CN101327218A - Immunoregulation novel use of cinobufagin - Google Patents
Immunoregulation novel use of cinobufagin Download PDFInfo
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- CN101327218A CN101327218A CNA2007100557797A CN200710055779A CN101327218A CN 101327218 A CN101327218 A CN 101327218A CN A2007100557797 A CNA2007100557797 A CN A2007100557797A CN 200710055779 A CN200710055779 A CN 200710055779A CN 101327218 A CN101327218 A CN 101327218A
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Abstract
Through a method of MTT, the individual effects of cinobufagin and the influence of combined effects of the cinobufagin and canavaline A or lipopolysaccharide on the reproduction or conversion of splenic lymphocytes of a normal mouse are detected; through the method of MTT and neutral red phagocytosis experiment, the influence of CBG on the energy metabolism level and the phagocytosis function of macrophages in the abdominal cavity is detected; through flow cytometry, the influence of the CBG on the splenic lymphocytes period of the normal mouse and the expression of the surface antigens CD4 and CD8 of the T lymphocyte in the spleen is detected. The invention validates that the cinobufagin has the effect of enhancing the immunity.
Description
One, technical field
The present invention relates to the immunoregulation effect of cinobufacin, belong to the field of medicaments scope.
Two, background technology
Cinobufacin (Cinobufagin CBG) is one of Bufo siccus effective ingredient, and chemistry is by name: 5beta-Bufa-20, and 22-dienolide,
14，15beta-epoxy-3beta，16beta-dihydroxy-，16-acetate(8CI)。Its structure is as follows:
The tool heart tonifying, boost, effect such as local anesthesia, antitumor, but its influence to body's immunity does not appear in the newspapers as yet.The present invention has studied cinobufacin to the immune function of mice regulating action, and tentatively illustrates the mechanism of its immunological enhancement, provides certain scientific basis for seeking new effective Chinese medicine immunostimulant.
Three, summary of the invention
Detect independent effect of CBG and collaborative con A (Con A) or lipopolysaccharide (LPS) effect influence by mtt assay to normal mice spleen lymphocytes proliferation, conversion, engulf experiment by mtt assay and dimethyl diaminophenazine chloride and detect the influence of CBG peritoneal macrophage (Peritonealmacrophage, PM Φ) energy metabolism level and phagocytic function; By the influence of Flow cytometry CBG to T lymphocyte surface antigen CD4, CD8 expression in normal mouse spleen lymphocyte cycle and the spleen.
Four, the specific embodiment
1 materials and methods
1.1 laboratory animal
Healthy Balb/c mice, male ♂, 6-8 age in week, body weight (20 ± 2) g purchases the Experimental Animal Center in preclinical medicine institute of Jilin University, the quality certification number: SCXK (lucky 2003-0001).
1.2 medicine and reagent
1.2.1 cinobufacin source
Cinobufacin is available from examining and determine institute in the Chinese biological goods.
1.2.2 reagent and preparation
The cinobufacin monomer is a fat-soluble medicine, uses DMSO (final volume＜0.1%) hydrotropy during preparation.Con A (Con A Type IV), lipopolysaccharide (LPS), tetramethyl azo tetrazolium bromide (MTT), propidium iodide (PI), trypan blue (Trypan Blue) are the Sigma product, (the RPMI-1640 complete medium preparation: 100UmL of RPMI-1640 culture medium
-1Penicillin, 100gL
-1Streptomycin, 1% glutamine, 10% deactivation new-born calf serum, pH7.2), trypsin is the Gibco product, CD4/L3T4-PE, CD8a/Lyt-2-FITC fluorescent-labeled antibody are Southern Biotech product.
1.3 mtt assay is measured the mouse spleen lymphocyte conversion ratio
The aseptic mice spleen of getting is prepared into 5 * 10
6Individual L
-1The lymphocyte suspension, every hole 100 μ L add 96 well culture plates, every again hole 100 μ L add the RPMI-1640 complete culture solution that contains variable concentrations CBG, make that final concentration is respectively 0.5,1.0,2.5,5.0,10,15mgL
-1(single medicine group), or add variable concentrations CBG (0.5,1.0,2.5,5.0,10,15mgL
-1) and ConA (5mgL
-1) or LPS (20mgL
-1) mixed liquor (composition of medicine group), establish the positive, feminine gender and blank group simultaneously, all establish 4 multiple holes for every group.37 ℃, 5%CO
2Continuous culture 44h in the incubator, every hole 20 μ L add MTT liquid, continue to cultivate 4h, measure absorbance A in the 570nm place with TECAN microplate reader (Austrian product), the result reflects the height of mice spleen lymphocytes proliferation, conversion ratio with stimulation index (SI): SI=medicine irritation hole A/ negative control hole A
1.4 the mensuration of Turnover of Mouse Peritoneal Macrophages energy metabolism level and phagocytic function
Preparation, purification mice PM Φ, counting cells density is 5 * 10
6Individual L
-1, every hole 100 μ L add 96 well culture plates, and every again hole 100 μ L add the RPMI-1640 complete culture solution that contains variable concentrations CBG, make that final concentration is respectively 0.5,2.5,10mgL
-1(single medicine group), or add variable concentrations CBG (0.5,2.5,10mgL
-1) and ConA (5mgL
-1) or LPS (20mgL
-1) mixed liquor (composition of medicine group), establish the positive, feminine gender and blank group simultaneously, all establish 4 multiple holes for every group.37 ℃, 5%CO
2Cultivate 20h in the incubator, every hole 20 μ L add MTT, continue to cultivate 4h, measure absorbance in the 570nm place.Other gets PM Φ, and the dosing method is the same, cultivates 24h, and 1800 * g is centrifugal, and 10min abandons supernatant, and every hole 100 μ L add dimethyl diaminophenazine chloride 1gL
-1Physiological salt liquid continues to cultivate 30min; PBS washes plate 2 times, every hole 100 μ L add cytolysate (acetic acid: dehydrated alcohol=1: 1 V: V), room temperature standing over night and in 540nm place mensuration absorbance.
1.5 T lymphocytic cell surface sign in the Flow cytometry mice spleen
The aseptic mice spleen of getting is prepared into 5 * 10
6Individual L
-1The lymphocyte suspension, every hole 1mL adds 24 well culture plates, every again hole 1mL adds the RPMI-1640 complete culture solution that contains variable concentrations CBG, makes that final concentration is respectively 0.5,2.5,10mgL
-1(single medicine group), or add contain different final concentration CBG (0.5,2.5,10mgL
-1) and Con A (5mgL
-1) or LPS (20mgL
-1) mixed liquor (composition of medicine group), and establish feminine gender, positive controls, all establish 3 multiple holes for every group.37 ℃, 5%CO
2Cultivate 48h in the incubator, collecting cell, CD4/L3T4-PE, CD8a/Lyt-2-FITC fluorescent labeling, last FACS Calibur type flow cytometer (U.S. Becton Dickinson company product) detects the T lymphocyte surface antigen.
1.6 the Flow cytometry mouse spleen lymphocyte cycle
The mouse boosting cell suspension is handled with 1.5, collecting cell, and PI dyeing, the up flow type cell instrument detects cell cycle.
1.7 statistical procedures
All data input computers carry out the t check with SPSS for windows 10.0 version softwares, and result of calculation is represented with x ± s.
2 results
2.1 cinobufacin is to the influence of mice spleen lymphocytes proliferation, conversion
As shown in Table 1, the SI value of the single medicine group of CBG and collaborative Con A or LPS medicine group all improves with respect to feminine gender and positive controls, and be a certain amount of effect relationship, SI value peak (P＜0.05 or P＜0.01) when doses, along with the increase of dosage, it reduces on the contrary to the facilitation that mouse spleen lymphocyte transforms; Wherein collaborative LPS effect group is obvious than other two groups to the facilitation of mice spleen lymphocytes proliferation, conversion.
Table 1. cinobufacin is to the influence of mouse spleen lymphocyte conversion ratio
Annotate: 1) negative control group, 2) 5mgL
-1Con A positive controls, 3) 20mgL
-1The LPS positive controls.Single medicine group compares with negative control group, and combination group and positive controls compare, and 4) P＜0.05,5) P＜0.01, every group 4 multiple hole.
2.2 cinobufacin is to the influence of Turnover of Mouse Peritoneal Macrophages
As show 2-3 and show, 0.5,2.5,10mgL
-1Under three dosage, single medicine group of CBG and collaborative Con A or LPS medicine group are to mice PM Ф metabolism MTT and engulf the dimethyl diaminophenazine chloride ability certain facilitation (P＜0.05 or P＜0.01) is all arranged, and at 2.5mgL
-1The facilitation of CBG peaks during dosage, and wherein collaborative LPS effect group is obvious than other two groups.
Table 2. cinobufacin is to the influence of Turnover of Mouse Peritoneal Macrophages energy metabolism level
Table 3. cinobufacin is engulfed the influence of dimethyl diaminophenazine chloride ability to Turnover of Mouse Peritoneal Macrophages
Annotate: handle with table 1.
2.3 cinobufacin is to the influence of mouse spleen lymphocyte cycle and T lymphocytic cell surface sign
As shown in Table 4,0.5,2.5,10mgL
-1Under the dosage, compare the single medicine group of CBG, CD4 in the mice spleen with negative control group
+CD8
+T lymphocyte subset group percentage rate all is significantly increased, CD4 in the middle and high dosage group of CBG
+CD8
-And CD4
+CD8
+T lymphocyte subset group percentage rate sum all is significantly increased the CD4 of CBG low dose group
+/ CD8
+Ratio be significantly increased (P＜0.05); Low, the high dose group of CBG, CD4
-CD8
+T lymphocyte subset group percentage rate all has obvious reduction (P＜0.05);
Compare with negative control group, CBG works in coordination with Con A effect, can significantly improve CD4 in the mice spleen
+CD8
+T lymphocyte subset group percentage rate increases CD4
+CD8
-And CD4
+CD8
+T lymphocyte subset group percentage rate sum (P＜0.05 or P＜0.01); But CD4
+CD8
-, CD4
-CD8
+T lymphocyte subset group percentage rate all has obvious reduction, in the collaborative Con A effect of CBG, low dose group CD4
+/ CD8
+Ratio also obviously reduces (P＜0.05 or P＜0.01);
Compare with negative control group, collaborative Con A of CBG or LPS effect group can obviously increase the cell percentage (P＜0.05 or P＜0.01) that mouse spleen lymphocyte enters the S phase; The S phase cell percentage of the single medicine group of CBG also increases, but not statistically significant.
The influence that table 4. cinobufacin is expressed mouse T lymphocyte CD4, CD8
Continuous table 4
Annotate: positive controls 5mgL
-1Con A stimulates.Compare 1 with negative control group) P＜0.05,2) P＜0.01, experiment repetitive operation 3 times.
Table 5. cinobufacin is to the influence in mouse spleen lymphocyte cycle
Annotate: positive controls 5mgL
-1Con A or 20mgL
-1LPS stimulates.Compare 1 with negative control group) P＜0.05,2) P＜0.01, experiment repetitive operation 3 times.
3 discuss
The splenocyte transformation experiment is the common method of research medicine to T, bone-marrow-derived lymphocyte effect, lymhocyte transformation rate is the basic index of reflection immunity of organism, therefore detect lymphocytic propagation, transform and to understand the influence of medicine, it is generally acknowledged that Con A mainly acts on the T lymphocyte and LPS mainly acts on bone-marrow-derived lymphocyte body's immunity; After exotic antigen is invaded body, antigen presenting cell such as huge biting carefully also play an important role in the nonspecific immune reaction of regulating body, the cytokine of activatory macrophage synthesis secretion such as IL-2 etc., have the important immunomodulating and the function of killing tumor cell, the activation of appropriateness regulation and control macrophage is also significant for the immunity that improves body.Above-mentioned immune indexes testing result shows, there is certain influence in CBG in the Venenum Bufonis chloroform extraction layer to immune system, in range of doses, can strengthen mouse spleen lymphocyte and to the respond of Con A or LPS exogenous stimulation, the drug effect of Chinese medicine immunological enhancement has certain dose-effect relationship, promptly there is a certain proper dosage, illustrates that CBG has certain forward regulating action to the cellular immunization and the humoral immunoresponse(HI) of body in range of doses; Independent effect of CBG and collaborative Con A or LPS effect also can enliven the energy metabolism level of Turnover of Mouse Peritoneal Macrophages and improve the ability that it engulfs the allogenic material dimethyl diaminophenazine chloride, and wherein the facilitation of collaborative LPS drugs with function group is the most obvious.
The T lymphocyte has multiple important immunologic function, the kind of the differentiation antigen (CD) on different immunologic functions and its cell membrane is associated, wherein CD4 and CD8 are crucial differentiation antigens, and it is the T cell is brought into play immunologic function outside thymus the two positive T cell subgroup (CD4 of biochemical basis CD4, CD8 that CD4 and CD8 combine with corresponding MHC antigen
+CD8
+Be called for short the DP cell) can produce the various kinds of cell factor, participate in cell-mediated immune responses and regulate the growth of Th, stimulate the growth of B cell and bring out the B cell and produce antibody, promptly has the complementary function that is similar to the Th cell, its model of action is that MHC-II is restrictive, and it also has the function of cytotoxic T cell concurrently, its cytotoxicity vigor and CD8 simultaneously
+Cell is suitable, and the DP cell also has the immunological memory function in addition, and its memory function is from CD4
+Cytogenetics is got off.
Flow cytometry result shows, compares with negative control group, and CBG acts on separately and collaborative Con A effect all can significantly promote CD4
+CD8
+T lymphocyte subset group percentage rate promptly increases the lymphocytic quantity of DP (T) (P＜0.05 or P＜0.01).Though except that the indivedual dosage of the single medicine group of CBG, after the effect of other medicines group, tool CD4
-CD8
+Phenotype and CD4
+CD8
-The t lymphocyte subset group percentage rate of phenotype all has obvious decline, but tool CD4
+CD8
+With CD4
+CD8
-T lymphocyte subset group percentage rate sum all has remarkable lifting (P＜0.05 or P＜0.01).CD4
+CD8
-The t lymphocyte subset group is the Th cell with helper-inducer function, and tool CD4
+CD8
+The DP of phenotype (T) cell also has the complementary function that is similar to the Th cell, and simultaneously it also has the cytotoxicity of Tc cell concurrently and from CD4
+Cytogenetic immunological memory function.Therefore, above-mentioned streaming testing result is pointed out us, and CBG may be by activation CD4
+T cellular expression CD8 α receptor, the quantity that increases DP (T) cell have auxiliary and cytotoxicity concurrently are brought into play immunological enhancement; And this immunologic enhancement may have memory function.
With reference to Chinese medicine immunocompetence experiment in vitro research both domestic and external, many medicines are by promoting tool CD4 in the T lymphocyte
+CD8
-The t lymphocyte subset group percentage rate and the CD4 of phenotype
+/ CD8
+Ratio bring into play immunological enhancement, promptly mainly be by promoting the quantity performance immunologic enhancement of Th cell.In this experiment, except that indivedual administration groups, CD4
+CD8
-The percentage rate of subgroup and CD4
+/ CD8
+Ratio certain decline is all arranged, that is to say to have the CD4 of miscellaneous function
+The quantity of single positive cell has reduced, and this may be because CD4 has been induced in the effect of medicine
+Single positive cell is expressed CD8 α receptor, shows as tool CD4
+CD8
+The t lymphocyte subset group percentage rate of two positive phenotypes increases.It is restricted that the DP cell can show its MHC-II by the CD4 molecule, and CD4
+CD8 on the T cell
+Antigen presentation is CD4
+The performance of T cell activation, therefore, CD4
+On behalf of the lymphocyte quantity with complementary function, the minimizing of single positive cell quantity reduced, just CD4
+Single positive cell is by drug-induced activation and expressed CD8 α receptor, and its complementary function does not disappear, and has increased the cytotoxicity that kills and wounds target cell.Discover CD4 behind the drug effect
+CD8
+And CD4
+CD8
-T lymphocyte subset group percentage rate sum significantly increases, so we infer that CBG may be by inducing CD4
+Single positive cell is expressed CD8 α receptor, increases the T lymphocyte quantity of the complementary function of tool, the performance immunologic enhancement.Also have both at home and abroad some similarly, by increasing the DP cell quantity; the drug research report of performance immunological enhancement; bacillus calmette-guerin vaccine immune rat such as thunderous Jianping; can significantly reduce morbidity and the mortality rate of the rat of being attacked by the tuberculosis mycobacteria, one of performance that it strengthens rat anti tuberculosis protectiveness cellular immunity is exactly DP (CD4
+CD8
+) rising of cell percentage.In addition, the research that has an immanoprotection action for the DP cell also has relevant report both at home and abroad.
Under conditions in vitro, we have carried out preliminary study to the immunocompetence of the cinobufacin in the Venenum Bufonis chloroform solvent extract layer, the result shows that CBG has certain immunological enhancement, get in touch existingly, point out we CBG may have antitumor and immunoregulatory dual function CBG Antitumor Effects report; Its immune regulation mechanism may be by promoting tool CD4
+CD8
+The t lymphocyte subset group percentage rate of two positive phenotypes promotes that mouse spleen lymphocyte enters the DNA synthesis stage, and activation also increases the immunological effect lymphocyte, and then the performance immunological enhancement.This experiment has certain directive significance to the immune active ingredient of Chinese medicine Venenum Bufonis as the research aspect of immunostimulant.
Claims (2)
1. one kind has following structure
The immunoregulation effect of cinobufacin.
2. immunoregulation effect as claimed in claim 1 is characterized in that the respond of enhancing body splenocyte, peritoneal macrophage having immunological enhancement, is used for the application at preparation treatment or prevention humans and animals disease of immune system preparation.
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| CNA2007100557797A CN101327218A (en) | 2007-06-20 | 2007-06-20 | Immunoregulation novel use of cinobufagin |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103599112A (en) * | 2013-12-05 | 2014-02-26 | 南京大学 | Application of bufanolide diene compound in preparation of medicines for treating sepsis immune paralysis |
| EP3412308A4 (en) * | 2016-02-02 | 2020-02-26 | Nitto Denko Corporation | Composition for immunity induction promotion and vaccine pharmaceutical composition |
-
2007
- 2007-06-20 CN CNA2007100557797A patent/CN101327218A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103599112A (en) * | 2013-12-05 | 2014-02-26 | 南京大学 | Application of bufanolide diene compound in preparation of medicines for treating sepsis immune paralysis |
| EP3412308A4 (en) * | 2016-02-02 | 2020-02-26 | Nitto Denko Corporation | Composition for immunity induction promotion and vaccine pharmaceutical composition |
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Open date: 20081224 |