CN107929307A - A kind of application of panax japonicus polysaccharides in the medicine for improving liver cancer patient immune function is prepared - Google Patents
A kind of application of panax japonicus polysaccharides in the medicine for improving liver cancer patient immune function is prepared Download PDFInfo
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Abstract
The invention belongs to the medical usage technical field of plant polyose, discloses a kind of application of panax japonicus polysaccharides in the medicine for improving liver cancer patient immune function is prepared.The application has found that panax japonicus polysaccharides can significantly inhibit the tumor growth of H22 liver cancer cells under doses first, panax japonicus polysaccharides are by increasing body thymus gland/spleen index and Spleen cell proliferation, the ratio of significant raising CD8+T and NK cells, and strengthen its cellular cytoxicity activity to Murine Hepatoma22 tumour cell.Show that panax japonicus polysaccharides can stimulate mouse immune response to play antitumor action, while do not detect the toxicity to tumor-bearing mice.Dependent interaction mechanism includes：Ability in the CD4+T Apoptosis that panax japonicus polysaccharides are induced by improving tumour transplatation；The immuno-stimulator raised in tumor-bearing mice body is horizontal, while reduces its immunosuppressive factor level；Tumor-bearing mice CD4+T cell differentiations Th1 is promoted to participate in antineoplastic immune stimulating activity；Immunostimulatory activity is produced so as to reach antitumous effect by the polarization of anti-inflammatory and adjusting TAMs.
Description
Technical field
The present invention relates to the medical usage technical field of plant polyose, and in particular to a kind of panax japonicus polysaccharides are preparing raising
Application in the medicine of liver cancer patient immune function.
Background technology
Liver cancer is a kind of malignant tumour for seriously endangering human health, its incidence is high and lethality occupies height not in recent years
Under.Onset of liver cancer reason is extremely complex, wherein, the missing of chronic persistent inflammation and tumor suppressor gene is the weight for causing liver cancer to occur
Want factor.At present, most tumor suppressor gene regulation and control hepatoma cell proliferations, the mechanism of apoptosis have been studied clear.In view of some press down
Oncogene can also express in immunocyte, have the scorching suppression cancer dual-use function of suppression, Jin Ertong concurrently in immunocyte and tumour cell
Signaling molecule common in targeting immunocyte and tumour cell is crossed, playing the scorching cancer transformation of efficiently blocking, (particularly inflammation is related
Liver cancer) occurrence and development be the application main Research Thinking.Therefore, the application mainly study panax japonicus polysaccharides by anti-inflammatory and
Immunestimulatory effect suppresses hepatocellular carcinoma, is carried for panax japonicus polysaccharides as immunologic adjuvant in the clinical practice for treating malignant disease
For theoretical foundation.
In the prior art there is not yet relevant report, the application will fill up this blank.
The content of the invention
For the deficiencies in the prior art, it is an object of the invention to provide a kind of panax japonicus polysaccharides to prepare raising
Application in the medicine of liver cancer patient immune function.
Compared with prior art, the present invention has the following advantages and effects：
1st, this method prepares that panax japonicus polysaccharides (PSPJ) simple process is easy, and controllability is strong.
2nd, the panax japonicus polysaccharides product purity that this method obtains is high, and yield is higher.
3rd, the application has found that PSPJ has prevention and protective effect to the LPS/D-GalN acute livers induced first,
And there is preferable dose dependent.
4th, the application has found that panax japonicus polysaccharides can significantly suppress the internal raw of H22 liver cancer cells under doses first
Long, panax japonicus polysaccharides are by increasing body thymus gland/spleen index and Spleen cell proliferation, the ratio of significant raising CD8+T and NK cells,
And strengthen its cellular cytoxicity activity to H22 hepatoma cellses.Being indicated above panax japonicus polysaccharides can stimulate mouse body to exempt from
Epidemic disease response plays antitumor action, while does not detect the toxicity to tumor-bearing mice.
Its relevant mechanism of action includes：In the CD4+T Apoptosis that PSPJ can be induced by improving tumour transplatation
Ability；The immuno-stimulator (IFN-γ and IL-2) raised in tumor-bearing mice body is horizontal, while reduces its immunosuppressive factor
(IL-4 and IL-10) is horizontal；Tumor-bearing mice CD4+T cell differentiations Th1 is promoted to participate in antineoplastic immune stimulating activity；Can be with anti-inflammatory
With the polarization for adjusting TAMs and produce immunostimulatory activity so as to reaching antitumous effect.
5th, in the clinical practice for the treatment of liver cancer, there is not been reported as immunologic adjuvant for panax japonicus polysaccharides.
Brief description of the drawings
Fig. 1 is Sephadex G-75 pillar layer separation panax japonicus Thick many candies elution curves in embodiment 1.
Fig. 2 is the panax japonicus polysaccharides HPLC figures after pillar layer separation in embodiment 1.
Chromatographic condition：Chromatographic column is Shodex OHpak SB-804HQ (8 × 300mm), shows poor photodetector, 0.1M
NaCl is mobile phase, flow velocity 0.5mL/min, 35 DEG C of column temperature, 20 μ L of sample size.
Fig. 3 is influence figure of the panax japonicus polysaccharides to CD8+T the and NK cells of tumor-bearing mice.
Scheme A and figure B：At the end of experiment, the spleen and lymph node of mouse are dissected.Spleen and lymph node are measured by FACS
Single cell suspension in CD8+ and NK cells percentage.Scheme C：CD8+T and NK cells are purified from the spleen of mouse.Measure NK
The cytotoxic activity of cell and CD8+T cells to H22 tumor-bearing mices.Compared with blank group：*p<0.05；**p<0.01.
Fig. 4 is influence figure of the panax japonicus polysaccharides to CD4+T cells in H22 tumor-bearing mice serum.
Scheme A：At the end of experiment, with the ratio of the CD4+T cells in the spleen and lymph node of FACS detection mouse.Scheme B
And C：At the end of experiment, CD4+T cells are purified from the spleen and lymph node of mouse.Lived with FACS (figure B) and caspase-3
Property measure (figure C) quantitative CD4+T cells apoptosis.Scheme D and E：CD4+T cells are purified from the spleen of mouse.TR-PCR (figure D) and
The transcription and translation that Western blotting (figure E) measures related gene respectively is horizontal.Compared with normal group：++p<0.01；With blank group
Compare：*p<0.05；**p<0.01.
Fig. 5 is influence figure of the panax japonicus polysaccharides to tumor-bearing mice TAMs.
Scheme A：The transcriptional level of related gene in quantitative PCR detection TAM.Scheme B：TAM24h is cultivated in serum free medium
Afterwards.With ELISA detect supernatant in IL-10 (left side), TGF-β (in) and PGE2 (right side) level.Scheme C：FACS detections lotus knurl is small
The number of the total macrophages of CD68+ and CD163+M2 polarization macrophages in mouse tumor tissues.Compared with blank group：**p<
0.01。
Fig. 6 is the survivorship curve figure that panax japonicus polysaccharides influence acute hepatic injury mice.
Fig. 7 is panax japonicus polysaccharides to mouse liver injury morphology and changes in histopathology figure.
Embodiment
Applicant in conjunction with specific embodiments will be described in further detail technical scheme below.Ying Li
Solution, herein below should not be construed as in any way the limitation for claims of the present invention being claimed scope.
Embodiment 1
The preparation of panax japonicus polysaccharides：
Raw material panax japonicus medicinal material is the dry of araliaceae ginseng plant panax japonicus (Panax japonicus C.A.Mey.)
Dry rhizome, plants panax japonicus for family, originates from Enshitujianationalitymiaonationality Autonomous Prefecture Xuanen County Chun Muying townshiies mud dam.
Step is as follows：
Dry panax japonicus medicinal powder is taken, adding 95% ethanol, (V/V, the percentage of ethanol described in this specification are dense
Degree refers both to concentration of volume percent, does not repeat hereinafter), flow back 3h at 60 DEG C, degreasing；
80% ethanol is added, flow back 2h at 60 DEG C, takes off monose and oligosaccharide；
The dregs of a decoction are placed in baking oven after drying, are extracted 3 times by the use of purified water as solvent refluxing, each 2h, merging filtrate is simultaneously
It is placed in Rotary Evaporators and is concentrated into liquid extract state, add ethanol, it is 80% to adjust concentration of alcohol, and 4 DEG C overnight, next day mistake
Filter, precipitation drying, up to panax japonicus crude product；
Gained panax japonicus crude product is taken, adds (60 DEG C) of appropriate warm water to dissolve,, will after the 48h that dialyses in flowing water after removing protein
Dialyzate vacuum freezedrying, up to panax japonicus Thick many candies.
By panax japonicus Thick many candies after DEAE-Sepharose FF column purifications 3 times；Again through Sephadex G-75 post separations
Purifying, obtains a main peak, will be dry after peak position eluent dialysis desalination, obtains panax japonicus polysaccharides, abbreviation PSPJ, related data
It is shown in Table 1.1.
Table 1.1
After testing, gained panax japonicus polysaccharides are mainly by glucose, galacturonic acid, galactolipin, glucuronic acid, sandlwood
Sugar, arabinose, xylose etc. form, its mass fraction is as shown in table 1.2.
Each monosaccharide component mass fraction table in 1.2 panax japonicus polysaccharides of table
Panax japonicus Thick many candies are after DEAE-Sepharose FF and Sephadex G-75 pillar layer separations, and elution curve is such as
Shown in Fig. 1.Further, confirm that panax japonicus polysaccharides after purification are homogeneity polysaccharide using HPLC chromatogram technology, see Fig. 2.
Embodiment 2
The influence of panax japonicus polysaccharides prepared by embodiment 1 to Murine Hepatoma22 tumor-bearing mice immune function
2.1 experiment material
2.1.1 animal and cell line
Kunming mouse：Half male and half female, 18~22g of weight, is provided by Disease Prevention Control Center, Hubei Prov, credit number
For：SCXK (Hubei Province)：2008-0003.
H22 cell lines are given by the old trip's wing teacher of pharmaceutical college of South-Center University For Nationalities, are stored in -80 DEG C of refrigerator.Recovery
When centrifuge, the DMSO in frozen stock solution is removed, with normal saline dilution into 1*106A/mL, mouse abdomen is inoculated in by every 0.2mL
Intracavitary, is passed on after seven days, takes third generation H22 ascites tumor mouse ascites, and 1 is pressed with physiological saline:10 dilutions, every 0.2mL inoculation
It is subcutaneous in mouse right fore armpit, establish H22 solid tumor models.
2.1.2 experiment reagent
Panax japonicus polysaccharides：It is prepared by embodiment 1；
Sodium chloride injection：Binghu Shuanghe Pharmaceutical Co., Ltd., Wuhan；
Concanavalin A (ConA) and lipopolysaccharides (LPS) purchased from Sigma-Adrich (St.Louis, Missouri,
USA)；
IFN-γ, IL-2, IL-4 and IL-10 assay ELISA kits are purchased from Nanjing and build up biological study institute；
Prostaglandin E2 (PGE2) assay ELISA kit is purchased from Cayman Chemical companies；
Caspase-1 activity detection kits are green skies biotechnology research institute；
The reagent consumptive materials such as the antibody and various types of cells factor enzyme linked immunological quantification kit of anti-CD4, CD8, CD49b albumen are equal
Purchased from the bright sub- biological art Co., Ltd in Wuhan.
2.2 experimental method
2.2.1 influence of the Spleen cell proliferation measuring panax japonicus polysaccharides to H22 tumor-bearing mice Spleen cell proliferations
After tumor-bearing mice is put to death, the spleen and thymus gland of mouse, weigh after rapid taking-up is put to death in an aseptic environment immediately.
Then, spleen is cut into slices, and is filtered by thin steel mesh, obtain the Spleen cell suspensions of homogenate.Use NH4Cl solution (0.15mol/
L, pH7.4) Spleen cell suspensions are incubated to remove red blood cell., will after the vigor of determination of trypan blue staining splenocyte is more than 95%
Each group splenocyte is seeded in 96 orifice plates (per hole 5 × 106A cell), then added into one group of experimental port and contain ConA (2 μ g/
ML 100 μ L of RPMI-1640 nutrient solutions), another group of experimental port add the 100 μ L of RPMI1640 nutrient solutions containing LPS (5 μ g/mL),
Negative control hole (adding isometric RPMI-1640 nutrient solutions) is set at the same time.5%CO is used at 37 DEG C2After when culture cell 72 is small,
10 μ LMTT solution (2.0mg/mL) are added in each hole, and incubate again 4 it is small when.Then, culture medium is discarded, and to each hole
150 μ LDMSO of middle addition, and be sufficiently mixed.Finally, the optical density in each hole is measured under 570nm wavelength.Stimulus index is real
The ratio verified with control wells optical density, representing ConA and LPS with stimulus index stimulates the multiplication capacity of lower immunocyte.Internal organs
Index=organ weights (mg)/mouse weight (g).
2.2.2 influence of the flow cytometry panax japonicus polysaccharides to H22 tumor-bearing mice NK cells and CD8+T cells
For the ratio of the immunocyte subgroup in quantitative analysis tumor-bearing mice spleen or lymph node, with flow cytometry point
Analyse the content of CD4+T cells, CD8+T cells and natural killer (NK) cell in suspension.By each group tumor-bearing mice spleen and leaching
Fawn on and single cell suspension and rat anti-mouse Abs FITC-CD4, PE-CD8 and PE-CD49b is made carries out incubation dyeing.Wash
After removing the antibody that is not combined with cell surface, analyzed with flow cytometer.By method of the prior art measure purifying
The ratio of apoptotic nucleus in CD4+T subgroups.Infiltration is measured to the macrophage in transplantation tumor by method of the prior art
The number of cell and M2 polarization macrophages.
2.2.3 influence of the panax japonicus polysaccharides to the H22 tumor-bearing mice serum cellular immunity factors
CD4+T is separated from the spleen single cell suspension of each group experiment mice with immunomagnetic beads method, CD8+T and NK cells are sub-
Group.Using Magnetic activated cell sorting (MACS) system for having rat anti-mouse CD4, CD8 and CD49b antibody and use mountain sheep anti mouse
Microballon.It is more than 95% with the purity of each immunocyte subgroup of flow cytomery.Immediately to CD8+T the and NK cells of purifying
Carry out determination of cytotoxic activity.Tumor-associated macrophage (TAM) is prepared from the tumour of transplanting.By the dense of CD4+T cells and TAM
Degree is adjusted to 1 × 106A cell/mL, with serum-free RPMI-1640 medium cultures.Nutrient solution supernatant is collected after 24h, is used
Enzyme-linked immunization measures the content of IFN-γ, IL-2, IL-4 and IL-10 in nutrient solution.Concrete operation method is according to corresponding
Kit specification carries out.
2.2.4 enzyme linked immunosorbent assay (ELISA) (ELISA) measure H22 tumor-bearing mices are immunized and inflammation-associated cytokine
IFN-γ, IL-2, IL-4, IL-10 and PGE2 content are measured with corresponding reagent box.Concrete operation method is according to corresponding
Kit specification carry out.
2.2.5 panax japonicus polysaccharides measure tumor-bearing mice Cell-mediated Immunity
CD8+T and NK is effector cell.By each group purifying in the DMEM culture mediums containing 10% hyclone (FBS)
The concentration of NK and CD8+T cells is adjusted to 107A/mL.H22 cells are target cells, will in the DMEM culture mediums with 10%FBS
Their concentration is adjusted to 105A/mL.100 μ L effector cells and 100 μ L target cells are sufficiently mixed in 96 orifice plates, per hole
To assess the cellular cytoxicity activity of effector cell, (effector is 100 with target ratio to 200 μ L of cumulative volume：1).By target cell (100 μ
L) incubated with together with the DMEM complete mediums of 100 μ L as negative control.After when incubation 5 is small in cell incubator, collect
Supernatant, with the activity of CytoTox96 non-radioactive cell toxicity test kit measurement lactic dehydrogenases come quantify CD8+T and
The Cell-mediated Immunity of NK cells.
2.2.6 panax japonicus polysaccharides are to Caspase-3 determinations of activity in H22 tumor-bearing mices CD4+T
With the activity of Caspase-3 in the CD4+T cells purified of Caspase-3 Activity Assay Kits measurement each group.Tool
Body operating method is carried out according to corresponding kit specification.
2.2.7RT-PCR method measures panax japonicus polysaccharides to tumor-bearing mice CD4+T gene expressions
Using extracting total serum IgE in the CD4+T immunocyte subgroups of Trizol reagent purifications.With agarose gel electrophoresis and bromine
After the integrality for changing the RNA of second ingot chromoscopy extraction, each sample takes about 5 μ g total serum IgEs, by ReverTraAce- α kits
Illustrate its 42 DEG C of reverse transcriptions obtaining cDNA in 30 minutes.Using obtained cDNA as template, glyceraldehyde 3-phosphate dehydro-genase is used
(glyceraldehyde-3-phosphatedehydrogenase, GAPDH) is internal reference, and Bcl- is detected by real-time quantitative PCR
The mRNA level in-site of 2, Bcl-xL, Mcl-1, IL-10, TGF-β and COX-2.Primer pair group for quantitatively real-time PCR is enumerated such as table
2.1：
The primer pair group that table 2.1 quantifies real-time PCR enumerates table (5' to 3')
2.2.8 Western blot detection panax japonicus polysaccharides are to the protein expression levels of H22 tumor-bearing mice related genes
Influence
Every group takes the H22 solid tumors of 0.25g to be cracked on ice with Tissue lysates, and 12000r/min centrifugation 15min, are collected
Supernatant.With Bradford determination of protein concentration kit measurement protein concentrations.The protein sample (30 μ g) of equivalent is taken through SDS-
PAGE is simultaneously transferred on nitrocellulose filter.With TBST prepare 5% skimmed milk power room temperature closing 2h after, and with Bcl-2, Bcl-
XL, Mcl-1 and β-actin antibody (1：1500 dilutions) 4 DEG C overnight after.Then, film 3 times, each 5min is washed with TBST, then
2h is incubated at room temperature with the secondary antibody of corresponding horseradish peroxidase mark, TBST washes film three times.The method to shine according to ECL sequentially adds
Luminous substrate, exposure, development.
2.2.9 data analysis
Experimental data is expressed as mean ± standard deviation (Mean ± SE), and application Sigma-plot10.0 statistical softwares carry out
Analysis.Examined using double tail t and carry out two groups of sample average comparative analysis, one-way analysis of variance distinguishes whether multigroup have statistics
Meaning, P<0.05 shows difference with statistical significance.
2.3 results and analysis
2.3.1 influence of the panax japonicus polysaccharides to H22 tumor-bearing mices Spleen cell proliferation and thymus gland/spleen index
External MTT experiment does not detect cytotoxic effects of the PSPJ to H22 cells, it is possible thereby to show that PSPJ does not press down
Make internal H22 cell growths, it may be possible to stimulate the immune response in tumor-bearing mice body.In order to verify one it is assumed that we have evaluated
Influences of the PSPJ to thymus gland/spleen index of tumor-bearing mice.The thymus gland of 5-FU tumor-bearing mices/spleen index declines, and PSPJ dosage according to
Rely thymus gland/spleen index of property up-regulation tumor-bearing mice.H22 tumor-bearing mices Spleen cell proliferation after ConA and LPS stimulations substantially drops
Low, the splenocyte stimulus index of PSPJ administration groups is then obviously improved relative to H22 lotus knurls model group, and lifted degree with
The increase of dosage and increase, but be administered 5-FU after Spleen cell proliferation ability do not change, experimental result is shown in Table 2.2.Thus table
Bright, PSPJ can substantially increase the spleen and thymus index of tumor-bearing mice, and improve its splenocyte under ConA/LPS stimulations
Multiplication capacity.
2.2 panax japonicus polysaccharides of table are to H22 tumor-bearing mices thymus gland/spleen index and ConA and the LPS Spleen cell proliferation induced
Influence (Mean ± SE, n=12)
Compared with blank group：ap<0.05,bp<0.01；Compared with normal group：cp<0.05
2.3.2 influence of the panax japonicus polysaccharides to NK cells and CD8+T cells in H22 tumor-bearing mices
NK and CD8+T cells are the immunocyte subgroups with the ability for directly destroying malignant cell.In spleen and lymph node
In, the ratio of NK and CD8+T cells remains unchanged after tumour transplatation.Cause CD8+T cells and NK ratios after PSPJ administrations
Dose dependent increases (Fig. 3 A and B).By detecting cell toxicants of the PSPJ to NK cells and CD8+T cells to H22 tumor-bearing mices
Property activity influence.Compared with normal group, the significant reduction of cellular cytoxicity activity of the NK and CD8+T cells of H22 tumor-bearing mices, gives
After medicine PSPJ, the cellular cytoxicity activity (Fig. 3 C) of NK and CD8+T cells is dose-dependently improved.
2.3.3 influence of the panax japonicus polysaccharides to H22 tumor-bearing mice serum cytokines
In order to evaluate influences of the PSPJ to serum cytokines, IFN-γ is have detected with immune enzyme-linked method, IL-2, IL-4,
With the content of IL-10.IFN-γ and IL-2 are classical immunostimulatory cells factors.IL-4 and IL-10 is classical is immunized
Inhibitor.As shown in table 2.3, be inoculated with H22 liver cancer cells after, in tumor-bearing mice serum IFN-γ and IL-2 levels significantly reduce and
IL-4's and IL-10 is horizontal significantly raised.Compared with blank group, after PSPJ (30 and 100mg/kg) is administered, tumor-bearing mice serum
The level of middle IFN-γ increases to 21.4 and 29.0pg/mL, and IL-2 levels are increased to 17.7 and 21.6pg/mL.In addition, in serum
The horizontal horizontal reductions by 37.3% and 63.9% for reducing by 24.6% and 40.0%, IL-10 of IL-4.Thus illustrate, panax japonicus is administered
After polysaccharide, the immuno-stimulator rise in tumor-bearing mice body, immunosuppressive factor is horizontal to be declined.
2.3 panax japonicus polysaccharides of table are on IFN-γ, the horizontal influence (Mean of IL-2, IL-4 and IL-10 in tumor-bearing mice serum
± SE, n=12)
Compared with normal group：ap<0.01；Compared in blank groupbp<0.01
2.3.4 influence of the panax japonicus polysaccharides to H22 tumor-bearing mice change of serum C D4+T cells
After being inoculated with H22 liver cells, the significant reduction of CD4+T cells ratios in the spleen and lymph node of tumor-bearing mice, administration
After PSPJ, raise (Fig. 4 A) to CD4+T cells ratio dosage accordance with tolerance.Compared with normal group, after being inoculated with H22 liver cells, CD4+T
The apoptosis rate rise of cell, after PSPJ is administered, the apoptosis rate dosage accordance with tolerance of CD4+T cells reduces (Fig. 4 B and C).Bcl-2,
Bcl-xL and Mcl-1 is three kinds of classical anti-apoptotic genes expressions.In model group CD4+T cells, mRNA and protein level are notable
Reduce.Strengthen to administration PSPJ post dose dependences the transcription and translation (Fig. 4 D and E) of these anti-apoptotic genes expressions.
After being inoculated with H22 liver cells, the cell factor of tumor-bearing mice CD4+T cells secretion is also changed.In model group
In, the significant reduction of the amount of IFN-γ and IL-2 in CD4+T cells, and the increase of the amount of IL-4 and IL-10.After PSPJ is administered,
The cytokine dysregulation situation of tumor-bearing mice CD4+T cells secretion is improved (table 2.4).
IFN-γ that 2.4 PSPJ of table secretes the CD4+T cells of H22 tumor-bearing mices, IL-2, IL-4 and IL-10 are horizontal
Influence (Mean ± SE, n=12)
Compared with normal group：ap<0.01；Compared with blank group：bp<0.01。
2.3.5 influence of the panax japonicus polysaccharides to H22 tumor-bearing mices TAMs
TAMs is the important component of tumor microenvironment.IL-10, TGF-β and COX- in PSPJ energy dose-dependent inhibitions TAM
2 transcription (Fig. 5 A).PSPJ is significant to reduce the level of IL-10 and TGF-β in serum-free TAM supernatants (Fig. 5 B, left with).
PGE2 is the catalysis metabolite of COX-2.The amount for the PGE2 that TAM is produced is significant after PSPJ is administered reduces (Fig. 5 B, right).Administration
After PSPJ, the total macrophages of CD68+ change unobvious to H22 tumor-bearing mices wetting capacity, and still, PSPJ can be dose-dependently
Reduce the quantity (Fig. 5 C) of CD163+M2 samples TAM.
2.4 discuss
Experimental result shows that PSPJ suppresses the tumor growth of H22 liver cancer cells under the dosage of 100mg/kg.PSPJ increases
Body thymus gland/spleen index and Spleen cell proliferation.In addition, the ratio of the also significant raising CD8+T and NK cells of PSPJ, and it is right to strengthen its
The cellular cytoxicity activity of H22 hepatoma cellses.This, which is indicated above PSPJ, can stimulate antitumor immunity of organism response.Panax japonicus
Content of the polysaccharide in medicinal material is 2.43%.The PSPJ (100mg/kg) of high dose takes in about 5.32g/kg's equivalent to mouse
Dry root-like stock.In terms of surface area, 0.43g/kg of the dosage equivalent to the mankind.That is 65 kilograms of people absorbs about daily
The panax japonicus medicinal material of 28g, this is normal dosage.5-FU is conventional antineoplastic.Relevant report 5-FU has serious
Adverse reaction.5-FU (10mg/kg) tumor-bearing mice serum alts afterwards are administered, AST, BUN and the horizontal of CRE are improved, and thymus gland/
Spleen index drastically declines.And panax japonicus polysaccharides do not detect the poison to tumor-bearing mice under the dosage of 100mg/kg yet
Property.These results of study demonstrate jointly suppresses hepatocellular carcinoma not by cytotoxicity in panax japonicus polysaccharides body.
In mammal, the important subgroup group of CD4+T cellularity immunocytes, can adjust extensively immune response with
Resist malignant cell, including HCC cells.Research shows that the CD4+T cell quantities of HCC patient are greatly decreased.In normal condition
Under, the quantity of CD4+T cells should strictly be controlled by a series of molecular mechanisms for adjusting its propagation and apoptosis.CD4+T is thin
An important factor for apoptosis of born of the same parents is immunologic escape and cancer progression.It is thin that CD4+T is promoted in spleen and lymph node, after tumour transplatation
Born of the same parents' apoptosis and the percentage for reducing CD4+T cells.Improve these exceptions to dependence after administration panax japonicus polysaccharides.Bcl-2, Bcl-
XL and Mcl-1 is three kinds of anti-apoptotic proteins of the integrality that can keep mitochondrial membrane, after being inoculated with H22 hepatocellular carcinomas, these three
Transcription and translation of the anti-apoptotic proteins in CD4+T cells is horizontal to be declined.After panax japonicus polysaccharides are administered, they are in CD4+T cells
The expression quantity rise of middle anti-apoptotic genes expression, shows in the CD4+T Apoptosis that panax japonicus polysaccharides can improve tumour transplatation induction
Ability.
The cell factor Th1 and Th2 of the two classes classics of CD4+T cells secretion.Th1 cells produce IFN-γ and IL-2.Th2
Produce IL-4 and IL-10.Clinical research shows that the HCC patient with the IL-2 (Th1 cell factors) of higher level can increase
CD8+T cytotoxic cells number and more favourable prognosis.Moreover it has been reported that IFN-γ, another Th1 cell factors,
Being capable of activating cytotoxic NK cells.In contrast, Th2 cell factors are the significant contribution persons of carcinoma cell immunization escape.Th1 and
The change of Th2 serum cytokines plays a crucial role during HCC develops.Panax japonicus polysaccharides increase Th1 cell factors
The serum levels of IFN-γ and IL-2, and reduce the level of Th2 cell factors IL-4 and IL-10.In addition, panax japonicus polysaccharides also may be used
Dose-dependently to change the functional status of the imbalance of cytotoxicity CD8+T and NK cell.This experiment is in order to verify panax japonicus
Effect of the polysaccharide to CD4+T cell differentiations, analyzes the cell factor from the CD4+T cells secretion of purifying.It is more that panax japonicus is administered
The IFN-γ of CD4+T cells and the horizontal increases of IL-2 after sugar, and the horizontal reduction of IL-4 and IL-10.Thus illustrate that panax japonicus is more
Sugar promotes tumor-bearing mice CD4+T cell differentiations Th1 to participate in antineoplastic immune stimulating activity.
In general, TAMs is infiltrated to the most abundant immune cell population in HCC microenvironments.TAMs is related in cancer
Play a significant role in inflammation and immune tolerance, promote the progress of HCC.TGF-β and IL-10 are that two kinds secreted by TAMs are common
Immuno-suppressing cytokine.Panax japonicus polysaccharides reduce their transcription and generation.PGE2 is to suppress Host Anti-tumor Immunity
The crucial proinflammatory inflammation factor of response.COX-2 is the rate-limiting enzyme for the approach for being catalyzed PGE2 biosynthesis.COX-2/PGE2 cascade signals
It can be adjusted by plant polyose, cause the significant improvement of Host Anti-tumor Immunity response.Panax japonicus polysaccharides can be with dose dependent
Ground suppresses the transcription of COX-2 and the generation of PGE2 in TAMs.In general, TAMs can be activated into M1 samples or the polarization of M2 samples.M1 samples
Polarization TAMs is conducive to antitumor Th1 immune responses, and M2 samples polarization TAM can promote Th2 immune responses, suppresses antineoplastic immune
Reaction.Panax japonicus polysaccharides can dose-dependently promote Th1 immune responses and suppress Th2 immune responses, polarize and produce to TAMs
It is raw to influence.Although the total macrophage quantity of CD68+ in tumor-bearing mice tumor tissues is administered after panax japonicus polysaccharides, infiltration
CD168+M2TAMs quantity but declines to a great extent.These result of study collectively show thats, panax japonicus polysaccharides can adjust the polarization of TAM and
Immunostimulatory activity is produced so as to reach antitumous effect.
Embodiment 3
The protective effect of panax japonicus polysaccharides prepared by embodiment 1 to LPS/D-GalN inducing mouse acute liver damages：
Using LPS/D-GalN inducing mouses acute liver damage as model, research panax japonicus polysaccharides are to acute liver damage
Prevention and protective effect simultaneously are attempted to inquire into its mechanism.
3.1 experimental animals and reagent
3.1.1 experimental animal
SPF grades of male mouse of kunming 48,18~22g of weight, it is dynamic purchased from Hubei Province Animal Experimental Study center, experiment
Thing production licence number：SCXK (Hubei Province) 2015-0018.
3.1.2 reagent
Panax japonicus polysaccharides：Embodiment 1 is prepared；Lipopolysaccharides (LPS) is purchased from Biosharp；Amine-galactose (D-GalN)
Purchased from Tokyo Chemical Industry；Alanine aminotransferase (ALT), aspartate aminotransferase (AST), go back
Prototype glutathione (GSH), superoxide dismutase (SOD) and malonaldehyde (MDA) assay kit are purchased from Nanjing and build up
Biological study institute；Tumor necrosis factor α (TNF-α), interleukin-1 beta (IL-1 β), interleukin-6 (IL-6) are containing measurement
Determine kit and be purchased from Shanghai Yuan Ye biotechnologies company.
3.2 method
3.2.1 packet administration and animal model
48 kunming mices are randomly divided into 4 groups, i.e. Normal group, model group, PSPJ administration groups, and gastric infusion is (normal
Control group and model group give isometric physiological saline, and administration group gives the PSPJ of 50,150mg/kg respectively), 1 time a day,
Successive administration 7 days.After last dose 2h, remaining each group mouse peritoneal injection LPS (8 μ g/kg) and D- in addition to Normal group
GalN (800mg/kg) prepares acute hepatic injury model.
3.2.2 the collection and processing of sample
Observed in 24h and record the death condition of mouse, and plucked eyeball when experiment mice will be dead and take blood, separate blood
Clearly.Mouse is dissected immediately after taking blood, and the hepatic tissue fritter for taking mouse right lobe of liver same area size identical is placed in 10% neutral first
It is fixed in aldehyde, observation pathology of hepar change is dyed for HE, -80 DEG C of preservations of remaining liver are used for biochemical analysis.
3.2.3 index of correlation detects
The death condition of each group mouse different time points in 24h is recorded, and draws survivorship curve.Liver is taken, is taken pictures simultaneously
Weigh, calculate liver index.According to kit specification, the level of ALT, AST, GSH, SOD and MDA in mice serum are detected；
TNF-α, IL-1 β and IL-6 levels are measured with ELISA method in mouse liver even slurry.
3.2.4 data analysis
Experimental data is expressed as mean ± standard deviation (Mean ± SE), and application Sigma-plot10.0 statistical softwares carry out
Analysis.Examined using double tail t and carry out two groups of sample average comparative analysis, one-way analysis of variance distinguishes whether multigroup have statistics
Meaning, P<0.05 shows difference with statistical significance.
3.3 results and analysis
3.3.1 influence of the panax japonicus polysaccharides to acute hepatic injury mice survival rate
The acute liver of LPS/D-GalN inductions can cause model mice (6h) mortality in a short time, lead to
Prevention and guarantor of the medicine to hepatic injury can preferably be evaluated by crossing life span and survival rate of the observation administration mouse after modeling
Shield acts on.Experimental result is shown：Model group mouse starts death in modeling 4h or so, and the time that peak mortality occurs is left for 5h
The right side, 24h survival rates are 16.7%.In administration group, the peak mortality time of PSPJ low dose group mouse is 8h or so, and 24h gives birth to
Deposit rate 50%；PSPJ high dose group dead mouse rush hours are 4.5 times of model group close to 10h, 24h survival rates, are compared
PSPJ low dose groups improve 25% (Fig. 6).This shows that PSPJ has prevention to the LPS/D-GalN acute livers induced
With protective effect, and there is preferable dose dependent.Then we have carried out tracing study to the time-to-live for the mouse that survives again,
The results show：The model group mouse limit time-to-live was at 2 days, and the limit time-to-live of PSPJ high dose groups can even reach
7 days.
3.3.2 panax japonicus polysaccharides are to acute hepatic injury mice liver morphology and pathological effect
Liver appearance and pathology form are to evaluate hepatic injury and medicine to the most directly perceived of damage prevention and protective effect
Index.Normal group mouse liver appearance color is light, and edge is lubricious, and matter is tough；In terms of liver histopathology form, cell size
Homogeneous, marshalling, structural integrity, it is central that nucleus is located at liver cell.Model group liver appearance color is dark red, has obvious swollen
Swollen, extravasated blood；Hepatic tissue pathology damage is serious, is full of small vacuole in degeneration of liver cells, endochylema, and have obvious liver bleeding and inflammation
Disease cellular infiltration.After PSPJ is administered, obvious improvement is presented in the appearance and color of mouse liver, and pathologic finding shows that PSPJ gives
Medicine group mouse liver cell gap reduces, and high dose group is congested and cell infiltration situation significantly improves, and it is normal to be bordering on recovery.Explanation
PSPJ can dose-dependently alleviate the liver appearance of LPS/D-GalN inductions and change (experimental result such as figure of micromorphology
Shown in 7).
3.3.3 influence of the panax japonicus polysaccharides to hepatic injury mouse ALT, AST and liver index level
Mouse liver index reflects liver damage degree indirectly, and the ALT and AST in mice serum can directly react mouse
Liver function.After LPS/D-GalN is stimulated, the level of ALT, AST increase 4.1 times and 3.1 respectively in model group mice serum
Times, mouse liver index also have conspicuousness rise (##P<0.01), illustrate that LPS/D-GalN result in hepatic injury, show as liver
The oedema and dysfunction of liver of tissue.Compared with model group, the level of ALT, AST and liver refer in PSPJ administration group mice serums
Number has different degrees of decline, and all indexs of wherein high dose group decrease beyond 40% (the experimental result such as institute of table 3.1
Show).Illustrate that PSPJ can dose-dependently improve the dysfunction of liver of LPS/D-GalN induced liver injury mouse and edema due to dysfunction of the liver swells.
Influence (Mean ± SE, n=12) of 3.1 panax japonicus polysaccharides of table to hepatic injury mouse liver function indexes and liver index
Compared with normal group#P<0.05,##P<0.01；The * P compared with model group<0.05, * * P<0.01, similarly hereinafter.
3.3.4 influence of the panax japonicus polysaccharides to hepatic injury mouse MDA, SOD and GSH level
Antioxidative Factors SOD, GSH and oxidative stress marker MDA reflection body oxidative stress status.Work as LPS/D-
After GalN inducing mouse hepatic injuries, SOD and GSH activity in its hepatic tissue are reduced, the increase of MDA contents.In model group, liver group
Knit the level of middle GSH and SOD does not reduce 3.2 times and 1.0 times compared with normal components, and MDA levels increase 0.97 times, and has aobvious
Work sex differernce (##P<0.01), illustrate that LPS/D-GalN inducing mouse hepatic injuries are related with oxidative stress.Compared with model group,
The horizontal rises of GSH and SOD in PSPJ administration group murine liver tissues, MDA is horizontal to be reduced, GSH and MDA changes wherein in high dose group
Significantly (* * P<0.01) (experimental result is as shown in table 3.2).Illustrate that PSPJ can improve LPS/D-GalN inducing mouse hepatic injuries
Oxidative stress status.
Influence (Mean ± SE, n=12) of 3.2 panax japonicus polysaccharides of table to hepatic injury mouse MDA, SOD and GSH level
Compared with normal group#P<0.05,##P<0.01；The * P compared with model group<0.05, * * P<0.01, similarly hereinafter.
3.3.5 influence of the panax japonicus polysaccharides to hepatic injury murine liver tissue inflammatory mediator TNF-α, IL-1 β and IL-6
The degree of acute liver has significantly with internal inflammatory mediator TNF-α, the concentration level of IL-1 β and IL-6
Positive correlation.Compared with normal group, TNF-α, IL-1 β and IL-6 concentration in model group murine liver tissue significantly rise (##P<
0.01), front and rear 10 times or so of difference.Illustrate experiment mice after LPS/D-GalN is stimulated, induce serious hepar damnification, promote
Into a large amount of releases of inflammatory factor, while also illustrate this experiment modeling success.Compared with model group, after PSPJ is treated, by
TNF-α, the concentration of IL-1 β and IL-6 reduce 0.44 times, 0.62 times and 0.31 times respectively in damage hepatic tissue, and with preferable
Dose-effect relationship (result is as shown in table 3.3).Illustrate that PSPJ can be suppressed by the release for the inflammatory factor for preventing murine liver tissue
Its inflammatory reaction and reach prevention and protective effect to LPS/D-GalN inducing mouse hepatic injuries.
Influence (Mean ± SE, n=of 3.3 panax japonicus polysaccharides of table to hepatic injury murine liver tissue TNF-α, IL-1 β and IL-6
12)
Compared with normal group#P<0.05,##P<0.01；Compared with model group*P<0.05,**P<0.01, similarly hereinafter.
3.4 discuss
Originally test result indicates that, PSPJ can extend survival rate and the time-to-live of experiment mice, improve the appearance of liver
And tissue morphology；Compared to model group, after PSPJ is administered, in mice serum AST, ALT and the equal conspicuousness of liver index decline (**P
<0.01), SOD and GSH is horizontal in liver tissue homogenate rises, the equal conspicuousness decline of MDA, TNF-α, the content of IL-1 β and IL-6 (**
P<0.01)。
Intraperitoneal injection LPS and D-GalN is the more easily method for building acute liver model.LPS is leather
Endotoxic main component secreted by gram-negative bacteria, LPS is by stimulating the immunocyte including macrophage to discharge
Inflammatory factor, so that apoptosis and necrosis can occur for liver cell.D-GalN can be pressed down by consuming the uridine triphosphate in liver
Large biological molecule of the system using RNA and protein as representative synthesizes, and causes inflammation reaction and the necrosis of liver cell diffusivity.
Under the synergistic effect of LPS and D-GalN, the liver cell mortality in a short time of experimental animal, liver physiology function seriously by
Damage.In this experiment, survival rate of the model group mouse in 24h is only that 16.7%, PSPJ group mouse 24h survival rates substantially carry
Height, the liver cell extent of damage significantly reduce, and Serum ALT and AST contents are substantially less than model group.Above experimental fact shows PSPJ
There is protection liver cell, to anti-liver injury.
The hepatic injury of resulted from chemical medicine generally changes with body oxidative stress status to be closely related.This can further cause
SOD and GSH contents reduce in liver, and MDA contents rise.Similarly, it has been found that in model group murine liver tissue, SOD and
GSH is horizontal to be reduced, the increase of MDA contents.These experimental results prompt liver oxidative stress to be damaged in LPS/D-GalN combined inductions liver
Important function is played during wound.For PSPJ high dose groups compared with model group, the activity of SOD, GSH have been respectively increased 4.02 times
With 1.58 times, MDA reduces 1.65 times, and difference has statistical significance.Show that PSPJ can improve the oxygen of mouse damaged liver
Change stress situation.
TNF-α, IL-1 β and IL-6 are the inflammatory factors secreted by hepatic macrophages.The inflammation such as TNF-α, IL-1 β, IL-6
On the one hand inflammation factor acts on surface of hepatocytes acceptor, cause necrosis of liver cells, and NF- κ B signals lead in another aspect active cell
Road, further increases its burst size to positive feedback, aggravates hepatocellular injury.It was found that the joint in LPS/D-GalN is made
Under, TNF-α, the content of IL-1 β and IL-6 are significantly raised in hepatic tissue, and this phenomenon can effectively be suppressed by PSPJ, prompt
Suppress liver inflammatory reaction and play key player in the hepatocyte protection activity of PSPJ.
In conclusion the result of study preliminary proof of the present embodiment Acute Hepatics of the PSPJ to LPS/D-GalN inducing mouses
Damage has clearly prevention and protective effect, its mechanism of action is probably the hair for suppressing response to oxidative stress and inflammatory reaction
It is raw, so as to prevent and prevent hepatic injury, improve the function of damaged liver, it is final to prevent conversion of the inflammation to liver cancer.
Claims (2)
1. application of the panax japonicus polysaccharides in the medicine for improving liver cancer patient immune function is prepared.
2. application of the panax japonicus polysaccharides as immunologic adjuvant in the medicine for improving liver cancer patient immune function is prepared.
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| CN116617247A (en) * | 2023-07-25 | 2023-08-22 | 青岛农业大学 | Roughhaired holly root polysaccharide prepared by organic ultrafiltration membrane purification and separation and application thereof |
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