Immunoregulation application of rhizoma bletillae polysaccharide based on NF-kappa B signal channel

Abstract

The invention relates to the field of plant extraction, in particular to immunoregulation application of rhizoma bletillae polysaccharide based on an NF-kappa B signal channel. The rhizoma bletillae polysaccharide is applied to regulation of protein expression of p65, p-p65, p-I kappa B alpha, COX-2 and iNOS in an NF-kappa B signal channel; the rhizoma bletillae polysaccharide is obtained by treating rhizoma bletillae through a water extraction and alcohol precipitation method. After the rhizoma bletillae polysaccharide intervenes RAW264.7 macrophages, the cell value-added rate is 90% or above. The immunomodulatory activity of the macrophage RAW264.7 is researched on the basis of the macrophage RAW264.7. Specifically, related cell factors generated by macrophages intervened by a target polysaccharide component are detected by adopting an enzyme linked immunosorbent assay kit, so that the immunocompetence of the polysaccharide component is verified. On the other hand, whether the bletilla striata polysaccharide generates NF-kappa B-based protein or not is verified by adopting WB, and a foundation is laid for subsequent research on the immune anti-inflammatory effect of the bletilla striata-polysaccharide.

Description

Technical Field

[0001] The invention relates to the field of plant extraction (IPC classification number: C12P), and in particular to an immunomodulatory application of bletilla striata polysaccharide based on NF-κB signaling pathway. Background Art

[0002] Natural polysaccharides are widely distributed in nature, and more and more studies have shown that polysaccharides have certain immunomodulatory activity, and other pharmacological effects of natural polysaccharides, such as anti-cancer and anti-tumor, lowering blood sugar, and anti-cell aging, are directly or indirectly achieved through immunomodulation to a certain extent. Existing studies have found that plant polysaccharides can stimulate the proliferation of macrophages, lymphocytes, natural killer cells and other immune cells in living organisms, either alone or together, and release cellular inflammatory factors and other substances to activate the cellular immunity of organisms, thereby directly or indirectly promoting humoral immune operations and the immune response of living tissues and organs. Therefore, while polysaccharides enhance the body's natural immune mechanism against pathogens, they also treat the body's immune damage to a certain extent. However, the effective role of polysaccharides in the immune mechanism is not yet clear.

[0003] The existing technology has studied the immune mechanism and found that activated macrophages mainly increase the secretion of IL-6 (interleukin 6), IL-β (interleukin β), TNF-α (tumor necrosis factor α), IFN-γ (interferon γ) and other cytokines, thereby regulating or promoting the production of immune effects. Among them, IL-β, IL-6 and other cytokines are mainly involved in regulating the recruitment of peripheral macrophages of diseased cells such as tumor cells in the body, directly inducing cellular immune responses, or enhancing or inducing the simultaneous occurrence of other innate and acquired immunity in the body through the expression of related cytokines, so they are important regulators of host defense responses; TNF-α is an important host regulatory molecule with tumor necrosis activity, and can further activate a variety of immune cells in the body on the original basis, thereby strengthening the body's immune response; IFN is a type of glycoprotein that can induce tumor cell apoptosis, and plays an important regulatory role in the body's cellular immunity and humoral immunity, and the polysaccharide component can promote macrophages to secrete IFN-γ and enhance the body's immune response. Therefore, detecting the relevant cytokines produced by macrophages after intervention with the target polysaccharide component can verify the immune activity of the polysaccharide component.

[0004] Chinese patent CN102641284B discloses the application of a spirulina polysaccharide, and Chinese patent CN105535011A discloses the application of a shiitake mushroom polysaccharide, but there are still differences in the immunomodulatory activity of polysaccharides from different sources, and the existing technology is still in the stage of exploration for polysaccharides from different sources. As a type of traditional Chinese medicine with the functions of promoting blood circulation, stopping bleeding, promoting tissue regeneration and promoting healing, the polysaccharide content in Bletilla striata can be as high as 50% or more, but there are currently very few studies on the immunomodulatory activity of Bletilla striata polysaccharides. Chinese patent CN110684812A-A Bletilla striata polysaccharide with immune activity and its preparation method and application, the method uses enzymatic hydrolysis to extract Bletilla striata polysaccharides, and verifies that Bletilla striata polysaccharides have a certain scavenging effect on DPPH·, ABTS+·, etc. The method also studies the effects of different concentrations of Bletilla striata polysaccharides on the survival rate of RAW264.7 cells, as well as the secretion of immune factor TNF-α induced by Bletilla striata polysaccharides to activate RAW264.7 cells. However, this technology is still very limited in verifying the immune activity of Bletilla striata polysaccharides, and the verification data is relatively weak. It cannot fully and objectively reflect the immune mechanism of Bletilla striata polysaccharides and accurately measure the immune regulatory activity of Bletilla striata polysaccharides.

[0005] Based on this, exploring the immunomodulatory activity of Bletilla striata neutral polysaccharides based on the NF-κB signaling pathway to provide preliminary research basis and reference basis for subsequent in vivo activity research experiments in animals has become a technical problem that needs to be urgently solved in this field. Summary of the invention

[0006] The present invention selects the isolated and purified polysaccharide component of Bletilla striata (Latin name Bletilla striata), and studies its immunomodulatory activity based on macrophage RAW264.7 on the one hand. Specifically, an enzyme-linked immunosorbent assay kit is used to detect the relevant cytokines produced by macrophages after intervention of the target polysaccharide component, thereby verifying the immune activity of the polysaccharide component. On the other hand, WB (protein immunoblotting) is used to verify whether Bletilla striata polysaccharide produces NF-κB-based proteins to produce immune anti-inflammatory effects, laying the foundation for subsequent research on the immune anti-inflammatory effects of Bletilla striata polysaccharides.

[0007] The present invention provides an immunomodulatory application of bletilla striata polysaccharide based on NF-κB signaling pathway. The bletilla striata polysaccharide is used for regulating the expression of p65, p-p65, p-IκBα, COX-2 and iNOS proteins in the NF-κB signaling pathway.

[0008] In some preferred embodiments, the polysaccharide of Bletilla striata is obtained by treating the medicinal material of Bletilla striata with water extraction and alcohol precipitation. The water extraction and alcohol precipitation method combines the two techniques of water extraction and alcohol precipitation. The present invention unexpectedly found that the method can collect Bletilla striata polysaccharide simply and efficiently, and the obtained Bletilla striata polysaccharide has a good anti-inflammatory effect and can perform immune regulation based on the NF-κB signaling pathway.

[0009] In some preferred embodiments, after the Bletilla striata polysaccharide intervenes in RAW264.7 macrophages, the cell proliferation rate of RAW264.7 macrophages is above 90%.

[0010] More preferably, after the Bletilla striata polysaccharide intervenes in RAW264.7 macrophages, the cell proliferation rate of RAW264.7 macrophages is above 110%.

[0011] In some preferred embodiments, after the Bletilla striata polysaccharide intervenes in RAW264.7 macrophages, the secretion amount of TNF-α cytokine in RAW264.7 macrophages is above 700 pg / mL.

[0012] In some preferred embodiments, after the Bletilla striata polysaccharide intervenes in RAW264.7 macrophages, the secretion amount of INF-γ cytokine in RAW264.7 macrophages is above 220 pg / mL.

[0013] In some preferred embodiments, after the Bletilla striata polysaccharide intervenes in RAW264.7 macrophages, the secretion amount of IL-6 cytokine in RAW264.7 macrophages is above 100 pg / mL.

[0014] In some preferred embodiments, after the Bletilla striata polysaccharide intervenes in RAW264.7 macrophages, the secretion amount of IL-β cytokine in RAW264.7 macrophages is above 65 pg / mL.

[0015] In some preferred embodiments, after the Bletilla striata polysaccharide acts on the NF-κB signaling pathway, the protein expression of p-p65 in the NF-κB pathway is detected by Western blotting to be a gray value of 1.2 or above.

[0016] In some preferred embodiments, after the Bletilla striata polysaccharide acts on the NF-κB signaling pathway, the protein expression of p-IκBα in the NF-κB pathway is detected by Western blotting to be a gray value of 1.5 or above.

[0017] In some preferred embodiments, after the Bletilla striata polysaccharide acts on the NF-κB signaling pathway, the protein expression of iNOS in the NF-κB pathway is detected by Western blotting to be a gray value of 1.5 or above.

[0018] In some preferred embodiments, after the Bletilla striata polysaccharide acts on the NF-κB signaling pathway, the protein expression of COX-2 in the NF-κB pathway is detected by Western blotting to have a gray value of 1.0 or above.

[0019] NF-κB is one of the most well-known components in the immune-related pathway. As a transcription factor, it consists of two NF-κB monomers combined with the inhibitory IκB protein. The stimulation of macrophages leads to a series of proteolysis and phosphorylation processes, through which the NF-κB / IκB complex is decomposed, NF-κB migrates to the nucleus, and activates the expression of pro-inflammatory mediator genes. Studies have found that IκB is a key molecule in regulating inflammatory responses. This experiment used western blot and other methods to observe the effects of BSP and BSP-2 on p65, p-p65, IκB (such as p-IκB, p-IκBα), COX-2, iNOS and other proteins in the NF-κB pathway, and to explore the mechanism of action of the anti-inflammatory effect of Bletilla striata polysaccharide at the molecular level.

[0020] The research significance of the present invention also lies in providing a research basis for some subsequent experimental studies. For example, more methods can be used to study the immune and anti-inflammatory effects of Bletilla striata polysaccharides in multiple dimensions, and necessary in vivo research experiments can be added. In vivo experimental studies can also be conducted on certain inflammations such as tracheitis, in order to further verify its immune and anti-inflammatory effects. At the same time, in the in vitro experimental studies of Bletilla striata polysaccharide components, human-related immune cells can be further supplemented to lay the foundation for the subsequent research and development of related drugs. The experimental research results of human cells are of irreplaceable importance for the possible clinical effectiveness of the drug in the next step.

[0021] Beneficial effects:

[0022] The present invention provides an immunomodulatory application of Bletilla striata polysaccharide based on NF-κB signaling pathway, which has the following advantages:

[0023] (1) As a widely used traditional Chinese medicine, Bletilla striata has a high popularity in both clinical application and scientific research. The present invention unexpectedly found that Bletilla striata polysaccharide may play an immune and anti-inflammatory role by helping the body to timely exert or restore immune function, which lays a reliable theoretical foundation for subsequent related research on Bletilla striata polysaccharide.

[0024] (2) The present invention conducted an in vitro study on the polysaccharide component BSP-2 of Bletilla striata, mainly by using substances such as LPS (lipopolysaccharide) to induce the activation of macrophage RAW264.7, and then compared it with an experimental group using a certain concentration of polysaccharide components, thereby verifying that the polysaccharide BSP-2 of Bletilla striata can exert anti-inflammatory immune effects in the body, and at the same time, it is speculated that its immune and anti-inflammatory effects may be based on the NF-κB signaling pathway.

[0025] (3) The research data showed that the cytokine secretion of macrophages after LPS stimulation was significantly increased, and the secretion amount was significantly different from that of the blank group (P<0.05). The gradient concentration of BSP and BSP-2 (500, 62.52, 15.525 μg mL -1 ) experimental groups can reduce the secretion of cellular inflammatory factors to a certain extent, and the secretion of inflammatory factors in the drug concentration experimental group was significantly different from that in the LPS group (P<0.05), which reflects the significant effect of Bletilla striata polysaccharide on immune cells.

[0026] (4) The present invention preliminarily verified through protein immunoblotting that the polysaccharide component BSP-2 of Bletilla striata can regulate the balance of pro-inflammatory factors and anti-inflammatory factors by affecting the iNOS, COX-2, P65, P-P65, IκBα and P-IκBα pathway proteins in the NF-κB signaling pathway, thereby exerting an immune and anti-inflammatory effect.

[0027] (5) Chinese herbal polysaccharides have certain anti-cancer activity by enhancing the body's immune ability. Among them, macrophages are one of the immune cells most closely related to the anti-tumor effect of Chinese herbal polysaccharides. The present invention has determined that Bletilla striata polysaccharides have the effect of enhancing the body's anti-inflammatory immunity. On this basis, it can be further explored whether the Bletilla striata polysaccharide components have a certain inhibitory effect on certain cancer tumors, thereby further expanding the research on the pharmacological effects of Bletilla striata polysaccharides. (6) The present invention explores the biological activity of the polysaccharide components in Bletilla striata medicinal materials and their mechanism of immune regulation, providing a feasible idea for the further development of active ingredients in Bletilla striata. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 .Growth curve of RAW264.7 macrophages within 7 days;

[0029] Figure 2 .The effect of different concentrations of Bletilla striata polysaccharide components on macrophage proliferation rate;

[0030] Figure 3 .The effect of Bletilla striata polysaccharide on the secretion of TNF-α cytokine by RAW264.7 macrophages;

[0031] Figure 4 .The effect of Bletilla striata polysaccharide on the secretion of INF-γ cytokine by RAW264.7 macrophages;

[0032] Figure 5 .The effect of Bletilla striata polysaccharide on the secretion of IL-6 cytokine by RAW264.7 macrophages;

[0033] Figure 6 .The effect of Bletilla striata polysaccharide on the secretion of IL-β cytokine by RAW264.7 macrophages;

[0034] Figure 7 .Samples immunoblotted for iNOS, COX-2, P65, p-p65, IκBα and P-IκBα proteins in the NF-κB pathway;

[0035] Figure 8 .Gray value test results of sample p-p65;

[0036] Fig. 9 . Gray value test results of sample P-IκBα;

[0037] Fig.10 . Gray value test results of sample iNOS;

[0038] Fig.11 .Gray value test results of sample COX-2. DETAILED DESCRIPTION

[0039] Example

[0040] This embodiment provides an immunomodulatory application of Bletilla striata polysaccharide based on the NF-κB signaling pathway, using IL-6, IL-β, TNF-α, and IFN-γ series cytokines secreted by RAW264.7 macrophages as detection indicators of the in vitro immunomodulatory activity of Bletilla striata polysaccharide components. iNOS, COX-2, P65, P-P65, IκBα, and P-IκBα are used as observation indicators of the regulatory effect of Bletilla striata polysaccharide on the NF-κB pathway. It is verified that Bletilla striata polysaccharide can perform immunomodulatory and anti-inflammatory effects based on the NF-κB signaling pathway, and the specific demonstration is as follows.

[0041] 1. Obtaining Bletilla striata polysaccharides

[0042] The polysaccharide of Bletilla striata in this embodiment is extracted from the laboratory of Guizhou University, and the specific steps include:

[0043] (1) Extraction: Take 1.5 kg of dried Bletilla striata medicinal material (origin: Shibing, Guizhou), add distilled water at a mass-to-volume ratio of 1:14 (g:mL), and extract in a 68°C water bath for 3 times, each time for 94 min. Filter through four layers of gauze and then vacuum filter. Combine the 3 filtrates and concentrate the filtrate by rotary evaporation to 1 / 5 of the original volume. Add anhydrous alcohol until the mass fraction of Bletilla striata polysaccharide reaches 85%, and place at 4°C overnight. Filter first, then freeze-dry the sediment, add distilled water and stir until completely dissolved. Place the sample solution in a dialysis bag with a molecular weight of 7000 Da, dialyze with distilled water, then collect the solution in the bag and spin at 4000 r.min -1 After centrifugation for 20 minutes, the supernatant was concentrated and dried to obtain the polysaccharide component BSP (raw sugar); after weighing, it was stored at -20°C for future use.

[0044] (2) Separation and purification: Take 2 g of crude sugar component BSP from white jelly and dissolve it in water, then heat it at 4000 r.min. -1 Centrifuge for 15 minutes, take 5 mL of supernatant; load the sample onto a filled anion exchange column (DEAE-650M, 55 mm x 30 cm), rinse with water, measure it with phenol, and recover the polysaccharide to obtain the crude product, which is then concentrated and freeze-dried to obtain BSP-1.

[0045] 500 mg of BSP-1 was completely dissolved in water and placed in a dialysis bag with a molecular weight of 7000 Da. After dialysis with distilled water, the solvent in the bag was collected and the mixture was heated at 4000 r.min- 1 Centrifuge for 20 minutes, collect the supernatant, concentrate the supernatant to 1 / 3 of the original volume, add it to a uniformly filled gel chromatography (Sepharose 6B, 45mmx90 cm), and then elute with H2O, 14mL in each small tube, and then determine the sugar-containing components using the sulfuric acid phenol method, and collect until there is no sugar; combine, concentrate and dialyze according to the elution curve drawn according to the elution amount; obtain Bletilla striata polysaccharide BSP-2 after freeze-drying for later use.

[0046] The experimental demonstration of using BSP and BSP-2 as Bletilla striata polysaccharide samples for immunomodulatory applications is as follows.

[0047] 2. Test methods

[0048] 2.1 Cultivation and activity assay of RAW264.7 macrophages

[0049] 2.1.1 Preparation and processing of cell culture related reagents

[0050] 1) Treatment of cell culture serum

[0051] Place the fetal bovine serum in a 37°C water bath to dissolve it completely, and then inactivate it in 56°C water. After inactivation, cool it at room temperature, and then place it in a 4°C refrigerator for 24 hours of equilibrium. Before use, filter the fetal bovine serum with a sterile filter; the filtered fetal bovine serum is stored sterile.

[0052] Note: 1. Do not open the seal of the newly purchased premium fetal bovine serum upon receipt. Allow it to dissolve completely at room temperature, and then place it in a water bath to dissolve; and, in order to completely dissolve the fetal bovine serum, shake it continuously during the entire process. 2. When filtering, ensure that each filter tip can filter more than 20 ml of blood, and the syringe cannot be retracted during the entire process, otherwise the filter membrane will be damaged and the filtration efficiency of the serum will be affected. 3. When sterile storage, use sterile containers of appropriate volume for batch storage to avoid repeated freezing and thawing of the serum during use, which makes it impossible to ensure the uniform quality of the serum used throughout the experiment.

[0053] 2) Preparation of cell culture medium

[0054] 5mL fetal bovine serum + 45mL DMEM high glucose cell culture medium (Shanghai Shenqi Biotechnology Co., Ltd., DMEM high glucose cell culture medium) + 0.5mL double antibody (penicillin and streptomycin).

[0055] 3) Preparation of cell freezing solution

[0056] 9 mL of fetal bovine serum + 18 mL of DMEM high-glucose medium + 3 mL of dimethyl sulfoxide. Filter with a sterile filter after preparation and prepare before use.

[0057] 2.1.2 Cultivation of RAW264.7 macrophages

[0058] 1) Recovery of RAW264.7 macrophages

[0059] Take out the RAW264.7 macrophages from the liquid nitrogen storage tube and quickly freeze them at -80℃ for 4 hours for activation. After activation, store the cells in a constant temperature tank at -80℃. When used, quickly shake and dissolve them in a constant temperature water bath at 37℃ to ensure that the entire thawing process does not exceed 1min. After thawing, disinfect the cryopreservation tube with alcohol and quickly move it into the clean bench for the next step. Transfer the cryopreservation tube completely to a 15mL centrifuge tube, add 9mL of cell culture medium, mix the cells and complete culture medium by blowing and mixing, and centrifuge at 1000r / min for 5min. Use a pipette to discard the supernatant in the centrifuge tube, then add 5 mL of the prepared complete culture medium. Use pipetting to mix the cells and the complete culture medium thoroughly. Transfer the mixture to the cell culture bottle and shake the culture bottle in an "8" shape to evenly cover the entire bottom of the cell bottle. Finally, observe under a microscope. If there is no problem, move it to a carbon dioxide incubator and culture it for 24 hours before replacing the cell culture medium.

[0060] Replace the medium: After discarding the culture medium with a pipette, gently rinse the attached cells with phosphate buffer. After removing the phosphate buffer, add 5 mL of cell culture medium (the cell culture medium needs to be preheated in a water bath before use to avoid irritating the cells) and culture for 2 days. Pay attention to observe the cell morphology under a microscope when appropriate.

[0061] 2) Passaging of RAW264.7 macrophages

[0062] After the cells were cultured in the incubator for two days, it was observed under a microscope that the cells had grown all over the bottom of the culture bottle and adhered to the wall, and the cell morphology was normal. The cell density was about 70%-90%, and the cell passage operation could be performed. RAW264.7 macrophages are easily stimulated to differentiate, and the cell morphology is seriously changed and the cells stop increasing. Therefore, trypsin was not used to digest the adherent cells during the passage process. The present invention adopts a cell scraping and passage method. The cells to be passaged are taken, the original culture medium is removed by a pipette, and the cells are gently washed with a phosphate buffer solution. The washing solution is discarded, and new phosphate buffer is added. The phosphate buffer can be pre-cooled in a 4°C refrigerator. The cells are scraped by a cell scraper. During the scraping process, the cells are completely scraped. The cell scraper is always scraped in the same direction to avoid scraping the cells back and forth, which will cause damage and stimulation to the cells. After scraping, transfer the phosphate buffer mixed with cells to a 15mL centrifuge tube and centrifuge at 1000r / min for 3min. After centrifugation, add cell culture medium and use a pipette to mix the cells and culture medium so that the cells are evenly distributed in the complete culture medium. Transfer the mixed culture medium into a cell culture bottle, add 1mL of mixed culture medium to each bottle, and then add 4mL of new complete culture medium. Passage according to the ratio of 1:2, shake in an "8" shape, and culture in a carbon dioxide incubator. Observe the cell status regularly and change the medium the next day.

[0063] 3) Cryopreservation of RAW264.7 macrophages

[0064] Observe the cells under a microscope. The cells are normal in morphology and cover more than 80% of the entire culture bottle. Separate and precipitate the cells according to the cell passage method. Add freezing solution according to the cell precipitation and break up the cell precipitation. Each 0.5mL freezing solution can be transferred to one cryopreservation tube. Cells should be cryopreserved in a gradient way, that is, the cryopreservation tubes are placed in a 4℃ refrigerator for 1h, a -20℃ refrigerator for 2h, and finally moved to a -80℃ refrigerator. If the cells do not recover in the short term and need to be stored for a long time, store the cryopreservation tubes in liquid nitrogen.

[0065] 2.2 Determination of the growth curve of RAW264.7 macrophages

[0066] Use a cell scraper to scrape the macrophages filled with culture flasks, then place them in a 15 ml centrifuge tube, centrifuge at 1000 rpm for 3 minutes, count them using a cell counter, and then add cell culture medium until the cell concentration reaches 1×10 4 / mL. Cell culture was performed on a 96-well plate. In order to avoid the influence of edges, no seeds were placed on the side holes of the 96-well plate. After the same amount of phosphate buffer was injected, the seeds were sown. The 96-well plate was transferred to a CO2 cell culture box and cultured for 1, 2, 3, 4, 5, 6, and 7 days. The OD value was measured every day using a CCK-8 kit to characterize the cell proliferation viability (the measurement time remained fixed).

[0067] Before culturing, a preliminary test was conducted to detect the changes in the OD value of the CCK-8 kit at different time periods. According to the test results of multiple time periods (see Table 1), it was determined that the OD value of the CCK-8 kit should be measured 1 hour after being added to the reaction solution. During this period, the culture medium was replaced every 2 days, and observations were made under a microscope regularly and records were kept.

[0068] The wells tested by the CCK-8 reagent package were thoroughly washed with phosphate buffer to prevent contamination of other wells. After the test, the cells in each group were cultured and the cell proliferation activity was measured by the kit to depict the cell growth curve of RAW264.7, so as to determine its logarithmic growth cycle, so as to screen suitable cells in subsequent cell tests and improve the accuracy of the test.

[0069] Test results see Figure 1 .

[0070] 2.3 Determination of proliferation activity of RAW264.7 macrophages

[0071] RAW264.7 macrophages in the logarithmic growth phase were collected and 1×10 5 / mL 96-well plates were placed in a CO2 incubator for 24 hours to adhere to the wall. The next day, the supernatant was removed with a pipette, and BSP (Bletillastriata Polysaccharide) and BSP-2 solutions with concentrations of 0, 1000, 500, 250, 125, 62.5, 31.3, and 15.6 μg / mL were used for intervention, respectively, and the addition amount was 0.2 mL. LPS (lipopolysaccharide) was used as a positive drug, 1 μg / mL, and the addition amount was the same as BSP. Six duplicate wells were opened in each test group (BSP, BSP-2) and control group (LPS). The reagent was transferred to a CO2 incubator. After 24 hours, a detection reagent was added to each well using a CCK-8 kit, and the culture was continued in a CO2 incubator at 37°C to allow the reagent to react for 1 hour, and then the OD value of each well was measured by an enzyme reader at 450nm. In order to further improve the accuracy of the test, the OD value was measured for 15 minutes, and the measured data were analyzed in parallel three times to take the average value. Calculation of cell proliferation rate: Macrophage proliferation rate = (OD - OD 空白 ) / OD 空白×100%. Finally, SPSS statistical software was used to determine the statistical significance of the data. Figure 2 .

[0072] RAW264.7 macrophages in the logarithmic growth phase were used for the following experiments.

[0073] 2.4 Effect of Bletilla striata polysaccharide on cytokine secretion of RAW264.7 macrophages

[0074] 2.4.1 Effect of Bletilla striata polysaccharide on TNF-α cytokine secretion by RAW264.7 macrophages

[0075] RAW264.7 macrophages were cultured at 1×10 5 / mL concentration was inoculated into a 96-well plate, and then the 96-well plate was transferred to a CO2 constant temperature incubator at 37°C for 24 hours. On the second day, all cells were checked under a microscope and found to be growing well, so further experiments could be continued.

[0076] Set up blank control group (corresponding to Figure 3-6 LPS), model control group (corresponding to Figure 3-6 Ctrl), drug-treated group (corresponding to Figure 3-6 BSP, BSP-2) and positive control group (corresponding to Figure 3-6 Dex in the literature) and conduct drug intervention based on this.

[0077] Blank control group: 1 μg / mL LPS culture medium.

[0078] Drug group: 1 μg / mL BSP and BSP-2 culture medium

[0079] Three doses were set: high, medium, and low. 20 μg / mL dexamethasone culture medium was added to the positive control, and 0.2 mL of culture medium was added to each well, and then transferred to a 37°C CO2 incubator. After centrifugation at 1000 r / min for 20 min at 24, 48, 72, and 96 hours, the supernatant was taken, and then the secreted TNF-α factor was determined. The supernatant was refrigerated at -20°C and the storage time was no more than 1 month. The determination of TNF-α factor was carried out according to the instructions of the TNF-α detection kit, and the standard substance was used for the test. The TNF-α standard substance was added in concentrations of 0, 5, 10, 20, 40, 80, 160, 320, and 640 pg / mL, respectively, and more than three measurements were performed on this basis to obtain a standard curve.

[0080] Model control group: 1 μg / mL culture medium

[0081] The TNF-α detection kit was used to determine the concentration of TNF-α in the supernatant of samples after BSP, BSP-2, dexamethasone and LPS intervention using TNF-α standard; TNF-α was determined using cell culture medium. The release amount was predicted by determining the concentration of TNF-α in the supernatant of each group of samples. The test results are shown in Figure 3 .

[0082] 2.4.2 Effect of Bletilla striata polysaccharide on INF-γ cytokine secretion by RAW264.7 macrophages

[0083] The test method and test group design are the same as 2.4.1. The INF-γ standard and related tool kits are used to determine the concentration of INF-γ in the supernatant of each group of samples and predict its release amount. The test results are shown in Figure 4 .

[0084] 2.4.3 Effect of Bletilla striata polysaccharide on IL-6 secretion by RAW264.7 macrophages

[0085] The test method and test group design are the same as 2.4.1. The IL-6 standard and related tool kits are used to determine the concentration of IL-6 in the supernatant of each group of samples and predict its release amount. The test results are shown in Figure 5 .

[0086] 2.4.4 Effect of Bletilla striata polysaccharide on IL-β cytokine secretion by RAW264.7 macrophages

[0087] The test method and test group design are the same as 2.4.1. The IL-β concentration in the supernatant of each group of samples is determined using IL-β standards and related tool kits to predict its release amount. The test results are shown in Figure 6 .

[0088] 2.4.5 Statistical analysis

[0089] The above tests were conducted three times, and the data were expressed as mean and standard deviation. SPSS software was used to process the data and statistically analyze the data between groups. The test of homogeneity of variance, the test of this study, the discarded commercial test and the single variance test were used.

[0090] 2.5 Effects of Bletilla striata polysaccharide components on the regulation of NF-κB pathway

[0091] In order to investigate the potential mechanism of the protective effect of BSP and BSP-2 at a concentration of 500 μg / mL on inflammation, the protein expressions of p65, NF-κB phosphorylated-p65 (p-p65), IκBα, and phosphorylated-IκBα (p-IκBα) in the NF-κB pathway were detected by Western blotting. Figure 7 -.

[0092] Note: 1. Unless otherwise specified, the solvents of the solutions, buffer solutions and other reagents in the test methods of the present invention are all water. 2. Unless otherwise specified, the operation procedures of the present invention adopt the common methods in the field, and the operation can be performed by referring to the relevant work package instructions.

[0093] 3. Test results

[0094] 3.1 Study on the immunomodulatory activity of Bletilla striata polysaccharide fractions in vitro

[0095] 3.1.1 Plotting of the growth curve of RAW264.7 macrophages

[0096] The preliminary experimental results are shown in Table 1. According to the analysis in Table 1, the OD values ​​of RAW264.7 macrophages measured 0.5h-1h after adding CCK-8 reagent were slightly different, and there was little difference between the 1st and 2nd hours. However, there was a large difference in the OD values ​​detected after 4h, and in general, the best OD value range was between 0.2-0.8. Considering the above situation and experimental efficiency factors, this experiment chose to measure the OD value after the 1st hour after adding CCK-8 reagent. To ensure the accuracy of the experimental data, all experimental measurement operations were completed within 15min.

[0097] Table 1 Changes in OD values ​​of CCK-8 kit at different time periods

[0098]

[0099] The growth curve of RAW264.7 macrophages within 7 days is shown in Figure 1 .according to Figure 1 The data showed that the growth activity of macrophages was optimal in the first 4 days, when they were in a logarithmic growth cycle and reached a peak on the fourth day. After the fourth day, the proliferation activity of macrophages decreased. Therefore, cells in a logarithmic growth cycle were selected for subsequent experiments to ensure the accuracy and effectiveness of the experiments.

[0100] 3.1.2 Results of the determination of the proliferation activity of RAW264.7 macrophages after intervention with Bletilla striata polysaccharide

[0101] The results of the effects of BSP and BSP-2 of Bletilla striata polysaccharide components on the proliferation activity of RAW264.7 macrophages are shown in Figure 2 The data showed that as the polysaccharide content increased, the inhibitory effect of each polysaccharide component on cells also increased to varying degrees. -1 ) was dose-dependent. When the polysaccharide content was 125 μg.mL -1In the subsequent study, the concentrations with high growth activity were selected in the range of high, medium and low concentrations, which were: 500, 62.52, 15.525μg.mL -1 The concentration of polysaccharide components.

[0102] 3.1.3 Effects of Bletilla striata polysaccharide components on cytokine secretion by RAW264.7 macrophages

[0103] The secretion of TNF-α, INF-γ, IL-6, and IL-β cytokines after BSP and BSP-2 treatment are shown in Figure 2 Figure 3-6 .from Figure 3-6 It can be seen that the effects of Bletilla striata polysaccharide on the secretion of TNF-α, INF-γ, IL-6, and IL-β cytokines by RAW264.7 macrophages are similar, all showing that lipopolysaccharide (LPS) induced a significant increase in the secretion of TNF-α in macrophages, and the secretion amount was significantly higher than that in the blank group (P<0.05). -1 ) Compared with the blank control group, the secretion of TNF-α, INF-γ, IL-6, and IL-β cytokines was significantly increased (P<0.05), but significantly lower than that of the lipopolysaccharide (LPS) group (P<0.05); the secretion of TNF-α, INF-γ, IL-6, and IL-β cytokines in the dexamethasone experimental group was significantly lower than that of the lipopolysaccharide (LPS) group and basically lower than that of each polysaccharide component concentration group; the secretion of TNF-α, INF-γ, IL-6, and IL-β cytokines in the BSP-2 group at each concentration (15.525, 62.5, 1000 μg·mL-1) was significantly higher than that of the blank group (P<0.05), but significantly lower than that of the lipopolysaccharide (LPS) group (P<0.05); the secretion of TNF-α, INF-γ, IL-6, and IL-β cytokines in the dexamethasone experimental group was significantly lower than that of the lipopolysaccharide (LPS) group and basically lower than that of each polysaccharide component concentration group.

[0104] 3.2 Effects of Bletilla striata polysaccharide components on the regulation of NF-κB pathway

[0105] The immunoblotting of 500 μg / mL samples (BSP and BSP-2, etc.) for iNOS, COX-2, P65, P-P65, IκBα, and P-IκBα proteins in the NF-κB pathway is shown in the figure. Figure 7 The test results are shown in Figure 8-11 The detection software was Image J.

[0106] By detecting the gray value of the results of protein immunoblotting experiments, the present invention found that the LPS group increased the protein expression of phosphorylated p65 and phosphorylated IκBα, while compared with the LPS treatment group, the protein expression of IκBα and phosphorylated-IκBα (p-IκBα) decreased after pretreatment with BSP and BSP-2. At the same time, BSP and BSP-2 also had a certain inhibitory effect on the expression of p-p65 and p-IκBα proteins. This result is consistent with the inhibition of the production of inflammatory cytokines, suggesting that the NF-κB signaling pathway may play a leading role in the activation of RAW264.7 cells.

[0107] Furthermore, the expression of iNOS and COX-2 in the samples was upregulated after LPS stimulation, while the expression of iNOS was significantly downregulated in the experimental group pretreated with BSP (p<0.05), and the expression of COX-2 was also significantly downregulated in the experimental group pretreated with BSP-2 (p<0.05). It can be seen that the expression of iNOS and COX-2 was significantly inhibited, which is also consistent with the inhibition of the production of inflammatory cytokines. Therefore, the experiment shows that BSP and BSP-2 achieve anti-inflammatory immune effects by inhibiting the NF-κB pathway.

Claims

1. An application of Bletilla striata polysaccharide, characterized in that The Bletilla striata polysaccharide is used for immunomodulation based on the NF-κB signaling pathway, specifically by regulating the expression of p65, p-p65, p-IκBα, COX-2 and iNOS proteins in the NF-κB signaling pathway; The Bletilla striata polysaccharide is obtained by treating the Bletilla striata medicinal material with a water extraction and alcohol precipitation method.

2. The use of Bletilla striata polysaccharide according to claim 1, characterized in that: After the Bletilla striata polysaccharide intervenes in RAW264.7 macrophages, the cell proliferation rate of RAW264.7 macrophages is above 90%.

3. The use of Bletilla striata polysaccharide according to claim 1, characterized in that: After the Bletilla striata polysaccharide intervenes in RAW264.7 macrophages, the secretion amount of TNF-α cytokine in the RAW264.7 macrophages is above 700 pg / mL.

4. The use of Bletilla striata polysaccharide according to claim 1, characterized in that: After the Bletilla striata polysaccharide intervenes in RAW264.7 macrophages, the secretion amount of INF-γ cytokine in the RAW264.7 macrophages is above 220 pg / mL.

5. The use of Bletilla striata polysaccharide according to claim 1, characterized in that: After the Bletilla striata polysaccharide intervenes in RAW264.7 macrophages, the secretion amount of IL-6 cytokine in the RAW264.7 macrophages is above 100 pg / mL.

6. The use of Bletilla striata polysaccharide according to claim 1, characterized in that: After the Bletilla striata polysaccharide intervenes in RAW264.7 macrophages, the secretion amount of IL-β cytokine in the RAW264.7 macrophages is above 65 pg / mL.

7. The use of Bletilla striata polysaccharide according to claim 1, characterized in that: After the Bletilla striata polysaccharide acts on the NF-κB signaling pathway, the protein expression of p-p65 in the NF-κB pathway is detected by Western blotting to be above a gray value of 1.

2.

8. The use of Bletilla striata polysaccharide according to claim 1, characterized in that: After the Bletilla striata polysaccharide acts on the NF-κB signaling pathway, the protein expression of p-IκBα in the NF-κB pathway is detected by Western blotting to be above a gray value of 1.

5.

9. The use of Bletilla striata polysaccharide according to claim 1, characterized in that: After the bletilla striata polysaccharide acts on the NF-κB signaling pathway, the protein expression of iNOS in the NF-κB pathway is detected by Western blotting to be above a gray value of 1.

5.

10. The use of Bletilla striata polysaccharide according to claim 1, characterized in that: After the Bletilla striata polysaccharide acts on the NF-κB signaling pathway, the protein expression of COX-2 in the NF-κB pathway is detected by Western blotting to be above a gray value of 1.0.

Images