CN115006425A - Application of acid polysaccharide of herba Sambuci Adnatae in preparing medicine for regulating immunity and treating tumor - Google Patents
Application of acid polysaccharide of herba Sambuci Adnatae in preparing medicine for regulating immunity and treating tumor Download PDFInfo
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- CN115006425A CN115006425A CN202210737346.4A CN202210737346A CN115006425A CN 115006425 A CN115006425 A CN 115006425A CN 202210737346 A CN202210737346 A CN 202210737346A CN 115006425 A CN115006425 A CN 115006425A
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- acid polysaccharide
- polysaccharide
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Abstract
The invention belongs to the technical field of plant extract preparation, and discloses application of acid polysaccharide of herba Paederiae in preparing medicines for immunoregulation and tumor treatment, wherein the medicine for improving the immunity of an organism is prepared by utilizing glycosyl ligand formed by combining acid polysaccharide SPS-1 of herba Paederiae with RAW264.7 macrophage surface receptor TRL 2. Under the condition of co-culture, the acid polysaccharide SPS-1 of the herba hyperici japonici inhibits migration, invasion and apoptosis of Lewis of lung cancer cells by regulating and controlling RAW264.7 cell polarization. The SPS-1 extracted by the invention is combined with a TLR2 receptor of a RAW264.7 cell to form a glycosyl ligand, a MyD88/TRAF-6 signal channel is activated, and MAPKs and NF-kappa B signal channels are continuously activated through specific signal regulation reaction, so that the secretion of a series of immune factors is promoted, and the improvement of the immunity of an organism is facilitated.
Description
Technical Field
The invention belongs to the technical field of plant extract preparation, and particularly relates to application of a herba Sambuci Adnatae acidic polysaccharide in preparation of medicines for immunoregulation and tumor treatment.
Background
The polysaccharide and its complex are widely distributed, and they are involved in the regulation of various vital activities of cells, and have various biological functions, such as immunoregulation and antitumor effect. However, bacterial polysaccharides and synthetic compounds have attracted considerable attention for their adverse reactions and side effects. Most of plant-derived polysaccharides have no adverse reaction and do not produce great side effect on organisms, so that the polysaccharides separated from plants draw great attention in biomedicine. Macrophages play a crucial role in the host's defense system against microbial infections and tumors, and they are one of the first lines of defense against infectious agents. The research shows that the plant polysaccharide activates macrophage immune response, and the macrophage immune response is realized by recognizing specific receptors on the membrane surface of the macrophage and combining with the polysaccharide. Recent studies have shown that natural polysaccharides can trigger a series of signaling cascades within macrophages by binding to TLR2 on the surface of macrophages, leading to transcriptional activation of messenger RNAs such as proinflammatory cytokines, and the like, and finally synthesis and secretion of immune cytokines.
The action mechanism of these active substances is also continuously developing, and the polysaccharide is more important to the non-specific induced immune mechanism. Plant polysaccharides are the most ideal immune candidate.
The herba Sambuci Adnatae is a common Chinese medicine and one of Tibetan medicinal plants, has pungent, astringent and warm natured taste, has effects of dispelling pathogenic wind, promoting diuresis, promoting blood circulation and dredging collaterals, and can be used for treating acute and chronic nephritis, rheumatalgia, etc. At present, the improvement of the body immunity and the action mechanism of the acidic polysaccharide of the herba hyperici japonici are not reported. ,
through the above analysis, the problems and defects of the prior art are as follows: the prior art does not research on the improvement of the body immunity and the action mechanism of the acid polysaccharide of the herba hyperici japonici. The acid polysaccharide SPS-1 of the herba Sambuci Adnatae prepared by the prior art has low purity, and the immunoregulation effect is not obvious, and the theoretical basis is lacked in the later stage of drug development.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the application of the acid polysaccharide of the herba hyperici japonici in preparing medicines for immunoregulation and tumor treatment.
The invention is realized by the application of the acid polysaccharide SPS-1 of the herba Sambuci Adnatae as an immunomodulator in preparing medicines for immunoregulation and tumor immunotherapy.
Another object of the present invention is to provide a pharmaceutical preparation for enhancing the immunological competence of the body, which is prepared by using the acid polysaccharide of herba Sambuci Adnatae SPS-1 as an immunomodulator, wherein the pharmaceutical preparation for enhancing the immunological competence of the body is prepared from a glycosyl ligand formed by binding the acid polysaccharide of herba Sambuci Adnatae SPS-1 with RAW264.7 macrophage surface receptor TRL 2.
The invention also aims to provide a medicament for inhibiting lung cancer, which is prepared by using the tetrandra acidic polysaccharide SPS-1 in the application as an immunomodulator, wherein the tetrandra acidic polysaccharide SPS-1 inhibits migration, invasion and apoptosis of lung cancer cell Lewis by regulating and controlling RAW264.7 cell polarization under a co-culture condition.
Another purpose of the invention is to provide an oral preparation for immune regulation and tumor immunotherapy, which is prepared by using the acid polysaccharide SPS-1 of the herba Sambuci Adnatae in the application as an immune regulator.
The oral preparation comprises tablets, capsules and soft capsules.
Another purpose of the invention is to provide an injection for immune regulation and tumor immunotherapy, which is prepared by using the acid polysaccharide SPS-1 of the herba Papaveris as an immune regulator.
The injection comprises oral liquid, mixture and syrup.
The invention also aims to provide the application of the hematuria acidic polysaccharide SPS-1 in preparing the livestock and poultry immunoregulation medicament.
The invention also aims to provide the application of the acid polysaccharide SPS-1 of the herba hyperici sampsonii in preparing the health care product with immunoregulation function.
The invention also aims to provide a construction method of a cell model for verifying the efficacy of the acid polysaccharide SPS-1 of the herba rhododendri daurici in the application, which comprises the following steps:
preparing a conditioned medium;
taking RAW264.7 cells in logarithmic growth phase, counting at 3X 10 5 Transplanting the wells into a six-well plate containing serum-free DMEM, adding different reagents according to groups, culturing for 24h, and collecting supernatant to obtain a conditioned medium for later use;
(2) grouping experiments: a control group (Lewis cells), a CM0 group (SPS-1+ Lewis cells), a CM1 group (RAW264.7+ IL-4 conditioned medium + Lewis cells), a CM2 group (RAW264.7+ SPS-1 conditioned medium + Lewis cells), and a CM3 group (RAW264.7+ IL-4+ SPS-1 conditioned medium + Lewis cells);
(3) inhibition of Lewis cells by SPS-1 under coculture conditions
And (3) treating each group of cells in groups according to the experiment for 24 hours, and collecting the cells to perform migration and invasion experiments. Invasion and migration experiments of cells in vitro were performed using 24-well Transwell chambers.
In combination with the technical solutions and the technical problems to be solved, please analyze the advantages and positive effects of the technical solutions to be protected in the present invention from the following aspects:
first, aiming at the technical problems existing in the prior art and the difficulty in solving the problems, the technical problems to be solved by the technical scheme of the present invention are closely combined with the technical scheme to be protected and the results and data in the research and development process, and some creative technical effects brought after the problems are solved are analyzed in detail and deeply. The specific description is as follows:
the invention firstly analyzes the primary structure characteristics of the acid polysaccharide SPS-1 of the herba Sambuci Adnatae in detail;
the invention discovers for the first time that SPS-1 activates the immunological activity of the SPS-1 by being combined with RAW264.7 macrophage surface receptor TRL 2;
the invention conjectures for the first time the molecular mechanism of SPS-1 possible to exert the immunostimulation activity: SPS-1 is combined with TLR2 receptor of RAW264.7 cell to form glycosyl ligand, MyD88/TRAF-6 signal channel is activated, MAPKs (Erk, p38, JNK) and NF-kappa B (I kappa B alpha, NF-kappa B p65) signal channel are continuously activated through specific signal regulation reaction, and then secretion of a series of immune factors (such as NO, IL-1 beta, IL-6, TNF-alpha and the like) is promoted, so that the immunity of organism is improved.
The invention firstly discovers that SPS-1 can regulate RAW264.7 cell polarization to inhibit migration, invasion and apoptosis of lung cancer cell Lewis under the co-culture condition.
Secondly, considering the technical scheme as a whole or from the perspective of products, the technical effect and advantages of the technical scheme to be protected by the invention are specifically described as follows:
the SPS-1 can be used as an immunomodulator, becomes a potential candidate drug for immune diseases, and lays a foundation for the research and development of subsequent immunoregulation and tumor immunotherapy drugs.
Third, as the inventive supplementary proof of the claims of the present invention, the expected profit and commercial value after the technical solution of the present invention is transformed are:
the purity of the acid polysaccharide SPS-1 of the herba Sambuci Chiensis prepared by the invention is more than 90%, the detailed structural information is analyzed, the immunoregulation effect is obvious, the regulation mechanism is clear, and the method has important economic and production values. SPS-1 can be made into oral preparation, injection, etc. Oral preparations such as conventional solid preparations such as tablets, capsules, soft capsules and the like; liquid preparation such as oral liquid, mixture, syrup, etc. SPS-1 can also be developed into livestock and poultry immunoregulation medicine for supplementing veterinary antibiotics to replace natural medicines after limited use.
Drawings
FIG. 1 is a flow chart of the method for extracting acid polysaccharide from herba Sambuci Adnatae provided in the embodiment of the present invention.
FIG. 2, physical and chemical properties of SPS-1. (a) HPGPC elution profile for SPS-1; (b) HPLC analysis of SPS-1 monosaccharide composition; (c) infrared Spectrum of SPS-1.
FIG. 3 is a one-dimensional nuclear magnetic resonance spectrum of SPS-1. (a) 1 H, spectrogram; (b) 13 and C, spectrum.
FIG. 4, two-dimensional NMR spectrum of SPS-1. (a) COSY spectrum of SPS-1; (b) HSQC spectrum of SPS-1; (c) HMBC spectrum of SPS-1; (d) NOESY spectrum of SPS-1.
FIG. 5, the repeating structural unit of SPS-1.
FIG. 6, effect of SPS-1 on RAW264.7 cells. (a) Effect of SPS-1 on RAW264.7 cell viability; (b) the effect of SPS-1 on phagocytosis of RAW264.7 cells; (c) the effect of SPS-1 on NO release from RAW264.7 cells; (d) the effect of SPS-1 on IL-1 β secretion by RAW264.7 cells; (e) the effect of SPS-1 on IL-6 secretion by RAW264.7 cells; (f) effect of SPS-1 on TNF-. alpha.secretion by RAW264.7 cells. Values are expressed as mean ± standard deviation (n ═ 3). P <0.05, p <0.01, compared to the negative control group.
FIG. 7, effect of different membrane receptor blockers on SPS-1 stimulation of NO (a), IL-1 β (b), IL-6(c) and TNF- α (d) secretion by RAW264.7 cells. Data are presented as mean ± standard deviation (n-3). P <0.05, p <0.01 compared to the SPS-1(400 μ g/mL) treated group.
FIG. 8, effects of SPS-1 on TLR2, MyD88, TRAF6, MAPKs and NF-. kappa.B expression. (a) The effect of SPS-1 on TLR2, MyD88, and TRAF6 expression; (b) effect of SPS-1 on expression of MAPKs pathway proteins; (c) effect of SPS-1 on NF-. kappa.B pathway protein expression. Data are presented as mean ± standard deviation (n ═ 3). P <0.05, p <0.01, compared to the negative control group.
FIG. 9, inhibition of Lewis cells by SPS-1 under co-culture conditions. a) The effect of SPS-1 on migration of Lewis cells; b) the effect of SPS-1 on Lewis cell invasion; c) the effect of SPS-1 on HIF-1 α secretion by Lewis cells; d) the effect of SPS-1 on VEGF secretion by Lewis cells; e) the effect of SPS-1 on Lewis apoptosis; f) effect of SPS-1 on Lewis cell metastasis and apoptosis-related protein expression. Denotes p <0.05, p <0.01 compared to control. Δ indicates Δ p <0.05 and Δ p <0.01, compared to CM 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
First, an embodiment is explained. This section is an explanatory embodiment expanding on the claims so as to fully understand how the present invention is embodied by those skilled in the art.
The embodiment of the invention provides application of a herba Sambuci Adnatae acidic polysaccharide SPS-1 as an immunomodulator in preparing medicines for immunoregulation and tumor immunotherapy.
The embodiment of the invention provides a medicine for improving the immunity of a human body, which is prepared by using the acid polysaccharide SPS-1 of the tetrandra lemongrass in the application as an immunomodulator, wherein the medicine for improving the immunity of the human body is prepared by using glycosyl ligand formed by the acid polysaccharide SPS-1 of the tetrandra lemongrass through being combined with a RAW264.7 macrophage surface receptor TRL 2.
The embodiment of the invention provides a medicine for inhibiting lung cancer, which is prepared by using the acid polysaccharide SPS-1 of the tetrandra lemongrass in the application as an immunomodulator, wherein under the co-culture condition, the acid polysaccharide SPS-1 of the tetrandra lemongrass inhibits migration, invasion and apoptosis of lung cancer cells Lewis by regulating and controlling RAW264.7 cell polarization.
The embodiment of the invention also provides an oral preparation for immunoregulation and tumor immunotherapy, which is prepared by using the acid polysaccharide SPS-1 of the herba hyperici sampsonii in the application as an immunoregulator.
The oral preparation comprises tablets, capsules and soft capsules.
Another purpose of the invention is to provide an injection for immune regulation and tumor immunotherapy, which is prepared by using the acid polysaccharide SPS-1 of the herba Papaveris as an immune regulator.
The injection comprises oral liquid, mixture and syrup.
The embodiment of the invention also provides application of the herba hyperici japonici acidic polysaccharide SPS-1 in preparing livestock and poultry immunoregulation medicaments.
The embodiment of the invention also provides application of the herba hyperici japonici acidic polysaccharide SPS-1 in preparing health care products with immunoregulation function.
The first embodiment is as follows: preparation of Sambucus acidic polysaccharide SPS-1
Drying and crushing the leaves of the herba Sambuci Adnatae, and refluxing with 3 times of petroleum ether and 95% industrial ethanol for 3 times (2 hr each time) to remove fat-soluble and alcohol-soluble impurities; drying the pretreated herba Sambuci Adnatae sample, extracting with distilled water at 89 deg.C under heating at a ratio of 1:26 for 114min, extracting for 4 times, mixing extractive solutions, concentrating to 1/10 of original volume, adding anhydrous ethanol into the concentrated solution to final concentration of 80%, precipitating with ethanol at 4 deg.C for 24h, centrifuging at 4000r/min for 10min, dissolving the precipitate with a small amount of distilled water, and freeze drying to obtain herba Sambuci Adnatae crude polysaccharide (CSP). Purifying CSP by Saveg method and DEAE Sepharose Fast Flow ion exchange chromatography (3.5 × 40cm) at Flow rate of 20mL/10min, collecting 0.2M sodium chloride eluate fraction, concentrating, and freeze drying to obtain herba Sambuci Adnatae acidic polysaccharide SPS. Purifying SPS with Sephacryl S-300HR gel chromatography column (2.5 × 100cm), eluting with 0.9% sodium chloride solution at flow rate of 10mL/20min, collecting polysaccharide fractions, concentrating, dialyzing, and freeze drying to obtain herba Sambuci Adnatae acidic polysaccharide SPS-1 (figure 1).
Example two: structural features of SPS-1
(1) Total sugar, protein and uronic acid content determination
The sugar content, protein content and uronic acid content of SPS-1 were determined to be 94.76%, 1.28% and 6.08% using the phenol-sulfuric acid method, the Bradford method and the m-hydroxybiphenyl method, respectively.
(2) Uniformity and molecular weight determination
The homogeneity and molecular weight of SPS-1 were determined by high performance gel chromatography. Chromatographic conditions are as follows: the chromatographic column is KS805-804-802 series gel column (7.8 × 300mm), the mobile phase is 0.2M sodium chloride, the flow rate is 0.8mL/min, the column temperature is 40 ℃, and the sample injection amount is 20 μ L. HPGPC analysis showed (FIG. 2a), SPS-1 was a single symmetrical peak, indicating a uniform molecular weight distribution; the molecular weight of the acid polysaccharide of herba Sambuci Adnatae SPS-1 is 138.52kDa, which is determined by a series of molecular weight dextrans (Mw5200, 11600, 148000, 273000, 410000) as standard curves.
(3) Monosaccharide composition analysis
The monosaccharide composition of SPS-1 was detected by HPLC, selection of pre-column derivatization. Chromatographic conditions are as follows: agilent1260 chromatography system, DAD-UV detector, isocratic elution, column C18(250mm × 4.6mm), mobile phase: v (0.1mol/L phosphate buffer pH 6.7) V (acetonitrile) 82: 18, flow rate 1.0mL/min, detection wavelength 250nm, column temperature 35 ℃, sample injection 10 u L.
SPS-1 mainly comprises L-rhamnose, D-glucuronic acid, D-galacturonic acid, D-galactose and L-arabinose at a molar ratio of 0.6:0.4:0.1:4.9:4.0 (figure 2 b).
(4) Infrared spectroscopic analysis
SPS-1 is mixed with a proper amount of potassium bromide, and the mixture is tabletted at 400-4000cm -1 And infrared spectrum scanning is carried out.
Infrared Spectrum of SPS-1 showed 3405.66cm -1 The broad and strong absorption peak is attributed to the stretching vibration of O-H, and is 2930.80cm -1 The peak is caused by C-H stretching vibration; 1732.89cm -1 Nearby vibrational peaks demonstrate the presence of uronic acid; 1073cm –1 The two signals at (A) are due to stretching vibrations of the C-O-C bond in the pyranose ring, which are characteristic signals of carbohydrates. 890cm -1 The absorption peak was attributed to the beta-type glycosidic bond in SPS-1, which confirmed that SPS-1 was a polysaccharide (FIG. 2 c).
(5) Methylation analysis
SPS-1 was subjected to methylation modification and GC-MS analysis was performed. GC-MS conditions: rxi-5 Sil MS column (30 m.times.0.25 mm.times.0.25 μm); the temperature programming conditions are as follows: the initial temperature is 120 ℃, the temperature is kept for 2min, and the temperature is increased to 250 ℃/min at the speed of 10 ℃/min; keeping for 5min, using helium as carrier gas, and setting flow rate at 1 mL/min.
Comprehensively analyzing retention time, a cracking mode and a standard map, and the SPS-1 mainly comprises the following connection modes: araf- (1 →, Rhap- (1 →, → 2) -Araf- (1 →, → 5) -Araf- (1 →, Glcp- (1 →, Galp- (1 →, → 2,4) -Rhap- (1 →, → 4) -Glcp- (1 →, → 4) -Galp- (1 →, → 6) -Galp- (1 → and → 3,6) -Galp- (1 →, the molar ratio being 14.1:1.0:1.4:3.9:3.8:1.8:3.1:3.0:3.7:1.9: 16.6.
(6) Nuclear magnetic resonance spectroscopy
Dissolving 50mg SPS-1 in heavy water for replacement, freeze-drying, repeating the operation for three times, and freeze-drying the sample for the last time by using P 2 O 5 Dried and dissolved in heavy water, and 1D and 2D NMR spectra were recorded at 25 ℃ using a Bruker AV500M NMR spectrometer, including 1 H(500MHz)、 13 C(126MHz)、 1 H- 1 H COSY, HSQC and HMBC. Data were processed and analyzed using MestReNowa software.
Of SPS-1 1 In the H NMR spectrum (FIG. 3a), 10 anomeric hydrogen signal peaks mainly exist in the resonance region of the anomeric hydrogen, the chemical shifts are respectively 5.14, 5.11, 4.98, 4.67, 4.61, 4.57, 4.41, 4.39 and 4.39ppm, and the sugar residues are numbered A to J according to the descending chemical shifts of the anomeric hydrogen. 13 The major anomeric carbon chemical shifts of SPS-1 in the C NMR spectrum (FIG. 3b) were 108.47, 106.71, 106.37, 103.30, 102.92, 102.90, 102.43, 102.11, 101.90 and 100.01ppm, respectively. According to the monosaccharide composition result, the polysaccharide contains small amount of glucuronic acid and galacturonic acid, 174.84ppm is the signal peak of C6 carboxyl of glucuronic acid, and 174.37ppm is the signal peak of C6 carboxyl of galacturonic acid. 1.15ppm in the H spectrum and 15.86ppm in the C spectrum are the H6 and C6 signal peaks for rhamnose.
The chemical shift of C, H in SPS-1 was assigned by analysis of the H-H COSY and HSQC data (FIGS. 4a-4b) with simultaneous reference to the same sugar residue correlation literature and methylation results. Analysis of the HSQC data showed that the anomeric carbon signal δ 108.47 correlated with the anomeric hydrogen signal δ 5.14, and further by H H-COSY analysis, it was concluded that H2, H3, H4, H5a and H5b were δ 4.12, 3.85, 4.02, 3.73 and 3.79, respectively, the corresponding carbon spectra were δ 80.75, 75.65, 80.35 and 65.80, respectively, and the residue was concluded to be a glycosidic bond → 5) - α -L-Araf- (1 →). Other sugar residues were assigned using the same method and in conjunction with the relevant literature.
The results of chemical shifts assigned to each sugar residue C, H are shown in Table 1.
TABLE 1 SPS-1 sugar residues 1 H and 13 chemical shift assignment of C
The connection mode of the polysaccharide was analyzed by combining HMBC and NOESY spectra (FIGS. 4c-4d), and SPS-1 repeat structural unit (FIG. 5) was obtained.
Example three: immunity study of SPS-1
Experimental cells: mouse mononuclear macrophage RAW264.7
(1) Cell viability assay
The MTT method is adopted to determine the influence of SPS-1 on the viability of RAW264.7 cells. RAW264.7 cells were treated at 5.0X 10 3 one/mL was inoculated in a 96-well cell culture plate, after overnight incubation at 37 ℃, different concentrations of SPS-1(12.5, 25, 50, 100, 200, 400, 800, 1600. mu.g/mL) were added, and cells treated with medium alone were used as negative controls, with 3 duplicate wells per group. In the presence of 5% CO 2 The cells were cultured in a 37 ℃ incubator for 24 hours, then 10. mu.L of MTT was added, and after incubation at 37 ℃ for 4 hours, the culture was terminated and the light absorption (A) value was measured at a wavelength of 568 nm. Cell viability was calculated according to the following formula: cell viability (%) ═ a Treatment group /A Negative control group ×100％。
The results show (fig. 6a) that the experimental concentration can stimulate the proliferation of RAW264.7 cells compared with the blank control, SPS-1 has a significant stimulation effect (p <0.05) on the RAW264.7 cells at 50 and 100 μ g/mL, and the stimulation effect reaches a very significant level (p <0.01) when the concentration is further increased.
(2) Phagocytosis assay
RAW264.7 cells in logarithmic growth phase were added to 6-well cell culture plates at a cell density of 5X 10 5 one/mL, 100. mu.L per well, 5% CO at 37 ℃ 2 The cells were incubated overnight in an incubator, different concentrations of SPS-1(200, 400, 800. mu.g/mL) were added, cells treated with medium alone served as negative control, cells treated with 1. mu.g/mL LPS served as positive control, 3 duplicate wells were provided for each group, 37 ℃ and 5% CO 2 After 24h incubation in an incubator, the supernatant was discarded and each group of cells was incubated with FITC-dextran (1mg/mL) at 37 ℃ with 5% CO 2 Incubating the culture box for 1h, centrifuging for 5min at 1000rpm after incubation, discarding supernatant, washing twice with PBS, and analyzing phagocytosis of FITC-dextran by RAW264.7 by flow cytometry.
The results show (fig. 6b) that the set experimental concentration can activate the phagocytosis of RAW264.7 cells and increase in a dose-dependent manner, and reaches a very significant level (p <0.01) at 800 μ g/mL, which indicates that SPS-1 can significantly improve the phagocytosis of macrophages.
(3) Determination of NO, IL-1 beta, IL-6 and TNF-alpha
RAW264.7 cells were pressed 10 6 one/mL was inoculated in a 96-well cell culture plate, and different drugs were added according to experimental groups (same 1.3.2), each group having 3 duplicate wells. In the presence of 5% CO 2 Culturing for 24h in an incubator at 37 ℃, respectively collecting the supernatant of RAW264.7 cell culture, detecting the content of NO by adopting a Griess method, and detecting the content of IL-1 beta, IL-6 and TNF-alpha by adopting an ELISA method.
The results show (figures 6c-6f) that SPS-1 can obviously improve the NO secretion amount of RAW264.7 cells, and when the concentration is 800 mu g/mL, the NO secretion amount is very obviously different from that of a blank control (p is less than 0.01); compared with blank control, SPS-1 can obviously improve the secretion amounts (p <0.05, p <0.01) of IL-1 beta, IL-6 and TNF-alpha of RAW264.7 cells, and is in a dosage effect. Thus, SPS-1 may enhance macrophage-mediated innate immune responses.
(4) Determination of recognition of receptors
Taking RAW264.7 cells in logarithmic growth phase, inoculating in 12-hole cell plate, placing at 37 deg.C and 5% CO 2 Culturing for 24h in an incubator, discarding cell supernatant, adding 1mL and 5 mug/mL of various antibody solutions (anti-TLR2, anti-TLR4, anti-GR, anti-MR, anti-CR3 and anti-SR which are all prepared by DMEM basal medium) respectively, adding DMEM basal medium into a negative control group, adding 1.0 mug/mL LPS into a positive control group, repeating the steps for 3 times, continuing culturing for 1h, carefully discarding supernatant of 6 groups of antibody groups, adding SPS-1(400 mug/mL) containing basal medium, and continuing culturing for 24h in the cell incubator. Collecting the supernatant of RAW264.7 cell culture according to the components, detecting the content of NO by adopting a Griess method, and detecting the content of IL-1 beta, IL-6 and TNF-alpha by adopting an ELISA method.
The results show (FIG. 7) that the secretion amounts of NO, IL-1 β, IL-6 and TNF- α of RAW264.7 cells co-treated with anti-TLR2 and SPS-1 were all very significantly decreased (p <0.01), anti-GR had a significant decrease in cytokine secretion (p <0.05), and anti-TLR4, anti-GR, anti-MR, anti-SR and anti-CR3 had little effect on the secretion of RAW264.7 cytokines, compared with the SPS-1 only (p >0.05), indicating that TLR2 and GR are recognition receptors for RAW264.7 cells to recognize SPS-1, and TLR2 is the main recognition receptor.
(5) Assay for MAPKs and NF- κ B pathway activation in RAW264.7 cells
The activity of MAPKs and NF-kB channels is detected by using a Western blot method. The RAW264.7 cells were treated with different concentrations of SPS-1(200, 400, 800 μ g/mL) for 24h, then trypsinized and collected separately, lysed with RIPA lysate for 30min, centrifuged at 12000r/min at 4 ℃ for 10min, and the supernatant was taken for protein concentration determination. After SDS-PAGE electrophoresis, membrane conversion and sealing for 1h, the sample is incubated overnight with primary antibody, washed 5 times with TBST, incubated with secondary antibody, developed with ECL kit, developed by an imaging system and analyzed for band gray values by BandScan software.
Western blot results (FIG. 8) show that compared with blank control group, SPS-1 treated RAW264.7 cells have significant or extremely significant up-regulated protein expression (P <0.05, P <0.01) of TLR2, MyD88, TRAF6, MAPKs (JNK, Erk, P38) and NF- κ B (I κ B α, NF- κ B P65), and are in dose-effect relationship.
In conclusion, the molecular mechanism underlying the potential of SPS-1 to exert immunostimulatory activity is presumed: SPS-1 is combined with TLR2 receptor of RAW264.7 cell to form glycosyl ligand, MyD88/TRAF-6 signal channel is activated, MAPKs (Erk, p38, JNK) and NF-kappa B (I kappa B alpha, NF-kappa Bp65) signal channel are continuously activated through specific signal regulation reaction, and then secretion of a series of immune factors (such as NO, IL-1 beta, IL-6, TNF-alpha and the like) is promoted, thereby improving the immunity of organism.
And II, application embodiment. In order to prove the creativity and the technical value of the technical scheme of the invention, the part is the application example of the technical scheme of the claims on specific products or related technologies.
The application of the acid polysaccharide SPS-1 of the herba Sambuci Adnatae extracted by the extraction method of the acid polysaccharide of the herba Sambuci Adnatae provided by the application embodiment of the invention in preparing immunomodulators for treating, relieving and preventing immune diseases, health products with immunoregulation, anti-inflammatory drugs and anti-tumor drugs.
And thirdly, evidence of relevant effects of the embodiment. The embodiment of the invention achieves some positive effects in the process of research and development or use, and has great advantages compared with the prior art, and the following contents are described by combining data, diagrams and the like in the test process.
The first application embodiment: SPS-1 inhibits migration, invasion and apoptosis of Lewis cells by regulating RAW264.7 cell polarization.
Experimental cells: mouse mononuclear macrophage RAW264.7, lung cancer cell Lewis
(1) Preparation of conditioned Medium
Taking RAW264.7 cells in logarithmic growth phase, counting at 3X 10 5 Each well was transplanted in a six-well plate containing serum-free DMEM, and different reagents (SPS-1 at a final concentration of 800. mu.g/mL or/and IL-4 at a final concentration of 20 ng/mL) were added in groups, and after 24 hours of culture, the supernatant was collected to obtain a conditioned medium for use.
(2) Grouping experiments: control group (Lewis cells), CM0 group (SPS-1+ Lewis cells), CM1 group (conditioned Medium for RAW264.7+ IL-4+ Lewis cells), CM2 group (conditioned Medium for RAW264.7+ SPS-1+ Lewis cells), and CM3 group (conditioned Medium for RAW264.7+ IL-4+ SPS-1+ Lewis cells)
(3) Inhibition of Lewis cells by SPS-1 under coculture conditions
And (3) treating each group of cells in groups according to the experiment for 24 hours, and collecting the cells to perform migration and invasion experiments. Invasion and migration experiments of cells in vitro were performed using 24-well Transwell chambers. Invasion assay 100. mu.g of matrigel was spread on top of the chamber. No matrigel is needed to be laid in a migration laboratory. And 3X 10 was added to the upper chamber of the Transwell cell 5 one/mL of 200. mu.L Lewis cells, after 24h of culture, the culture medium was aspirated and the cells that did not invade or migrate in the upper layer of the chamber were carefully wiped off with a cotton swab. PBS was washed 2 times and fixed in 10% methanol for 30 min. Then stained with 5% crystal violet for 20 min. And finally, photographing and counting, and performing semi-quantification.
Lewis cells were pressed 10 6 Inoculating each/mL of the cells in a 96-well cell culture plate, adding different culture media according to experimental groups for culturing for 24 hours, collecting supernatant, and detecting the content of HIF-1 alpha and VEGF-A by adopting an ELISA method.
And (3) collecting and culturing the Lewis cells for 24h, and detecting the Lewis cell apoptosis by using an annexin V-APC/7-AAD apoptosis detection kit.
The expression level of tumor cell metastasis and apoptosis related proteins (E-Cadherin, N-Cadherin, Vimentin, Bax, Bcl-2) is detected by using a Western Blot technology, and the relative expression level of the target protein is expressed by the ratio of the gray value of a target protein band to that of an internal reference GAPDH protein band.
The effect of SPS-1 on the transfer capacity of Lewis cells under the co-culture condition is shown in figures 9a-9b, compared with a control group, the groups CM0, CM2 and CM3 can reduce the transfer capacity of Lewis cells to different degrees, compared with the group CM1, the groups CM0 and CM2 can reduce the transfer capacity of Lewis cells to a great extent (p is less than 0.01), and the group CM3 can also reduce the transfer capacity of Lewis cells to a great extent or a great extent (p is less than 0.05 or 0.01). Analysis of HIF-1 alpha and VEGF secretion and expression levels of N-Cadherin, E-Cadherin and Vimentin in Lewis cells by ELISA and WB experiments (FIGS. 9c-9d, 9f) showed that the groups CM0, CM2 and CM3 all reduced HIF-1 alpha and VEGF secretion and N-Cadherin and Vimentin expression significantly or very significantly, whereas E-Cadherin expression increased significantly (p < 0.01). The results show that SPS-1 can inhibit the migration capacity of Lewis cells by inducing RAW264.7 cells to be polarized into M1 subtype.
The result of detecting Lewis cell apoptosis by flow cytometry is shown in figure 9e, compared with the control group and the CM1 group, the Lewis cell apoptosis rate of the co-culture system of the CM0 group, the CM2 group and the CM3 group is obviously increased (p is less than 0.01). The WB experiment is further used for detecting the expression levels of apoptosis-related proteins Bcl2 and Bax in Lewis cells (figure 9f), and the results show that the expression of the anti-apoptotic protein Bcl2 in the Lewis cells and the expression of the pro-apoptotic protein Bax can be obviously reduced by SPS-1 treatment, and the results show that the SPS-1 can induce RAW264.7 cell polarization to promote the apoptosis of the Lewis cells.
In conclusion, SPS-1 can be developed into an immunotherapy medicament for non-small cell lung cancer and other cancers by regulating macrophage polarization to inhibit the transfer of Lewis cells and promote the apoptosis of Lewis cells.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. Use of herba Sambuci Adnatae acidic polysaccharide SPS-1 as immunomodulator in preparing medicine for immunoregulation and tumor immunotherapy is provided.
2. A pharmaceutical preparation for improving the immunological competence of the body, which is prepared by using the acid polysaccharide of herba Sambuci Adnatae SPS-1 for the use according to claim 1 as an immunomodulator, wherein the pharmaceutical preparation for improving the immunological competence of the body is prepared from a glycosyl ligand formed by binding the acid polysaccharide of herba Sambuci Adnatae SPS-1 with a RAW264.7 macrophage surface receptor TRL 2.
3. A medicament for inhibiting lung cancer prepared by using the acid polysaccharide of tetrandra SPS-1 as an immunomodulator in the use of claim 1, wherein the acid polysaccharide of tetrandra SPS-1 inhibits migration, invasion and apoptosis of lung cancer cells Lewis by controlling RAW264.7 cell polarization under co-culture conditions.
4. An oral preparation for immunomodulation and tumor immunotherapy prepared by using the acid polysaccharide SPS-1 of Sambucus nigra for the use as described in claim 1 as an immunomodulator.
5. The oral formulation of claim 4, wherein the oral formulation comprises a tablet, capsule, or softgel.
6. An injection for immunomodulation and tumor immunotherapy prepared by using the acid polysaccharide SPS-1 of herba Sambuci Adnatae as an immunomodulator according to the use of claim 1.
7. The injection of claim 5, wherein the injection comprises an oral liquid, a mixture, or a syrup.
8. An application of herba Sambuci Adnatae acidic polysaccharide SPS-1 in preparing immunoregulation medicine for livestock and poultry is provided.
9. An application of herba Sambuci Adnatae acidic polysaccharide SPS-1 in preparing health product with immunoregulation effect is provided.
10. A method for constructing a cell model for verifying the efficacy of the acid polysaccharide of tetrandra paniculata SPS-1 for the use according to claim 1, said method comprising:
preparing a conditioned medium;
counting RAW264.7 cells in logarithmic growth phase at 3 × 10 5 Transplanting into six-well plate containing serum-free DMEM, adding according to different groupsCulturing the reagent for 24h, and collecting the supernatant to obtain a conditioned medium for later use;
(2) grouping experiments: a control group (Lewis cells), a CM0 group (SPS-1+ Lewis cells), a CM1 group (RAW264.7+ IL-4 conditioned medium + Lewis cells), a CM2 group (RAW264.7+ SPS-1 conditioned medium + Lewis cells), and a CM3 group (RAW264.7+ IL-4+ SPS-1 conditioned medium + Lewis cells);
(3) inhibition of Lewis cells by SPS-1 under coculture conditions
And (3) treating each group of cells in groups according to the experiment for 24 hours, and collecting the cells to perform migration and invasion experiments. Invasion and migration experiments of cells in vitro were performed using 24-well Transwell chambers.
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| CN112138018A (en) * | 2020-09-24 | 2020-12-29 | 合肥工业大学 | Application of dendrobium officinale polysaccharide in preparation of medicine for treating tumors by targeting tumor-related macrophages |
| CN112516157A (en) * | 2020-12-07 | 2021-03-19 | 上海中医药大学附属岳阳中西医结合医院 | Application of safflower polysaccharide in preparing medicine for treating melanoma |
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