CN111358805A - Application of platycodon grandiflorum polysaccharide in antagonizing fumonisin B1-induced apoptosis through autophagy - Google Patents

Abstract

The invention discloses application of platycodon grandiflorum polysaccharide in antagonizing fumonisin B1-induced apoptosis through autophagy. The research of the invention finds that the toxic effect of FB1 on alveolar macrophages is mainly reflected in the induction of apoptosis. The platycodon grandiflorum polysaccharide induces autophagy through two signal paths of PI3K/Akt/mTOR and MEK/ERK, and further antagonizes FB1 induced alveolar macrophage apoptosis through autophagy, so that the protection effect on alveolar macrophages is realized. The invention clarifies the regulation mechanism of the crosstalk between the apoptosis and autophagy of the alveolar macrophages by the platycodon grandiflorum polysaccharide, and provides a theoretical basis for developing the treatment medicines of corresponding diseases.

Description

Application of platycodon grandiflorum polysaccharide in antagonizing fumonisin B1-induced apoptosis through autophagy

Technical Field

The invention belongs to the field of medical application of platycodon grandiflorum polysaccharide, and particularly relates to application of platycodon grandiflorum polysaccharide in antagonizing fumonisin B1-induced apoptosis through autophagy.

Background

Fumonisins B1(FB1) are mycotoxins produced by Fusarium (Fusarium verticillioides) and are commonly associated with corn. It can produce species-and organ-specific toxicity, including equine leukoencephalomalacia, porcine pulmonary edema, and liver or kidney damage in most animal species.

Alveolar macrophage is an important component of lung resisting pathogenic infection, when the alveolar macrophage is contacted with pathogenic factors, the alveolar macrophage stretches out of a prosthetic foot, the alveolar macrophage surrounds the prosthetic foot until a closed corpuscle is formed, then the corpuscle is separated from a cell membrane to form a phagocyte of cytoplasm, and finally the alveolar macrophage is fused with lysosome in cells, and the lysosome contains various proteases capable of digesting the pathogenic bacteria, so that the aim of killing the pathogenic factors is fulfilled.

FB1 can cause toxicity to alveolar macrophages, so that the research on how to antagonize FB 1-induced alveolar macrophage injury has great significance.

Apoptosis may be triggered by extrinsic stimulation of cell surface death receptors, such as TNF- α (tumor necrosis factor- α), Fas (CD95/APO1) and TRAIL (TNF-related apoptosis-inducing ligand) receptors, or by intrinsic stimulation of the mitochondrial signaling pathway in both cases, activation of caspases leads to mitochondrial membrane permeability, chromatin condensation and DNA fragmentation, events that give apoptotic cells unique characteristic morphologies including cell aggregation, chromatin condensation, nuclear fragmentation and apoptotic body shedding, cytoplasmic-containing vacuoles and intact organelles.

Autophagy is one of the most important degradation systems in cells, where damaged or unwanted proteins and organelles are transported to lysosomes and degraded. Autophagy is of about three types, including macroautophagy, microautophagy, and chaperone-mediated autophagy. Apoptosis and autophagy are two evolutionarily conserved processes that maintain homeostasis, and functional crosstalk between apoptosis and autophagy is complex. Under certain cellular environments, autophagy acts as a stress response to inhibit apoptosis and promote cell survival. However, in other cases, autophagy may be a mechanism of caspase-dependent or independent cell death. The research on the molecular mechanism of apoptosis and autophagy crosstalk has important significance for the treatment of various diseases.

Platycodon grandiflorum (Jacq) a.dc) is a perennial herb belonging to the genus Platycodon of the family platycodonaceae. The main chemical components of the platycodon include platycodin, volatile oil, steroids, flavonoids, polysaccharide and the like. Radix Platycodi can be used for treating pharyngalgia, hoarseness, cough with excessive phlegm, chest distress, pyocutaneous disease with no ulcer, sore throat relieving, lung ventilating, phlegm eliminating, and pus discharge. The radix Platycodi can also be used as food, and has rich nutrition, and contains various essential microelements and amino acids. Modern pharmacological researches find that platycodon grandiflorum has various biological activities, such as improvement of insulin resistance, oxidation resistance, inflammation diminishing, enhancement of body immune function, tumor resistance and the like.

The platycodon grandiflorum polysaccharide is one of main active ingredients of platycodon grandiflorum, and early researches show that the total polysaccharide of platycodon grandiflorum can resist apoptosis induced by carbonyl cyanide metachlorophenylhydrazone (CCCP). However, on the one hand, the molecular weight, chemical composition, glycosidic bond, conformation, degree of branching, etc. of the polysaccharide all have an effect on the activity of the polysaccharide, and thus, the activity of the polysaccharide is differential and unpredictable; on the other hand, different kinds of inducers have different mechanisms of cell damage, and therefore, even the effect of the same substance on cell damage induced by different inducers is unpredictable. At present, reports on apoptosis induced by platycodon grandiflorum polysaccharide through autophagy antagonism to fumonisin B1 are not found.

Disclosure of Invention

In view of the prior art, the invention aims to provide a new application of platycodon grandiflorum polysaccharide in antagonizing fumonisin B1-induced apoptosis through autophagy.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, the use of platycodon grandiflorum polysaccharide in the preparation of modulators of PI3K/Akt/mTOR and MEK/ERK signaling pathways is provided.

In a second aspect of the invention, the application of platycodon grandiflorum polysaccharide in preparing an autophagy activator of cells by regulating the activity of PI3K/Akt/mTOR and MEK/ERK signaling pathways is provided.

In the application, the platycodon grandiflorum polysaccharide is time-dependent on the regulation and control of the activity of PI3K/Akt/mTOR and MEK/ERK signaling pathways, and specifically comprises the following steps: during the first 2h of platycodon polysaccharide treatment, the platycodon polysaccharide activates PI3K/Akt/mTOR and MEK/ERK signaling pathways; after 4h of platycodon polysaccharide treatment, the platycodon polysaccharide inhibits PI3K/Akt/mTOR and MEK/ERK signaling pathways.

In the above use, the cell is a porcine alveolar macrophage.

In a third aspect of the invention, the application of platycodon grandiflorum polysaccharide in preparing a medicament for antagonizing apoptosis induced by FB1 through activating autophagy is provided.

In the above use, the cell is a porcine alveolar macrophage.

Preferably, in each of the above uses, the platycodon grandiflorum polysaccharide is prepared by the following method:

slicing radix Platycodi root, soaking in 8-10 times of water at 90-100 deg.C overnight, leaching for 2-4 hr, filtering, concentrating the filtrate to relative density of 1.05-1.15 (measured at 50 deg.C), centrifuging to remove impurities to obtain extractive solution;

adding 95 vol% ethanol into the leaching solution while stirring to make ethanol final concentration 70%, standing, centrifuging, separating precipitate, drying at 60 deg.C to obtain PGPS₇₀(ii) a Adding 95 vol% ethanol into the supernatant, stirring to reach final concentration of 50%, standing, centrifuging, separating precipitate, drying at 60 deg.C, and making into PGPS₅₀(ii) a Mixing PGPS₇₀And PGPS₅₀And uniformly mixing to obtain the platycodon grandiflorum polysaccharide.

More preferably, the standing time is 24 hours.

Preferably, the concentration of the platycodon grandiflorum polysaccharide is 20-100 mu g/mL.

The invention has the beneficial effects that:

the research of the invention finds that the toxic effect of FB1 on alveolar macrophages is mainly reflected in the induction of apoptosis. The platycodon grandiflorum polysaccharide induces autophagy through two signal paths of PI3K/Akt/mTOR and MEK/ERK, and further antagonizes FB1 induced alveolar macrophage apoptosis through autophagy, so that the protection effect on alveolar macrophages is realized. The invention clarifies the regulation mechanism of the crosstalk between the apoptosis and autophagy of the alveolar macrophages by the platycodon grandiflorum polysaccharide, and provides a theoretical basis for developing the treatment medicines of corresponding diseases.

Drawings

FIG. 1: effect of PGPSt on 3D4/21 cell proliferation and FB 1-induced cell injury; in the figure, A: 3D4/21 cells were treated with PGPSt at different concentrations for 24h to study the effect on cell proliferation rate; b: 3D4/21 cells were treated with different concentrations of FB1 for 12h, to study the effect on cell viability; c: 3D4/21 cells were treated with different concentrations of FB1 for 24h, to study the effect on cell viability; d: the effect on cell viability was investigated by co-treating 3D4/21 cells with 100. mu.g/mL PGPSt and various concentrations of FB1 for 24 h.

FIG. 2: protection of FB 1-induced apoptosis of 3D4/21 cells by PGPSt; in the figure, A: 3D4/21 cells were co-treated with 10. mu.g/mLFB 1 for 24h with 100. mu.g/mL PGPSt to analyze the protein levels of Caspase-9, cleared Caspase-3 and Bcl-2; a fluorescence micrograph of Hoechst33342 staining shows the morphology of cultured 3D4/21 cells for 24h in the Control group, 10. mu.g/mL FB 1-treated group, 100. mu.g/mL PGPSt-treated group, and 10. mu.g/mLFB 1+ 100. mu.g/mL PGPSt-combination-treated group.

FIG. 3: PGPSt activated autophagy in 3D4/21 cells; in the figure, A: western blot analysis of LC3, Beclin-1 and SQSTM1 expression in 3D4/21 cells after treatment of 3D4/21 cells with different concentrations of PGPSt (4 h); b: formation of autophagosomes following PGPSt treatment (4 hours) was observed using a transmission electron microscope, with arrows indicating representative double membrane vacuoles; c: 3D4/21 cells were transfected with the GFP-LC3 plasmid for 48 hours, treated with PGPSt (100. mu.g/mL) for 4 hours, and the GFP-LC3 spots were observed by confocal microscopy; d: 3D4/21 fluorescence microscopy showed LC3 distribution and tubulin-labeled cytoskeleton, and co-staining of cells with Hoechst33342 revealed punctate aggregation of LC 3.

FIG. 4: autophagy inhibitor 3-methyladenine (3-MA) inhibits PGPSt-induced autophagy; in the figure, A: western blot analysis of LC3, Beclin-1 and SQSTM1 expression in 3D4/21 cells after PGPSt (20, 100. mu.g/mL) treatment with or without 3-MA (4 h); b: confocal microscopy revealed punctate aggregation of LC3 in 3D4/21 cells after PGPSt (100. mu.g/mL) treatment with or without 3-MA.

FIG. 5: the autophagy inhibitor Bafilomycin a1 inhibits PGPSt-induced autophagy; in the figure, A: western blot analysis of LC3, Beclin-1 and SQSTM1 expression in 3D4/21 cells 4h after PGPSt (20, 100. mu.g/mL) treatment with or without BafA 1; (B) confocal microscopy observations showed punctate aggregation of LC3 in 3D4/21 cells after PGPSt (100. mu.g/mL) treatment with or without Bafilomycin A1.

FIG. 6: the PGPSt regulates and controls a PI3K/Akt/mTOR pathway; in the figure, A: PGPSt (20, 100. mu.g/mL) treated 3D4/21 cells for 2h and 4h, and Akt and phosphorylated Akt expression were measured; b: PGPSt (20, 100. mu.g/mL) treated 3D4/21 cells for 2h and 4h, and mTOR and phosphorylated mTOR expression was determined; c: observing the p-Akt distribution and the cell skeleton marked by tubulin by a confocal microscope; cells were co-stained with Hoechst 33342.

FIG. 7: modulation of the MEK/ERK pathway by PGPSt; in the figure, A: PGPSt (20, 100. mu.g/mL) treated 3D4/21 cells for 2h and 4h, and MEK and phosphorylated MEK expression were measured; b: PGPSt (20, 100. mu.g/mL) treated 3D4/21 cells for 2h and 4h, and ERK and phosphorylated ERK expression were measured; c, observing p-MEK distribution and tubulin marked cytoskeleton by a confocal fluorescence microscope; cells were co-stained with Hoechst 33342.

FIG. 8: PGPSt induces autophagy through PI3K/AKT/mTOR and MEK/ERK pathways; in the figure, A: western blot analysis of LC3 expression in 3D4/21 cells with or without LY294002 and PD98059 in PGPSt (100 μ g/mL) treatment (4 h); b: confocal fluorescence microscopy was used to observe LC3 distribution and tubulin-labeled cytoskeleton; cells were co-stained with Hoechst 33342.

FIG. 9: PGPSt protects 3D4/21 cells from FB 1-induced apoptosis by autophagy; in the figure, A: cells were co-treated with 10. mu.g/mL FB1 or 100. mu.g/mL PGPSt for 24 hours; b: western blot analysis of Caspase-9 expression in 3D4/21 cells after FB1 (10. mu.g/mL) or co-treatment with 100. mu.g/mLPGPSt.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As described in the background section, FB1 can cause toxicity to alveolar macrophages, which damages lung airways and alveoli, and therefore, it is very important to study how to antagonize FB 1-induced alveolar macrophage damage. The inventor finds that the platycodon grandiflorum total polysaccharide can antagonize apoptosis induced by CCCP in previous research. However, due to the difference in the toxic action mechanism of FB1 and CCCP on cells and the difference in the activity of platycodon grandiflorum polysaccharides prepared by different methods, it is difficult to predict whether platycodon grandiflorum polysaccharides can exert an effect on FB 1-induced cell damage, and by which method platycodon grandiflorum polysaccharides can exert an effect on FB 1-induced cell damage.

The research of the invention finds that the toxic effect of FB1 on alveolar macrophages is mainly expressed in the form of apoptosis. Therefore, how to antagonize FB 1-induced apoptosis is critical to achieving protection of alveolar macrophages.

Functional crosstalk between apoptosis and autophagy is complex. Under certain cellular environments, autophagy acts as a stress response to inhibit apoptosis and promote cell survival. However, in other cases, autophagy may be a mechanism of caspase-dependent or independent cell death. Therefore, by studying modulators of crosstalk between apoptosis and autophagy, antagonism of FB 1-induced apoptosis could be achieved by activating normal autophagy flow.

Activation of autophagy is a very complex process, regulated by multiple signaling pathways. The invention discovers for the first time that platycodon grandiflorum polysaccharide has a regulation effect on two signal pathways of PI3K/Akt/mTOR and MEK/ERK, the regulation shows time dependence, the two signal pathways of PI3K/Akt/mTOR and MEK/ERK can be activated in the first 2h, and the two signal pathways of PI3K/Akt/mTOR and MEK/ERK are inhibited after 4 h.

The invention further discovers that the platycodon grandiflorum polysaccharide realizes the activation of alveolar macrophage autophagy by regulating the activities of two signal pathways of PI3K/Akt/mTOR and MEK/ERK. The two signal pathways are mutually cooperated and play a role in the activation of autophagy, wherein the activation of MEK/ERK signal pathway is mainly used in the early stage, and the inhibition of PI3K/Akt/mTOR signal pathway is mainly used in the later stage.

In addition, simply activating autophagy does not guarantee that antagonism of apoptosis is achieved, since apoptosis may be accelerated if autophagy flow is damaged. The autophagy flow of autophagy of cells activated by platycodon grandiflorum polysaccharide is smooth, antagonism on apoptosis of alveolar macrophages induced by FB1 is realized by the activation of autophagy, and further the protection effect on the alveolar macrophages is realized.

In addition, as mentioned above, the platycodon grandiflorum polysaccharides prepared by different methods have different chemical compositions and molecular weights, and their activities also vary. In order to research the difference of the protective effect of the platycodon grandiflorum polysaccharide prepared by different methods on FB 1-induced cell damage, the invention adopts a plurality of methods to prepare the platycodon grandiflorum polysaccharide, including:

preparing the platycodon grandiflorum total polysaccharide by a one-step alcohol precipitation method: slicing radix Platycodi root, soaking in 8 weight times of water overnight, leaching at 80 deg.C for 4 hr, filtering, heating and concentrating the filtrate to relative density of 1.05-1.15 (measured at 50 deg.C), cooling, centrifuging at 3000rpm for 10min to remove impurities to obtain leaching solution;

slowly adding 95 vol% ethanol into the leaching solution while stirring to make the final concentration of ethanol be 80%, standing for 24 hr, centrifuging, separating precipitate, and vacuum drying at 60 deg.C to obtain radix Platycodi total polysaccharide.

Preparing graded platycodon grandiflorum polysaccharide by a step-by-step alcohol precipitation method: slicing radix Platycodi root, soaking in 10 weight times of water overnight, leaching at 90 deg.C for 3 hr, filtering, concentrating the filtrate to relative density of 1.05-1.15 (measured at 50 deg.C), centrifuging to remove impurities to obtain leaching solution;

adding 95 vol% ethanol into the leaching solution while stirring to make ethanol final concentration 70%, standing, centrifuging, separating precipitate, drying at 60 deg.C to obtain PGPS₇₀(ii) a Adding 95 vol% ethanol into the supernatant, stirring to reach final concentration of 50%, standing, centrifuging, separating precipitate, drying at 60 deg.C, and making into PGPS₅₀(ii) a Mixing PGPS₇₀And PGPS₅₀And uniformly mixing to obtain the platycodon grandiflorum polysaccharide PGPSt.

And PGPS prepared by the similar method₈₀And PGPS₆₀。

The platycodon grandiflorum polysaccharide (100. mu.g/mL) prepared by the different methods and FB1 (10. mu.g/mL) were co-treated with alveolar macrophages, and the protective effect on cells was examined. The results are as follows:

group of	Cell survival rate (%)
		Blank control	100
FB1 alone	62
		FB1+ Platycodon Total polysaccharide	70
FB1+PGPS70	64
		FB1+PGPS50	65
FB1+PGPSt	85
		FB1+PGPS80+PGPS60	72

The above results show that PGPS prepared by fractionation₇₀And PGPS₅₀The PGPSt mixture has the optimum protective effect on FB 1-induced cell damage and a significant synergistic effect, and therefore PGPSt was used as a platycodon polysaccharide for the test in the subsequent tests.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available. Wherein:

RPMI 1640 is purchased from Marine clone of Rogen, USA LY294002(HY-10108), PD98059(HY-12028), 3-MA (HY-19312) is provided by Med Chem Express, USA BAfilomycin A1(A8510) is purchased from Solarbio (Beijing, China), Anti- β -actin, Anti-pho-MEK 1/2Ser217/221 and Anti-total MEK1/2, Anti-pho-Erk 1(Thr202/Tyr204)/Erk2(Thr185/Tyr187) and Anti-total ERK1/2 antibodies, Caspase9 is purchased from Beyoday biotech (Beyome) biotech (Begonite), Cassomoto Hippon, China LC3 antibody (229327) and Caspase-3 (Caspase-49822) are purchased from Campyloric Biotech, goat Anti-mTOR H9206), Anti-goat Anti-mTOR IgG 2976 (goat) and Anti-goat Anti-mTOR (SALT) No. 5, Anti-goat Anti-5 (Ak) and Anti-goat Anti-5 (Ak) antibodies, Anti-goat Anti-5, Anti-goat Anti-5 (Ak) and Anti-goat antibodies (Ak) 6172, Anti-goat 6172 (Ak) antibodies, Anti-goat 6172, Anti-goat.

Example 1: preparation of platycodon grandiflorum polysaccharide PGPSt

Slicing radix Platycodi root, soaking in 10 weight times of water overnight, leaching at 90 deg.C for 3 hr, filtering, concentrating the filtrate to relative density of 1.05-1.15 (measured at 50 deg.C), centrifuging to remove impurities to obtain leaching solution;

adding 95 vol% ethanol into the leaching solution while stirring to make ethanol final concentration 70%, standing, centrifuging, separating precipitate, drying at 60 deg.C to obtain PGPS₇₀(ii) a Adding 95 vol% ethanol into the supernatant, stirring to reach final concentration of 50%, standing, centrifuging, separating precipitate, drying at 60 deg.C, and making into PGPS₅₀(ii) a Mixing PGPS₇₀And PGPS₅₀And uniformly mixing to obtain the platycodon grandiflorum polysaccharide PGPSt.

Example 2: research on 3D4/21 apoptosis of platycodon grandiflorum polysaccharide induced by autophagy antagonism fumonisin B1

1. The test method comprises the following steps:

1.1, preparing a platycodon grandiflorum polysaccharide solution:

the platycodon grandiflorum polysaccharide prepared in example 1 was prepared into a platycodon grandiflorum polysaccharide solution having concentrations of 20. mu.g/ml, 50. mu.g/ml, 100. mu.g/ml and 200. mu.g/ml using RPMI 1640 medium, respectively.

1.2 cell culture:

the porcine alveolar macrophage 3D4/21 cell line was cultured in RPMI 1640 medium containing 10% fetal bovine serum and 100U/mL penicillin-streptomycin at 37 ℃ with 5% CO₂Culturing in a humidified environment.

1.3 cell treatment:

culture in 96-well plates3D4/21 cells (5 × 10)³Cells/well), PGPSt (20, 50, 100 and 200 μ g/mL) at various concentrations was added to the well plate for 24h of incubation, and then cell proliferation activity was analyzed by SRB method.

1.4 cell viability assay:

3D4/21 cells cultured in 96-well plates (5 × 10)³Cells/well), different concentrations of FB1(0.5, 1, 5, and 10 μ g/mL) were added to the well plate and incubated for 12h and 24h, respectively.

3D4/21 cells cultured in 96-well plates (5 × 10)³Cells/well), cells were treated into 5 groups: the Control group does not process; 5. mu.g/mL FB1 treatment, 10. mu.g/mL FB1 treatment, 5. mu.g/mLFB 1+ 100. mu.g/mL PGPSt co-treatment, 10. mu.g/mLFB 1+ 100. mu.g/mL PGPSt co-treatment.

1.5 immunoblot detection

Cells were lysed in a low temperature non-denaturing cell lysate. Protein lysates were denatured in protein sample buffer with boiling water and subjected to SDS polyacrylamide gel electrophoresis. Proteins were transferred to PVDF membrane and detected with specific antibodies. And (3) carrying out visual detection on the membrane by adopting the hypersensitive luminescent liquid ECL. (cells or lung tissue dissolved in 50mm Tris-HCl (pH 7.4), 150mm NaCl, 1% NP-40, 0.5% sodium deoxycholate, 2mm sodium fluoride, 2mm EDTA, 0.1% SDS and PMSF RIPA buffer solution protein concentration by BCA protein detection kit (Comwin, Beijing, China) fractionation of total protein (20 ug) 10% SDS polyacrylamide gel electrophoresis and transfer to PVDF membrane, at room temperature with 5% BSA in TBS-Tween closed cell membrane for 1 hours, and at 4 degrees C with the appropriate primary antibody incubated overnight, with horseradish peroxidase coupled secondary antibody incubation, using the ECL to develop PVDF membrane, using Image J software analysis of protein gray.

1.6GFP-LC3 plasmid transfection:

2 × 10 in 24-well plate⁵Cell/well density 3D4/21 cells were cultured. The GFP-LC3 plasmid was transfected using Lipofectamine 3000(Invitrogen, Carlsbad, Calif., USA) as described and maintained on a 24-well plate. 24h after transfection, cells were treated with PGPSt at the indicated concentrations for 4 h. DAPI (Sigma, St Louis, MO, USA) was used to stain nuclei. The image of the cell isObtained by Leica fluorescence microscope. Autophagy is detected by monitoring autophagosome fluorescent spot formation.

1.7Hoechst 33342 nuclear staining:

3D4/21 cells were cultured in a 24-well plate, treated with FB1 and PGPSt, washed 2 times with PBS, permeabilized with 100. mu.L of Hoechst33342 for 10min, and the nuclear morphology of stained cells was observed with a Leica TCS SPE confocal microscope.

1.8 Transmission Electron microscopy:

the treated cells were collected by centrifugation, observed with a transmission electron microscope and imaged.

1.9 immunofluorescence assay:

3D4/21 cells were cultured on polylysine pretreated slides, incubated with various concentrations of polysaccharide or inhibitor, and then fixed with paraformaldehyde (4% wt/vol). And (3) adopting a rabbit anti-mouse LC3B monoclonal antibody and an anti-mouse Actin monoclonal antibody, sequentially incubating the antibodies, staining the antibodies by Hoechst33342, and identifying LC3B and Actin in 3D4/21 cells by an immunofluorescence method.

1.10 statistical analysis

Data were expressed as Mean ± standard deviation (Mean ± SE) and One-Way ANOVA statistical analysis was performed on the data using SPSS 24.0 biometric software. Meanwhile, by adopting Graph Pad Prism 7.0 software for drawing, P <0.05 indicates that the difference is obvious, and P <0.01 indicates that the difference is extremely obvious.

2. And (3) test results:

2.1 Effect of PGPSt on 3D4/21 cell proliferation and FB 1-induced cell injury:

as shown in FIG. 1A, PGPSt did not inhibit the proliferation of 3D4/21 cells within 24 hours at the concentration range used. 20 and 100. mu.g/mL PGPS_tThe proliferation rate of 3D4/21 cells treated for 24 hours was increased by 5% and 11%, respectively. Therefore, 20. mu.g/mL and 100. mu.g/mLPGPSt concentrations were selected for subsequent experiments.

As shown in fig. 1B and fig. 1C, FB 1-induced apoptosis was time and dose dependent. The 3D4/21 cytostatic rate of 10. mu.g/mL FB1 treatment for 24h was 38%. Therefore, 100. mu.g/mL FB1 was selected for 24 hours for subsequent experiments. To study the remission of FB1 pairsCytotoxicity of 3D4/21 cells, toxicity concentration according to FB1 and PGPS_tFB1 and PGPSt were co-treated with 3D4/21 cells. FIG. 1D shows that the cytotoxicity of FB1 was significantly inhibited when treated with 100. mu.g/mL PGPSt (P)<0.05). These results indicate that PGPSt as a protective agent significantly ameliorated FB 1-induced cell damage.

2.2PGPSt can antagonize FB 1-induced apoptosis of 3D4/21 cells:

apoptosis plays an important role in the cytotoxicity of FB 1. To determine the protective effect of PGPSt, the present invention analyzed apoptotic pathways. As central molecules of the apoptotic cascade, the clear caspase-3, caspase-9 and the anti-apoptotic protein Bcl-2 were also studied. As shown in FIG. 2A, the expression of clear Caspase-3 and Caspase-9 was significantly increased in FB1 treated cells (P <0.05), while the levels of clear Caspase-3 and Caspase-9 were significantly decreased with 100. mu.g/mL PGPSt co-treatment. Bcl-2 expression was significantly reduced after FB1 treatment (P < 0.05). Bcl-2 expression was significantly increased after 100. mu.g/mL PGPSt co-treatment (P < 0.05).

To further investigate the protective role of PGPSt in FB 1-induced apoptosis, nuclear morphology was examined by immunofluorescence microscopy. As shown in FIG. 2B, after co-treatment with PGPSt, the nuclei were hyperchromic, and the nuclei of nuclear invagination and irregular shape were significantly reduced (P < 0.05).

2.3PGPSt activates autophagy in 3D4/21 cells:

the present study found that PGPSt (100. mu.g/mL) activated the autophagy process in a dose-dependent manner at 4h, and that the expression of LC3-II, p62 and Beclin1 was significantly increased in Western-blot analysis (FIG. 3A).

To further study autophagosomes, 3D4/21 cells were observed by transmission electron microscopy. The control 3D4/21 cells showed intact structure with normal organelles in the cytoplasm as shown in FIG. 3B. Autophagosomes were observed in the PGPSt group (100. mu.g/mL). The effect of PGPSt treatment on autophagosome formation was then assessed using transient transfection and immunofluorescence (fig. 3C, D). Compared with control cells, 3D4/21 cells treated with 100. mu.g/mL PGPSt for 4h had green fluorescent spot aggregates, similar to rapamycin (positive control), indicating that PGPSt can successfully induce autophagy.

2.4 autophagy inhibitor 3-MA inhibits PGPSt-induced autophagy:

autophagosome formation As shown in FIG. 4A, after 3-MA dry-pretreatment, the PGPSt group was observed to significantly reduce the expression of LC3-II protein, inhibiting the degradation of p62, but having little effect on the expression of Beclin1 protein. In FIG. 4B, the punctate distribution of LC3 was significantly increased in 3D4/21 cells after 4h treatment with 100. mu.g/mLPGPSt, whereas the 3-MA treated cells showed less punctate distribution.

2.5 autophagy inhibitor baveromycin a1 inhibits PGPSt-induced autophagy:

the autophagy inhibitor baveromycin a1 differs from 3-MA in that it prevents the process of fusion between autophagosome and lysosome. In the presence of PGPSt, a significant increase in the expression of LC3-II and p62 proteins was observed following baverromycin A1 intervention, and the effect on Beclin1 was also minimal (FIG. 5A). Cells treated with baverromycin a1 showed more punctate distribution of LC3 (fig. 5B).

The effect of autophagy inhibitor 3-MA, the autophagy inhibitor baverromycin a1 on PGPSt-induced autophagy can be seen: PGPSt-induced autophagy flow was intact and unobstructed.

2.6 Regulation of PI3K/Akt/mTOR and MEK/ERK pathways by PGPSt:

protein expression was measured for p-mTOR, mTOR, p-AKT, AKT, p-MEK, MEK, p-ERK and ERK after treatment with PGPSt (20, 100. mu.g/mL) at various time points (2, 4 h). Indicating PGPS compared to the control group_tTreatment for 2h up-regulated the ratio of p-Erk/Erk, p-MEK/MEK and p-AKT/AKT in the cells. PGPSt treatment at 4h significantly reduced the ratio of p-Erk/Erk, p-MEK/MEK, p-mTOR/mTOR and p-AKT/AKT compared to 2h treatment (FIGS. 6A, B and 7A, B).

Immunofluorescent staining showed a significant decrease in p-Akt and p-MEK (green) fluorescence intensity in 4h of 3D4/21 cells treated with 100. mu.g/mL PGPSt.

The results show that the platycodon grandiflorum polysaccharide has a regulation effect on two signaling pathways of PI3K/Akt/mTOR and MEK/ERK, and the regulation shows time dependence, the platycodon grandiflorum polysaccharide can activate the two signaling pathways of PI3K/Akt/mTOR and MEK/ERK in the first 2h, and can inhibit the two signaling pathways of PI3K/Akt/mTOR and MEK/ERK after 4 h.

2.7PGPSt induces autophagy through PI3K/AKT/mTOR and MEK/ERK pathways

To investigate whether the PI3K/Akt/mTOR and MEK/ERK pathways play a role in inducing PGPSt-induced autophagy, Akt (LY294002) and MEK inhibitor (PD98059) were used and LC3 expression was detected by western blot. Western blot analysis showed that under the inhibition conditions, the expression of LC3II could be significantly reduced (FIG. 8A).

These results show that: PGPSt enhances autophagy by activating the MEK/ERK pathway at a pre-stage and inhibiting the PI3K/Akt/mTOR pathway at a post-stage. The effect of PGPSt-induced autophagy with a single inhibitor was insufficient, therefore, the present invention co-treated with LY294002 and PD98059, and the treated cells showed less punctate distribution of LC3 (fig. 8B).

2.8PGPSt protects 3D4/21 cells from FB 1-induced apoptosis by autophagy

In order to analyze whether autophagy is related to apoptosis inhibition, the invention adopts a western blot method to detect the expression of autophagy-related protein LC3-II and apoptosis-related protein caspase-9. PGPSt resulted in a significant reduction in the conversion of LC3 protein after treatment with FB1 (FIG. 9A). As shown in FIG. 9B, Caspase-9 expression was significantly increased in FB 1-treated cells after co-treatment with PGPSt and 3-MA. These results indicate that PGPSt protects 3D4/21 from FB 1-induced apoptosis by autophagy.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims (9)

1. Use of platycodon grandiflorum polysaccharide in preparation of modulators of PI3K/Akt/mTOR and MEK/ERK signaling pathways.

2. The application of platycodon grandiflorum polysaccharide in preparing autophagy activator of cells by regulating the activity of PI3K/Akt/mTOR and MEK/ERK signaling pathway.

3. The use according to claim 2, wherein the platycodon grandiflorum polysaccharide is time-dependent on the regulation of PI3K/Akt/mTOR and MEK/ERK signaling pathway activities, in particular: during the first 2h of platycodon polysaccharide treatment, the platycodon polysaccharide activates PI3K/Akt/mTOR and MEK/ERK signaling pathways; after 4h of platycodon polysaccharide treatment, the platycodon polysaccharide inhibits PI3K/Akt/mTOR and MEK/ERK signaling pathways.

4. Use according to claim 2, wherein the cell is a porcine alveolar macrophage.

5. Use of platycodon grandiflorum polysaccharide in the preparation of a medicament for antagonizing apoptosis induced by FB1 by activating autophagy.

6. Use according to claim 5, wherein the cell is a porcine alveolar macrophage.

7. The use according to claim 1, 2 or 5, wherein the platycodon grandiflorum polysaccharide is prepared by the following method:

slicing radix Platycodi root, soaking in 8-10 times of water at 90-100 deg.C overnight, leaching for 2-4 hr, filtering, concentrating the filtrate to relative density of 1.05-1.15 (measured at 50 deg.C), centrifuging to remove impurities to obtain extractive solution;

adding 95 vol% ethanol into the leaching solution while stirring to make ethanol final concentration 70%, standing, centrifuging, separating precipitate, drying at 60 deg.C to obtain PGPS₇₀(ii) a Adding 95 vol% ethanol into the supernatant, stirring to reach final concentration of 50%, standing, centrifuging, separating precipitate, drying at 60 deg.C, and making into PGPS₅₀(ii) a Mixing PGPS₇₀And PGPS₅₀And uniformly mixing to obtain the platycodon grandiflorum polysaccharide.

8. Use according to claim 7, characterized in that the time of resting is 24 h.

9. The use according to claim 7, wherein the concentration of platycodon grandiflorum polysaccharide is 20-100 μ g/mL.

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