CN117959329A - Application of swimming bladder heparinoids in preparation of bi-directional immunomodulator - Google Patents
Application of swimming bladder heparinoids in preparation of bi-directional immunomodulator Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, and relates to application of a swimming bladder heparinoid compound in preparation of a bidirectional immunomodulator. Experiments prove that the swimming bladder heparinoids have bidirectional immunoregulation activity, and play a bidirectional immunoregulation role through multiple targets and multiple ways, so that the organism can recover the immune homeostasis. The swimming bladder heparinoids are combined with receptor protein TLR4 through hydrogen bonds to trigger MAPK and NF- κB signal paths, so as to promote the expression of related proteins; regulate the secretion of various cytokines mediated by macrophages, thereby exerting the immune function of the body. The swimming bladder heparinoids can be used as an immunostimulant or bi-directional immunomodulator to enhance immune response, and have potential application prospects in the fields of immune disease treatment or functional food processing and the like.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to application of a swimming bladder heparinoid compound in preparation of a bidirectional immunomodulator.
Background
The immunomodulator is a medicine for regulating the immune function of organisms, has important functions in diagnosis and treatment processes of tumors, autoimmune diseases and other immune dysfunction diseases or organ transplantation and the like which need to artificially regulate the immune function, and has wide clinical application. Immunomodulators are classified by function and mainly include immunopotentiators, immunosuppressants, and immunobidirectional modulators. The immunobidirectional regulator is a substance with bidirectional immunoregulation function, such as Lipopolysaccharide (LBP), water-soluble yeast beta-glucan, ginseng polysaccharide, etc. The bidirectional immunomodulator can up-regulate the immune function of the organism by protecting immune organs, promoting lymphocyte proliferation, increasing cytokine release and the like; meanwhile, the proliferation of partial immune cells in the body and the over-expression of cytokines are inhibited, so that the body immunity maintains normal level. The medicine with the function of regulating immunity in two directions can act on various immune tissues and organs, immune cells and immune molecules of the organism to regulate the immunity functions of different levels of the organism in two directions, and the immune systems of the organism interact, coordinate and regulate each other, so that the immune response of the organism is accurately controlled. Currently, bi-directional immunomodulatory drugs have been widely used in the treatment of immunodeficiency diseases, neoplastic diseases, allergic diseases, and graft rejection.
The swim bladder is rich in active substances such as collagen, polypeptide, mucopolysaccharide and the like, is a precious biological resource, but is not effectively developed and utilized at present. The heparinoids (heparinoid from swimming bladder, HSB) extracted from swim bladder are mainly composed of repeating disaccharide units [. Fwdarw.4GlcUAβ1→3GalNAc (4S). Beta.1→ ]. The related literature describes the isolation, purification and structural identification of the swim bladder heparinoids conjugate HSB and the evaluation of the anticoagulant and anti-inflammatory activity of the swim bladder heparinoids of different molecular weights (Zhou Siyi. Isolation of the swim bladder heparinoids, structural characterization and evaluation of activity [ D ]. University of guangdong ocean, 2019). However, whether HSB has an immunomodulatory function, in particular, a bidirectional immunomodulatory function has not been studied.
Disclosure of Invention
The invention aims to expand the application of the swimming bladder heparinoids in the aspect of immunoregulation, and particularly to clarify that the swimming bladder heparinoids have bidirectional immunoregulation function.
The invention provides an application of a swimming bladder heparinoid compound in preparing an immunoregulation medicament.
The preparation of the swimming bladder Heparinoids (HSB) refers to the prior literature, such as separation, purification and structural identification of swimming bladder heparinoids, zhou Siyi, and the like, food science, volume 40. The swimming bladder heparinoids have the following structure.
The invention further provides application of the swimming bladder heparinoids in preparing bi-directional immunoregulation medicines.
Experiments prove that the swimming bladder heparinoids promote proliferation of macrophages.
Experiments prove that the swimming bladder heparinoids promote phagocytosis of macrophages and enhance immune response of the macrophages to pathogens.
Experiments prove that the swimming bladder heparinoids promote macrophages to release cytokines, inflammatory mediators and active oxygen.
Preferably, according to an embodiment of the invention, the cytokines include TNF- α and IL-10 and the inflammatory mediators include NO.
Experiments prove that the swimming bladder heparinoids promote the expression of receptor protein TLR 4.
Experiments prove that the swimming bladder heparinoids promote the phosphorylation expression of key proteins JNK, ERK1/2, p38 and p65 proteins in MAPK and NF- κB signal paths.
Experiments prove that key signal paths of the swimming bladder heparinoids for playing immune activities comprise MAPK and NF- κB signal paths.
Also, the present invention provides an immunomodulatory drug comprising a swim bladder heparinoid compound.
Compared with the prior art, the technical scheme provided by the invention has at least the following beneficial effects or advantages.
The invention provides a new application of a swimming bladder heparinoid compound, namely the swimming bladder heparinoid compound has bidirectional immunoregulation function and can be used for preparing a bidirectional immunoregulator. According to the invention, in vivo experiments and in vitro experiments prove that the swimming bladder heparinoids can be used as a bidirectional immunomodulator, and through the combination of hydrogen bonds and TLR4 receptors, MAPK and NF- κB signal paths are triggered, and the expression of related proteins is promoted; regulate macrophage-mediated secretion of various cytokines, thereby exerting the immune function of the organism.
The invention verifies that the swimming bladder heparinoids compound does not obstruct the normal growth of macrophages at the concentration of 0.25-2mg/mL, has no toxic effect on cells, and can promote the phagocytosis of macrophages
The invention proves that the swimming bladder heparinoids can play a bidirectional immunoregulation role through multiple targets and multiple ways, and help organisms to restore immune homeostasis.
Drawings
In order to more clearly illustrate the technical solutions of the present invention, the drawings referred to in the description of the embodiments will be briefly described below, it being obvious that the drawings in the description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the results of an experiment of the effect of HSB on the cell viability and phagocytic activity of macrophage RAW 264.7. Wherein A is a graph of the effect of HSB on the cell viability of the macrophage RAW 264.7; b is a graph of phagocytosis index results of HSB on macrophage RAW 264.7.
FIG. 2 is a graph showing the results of an experiment in which HSB promotes the release of TNF-alpha, IL-10 and NO by macrophage RAW 264.7. Wherein, A is a graph of test results of HSB promoting macrophage RAW264.7 to release cytokine TNF-alpha; b is a graph of test results of HSB promoting macrophage RAW264.7 to release cytokine IL-10; c is a graph of test results of HSB promoting macrophage RAW264.7 to release inflammatory mediator NO.
FIG. 3 is a graph showing the results of an assay for the production of ROS by HSB-activated macrophages RAW 264.7. Wherein, A is a fluorescence microscope image of ROS generated by macrophage RAW 264.7; b is a graph of the fluorescence intensity measurement of ROS produced by macrophage RAW 264.7.
FIG. 4 is a graph showing the results of an imaging experiment of HSB affecting key proteins in the TLR4-MAPK signaling pathway, NF- κB signaling pathway.
Figure 5 is a graph showing results of molecular docking assays for HSB, CSC and receptor protein TLR 4. Wherein, A is the crystal structure of TLR4 and the binding site of HSB; b is a two-dimensional graph of HSB and TLR4 butt joint; c is the crystal structure of TLR4 and its binding site to CSC; d is a two-dimensional map of CSC docking to TLR 4.
In FIGS. 1 to 5, HSB represents a test group treated with a swimming bladder heparinoid compound, CSC represents a test group treated with 6-chondroitin sulfate, LPS represents a positive control group treated with lipopolysaccharide (1. Mu.g/mL), and control represents a blank control group; the different letters indicate significant differences for each group (P < 0.05).
Detailed Description
The following describes the technical aspects of the present invention with reference to examples, but the present invention is not limited to the following examples. The experimental methods and the detection methods in each embodiment are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.
The preparation of the swimming bladder Heparinoids (HSB) used in the examples of the present invention is described in the prior art ("separation and purification and structural identification of swimming bladder heparinoids", zhou Siyi et al, food science, volume 40). Chondroitin-6-sulfate (CSC) was purchased from Shanghai microphone Biochemical technologies Co., ltd. Lipopolysaccharide (LBP) was purchased from beijing solebao biotechnology limited. Macrophage RAW264.7 was purchased from the national academy of sciences.
Example 1
This example provides an assay for the effect of HSB on cell viability and phagocytic activity of macrophages.
(1) Cell viability assay
Cell viability was determined using the CCK-8 method. Macrophage RAW264.7 in logarithmic growth phase was adjusted to 1×10 4 cells/mL with Gibco DMEM medium (purchased from the company of the sciences, sameimer) and added to 96-well plates, 100 μl of 5% CO 2 was added to each well plate, incubated at 37 ℃ for 24 hours, and the medium was discarded; 100. Mu.L of HSB and CSC diluted with Gibco DMEM medium and having a concentration gradient of 1, 0.5 and 0.25 mg/mL were added to each well as a sample group, a control group was set, and after incubation for 24 hours, 10. Mu.L of CCK-8 solution was added to each well and absorbance was measured at a wavelength of 450 nm using an enzyme-labeled instrument. Cell viability is expressed as a percentage of the control group. The cell viability was calculated as follows:
，
Wherein A1 is the absorbance of the sample group and A0 is the absorbance of the control group.
The test results are shown as a in fig. 1. HSB and CSC did not hinder normal growth of macrophage RAW264.7 and did not have toxic effects on macrophage RAW264.7 at concentrations of 0.25-2mg/mL compared to control. HSB significantly promoted proliferation of RAW264.7 cells (P < 0.05) compared to CSC, cell viability was reduced at 2mg/mL, but there was no significant change compared to control.
(2) Phagocytic activity assay
Phagocytic capacity of macrophages was determined according to the method of Shi et al （Shi, M., Yang, Y., Hu, X.,&Zhang, Z. (2014). Effect of ultrasonic extraction conditions on antioxidative and immunomodulatory activities of a ganoderma lucidum polysaccharide originated from fermented soybean curd residue. Food Chemistry, 155(15), 50-56.）. Macrophages RAW 264.7 (cell concentration 1X 10 4 cells/mL) were seeded in 96-well plates and stimulated with 24 h under HSB, CSC (0.25, 0.5 and 1 mg/mL) and LPS (1. Mu.g/mL). Neutral red staining solution was added to the cells and incubated with 2 h. The supernatant was discarded and the cells were washed with PBS to remove excess dye. Then 100. Mu.L of cell lysate was added to lyse the cells. After shaking at room temperature for 30 min, absorbance was measured at 540, 540 nm. The calculation formula of the phagocytosis index is as follows:
Wherein A1 is the absorbance of the sample group and A0 is the absorbance of the control group.
Phagocytosis of macrophages is one of the most fundamental defense mechanisms in organisms and is often used as an indicator of immune function activation. As shown in B in fig. 1, macrophages after HSB treatment showed stronger phagocytic activity than CSC groups. At a concentration of 0.25-1mg/mL (P < 0.05), phagocytosis of macrophage RAW264.7 was significantly stimulated, indicating that HSB may promote phagocytosis of macrophages, increasing its innate immune response to invading pathogens. Meanwhile, the present example also found that at a concentration of 0.25mg/mL, HSB at a low concentration showed stronger phagocytic activity (P < 0.05) than LPS in the positive control group.
The test results show that HSB has bidirectional immunoregulation effect.
Example 2
This example provides an assay in which HSB promotes macrophage release of cytokines, inflammatory mediators, and Reactive Oxygen Species (ROS).
Immune cells secrete cytokines such as TNF- α, IL-10, etc. Such cytokines play an important role in cell growth, differentiation and immunity. Macrophages can regulate the immune homeostatic environment in the body by releasing cytokines (e.g., TNF- α and IL-10) or inflammatory mediators (e.g., NO), thereby killing pathogens either directly or indirectly.
The secretion levels of NO, TNF-. Alpha.and IL-10 in macrophage RAW 264.7 were determined using Griess kit and ELISA kit, respectively. Macrophages (2.5X10 5 cells/well) were seeded in 6-well plates and after 24h incubation, gibco DMEM medium was discarded and HSB and CSC were added to each diluted with Gibco DMEM medium at a concentration gradient of 1, 0.5, 0.25 mg/mL and incubated together. After 24h incubation, macrophage culture fluid was collected and treated according to ELISA protocol. The secretion levels of NO, TNF- α and IL-10 were calculated using calibration curves. The test results are shown in FIG. 2.
As can be seen from FIGS. 2A and B, HSB significantly increased macrophage RAW264.7 secretion of cytokines TNF- α and IL-10 (P < 0.05) at concentrations of 0.25-1mg/mL compared to the CSC group. At an HSB concentration of 0.25mg/mL, the secretion amounts of cytokines TNF- α and IL-10 peaked, at 1450.06pg/mL and 1155.6pg/mL, respectively. The results show that HSB has an immunoregulatory effect and can promote the secretion of cytokines TNF-alpha and IL-10.
Nitric Oxide (NO) is an inflammatory mediator produced by arginine-catalyzed nitric oxide synthase (iNOS), which may destroy microorganisms and tumor cells and play a key role in the immune system. As can be seen from C in figure 2, both HSB and CSC promote NO secretion. HSB has a weaker promotion of NO secretion by macrophage RAW264.7 than CSC group (P < 0.05). At 0.25mg/mL, the NO secretion of macrophage RAW264.7 is highest, 42.16. Mu. Mol; at 1mg/mL, the lowest is 24.96. Mu. Mol, which is presumably related to the bi-directional immunomodulatory or anti-inflammatory activity of HSB.
It is widely recognized by those skilled in the art that iNOS regulates the secretion of NO. Further experiments found that HSB promoted iNOS expression in macrophage RAW264.7 far less than CSC (fig. 4) in the concentration range of 0.25-1mg/mL, consistent with the results of ELISA assay of NO content in cell supernatants.
Macrophage RAW264.7 (1X 10 4 cells/well) was seeded in 96-well plates, treated with HSB, CSC or LPS respectively for 24h, then washed 2 times with PBS and incubated with 10. Mu. Mol/L DCFH-DA (2, 7-dichlorofluorescein diacetate) 30 min at 37 ℃. After staining with DCFH-DA, the cells were washed 2 times with PBS and the fluorescence intensity was measured in the excitation wavelength range of 485-525nm, and the test results are shown in FIG. 3.
Reactive Oxygen Species (ROS) are involved in the regulation of immune function in the body, act as secondary messengers for immune cell signaling, activate NF- κB and MAPKs signaling pathways in macrophages, and enhance cytokine expression. As can be seen from fig. 3, the HSB-treated macrophages RAW264.7 showed a significant increase in green fluorescence intensity, as well as a significant increase in average fluorescence intensity (P < 0.05) compared to the control and CSC groups. Therefore, HSB activates macrophage RAW264.7 to generate ROS, which are involved in regulating cell signaling pathways, exerting immunomodulatory effects.
Example 3
This example provides an assay for the effect of HSB on TLR4, MAPK and NF- κB signaling pathways in macrophages.
TLR4 is located on the surface of cell membranes and is an important target for initiating the immune response mechanism of macrophages by recognizing molecular patterns associated with pathogens, such as lipoproteins, activating NF- κb signaling cascades, thereby generating an immune response. It was found that polysaccharides can exert an immunomodulatory effect by activating MAPKs and NF- κB signaling pathways, thereby inducing immune cell activation. MAPKs, including ERKs, JNKs and p38, play a key role in regulating cell growth, differentiation and cytokine secretion and cell stress response. Immunoblotting is used for detecting the expression condition of TLR4 receptor protein in macrophage and the expression of JNK, ERK1/2, p38 and NF-kappa B p protein phosphorylation in MAPK and NF-kappa B signal channels so as to explore the immunoregulation effect of HSB on macrophage RAW 264.7 and possible molecular mechanism.
Macrophages were treated with HSB, CSC (0.25, 0.5 and 1 mg/mL) and LPS (1. Mu.g/mL) for 24h, respectively, and cells were extracted in lysis buffer. Protein samples were separated by 10% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes. PVDF membrane was blocked with 5% bovine serum albumin for 1h, shaken at room temperature, incubated overnight with primary antibodies (iNOS, TLR4, JNK, p-JNK, ERK1/2, p-38, p-p38, β -actin, NF- κ B p65, p-NF- κ B p65, GAPDH) and then incubated with secondary antibodies (enzyme labeled-commonly HRP) at room temperature for 60min. Protein bands were detected using super ECL Plus solution. The Bio-Rad image analysis system (Bio-Rad laboratories, dali god, calif.) was used to image protein bands. The test results are shown in FIG. 4.
As can be seen from fig. 4, HSB and CSC significantly promoted the expression of TLR4 receptor protein, while promoting the phosphorylated expression of the key proteins JNK, ERK1/2, p38 and p65 in the MAPK and NF- κb signaling pathways, which suggests that MAPK and NF- κb signaling pathways are key signaling pathways for HSB to exert immune activity in macrophages, as compared to the control group. HSB may exert an immunomodulatory effect by targeting TLR4 receptors on the surface of macrophages, activating MAPK and NF- κb signaling pathways, regulating the secretion of NO, TNF- α, IL-10 and ROS in macrophages.
Example 4
This example provides a molecular docking simulation of HSB binding to TLR4 receptors.
Molecular docking simulation technology is an effective and convenient tool for in-depth understanding the interaction between small molecules and targets and the binding energy thereof. The polysaccharides HSB, CSC and TLR4 receptor proteins were subjected to docking studies using Autodock Vina.1.2 software and the test results are shown in figure 5.
As can be seen from fig. 5, amino acid residues 286 GLU, 287 PHE, 288 ARG, 312 SER, 332 SER, 333 LEU and 334 SER of the receptor protein TLR4 form hydrogen bonds with HSB, and amino acid residue 336 ILE of the receptor protein TLR4 forms a hydrophobic bond with HSB. Furthermore, amino acid residues 382 ALA, 406 PHE, 409 ALA, 429 HIS and 456 ASN of the receptor protein TLR4 form hydrogen bonds with CSCs. These hydrogen bonds are important forces for the interaction of the polysaccharides HSB and CSC with the receptor protein TLR 4. Studies have shown that binding is easier to induce when the binding affinity is less than-5 kcal/mol. In this example, the binding energies of HSB, CSC and receptor protein TLR4 were-6.5 and-8.5 Kcal/mol, respectively, indicating the presence of interaction sites between the polysaccharides HSB, CSC and receptor protein TLR4, it could be verified that HSB and CSC might be through modulation of TLR4 protein expression, thereby activating MAPK and NF- κb signaling pathways.
In conclusion, the test of the application shows that HSB can play a bidirectional immunoregulation role through multiple targets and multiple ways, so that the organism can recover the immune homeostasis. HSB is combined with receptor protein TLR4 through hydrogen bond acting force, activates macrophages to generate ROS, starts MAPK and NF- κB signal paths, promotes the phosphorylation expression of key proteins JNK, ERK1/2, p38 and p65 proteins, and accordingly stimulates RAW 264.7 cells to produce cytokines such as TNF-alpha, IL-10, NO and the like. At the same time, this process also promotes phagocytosis by macrophages, increasing their innate immune response to foreign pathogens. HSB can activate macrophages through TLR 4-mediated MAPK and NF- κb signaling pathways to exert an immunomodulatory effect, suggesting that HSB may act as an immunostimulant or bi-directional immunomodulator to enhance immune responses and may be a potential application target for immune diseases or functional foods.
The embodiments described above are some, but not all, embodiments of the invention. The detailed description of the embodiments of the invention is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments obtained without inventive effort by a person skilled in the art, which are related deductions and substitutions made by the person skilled in the art under the condition of the inventive concept, are within the scope of protection of the present invention.
Claims (10)
1. Application of swimming bladder heparinoids in preparing immunomodulating drugs.
2. Application of swimming bladder heparinoids in preparing bi-directional immunoregulation medicine.
3. The use according to claim 1 or 2, wherein the swim bladder heparinoids promote proliferation of macrophages.
4. The use according to claim 1 or 2, wherein the swim bladder heparinoids promote phagocytosis of macrophages and enhance the immune response of macrophages to pathogens.
5. The use according to claim 1 or 2, wherein the swim bladder heparinoids promote the release of cytokines, inflammatory mediators and reactive oxygen species by macrophages.
6. The use of claim 5, wherein the cytokines include TNF- α and IL-10 and the inflammatory mediators include NO.
7. The use according to claim 1 or 2, wherein the swim bladder heparinoids promote expression of the receptor protein TLR 4.
8. The use according to claim 1 or 2, wherein the swim bladder heparinoids promote the phosphorylated expression of the key proteins JNK, ERK1/2, p38 and p65 proteins in MAPK and NF- κb signaling pathways.
9. The use according to claim 1 or 2, wherein the key signaling pathways for the immune activity of the swim bladder heparinoids include MAPK and NF- κb signaling pathways.
10. An immunomodulatory drug, wherein the immunomodulatory drug comprises a swim bladder heparinoid compound.
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