization, enhance macrophage proliferation activity, NO release, phagocytic ability, and release of M1 type markers, and simultaneously produce differential metabolites asparagine Asn, exerting the anti-tumor effect of seaweed polysaccharides;

[0029] The downregulation of the migration ability of liver cancer cells in the immune regulation index 2, seaweed polysaccharides directly act on liver cancer cells, restrict the migration of liver cancer cells, and exert the anti-tumor effect of seaweed polysaccharides;

[0030] The immune indicator 3, the medium produced by macrophages activated by seaweed polysaccharides, inhibits the proliferation activity and cell migration ability of liver cancer cells, and at the same time produces differential metabolites γ-aminobutyric acid GABA, which regulates the expression of apoptotic proteins, induces apoptosis of liver cancer cells, and exerts the anti-tumor effect of seaweed polysaccharides;

[0031] The immune index 4 is upregulated, and the mediator produced after seaweed polysaccharides act on liver cancer cells induces macrophage M1 polarization, enhances macrophage proliferation activity, NO release, phagocytic ability, and release of M1 type markers, and exerts the anti-tumor effect of seaweed polysaccharides;

[0032] The concentration of seaweed polysaccharides in the immunoregulatory index 1, immunoregulatory index 2, immunoregulatory index 3 and immunoregulatory index 4 is 80-320 μg / mL;

[0033] The seaweed polysaccharide in vivo immune regulation relationship is a bidirectional regulatory relationship in which the seaweed polysaccharide mediates the polarized immune response of macrophages M1 to induce apoptosis of liver cancer cells, and the seaweed polysaccharide mediates the immune response of liver cancer cells to induce the polarization of macrophages M1.

[0034] Furthermore, the macrophage conditioned medium RAW-CM is obtained by collecting the secretion fluid obtained by treating macrophages with seaweed polysaccharides, centrifuging, taking the supernatant, and mixing it with the complete medium in a ratio of 1:1;

[0035] The liver cancer cell conditioned medium Hepa-CM is obtained by collecting the secretion fluid obtained by treating liver cancer cells with seaweed polysaccharides, centrifuging, taking the supernatant, and mixing it with the complete medium in a ratio of 1:1;

[0036] The complete culture medium is high-glucose DMEM+10% fetal bovine serum+1% double antibody (v / v).

[0037] Furthermore, the expression index of apoptosis protein in the immune regulation index 3 was analyzed and compared, and the effects of complete culture medium, complete culture medium added with 320 μg / mL seaweed polysaccharide, macrophage conditioned medium RAW-CM with 0 seaweed polysaccharide and macrophage conditioned medium RAW-CM with 320 μg / mL seaweed polysaccharide on the apoptosis of liver cancer cells were analyzed and compared.

[0038] Macrophages are one of the most important immune cells in the natural immune system and play an important role in clearing tumor cells. Activated macrophages can enhance immune responses, and the activity of macrophages can be used as an indicator of immunomodulatory effects. Therefore, RAW264.7 macrophages were used to explore the immunomodulatory effects of seaweed polysaccharides. First, the present invention used the CCK-8 kit to explore whether seaweed polysaccharides can activate macrophages. Studies have shown that seaweed polysaccharides can activate RAW264.7 cells, and when the concentration reaches 320 μg / mL, the cell viability reaches the maximum value. In subsequent experiments, 80-320 μg / mL of seaweed polysaccharides were selected to treat macrophages.

[0039] One of the most notable characteristics of activated macrophages is the increase in phagocytic ability. Therefore, the phagocytic ability of macrophages treated with seaweed polysaccharides was tested by neutral red phagocytosis experiment. It was found that compared with the control group, seaweed polysaccharides can significantly enhance the phagocytic ability of macrophages, and the higher the concentration of seaweed polysaccharides, the stronger the phagocytic ability of macrophages, which promotes cell proliferation and phagocytosis.

[0040] In addition, activated macrophages can secrete various cytokines and promote the expression of TNF-α in cells; induce RAW264.7 cells to secrete proinflammatory factors and upregulate the expression of proinflammatory genes.

[0041] Activated macrophages are widely divided into M1 macrophages (anti-tumor) and M2 macrophages (pro-tumor). CD86 is a highly glycosylated transmembrane protein. In the study of macrophages, it is often used as a detection indicator for macrophage M1 polarization markers. The results not only showed that macrophages stimulated by seaweed polysaccharides have anti-tumor effects, but also that seaweed polysaccharides can promote the polarization of M1 macrophages. At the same time, liver cancer cell conditioned medium Hepa-CM can also promote the M1 polarization of macrophages, exerting the anti-tumor effect of macrophages. At the same time, seaweed polysaccharides directly act on macrophages to produce differential metabolites asparagine Asn, and various cytokines secreted by asparagine Asn also have anti-tumor effects.

[0042] The present invention explores whether the medium produced by macrophages activated by seaweed polysaccharides, RAW264.7 cell conditioned medium, can inhibit the growth of tumor cells. The inhibition of tumor cell growth is reflected in two aspects: inhibiting cell proliferation and inducing cell apoptosis. RAW264.7 cell conditioned medium can inhibit the proliferation of liver cancer cells, reduce cell viability, inhibit cell migration, increase cell apoptosis rate, and promote the expression of apoptotic protein Bax. Seaweed polysaccharides stimulate RAW264.7 cells to produce an immune response, induce the secretion and expression of inflammatory cytokines, inhibit the proliferation of liver cancer cells, and induce cell apoptosis.

[0043] Studies have shown that not only can seaweed polysaccharides directly induce macrophage polarization to M1 type, but seaweed polysaccharides can also act on the conditioned medium of liver cancer cells, that is, the medium produced by liver cancer cells activated by seaweed polysaccharides, to induce macrophage polarization to M1 type. Macrophages polarized to M1 type play a pro-inflammatory role, thereby affecting the migration ability of liver cancer cells and promoting liver cancer cells to produce differential metabolites GABA. GABA metabolism activates GABAB receptors, enhances the expression of pro-apoptotic protein Bax, reduces the expression of anti-apoptotic protein Bcl2, and activates Caspase3, thereby promoting apoptosis of liver cancer cells.

[0044] At the same time, not only can seaweed polysaccharides directly act on liver cancer cells and limit the migration of liver cancer cells, seaweed polysaccharides act on the conditioned medium of macrophages, that is, the medium produced by macrophages activated by seaweed polysaccharides, promoting liver cancer cells to produce differential metabolites asparagine Asn, and asparagine Asn promotes the apoptosis of liver cancer cells.

[0045] In summary, the present invention studies the interaction between macrophages and liver cancer cells regulated by seaweed polysaccharide CRVP. Specifically, CRVP promotes macrophage polarization to M1 type, exerts a pro-inflammatory effect, thereby affecting the migration ability of liver cancer cells, and promotes liver cancer cells to produce differential metabolites GABA, while inducing apoptosis of liver cancer cells. In addition, CRVP can also act on liver cancer cells, further affecting the proliferation and phagocytic ability of macrophages, and inducing their polarization to M1 type.

[0046] The second aspect of the present invention studies the use of a group of immune indicators in the preparation and evaluation of the effect of seaweed polysaccharides on macrophage M1 polarization, wherein the immune indicators are immune regulation indicators 1 including: seaweed polysaccharides on macrophage proliferation activity, NO release, phagocytic ability, secreted cytokine amount, M1 marker transcription level and cytokine transcription level, and differential metabolite asparagine Asn index;

[0047] The transcription levels of the differential metabolites asparagine Asn and M1 type markers increased after seaweed polysaccharides directly acted on macrophages.

[0048] The third aspect of the present invention studies the use of a group of immune indicators in the preparation of an evaluation of the effect of mediators produced by macrophages activated by seaweed polysaccharides on apoptosis of liver cancer cells, wherein the immune indicators are immune regulation indicators 3 including: macrophage conditioned medium RAW-CM on the proliferation activity of liver cancer cells, cell migration ability, the amount of secreted cytokines, differential metabolites γ-aminobutyric acid GABA, the effect of apoptosis inhibitor Z-VAD-FMK on liver cancer cells, the expression of apoptotic proteins, and indicators of Annexin V-FITC / PI double staining analysis;

[0049] The differential metabolite γ-aminobutyric acid (GABA) increased after macrophage conditioned medium RAW-CM acted on liver cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a diagram showing the effect of the seaweed polysaccharide on the proliferation of RAW264.7 cells;

[0051] Figure 2 This is the effect of the seaweed polysaccharide on NO release from RAW264.7 cells;

[0052] Figure 3 This is a diagram showing the effect of the seaweed polysaccharide on the phagocytic ability of RAW264.7 cells;

[0053] Figure 4 This is a graph showing the effect of the seaweed polysaccharide on the secretion of IL-6, TNF-α, and IL-1β by RAW264.7 cells;

[0054] Figure 5This is a diagram showing the effect of the seaweed polysaccharide on the proliferation of Hepa1-6 cells;

[0055] Figure 6 This is a diagram showing the effect of the seaweed polysaccharide on Hepa1-6 cell migration;

[0056] Figure 7 This is a graph showing the effect of the seaweed polysaccharide on the secretion of IL-6, TNF-α, and IL-1β by Hepa1-6 cells;

[0057] Figure 8 This is a diagram showing the effect of macrophage conditioned medium on the proliferation of liver cancer cells;

[0058] Fig. 9 This is a diagram showing the effect of macrophage conditioned medium on the migration of liver cancer cells;

[0059] Fig.10 This is a diagram showing the effect of macrophage conditioned medium on the secretion of IL-6, TNF-α, and IL-1β by liver cancer cells;

[0060] Fig.11 The effect of liver cancer cell conditioned medium on macrophage proliferation;

[0061] Fig.12 This is a graph showing the effect of liver cancer cell conditioned medium on the release of NO by macrophages;

[0062] Fig.13 This is a diagram showing the effect of liver cancer cell conditioned medium on the phagocytic ability of macrophages;

[0063] Fig.14 This is a diagram showing the effect of the liver cancer cell conditioned medium on the secretion of IL-6, TNF-α, and IL-1β by macrophages;

[0064] Fig.15 Targeted amino acid metabolomics

[0065] Fig.16 Figure 2 shows the effect of death inhibitors on the vitality of liver cancer cells.

[0066] Fig.17 This is the result of western blot detection. The concentration of seaweed polysaccharide is 80, 160, 320 μg / mL

[0067] Fig.18 Annexin V-FITC / PI double staining flow cytometry detection of cell death

[0068] Fig.19 Western blot was used to measure the protein level. Correlation between macrophage conditioned medium-induced hepatocellular carcinoma cell apoptosis and GABA

[0069] Fig. 20 RT-qPCR was used to evaluate the expression of transcriptional levels of apoptosis-related targets in liver cancer cells after RAW-CM was applied

[0070] Fig.21 RT-qPCR was used to evaluate the expression of M1 markers and cytokine transcription levels in macrophages after CRVP treatment

[0071] Fig. 22 RT-qPCR evaluation of the expression of M1 markers and cytokine transcription levels after Hepa-CM treatment of macrophages DETAILED DESCRIPTION

[0072] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0073] The above is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited to the above content. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0074] Embodiment 1:

[0075] 1. The preparation steps of seaweed polysaccharide are as follows:

[0076] S1. Mixed material extraction: Take naturally dried Pteris racemosus shield leaf variant as raw material, crush it and mix it evenly with distilled water at a solid-liquid ratio of 1:30 (g / mL), stir evenly, and extract at 85°C for 3 hours, repeat the extraction twice;

[0077] S2. Filtration and concentration: The extract obtained in step S1 was filtered through gauze, and the filtrate was rotary evaporated at 65°C to 5% of the original volume. The concentrate was centrifuged at 12000 rpm / min for 10 min, and the supernatant was collected;

[0078] S3. Add anhydrous ethanol to the supernatant obtained in step S2 until the final ethanol concentration is 75%, and then place in a refrigerator at 4°C overnight for alcohol precipitation. Collect the precipitate by centrifugation;

[0079] S4. Remove free protein: Add an appropriate amount of grade tertiary water to the precipitate for redissolution. Add ammonium sulfate to the redissolved polysaccharide-protein solution until saturated, then add an equal volume of tert-butyl alcohol. Use the three-phase method to remove protein. Stir well on a magnetic stirrer for 60 minutes, then centrifuge at 5000rpm / min for 10 minutes. After centrifugation, it is divided into three layers: the upper layer is the tert-butyl alcohol organic layer, the middle layer is the protein layer, and the lower layer is ammonium sulfate and polysaccharide. Collect the lower layer liquid and repeat the protein removal operation until the middle layer disappears;

[0080] S5. Dialysis freeze-drying: The lower layer of liquid was reconstituted with water and transferred to a dialysis bag with a molecular weight cutoff of 8KDa for dialysis. During the dialysis process, tap water was first used for 48 hours, and then laboratory grade 3 water (conductivity ≦0.50mS / m) was used for 24 hours. The dialysis water was replaced every four hours. Finally, the retentate was taken out and placed in a vacuum freeze dryer for freeze drying to obtain a crude polysaccharide sample of Pteris racemosus peltata variant. The seaweed polysaccharide was named CRVP.

[0081] Embodiment 2:

[0082] Experimental materials: Mouse-derived mononuclear macrophage RAW264.7 cells and mouse-derived liver cancer cell Hepa1-6 cells were preserved for this experiment.

[0083] RAW264.7 cells were cultured in DMEM medium with 10% (v / v) fetal bovine serum and 1% (v / v) dual antibody added to serve as the complete culture medium for RAW264.7 cells.

[0084] The complete culture medium formula of Hepa1-6 cells is high-glucose DMEM+10% fetal bovine serum+1% double antibody (v / v).

[0085] Preparation of RAW264.7 cell conditioned medium (RAW-CM): RAW264.7 cells were cultured with complete culture medium for 24 hours, and the supernatant culture medium was discarded after 24 hours. Different concentrations of CRVP samples (concentrations were 20μg / mL, 40μg / mL, 80μg / mL, 160μg / mL, and 320μg / mL) were added. A blank control group (con group) was set up at the same time. Cells and complete culture medium were added to the blank control group. The cells were placed in an incubator and cultured for 24 hours. After 24 hours, the supernatant was collected, centrifuged at 3000rpm for 5 minutes, and the supernatant: fresh culture medium = 1:1 was made into conditioned medium, and stored at -20°C.

[0086] Preparation of Hepa1-6 cell conditioned medium (Hepa-CM): Hepa1-6 cells were cultured with complete culture medium for 24 hours, and the supernatant culture medium was discarded after 24 hours. Different concentrations of CRVP samples (concentrations were 20μg / mL, 40μg / mL, 80μg / mL, 160μg / mL, and 320μg / mL) were added. A blank control group (con group) was set up at the same time. Cells and complete culture medium were added to the blank control group. The cells were placed in an incubator and cultured for 24 hours. After 24 hours, the supernatant was collected and centrifuged at 3000rpm for 5 minutes. The supernatant: fresh culture medium = 1:1 was made into conditioned medium and stored at -20°C.

[0087] 2. Effect of CRVP on the proliferation of RAW264.7 cells

[0088] CCK-8 kit was used to measure the effect of CRVP on the proliferation of RAW264.7 cells. The density of RAW264.7 cells was adjusted to 1.0×10 5 / mL, and cultured in an incubator for 24 hours. After 24 hours, the supernatant culture medium was discarded, and different concentrations of CRVP samples (concentrations were 20μg / mL, 40μg / mL, 80μg / mL, 160μg / mL, and 320μg / mL) were added. At the same time, a blank control group (con group) was set up, and cells and complete culture medium were added to the blank control group. It was placed in an incubator for another 24 hours, and 10μL CCK-8 reagent was added to each well. After incubation in the dark for 1 hour, the absorbance was recorded at a wavelength of 450nm using an enzyme reader.

[0089] The experimental results are as follows Figure 1 As shown. Figure 1 It can be seen that compared with the blank control group (con group), CRVP can promote the proliferation of RAW264.7 cells in the concentration range of 10-160 μg / mL, and the effect of promoting proliferation is better as the concentration increases. When the CRVP concentration is higher than 320 μg / mL, the proliferation effect decreases, which may be caused by excessive cell proliferation competing for nutrients or excessive production of metabolites. However, within the concentration range of 10-320 μg / mL, the overall rate is not less than 100%, indicating that CRVP is non-toxic to RAW264.7 cells within this concentration range.

[0090] 3. Effect of CRVP on NO release in RAW264.7 cells

[0091] When CRVP acts on RAW264.7 cells, it can induce the occurrence of a variety of cellular immune responses. As an active substance in the body, NO release is an important indicator for determining whether the immunoregulatory activity of RAW264.7 cells is enhanced. The NO kit was used to determine the effect of seaweed polysaccharide disease-resistant inducer CRVP on the release of NO from RAW264.7 cells. RAW264.7 cells were cultured with complete culture medium for 24 hours. After 24 hours, the supernatant culture medium was discarded, and CRVP-samples (concentrations of 80μg / mL, 160μg / mL, and 320μg / mL, respectively) were added. At the same time, a blank control group (con group) and a positive control group (LPS group) were set up. The blank control group was added with an equal volume of complete culture medium, and the positive control group was added with an equal volume of 1μg / mL LPS solution. The cells were placed in an incubator for 24 hours, the supernatant was collected, and the NO release was detected using a NO kit.

[0092] Depend on Figure 2It can be seen that compared with the blank control group (con group), different concentrations of CRVP (80μg / mL, 160μg / mL, and 320μg / mL) treatment groups can significantly promote the release of NO (p<0.01), and show a dose-dependent manner, but it is still less than the NO release of the positive control group (LPS group). The significant increase in NO release indicates that CRVP has a strong immunostimulatory effect on RAW264.7 cells.

[0093] 4. Effect of CRVP on the phagocytic ability of RAW264.7 cells

[0094] Take macrophage RAW264.7 in the logarithmic growth phase, set up a negative control group and a sample group to be tested. After the cells adhere to the wall, discard the original culture medium, add different concentrations of CRVP (0, 20, 40, 80, 160, 320 μg / mL) and LPS (1 μg / mL) drug-containing complete culture medium, and culture for 24 hours. Discard the culture medium and incubate with 100 μL 0.1% neutral red solution for 1 hour. Wash three times with PBS to remove excess neutral red dye, add 100 μL cell lysis solution (glacial acetic acid: anhydrous ethanol-1:1, v / v) to each well, incubate at room temperature for 2 hours, and then use an enzyme reader to detect the OD value at 540nm.

[0095] like Figure 3 As shown in the figure, after 24h of CRVP treatment, the phagocytic ability of RAW264.7 cells increased in a dose-dependent manner under treatment with different concentrations of CRVP (20, 40, 80 and 160 μg / mL) compared with the blank control group. When the CRVP concentration was higher than 160 μg / mL, the phagocytic ability decreased, but it was not lower than 100% overall, and was significantly different from the blank control group, indicating that CRVP can enhance the phagocytic ability of RAW264.7 cells within this concentration range.

[0096] 5. Effect of CRVP on cytokine secretion in RAW264.7 cells

[0097] When CRVP acts on RAW264.7 cells, it can induce the occurrence of a variety of cellular immune responses and achieve immunomodulatory effects by promoting the production of cytokines by cells. RAW264.7 cells are stimulated by CRVP to promote the secretion of cytokines, such as tumor necrosis factor (TNF) and interleukin (IL), which can regulate the body's immune function to a certain extent and play an important role in immune responses; lipopolysaccharide (LPS) is the main component of the cell wall of Gram-negative bacteria, which is toxic to the host and has the effect of inducing cells to produce immune responses. It is often used as a positive control for the evaluation of immunomodulatory activity. The ELISA kit was used to determine the effect of CRVP on the release of cytokines by RAW264.7 cells. RAW264.7 cells were cultured with complete culture medium for 24 hours. After 24 hours, the supernatant culture medium was discarded, and different concentrations of CRVP samples were added, with concentrations of 80, 160 and 320 μg / mL. A blank control group (con group) and a positive control group (LPS group) were set up at the same time. The blank control group was added with an equal volume of complete culture medium, and the positive control group was added with an equal volume of 1 μg / mL LPS solution. The cells were placed in an incubator and cultured for another 24 hours, and the supernatant was collected. The secretion of TNF-α, IL-1β and IL-6 was detected using TNF-α, IL-1β and IL-6 kits, respectively.

[0098] like Figure 4 As shown in the figure, compared with the blank control group, the secretion of cytokines (TNF-α, IL-1β and IL-6) increased in a dose-dependent manner under the treatment of different concentrations of CRVP (80, 160 and 320 μg / mL). After 24 hours of CRVP treatment, RAW264.7 cells significantly promoted the secretion of TNF-α compared with the blank control group, and it was dose-dependent. Among them, under the treatment of different concentrations of CRVP (80, 160 and 320 μg / mL), the secretion of TNF-α was extremely significantly different from that of the blank control group (p<0.01). After 24 hours of CRVP treatment, there was no significant difference in the secretion of IL-1β of RAW264.7 cells under the intervention of CRVP (80, 160 μg / mL) compared with the blank control group, and there was a significant difference in the secretion of IL-1β of RAW264.7 cells under the intervention of 320 μg / mL compared with the blank control group (p<0.05). Compared with the blank control group, RAW264.7 cells significantly promoted the secretion of IL-6, and it was dose-dependent. Among them, under the treatment of different concentrations of CRVP (80, 160 and 320 μg / mL), the secretion of IL-6 was significantly different from that of the blank control group (p<0.01). The above results show that seaweed polysaccharide anti-tumor immunomodulator (CRVP) can significantly promote the secretion of cytokines by RAW264.7 cells, that is, seaweed polysaccharide anti-tumor immunomodulator (CRVP) can affect the cellular immune response.

[0099] Embodiment 3:

[0100] 6. Effect of CRVP on the proliferation of Hepa1-6 cells

[0101] The CCK-8 kit was used to determine the effect of CRVP on the proliferation of Hepa1-6 cells. Hepa1-6 cells were cultured with complete culture medium for 24 hours. After 24 hours, the supernatant culture medium was discarded and different concentrations of CRVP samples were added (the concentrations were 20μg / mL, 40μg / mL, 80μg / mL, 160μg / mL, and 320μg / mL). At the same time, a blank control group (con group) was set up, and cells and complete culture medium were added to the blank control group. Then, the cells were placed in an incubator and cultured for 48 hours. 10μL of CCK-8 reagent was added to each well. After incubation in the dark for 1 hour, the absorbance was recorded at a wavelength of 450nm using an enzyme marker.

[0102] The experimental results are as follows Figure 5 As shown. Figure 5 It can be seen that compared with the blank control group (con group), CRVP had no significant effect on the proliferation of Hepa1-6 cells within the concentration range of 10-320 μg / mL.

[0103] 7. Effect of CRVP on Hepa1-6 cell migration

[0104] The effect of CRVP on the migration ability of Hepa1-6 cells was detected by scratch test. A blank group and a CRVP treatment group were set up, and the treatment methods of each group were as follows: serum-free medium (complete medium without fetal bovine serum) was added to the blank group, and serum-free medium with a CRVP concentration of 320 μg / mL was added to the CRVP treatment group. The 6-well plate was turned over to the back and the position for fixed-point photography was marked with a marker pen. Then the cells were inoculated into the 6-well plate at a density of 500,000 per well, 2 mL of complete medium was added to each well, and the cells were placed in an incubator and cultured for 24 hours until the cell state was stable. A 10ul gun tip was used to scratch a blank scratch area on the cell layer at the bottom of the well plate, keeping the force consistent, discarding the old medium and washing with PBS 2-3 times, and then serum-free medium was added according to the treatment method of each group, placed in an incubator, and fixed-point photography was taken at 0, 12, and 24 hours to record the changes in the blank scratch area. The obtained images were processed by the software ImageJ v1.53t, the edge of the scratch area was marked with a red curve, and the area A of the scratch area was measured. The scratch area (A0) at 0h was used as a reference for each position, and the scratch closure rate at 12h and 24h was calculated, that is, scratch closure rate (%) = (A0–A) / A0. Figure 6As shown in the figure, the red curve is the edge of the scratch area drawn by the software ImageJ v1.53t. It can be seen that more cells in the con group migrate to the blank scratch area over time, and the red curve gradually becomes tortuous. Under the treatment of 320μg / mL CRVP, there are fewer cells in the scratch area at 24h compared with the con group, and the red curve is relatively straight. The area of ​​the scratch area was calculated by the software ImageJ v1.53t, and it was found that the scratch closure rate of the Control group at 24h was about 26%, while the CRVP treatment group was only about 16%, a difference of about 1.5 times. The above experiments show that CRVP can inhibit the migration of Hepa1-6 cells.

[0105] 8. Effect of CRVP on cytokine secretion in Hepa1-6 cells

[0106] As an important component of the immune system interaction network and tumor microenvironment, cytokines regulate inflammation and immune responses and play an indispensable role in tumorigenesis and invasion. Excessive secretion of proinflammatory cytokines (such as interleukin (IL)-2, IL-6, tumor necrosis factor (TNF-α and IL-1β)) can cause severe inflammation and induce chronic autoimmune diseases such as rheumatoid arthritis. Persistent inflammation can aggravate malignant tumors, and proinflammatory cytokines reflect the strong correlation between the inflammatory environment and cancer. ELISA kits were used to determine the effect of CRVP on the release of cytokines from Hepa1-6 cells. Hepa1-6 cells were cultured with complete culture medium for 24 hours. After 24 hours, the supernatant culture medium was discarded and different concentrations of CRVP samples were added, with concentrations of 80, 160 and 320 μg / mL. A blank control group (con group) and a positive control group (LPS group) were set up at the same time. The blank control group was added with an equal volume of complete culture medium, and the positive control group was added with an equal volume of 1 μg / mL LPS solution. The cells were then placed in an incubator for culture for 48 hours, and the supernatant was collected. The secretion of TNF-α, IL-1β and IL-6 was detected using TNF-α, IL-1β and IL-6 kits, respectively.

[0107] like Figure 7 As shown, compared with the blank control group, the CRVP treatment group did not significantly promote the secretion of TNF-α and IL-6. When the CRVP concentration was 160 μg / mL, it could significantly promote the secretion of IL-1β.

[0108] Embodiment 4:

[0109] 9. Effect of RAW264.7 cell conditioned medium on Hepa1-6 cell proliferation

[0110] The most basic characteristic of cancer cells is that they can proliferate indefinitely, so the activity screening of drugs on cancer cells generally first determines whether they can inhibit cell proliferation. The CCK-8 method is used to detect cell viability values ​​to indicate the percentage of live cells in total cells. Hepa1-6 cells were cultured with complete culture medium for 24 hours. After 24 hours, the supernatant culture medium was discarded, and different concentrations of macrophage conditioned medium (RAW-CM) were added. After 48 hours of culture, the OD value was detected at 450nm.

[0111] like Figure 8 As shown in the figure, after 48 hours of CRVP treatment, the proliferation of Hepa1-6 cells was inhibited in a dose-dependent manner under treatment with different concentrations of RAW264.7 cell conditioned medium compared with the blank control group, and there was a significant difference. Therefore, it can be seen that macrophage conditioned medium (RAW-CM) has an inhibitory effect on liver cancer cells.

[0112] 10. Effect of RAW264.7 cell conditioned medium on Hepa1-6 cell migration

[0113] The effect of RAW-CM on the migration ability of Hepa1-6 cells was detected by scratch test. RC group and RT treatment group were set up, and the treatment methods of each group were as follows: RC group was added with macrophage conditioned medium without drug administration (CRVP), and RT treatment group was added with macrophage conditioned medium with CRVP concentration of 320μg / mL. The measurement method was the same as in 7. Fig. 9 As shown in the figure, the red curve is the edge of the scratch area drawn by the software ImageJ v1.53t. It can be seen that more cells in the RC group migrate to the blank scratch area over time, and the red curve gradually becomes tortuous. In the RT group, there are fewer cells in the scratch area at 24 hours than in the RC group, and the red curve is relatively straight. The area of ​​the scratch area was calculated by the software ImageJ v1.53t, and it was found that the scratch closure rate of the RC group at 24 hours was about 28%, while the RT group was only about 16%, a difference of about 1.7 times. The above experiments show that the conditioned medium after CRVP acts on macrophages can inhibit the migration of Hepa1-6 cells.

[0114] 11. Effect of RAW-CM on cytokine secretion in Hepa1-6 cells

[0115] ELISA kits were used to determine the effect of RAW-CM on the release of cytokines from Hepa1-6 cells. Hepa1-6 cells were cultured with complete culture medium for 24 hours, the supernatant was discarded after 24 hours, different concentrations of RAW-CM were added, and the cells were placed in an incubator for 48 hours. The supernatant was collected and the secretion of TNF-α, IL-1β and IL-6 was detected using TNF-α, IL-1β and IL-6 kits, respectively. Fig.10 As shown in Figure 2, since RAW-CM is prepared in a 1:1 ratio, the amount of cytokine secretion in it is half of that in the original supernatant. Figure 4 In contrast, RAW-CM did not significantly promote the secretion of cytokines in liver cancer cells after acting on liver cancer cells.

[0116] Embodiment 5:

[0117] 12. Effect of Hepa-CM on the proliferation of RAW264.7 cells

[0118] The CCK-8 kit was used to determine the effect of Hepa-CM on the proliferation of RAW264.7 cells. RAW264.7 cells were cultured with complete culture medium for 24 hours. After 24 hours, the supernatant culture medium was discarded and Hepa-CM treated with different concentrations of CRVP was added. Six replicate wells were set up in each group. The cells were then placed in an incubator for 24 hours, and 10 μL of CCK-8 reagent was added to each well. After incubation in the dark for 1 hour, the absorbance was recorded at a wavelength of 450 nm using an enzyme reader.

[0119] The experimental results are as follows Fig.11 As shown. Fig.11 It can be seen that compared with the Hepa-CM treatment group without CRVP, Hepa-CM after CRVP treatment of Hepa1-6 cells can promote the proliferation of RAW264.7 cells, and the effect of promoting proliferation is better with the increase of concentration. It is speculated that after CRVP acts on liver cancer cells, the secreted mediators cause the proliferation of macrophages.

[0120] 13. Effect of Hepa-CM on NO release from RAW264.7 cells

[0121] The NO kit was used to determine the effect of Hepa-CM on NO release from RAW264.7 cells. RAW264.7 cells were cultured with complete culture medium for 24 hours, the supernatant was discarded after 24 hours, and different concentrations of Hepa-CM were added. The cells were then placed in an incubator and cultured for another 24 hours, the supernatant was collected, and the NO kit was used to detect NO release.

[0122] Depend on Fig.12 It can be seen that compared with the Hepa-CM treatment group without CRVP, Hepa-CM after CRVP treatment of Hepa1-6 cells can promote the release of NO in RAW264.7 cells. And there is a significant difference. The significant increase in NO release indicates that Hepa-CM has a strong immunostimulatory effect on RAW264.7 cells.

[0123] 14. Effect of Hepa-CM on the phagocytic ability of RAW264.7 cells

[0124] Take macrophage RAW264.7 in the logarithmic growth phase and culture them. After the cells adhere to the wall, discard the original culture medium and add different concentrations of Hepa-CM for 24 hours. Discard the culture medium and incubate with 100μL 0.1% neutral red solution for 1 hour. Wash three times with PBS to remove excess neutral red dye, add 100μL cell lysis solution (glacial acetic acid: anhydrous ethanol = 1:1, v / v) to each well, incubate at room temperature for 2 hours, and then use an enzyme marker to detect the OD value at 540nm.

[0125] like Fig.13 As shown, after 24 hours of Hepa-CM treatment, compared with the Hepa-CM treatment group without CRVP, Hepa-CM after CRVP treatment of Hepa1-6 cells at concentrations (160μg / mL, 320μg / mL) can promote the phagocytic ability of RAW264.7 cells, and there is a significant difference with the blank control group. The phagocytic index decreases above 320μg / mL, but is not lower than 100%. Therefore, it shows that Hepa-CM can enhance the phagocytic ability of RAW264.7 cells within this concentration range.

[0126] 15. Effect of Hepa-CM on cytokine secretion of RAW264.7 cells

[0127] ELISA kits were used to determine the effect of Hepa-CM on cytokine secretion by RAW264.7 cells. RAW264.7 cells were cultured with complete culture medium for 24 hours, and the supernatant culture medium was discarded after 24 hours. Different concentrations of Hepa-CM were added. The cells were cultured in an incubator for another 24 hours, and the supernatant was collected. The secretion of TNF-α, IL-1β and IL-6 was detected using TNF-α, IL-1β and IL-6 kits, respectively.

[0128] like Fig.14 As shown in the figure, after the RAW264.7 cells were treated with Hepa-CM, there was no obvious secretion of IL-6, but TNF-α promoted it in a dose-dependent manner. Compared with the blank group, IL-1β showed significant difference under the action of Hepa-CM at a concentration of 320 μg / mL. Figure 4 By comparing the analysis of cytokines in RAW264.7 cells after CRVP treatment, it can be inferred that Hepa-CM contains very little or even no CRVP. Therefore, Hepa-CM after CRVP treatment of liver cancer cells can promote the release of TNF-α in RAW264.7 cells, and under the action of Hepa-CM at a concentration of 320μg / mL, it can promote the release of IL-1β in RAW264.7 cells.

[0129] Embodiment 6:

[0130] 16. Targeted amino acid metabolomics analysis

[0131] More than 5 mL of culture medium of adherent cells was aspirated, centrifuged at 1000 g and 4 °C for 1 min, all supernatant was collected, quick-frozen in liquid nitrogen for 15 min, and stored in a -80 °C refrigerator. Ultra-high performance liquid chromatography-tandem mass spectrometry was used to detect and collect data and analyze changes in extracellular metabolites.

[0132] The data obtained from the analysis of targeted amino acid metabolomics were subjected to differential expression analysis. After the data were preprocessed by Paretoscaling, unsupervised and supervised multidimensional statistical analysis were performed using PCA and OPLS-DA. The ratio of metabolite abundance in the two groups of biological samples was calculated according to the fold change (FC) to evaluate and quantify metabolic changes. Metabolites with both multidimensional statistical analysis log2(FC)>1 and univariate statistical analysis p value<0.05 were selected as metabolites with significant differences.

[0133] like Fig.15 As shown, the differential metabolites obtained by comparing the 320μg / mL polysaccharide treatment group (RT) of macrophages and the 0 polysaccharide control group (RC) of macrophages are Asn, GABA, Asp, Glu, etc. Among them, the metabolite with log2(FC)>1 and p value<0.05 is asparagine (Asn). The differential metabolites obtained by comparing the 320μg / mL polysaccharide macrophage conditioned medium treatment group (HT) of liver cancer cells and the 0 polysaccharide macrophage conditioned medium control group (HC) of liver cancer cells are GABA, Asp, Ser, Glu, etc. Among them, the metabolite with log2(FC)>1 and p value<0.05 is γ-aminobutyric acid (GABA).

[0134] 17. Effects of death inhibitors on the viability of liver cancer cells

[0135] The CCK-8 kit was used to determine the effect of cell death inhibitors on the proliferation of Hepa1-6 cells. Hepa1-6 cells were cultured with complete culture medium for 24 hours, and the supernatant culture medium was discarded after 24 hours. The treatment methods included: the blank group was added with complete culture medium; the CRVP treatment group was added with complete culture medium with a CRVP concentration of 320μg / mL; the cell death inhibitor group was added with apoptosis inhibitor Z-VAD-FMK (0.4μM), ferroptosis inhibitor Fer-1 (0.2μM), and ferroptosis inhibitor Lip-1 (0.1μM); the CRVP+cell death inhibitor group was added with complete culture medium with a CRVP concentration of 320μg / mL, and Z-VAD-FMK (0.4μM), ferroptosis inhibitor Fer-1 (0.2μM), and ferroptosis inhibitor Lip-1 (0.1μM) were added. Six replicate wells were set for each group. The cells were then placed in an incubator and cultured for 48 h. 10 μL CCK-8 reagent was added to each well. After incubation in the dark for 1 h, the absorbance was recorded at a wavelength of 450 nm using an enzyme reader.

[0136] like Fig.16 As shown, the CRVP+ apoptosis inhibitor Z-VAD-FMK group had a certain rescue effect on the death of liver cancer cells compared with the CRVP treatment group, while the ferroptosis inhibitor Fer-1 and the ferroptosis inhibitor Lip-1 had no obvious effect, indicating that the death of liver cancer cells induced by CRVP polysaccharide belongs to the apoptosis mode of Z-VAD-FMK.

[0137] 18. Western blot to measure protein levels

[0138] Western blot was used to determine the protein levels of Bcl2, Bax and Caspase3 in liver cancer cells Hepa1-6. The treatment methods included: the blank group was added with complete medium, and the macrophage conditioned medium treatment group was added with complete medium with CRVP concentrations of 80, 160, and 320 μg / mL.

[0139] (1) Seeding: Macrophages were seeded at a density of 400,000 per well; liver cancer cells were seeded at a density of 200,000 per well.

[0140] (2) Adding drugs: discard the old culture medium in the macrophages, add complete culture medium with CRVP concentrations of 80, 160, and 320 μg / mL, respectively, and treat in an incubator for 24 hours. Collect the macrophage supernatant, centrifuge at 3000 rpm for 5 minutes, and make the supernatant: fresh culture medium = 1:1 to make conditioned medium. Discard the old culture medium of liver cancer cells, add macrophage conditioned medium, and place in an incubator for 48 hours. Protein extraction and denaturation, electrophoresis and membrane transfer, blocking and antibody incubation, development and image processing.

[0141] like Fig.17 As shown in the results, the expression of anti-apoptotic protein Bcl2 in liver cancer cells Hepa1-6 was reduced, the expression of pro-apoptotic protein Bax was increased, and the level of Caspase3, the main execution gene in cell apoptosis, was upregulated. It can be inferred that macrophage conditioned medium induced Hepa1-6 cell apoptosis.

[0142] 19. Annexin V-FITC / PI double staining flow cytometry detection of cell death

[0143] Annexin V-FITC / PI apoptosis detection kit was used, and flow cytometry was used to verify the way in which macrophage conditioned medium induced Hepa1-6 cell death. The treatment methods include:

[0144] (1) Seeding plate: macrophages were seeded at a density of 400,000 per well; hepatoma cells were seeded at a density of 200,000 per well;

[0145] (2) Adding drugs: discard the old culture medium in the macrophages, add complete culture medium with CRVP concentrations of 80, 160, and 320 μg / mL, respectively, and treat in an incubator for 24 hours. Collect the macrophage supernatant, centrifuge at 3000 rpm for 5 minutes, and make the supernatant: fresh culture medium = 1:1 to make conditioned medium. Discard the old culture medium of liver cancer cells, add macrophage conditioned medium, and place in an incubator for 48 hours;

[0146] (3) Cell sample preparation: Collect the cell culture medium into a centrifuge tube for later use. Digest the cells with trypsin, add the collected cell culture medium for about 1 minute, blow off all the adherent cells, and collect them into a centrifuge tube. Centrifuge the cells at about 1000 rpm, remove the supernatant, add 1 ml of 4°C pre-cooled PBS, resuspend the cells, centrifuge again to precipitate the cells, and remove the supernatant;

[0147] (4) Dilute the Binding Buffer with deionized water at a ratio of 1:9.

[0148] (5) Resuspend the cells in 1x binding buffer and adjust the concentration to 1-5×10 6 / ml;

[0149] (6) Take 100 μl of cell suspension into a 5 ml flow cytometry tube, add 5 μl of Annexin V / FITC, mix well, and incubate at room temperature in the dark for 5 min;

[0150] (7) Add 5 μl of PI and 400 μl of PBS, and immediately perform flow cytometry. The flow cytometry data were processed and plotted using FlowJov10.3 software.

[0151] like Fig.18As shown in the figure, the cells will be divided into three subgroups: Q2 area is late apoptotic or necrotic cells (Annexin V-FITC + / PI + ), Q3 area is early apoptotic cells (Annexin V-FITC + / PI - ), Q4 area is normal cells (Annexin V-FITC - / PI - ), most of the points in the flow cytometry scatter plot of the Control group were located in the Q4 area, and were highly concentrated in a certain area. After treatment with 320μg / mL CRVP, the points in the Q4 area gradually dispersed to the Q2 and Q3 areas. After counting the proportion of cells in the Q4 area, we found that the number of normal cells decreased from 88.6% in the Control group to 68% in the 320μg / mL concentration group. This shows that Hepa1-6 induced by macrophage conditioned medium undergoes apoptosis.

[0152] 20. Western blot to measure protein levels

[0153] Western blot was used to determine the protein levels of Bcl2, Bax, and Caspase3 in liver cancer cells Hepa1-6. The determination method was the same as above, and the treatment methods included: CON added complete medium, CRVP group added complete medium with 320μg / mL CRVP, RC group added RAW-CM with 0 CRVP, and RT group added RAW-CM with 320μg / mL CRVP. The metabolites of these four groups were compared, and the metabolite with difference was GABA, and the group with the highest expression was the RT group.

[0154] (1) Seeding: Macrophages were seeded at a density of 400,000 per well; liver cancer cells were seeded at a density of 200,000 per well.

[0155] (2) Adding drugs: discard the old culture medium in the macrophages, add complete culture medium with CRVP concentrations of 80, 160, and 320 μg / mL, respectively, and treat in an incubator for 24 hours. Collect the macrophage supernatant, centrifuge at 3000 rpm for 5 minutes, and make conditioned medium with supernatant: fresh culture medium = 1:1. Discard the old culture medium of liver cancer cells, add complete culture medium to CON, add complete culture medium with 320 μg / mL CRVP to CRVP group, add RAW-CM with 0 CRVP to RC group, and add RAW-CM with 320 μg / mL CRVP to RT group. Culture for 48 hours, protein extraction and denaturation, electrophoresis and membrane transfer, blocking and antibody incubation, development and image processing. The results are shown in Fig.19As shown, the expression of anti-apoptotic protein Bcl2 in the RT group of Hepa1-6 liver cancer cells was reduced, the expression of pro-apoptotic protein Bax was increased, and the level of Caspase3, the main execution gene in cell apoptosis, was upregulated. It can be inferred that the apoptosis of liver cancer cells induced by macrophage conditioned medium may be related to GABA.

[0156] 21. RT-qPCR evaluation of the expression of transcriptional levels of apoptosis-related targets in liver cancer cells after RAW-CM treatment

[0157] RT-qPCR was used to evaluate the effect of RAW-CM on the transcription levels of Bcl2, Bax, Caspase3 and GABAB type receptor subunit 1 (Gabbr1) after treatment of liver cancer cells.

[0158] (1) Seeding plate: macrophages were seeded at a density of 400,000 per well; hepatoma cells were seeded at a density of 200,000 per well;

[0159] (2) Adding drugs: discard the old culture medium in the macrophages, add complete culture medium with CRVP concentrations of 80, 160, and 320 μg / mL, respectively, and treat in an incubator for 24 hours. Collect the macrophage supernatant, centrifuge at 3000 rpm for 5 minutes, and make the supernatant: fresh culture medium = 1:1 to make conditioned medium. Discard the old culture medium of liver cancer cells, add macrophage conditioned medium, and place in an incubator for 48 hours;

[0160] (3) Primer design and synthesis: According to the name and species of the target gene, qPCR primers were designed using Primer Premier6 software. No specific amplification was found after comparison using the Primer-Blast tool in NCBI. In this study, all primers were synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. The primer sequences are shown in Table 1.

[0161] Table 1 Primer sequences for real-time fluorescence quantitative PCR amplification

[0162]

[0163]

[0164] (4) RNA extraction: RNA was extracted using TRlzol reagent.

[0165] (5) Reverse transcription: using Yisheng III 1st Strand cDNA Synthesis SuperMix forqPCR kit product. According to the kit instructions, the reverse transcription system was configured as shown in Table 2, and the reverse transcription was performed using the standard reverse transcription program settings recommended in the kit instructions.

[0166] Table 2 Reverse transcription reaction system

[0167]

[0168] (6) Real-Time PCR reaction: using Hieff Universal Blue qPCR SYBRGreen Master Mix Kit product. The configured real-time fluorescence quantitative PCR system is shown in Table 3 below, using the PCR reaction fast program.

[0169] Table 3 Real-time fluorescence quantitative PCR reaction system

[0170]

[0171] The relative quantitative method was used to calculate the gene expression multiple, with β-Actin as the internal reference, and the results were expressed as relative expression, with the formula 2 -ΔΔCt The results are as follows Fig. 20 As shown in the results, in the liver cancer cells Hepa1-6, the transcriptional expression of Bcl2 was reduced, and the transcriptional expression of Bax, Caspase3, Gabbr1 and increased. The transcriptional expression of Bcl2, Bax, and Caspase3 was consistent with the results of WB. Some studies have shown that the activation of GABAB receptors can inhibit the migration of cancer cells and induce apoptosis of cancer cells. Based on this, it can be speculated that the apoptosis of liver cancer cells induced by macrophage conditioned medium may be through GABA metabolism to activate GABAB receptors, enhance the expression of pro-apoptotic protein Bax, reduce the expression of anti-apoptotic protein Bcl2, and activate Caspase3, thereby promoting the apoptosis of liver cancer cells.

[0172] 22. RT-qPCR evaluation of the expression of M1 markers and cytokine transcription levels in macrophages after CRVP

[0173] RT-qPCR was used to evaluate the effect of CRVP on the transcriptional levels of cytokines TNF-α, IL-1β and IL-6 and M1 macrophage marker CD86. The treatment methods included: the blank group was added with complete culture medium, and the treatment group was added with complete culture medium at CRVP concentrations of 80, 160, and 320 μg / mL.

[0174] (1) Seeding: Macrophages were seeded at a density of 400,000 per well;

[0175] (2) Administration: Complete culture medium was added to the blank group, and complete culture medium with CRVP concentrations of 80, 160, and 320 μg / mL was added to the treated group for 24 h.

[0176] like Fig.21As shown, CRVP can increase the transcription levels of TNF-α, IL-1β, IL-6 and CD86 in macrophages, that is, CRVP can induce macrophage polarization to M1 type and release pro-inflammatory factors.

[0177] 23. RT-qPCR evaluation of the expression of M1 markers and cytokine transcription levels after Hepa-CM treatment of macrophages

[0178] RT-qPCR was used to evaluate the effect of Hepa-CM on the transcriptional levels of cytokines TNF-α, IL-1β and IL-6 and M1 macrophage marker CD86 in RAW264.7 macrophages. RAW264.7 cells were cultured with complete culture medium for 24 hours, the supernatant was discarded, different concentrations of Hepa-CM were added, and the cells were cultured in an incubator for another 24 hours. RNA was extracted, reverse transcribed, and Real-Time PCR was performed. The results are shown in Fig. 22 As shown in the figure, Hepa-CM can increase the transcription levels of TNF-α, IL-1β, IL-6 and CD86 in macrophages, which means that Hepa-CM can induce macrophage polarization to M1 type and release pro-inflammatory factors to a certain extent.

Claims

1. A method for predicting the immune regulation relationship of seaweed polysaccharides in vivo in vitro, characterized in that: The method comprises: Step 1: preparing seaweed polysaccharides; Step 2: Obtain the immune regulation index 1 of seaweed polysaccharides on macrophages, analyze the direct effect of seaweed polysaccharides on macrophages to induce macrophage M1 polarization, and produce the metabolic difference product asparagine Asn; Obtain the immune regulation index 2 of seaweed polysaccharides on liver cancer cells, and analyze the direct effect of seaweed polysaccharides on liver cancer cells to limit the migration of liver cancer cells; Obtain the immune regulation index 3 of macrophage conditioned medium RAW-CM on liver cancer cells, analyze the effect of mediators produced by macrophages activated by seaweed polysaccharides on the apoptosis of liver cancer cells, and produce differential metabolites γ-aminobutyric acid GABA; Obtain the macrophage immunoregulatory index 4 of liver cancer cell conditioned medium Hepa-CM, and analyze the mediator produced by liver cancer cells activated by seaweed polysaccharides to induce macrophage M1 polarization; Step 3: Analyze and compare the relationships among immunoregulatory index 1, immunoregulatory index 2, immunoregulatory index 3 and immunoregulatory index 4, derive the immunoregulatory relationship among seaweed polysaccharides, macrophages and liver cancer cells, and predict the immunoregulatory relationship of seaweed polysaccharides in vivo.

2. The method for predicting the in vitro immune regulation relationship of seaweed polysaccharides in vivo according to claim 1, characterized in that: The immunomodulatory index 1 includes: seaweed polysaccharide on the proliferation activity of macrophages, NO release, phagocytic ability, secreted cytokine amount, M1 marker transcription level and cytokine transcription level, and the index of differential metabolite asparagine Asn; The immunomodulatory index 2 includes: the proliferation activity of seaweed polysaccharides on liver cancer cells, cell migration ability and the amount of secreted cytokines; The immunomodulatory index 3 includes: the proliferation activity of macrophage conditioned medium RAW-CM on liver cancer cells, cell migration ability, the amount of secreted cytokines, the differential metabolite γ-aminobutyric acid GABA, the effect of apoptosis inhibitor Z-VAD-FMK on liver cancer cells, the expression of apoptosis proteins, and the index of Annexin V-FITC / PI double staining analysis; The immunoregulatory index 4 includes: indicators of the proliferation activity of liver cancer cell conditioned medium Hepa-CM on macrophages, NO release, phagocytic ability, secreted cytokine amount, and M1 marker transcription level and cytokine transcription level.

3. The method for predicting the in vitro immune regulation relationship of seaweed polysaccharides in vivo according to claim 2, characterized in that: The immune regulation index 1 is upregulated, and seaweed polysaccharides directly act on macrophages, induce macrophage M1 polarization, enhance macrophage proliferation activity, NO release, phagocytic ability, and release of M1 type markers, and simultaneously produce differential metabolites asparagine Asn, exerting the anti-tumor effect of seaweed polysaccharides; The downregulation of the migration ability of liver cancer cells in the immune regulation index 2, seaweed polysaccharides directly act on liver cancer cells, restrict the migration of liver cancer cells, and exert the anti-tumor effect of seaweed polysaccharides; The immune indicator 3, the medium produced by macrophages activated by seaweed polysaccharides, inhibits the proliferation activity and cell migration ability of liver cancer cells, and at the same time produces differential metabolites γ-aminobutyric acid GABA, which regulates the expression of apoptotic proteins, induces apoptosis of liver cancer cells, and exerts the anti-tumor effect of seaweed polysaccharides; The immune index 4 is upregulated, and the mediator produced after seaweed polysaccharides act on liver cancer cells induces macrophage M1 polarization, enhances macrophage proliferation activity, NO release, phagocytic ability, and release of M1 type markers, and exerts the anti-tumor effect of seaweed polysaccharides; The concentration of seaweed polysaccharides in the immunoregulatory index 1, immunoregulatory index 2, immunoregulatory index 3 and immunoregulatory index 4 is 80-320 μg / mL; The seaweed polysaccharide in vivo immune regulation relationship is a bidirectional regulatory relationship in which the seaweed polysaccharide mediates the polarized immune response of macrophages M1 to induce apoptosis of liver cancer cells, and the seaweed polysaccharide mediates the immune response of liver cancer cells to induce the polarization of macrophages M1.

4. The method for predicting the in vitro immune regulation relationship of seaweed polysaccharides in vivo according to claim 2, characterized in that: The macrophage conditioned medium RAW-CM is obtained by collecting the secretion fluid obtained by treating macrophages with seaweed polysaccharides, centrifuging, taking the supernatant, and mixing it with the complete medium in a ratio of 1:1; The liver cancer cell conditioned medium Hepa-CM is obtained by collecting the secretion fluid obtained by treating liver cancer cells with seaweed polysaccharides, centrifuging, taking the supernatant, and mixing it with the complete medium in a ratio of 1:1; The complete culture medium is high-glucose DMEM+10% fetal bovine serum+1% double antibody (v / v).

5. The method for predicting the in vitro immune regulation relationship of seaweed polysaccharides in vivo according to claim 4, characterized in that: The expression index of apoptosis protein in the immune regulation index 3 is analyzed and compared, and the effects of complete culture medium, complete culture medium added with 320 μg / mL seaweed polysaccharide, macrophage conditioned medium RAW-CM with 0 seaweed polysaccharide and macrophage conditioned medium RAW-CM with 320 μg / mL seaweed polysaccharide on the apoptosis of liver cancer cells are analyzed and compared.

6. The method for predicting the in vitro immune regulation relationship of seaweed polysaccharides in vivo according to claim 1, characterized in that: The seaweed polysaccharide is prepared from C.racemosa varpeltata as raw material, and is subjected to water extraction, filtration, concentration, taking the supernatant, alcohol precipitation, protein removal, dialysis in a dialysis bag with a molecular weight cutoff of 8KDa, and freeze-drying to obtain the seaweed polysaccharide.

7. The method for predicting the in vitro immune regulation relationship of seaweed polysaccharides in vivo according to claim 6, characterized in that: The preparation steps of the seaweed polysaccharide are as follows: S1. Mixed material extraction: Take naturally dried Pteris racemosus shield leaf variant as raw material, crush and mix evenly with distilled water at a solid-liquid ratio of 1:30 (g / mL), stir evenly, and extract at 60-85°C for 2-3 hours, repeat the extraction twice to obtain an extract solution; S2. Filtration and concentration: The extract solution obtained in step S1 was filtered through gauze, and the filtrate was rotary evaporated at 65°C to 5% of the original volume. The concentrate was centrifuged at 12000 rpm / min for 10 min, and the supernatant was collected; S3. Alcohol precipitation: anhydrous ethanol was added to the supernatant obtained in step S2 to a final ethanol concentration of 75%, and then placed in a refrigerator at 4°C overnight for alcohol precipitation, and the precipitate was collected by centrifugation; S4. Removal of free protein: add water to the precipitate to redissolve it, then add ammonium sulfate until saturated, then add tert-butyl alcohol, stir thoroughly on a magnetic stirrer, then centrifuge, collect the lower layer of liquid, and repeat the protein removal operation until the middle layer disappears; S5. Dialysis freeze-drying: The precipitate obtained by alcohol precipitation is re-dissolved in water and transferred to a dialysis bag with a molecular weight cut-off of 8KDa for dialysis. The retentate is taken out and placed in a vacuum freeze dryer for freeze-drying to obtain a seaweed polysaccharide sample.

8. Application of a group of immune indicators in the preparation and evaluation of the effect of seaweed polysaccharides on macrophage M1 polarization, characterized in that: The immune index is an immune regulation index 1 including: seaweed polysaccharide on macrophage proliferation activity, NO release, phagocytic ability, secreted cytokine amount, M1 marker transcription level and cytokine transcription level, and differential metabolite asparagine Asn index; The transcription levels of the differential metabolites asparagine Asn and M1 type markers increased after seaweed polysaccharides directly acted on macrophages.

9. Application of a group of immune indicators in the preparation of a method for evaluating the effect of mediators produced by macrophages activated by seaweed polysaccharides on apoptosis of liver cancer cells, characterized in that: The immune index is immune regulation index 3 including: the proliferation activity of macrophage conditioned medium RAW-CM on liver cancer cells, cell migration ability, the amount of secreted cytokines, the differential metabolite γ-aminobutyric acid GABA, the effect of apoptosis inhibitor Z-VAD-FMK on liver cancer cells, the expression of apoptosis proteins, and the index of AnnexinV-FITC / PI double staining analysis; The differential metabolite γ-aminobutyric acid (GABA) increased after macrophage conditioned medium RAW-CM acted on liver cancer cells.

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