CN115154474A - Application of cycloartane type triterpenoid saponin compound in preparation of T cell immunosuppressive agent - Google Patents
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Abstract
The invention relates to application of cycloartane-type triterpenoid saponin compounds (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside, which are separated from Chinese medicinal herb of radix aconiti szechenyiani, in preparing T cell immunosuppressant. The compound can remarkably inhibit anti-CD3/CD28 mAbs induced CD3 + Proliferation of T cells and CD3 + Expression level of CD69 and CD25 in T cells, and reduction of CD4 in mouse spleen lymphocytes stimulated by anti-CD3/CD28 mAbs + /CD8 + In a ratio of (a) significantly inhibits anti-CD3/CD28 mAbs-induced mouse CD4 + Cytokines IL-2, IFN-gamma, IL-4 and IL-10 in T cells and inhibition of anti-CD3/CD28 mAbs induced mouse CD4 through JNK, ERK and PI3K/AKT signaling pathways + T cells proliferate.
Description
Technical Field
The invention belongs to the field of compound medicine, and particularly relates to an application of a cycloartane-type triterpenoid saponin compound (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside, which is separated from a traditional Chinese medicine of radix aconiti szechenyiani, in preparing a T cell immunosuppressive agent.
Technical Field
Autoimmune disease (AID) is a typical multi-etiologic disease characterized by intolerance of the autoimmune system to its components, attacking single or multiple self-normal tissues and causing tissue damage and dysfunction. Including Type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic Lupus Erythematosus (SLE), and the like, most AIDs pathogenesis is not clear, and disease progression is influenced by heredity and immune regulation.
At present, the classical drugs for clinically treating immune dysregulation comprise glucocorticoid, rapamycin, tacrolimus, cyclosporine A and the like, and the glucocorticoid preparation has wide action, lacks selectivity and has obvious toxic and side effects. Immunosuppressants such as cyclosporin A, tacrolimus and sirolimus affect the whole process from activation to effect of T cells, influence the self structure of thymus besides inhibiting the activation and function of T cells, have no selectivity to the whole T cell subset, and are not selective inhibitors in the true sense. The long-term use of the medicine can still cause the reduction of the anti-infection ability of the organism, the nephrotoxicity, the neurotoxicity, the increase of the tumor incidence rate and the like. Therefore, the development of a novel immunoregulation medicament with low toxic and side effects and good treatment effect has wide application prospect and important application value.
The plant of the genus Lycoris chinensis of the family Cimicifuga of the family Ranunculaceae [ Beesia calthaefolia (Maxim.) Ulhr ], which is widely distributed in the southwest of Yunnan, sichuan, guizhou, northern Guangxi, western of Hunan, western of Hubei, southern of Shaanxi and southern of Gansu of China, is a common herb in China and is a specific medicinal plant in China. The rhizome or the whole herb of the traditional Chinese medicine is used for medicine, and has the effects of clearing away heat and toxic materials, cooling blood, activating blood circulation, reducing swelling, easing pain and dispelling wind and cold. It is used for treating wind-cold type common cold, rheumatic arthralgia, red and white dysentery, sore throat, headache, toothache, etc., and can be used for external application for treating skin ulcer, furuncle, and venomous snake bite. The radix aconiti szechenyiani contains characteristic cycloartane triterpenoid saponin.
Disclosure of Invention
The invention discloses application of (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside in preparation of a T cell immunosuppressant for the first time.
The experiment of the invention shows that (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside can obviously inhibit the anti-CD3/CD28 mAbs induced CD3 + Proliferation of T cells and CD3 + Expression levels of CD69 and CD25 in T cells.
The experiment shows that 20S, 24R-20, 24-epoxy-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside can significantly reduce the CD4 in the mouse spleen lymphocyte stimulated by anti-CD3/CD28 mAbs + /CD8 + The ratio of (a) to (b).
The experiment shows that 20S, 24R-20, 24-epoxy-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside can remarkably inhibit mouse CD4 induced by anti-CD3/CD28 mAbs + Cytokines IL-2, IFN-. Gamma., IL-4 and IL-10 in T cells.
The experiment shows that 20S, 24R-20, 24-epoxy-9, 19-cyclojackane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside can inhibit mouse CD4 induced by anti-CD3/CD28 mAbs through JNK, ERK and PI3K/AKT signal pathways + T cells proliferate.
The (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside is a pharmaceutical active ingredient, and can be prepared into any dosage form suitable for clinical use, such as tablets, capsules, granules, oral liquid preparations, injections and the like, by taking the compound as a main active ingredient and adding conventional excipients, flavoring agents, disintegrating agents, preservatives, lubricants, wetting agents, adhesives, solvents, thickening agents and solubilizing agent pharmaceutical excipients according to the conventional preparation process.
Since the invention discloses the application of (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside in the preparation of T cell immunosuppressant for the first time, the invention belongs to the protection scope of the invention as long as the compound is used for treating autoimmune diseases by being prepared into a medicament alone or in combination with other active components or auxiliary materials.
The (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside has the function of treating autoimmune diseases when being prepared into any dosage form. Any medicament, if the component contains (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside, the single component is prepared into the medicament, and the medicament is also within the protection scope of the invention as long as the medicament is marked or suggested to have the effect of treating autoimmune diseases on the package, the instruction book and the like of the medicament or any other propaganda product.
(20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 β,12 β,16 β, 25-tetrol-3-O- β -D-xylopyranoside is prepared by the following method:
pulverizing the whole plant of radix Trigonellae Terebinthinae, extracting with 95% ethanol and 50% ethanol twice, and recovering ethanol under reduced pressure to obtain extract. After the extract extracted by 50 percent ethanol is dispersed by proper warm water, petroleum ether, chloroform, ethyl acetate and water saturated n-butanol are used for extraction in sequence. Separating the chloroform part by repeated 200-300 mesh silica gel chromatography to obtain the target product.
Drawings
FIG. 1 shows the chemical structure of compound (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside.
FIG. 2 shows the effect of compound (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 β,12 β,16 β, 25-tetrol-3-O- β -D-xylopyranoside on the proliferation of mouse T cells induced by anti-CD3/CD28 mAbs.
FIG. 3 shows the compound (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside on CD3 + Effect of CD25 and CD29 ratio in T cells.
FIG. 4 shows the compound (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside vs. CD3 + CD4 in T cells + And CD8 + The effect of the subtype.
FIG. 5 shows the induction of CD4 by the compound (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside against anti-CD3/CD28 mAbs + The effects of cytokines IL-2, IFN- γ, IL-17, IL-4 and IL-10 in T cells.
FIG. 6 shows the compound (20S, 24R) -20, 24-ringOxygen-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside CD4 induced by anti-CD3/CD28 mAbs + Effects of JNK, ERK and PI3K/AKT signaling pathways in T cells.
Detailed Description
Example 1: extraction and separation of (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside
1. Apparatus and materials
1100 series LC-MS T rapsl Mass spectrometer (Ag ilent, USA); JNN-ECA-400 type nuclear magnetic resonance apparatus (Japan electronic Co., ltd.); a lltech preparative high performance liquid chromatograph (a lltech corporation, usa); YMC ODS column (20 mm. Times.250mm, 10 μm); an Alltech uvis 200 detector. Thin layer chromatography silicon (GF) 254 ) And column chromatography silica gel (Qingdao ocean chemical Co., ltd.); sephadex LH-20 (Pharm ac ia B io tech, switzerland). The whole grass of the gong chufa collected in 12 months in 2010 was collected from Wudang region of Guiyang city, guizhou province, and collected and identified as [ Beesia calthaefolia (Maxim.) Ulhr ] by New Production of Natural products in major laboratories of Chinese academy of sciences, guizhou province.]The specimens are reserved in the basic research room of pharmacy of the general hospital of the people's liberation army of China.
2. Extraction and separation
Pulverizing herba Trigonellae Ternati 6.8kg, extracting with 95% ethanol and 50% ethanol twice, and distilling under reduced pressure to obtain 202995% ethanol extract and 1.0kg of 50% ethanol extract. Dispersing 50% ethanol extract with warm water, extracting with petroleum ether, chloroform, ethyl acetate, and water saturated n-butanol, and concentrating under reduced pressure to obtain concentrated extract. The chloroform extract (170 g) was applied to a silica gel column and eluted sequentially with Pe-EtOAc = 1: 4-1: 9, etOAc-MeOH = 20: 1-10: 1 to give 9 fractions (A-I) wherein fraction F was applied to a silica gel column and eluted with CHCl 3 Gradient elution with-MeOH = 40: 1-8: 1 to give compound (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 β,12 β,16 β, 25-tetrol-3-O- β -D-xylopyranoside.
3. Results
White amorphous powder; liebermann-Burchard and Molish reactionsShould be positive; 10% of 2 SO 4 The absolute ethyl alcohol is purple red in color, which indicates that the compound is possible to be a triterpenoid saponin compound. ESI-MS gives m/z 645.6[ 2 ] M + Na] + And m/z 657.6[ m ] +Cl] - Indicating molecular weight 622. Bonding of 1 H-NMR、 13 C-NMR spectrum to confirm the molecular formula as C 35 H 58 O 9 。 1 H-NMR(C 5 D 5 N,400 MHz) δ:1.64 (3H, s, 18-H), 1.64 (3H, s, 21-H), 1.54 (3H, s, 26-H), 1.30 (3H, s, 27-H), 1.26 (3H, s, 28-H), 0.97 (3H, s, 29-H), 0.94 (3H, s, 30-H), 0.30 (1H, d, J =4.0, 19-Ha), 0.51 (1H, d, J =4.0, 19-Hb), 3.90 (1H, t, J =7.2, 24-H), 2.72 (1h, d, J =7.2, 17-H), 4.79 (1h, d, J =7.6, xyl-H-1), 3.98 (1h, t, J =8.0, xyl-H-2), 4.10 (1h, t, J =8.8, xyl-H-3), 4.21 (1h, m, xyl-H-4), 4.32 (1h, dd, J =11.2,5.2, xyl-H-5 a), 3.67 (1h, t, J =10.0, xyl-H-5 b); the hydrogen and carbon spectra and literature were analyzed (Jian-hua Ju, dong Liu, geng Lin, et al. Beesiosis A-F, six New Cycloartane Triterpen carbohydrates from Beesia calthaea [ J ] J]Pro nat 2002, 65: 42-47.), and was essentially identical, thus identifying the compound as (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 β,12 β,16 β, 25-tetrol-3-O- β -D-xylopyranoside (compound 1).
Of Table 1 Compound 1 13 C NMR data (125MHz, pyridine-d) 5 ). [a][b]
[a] Assignments based on TOCSY，HMQC and HMBC experiments.
[b] Overlapped signals are reported without designating multiplicity.
Example 2: CD3 of (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside + T cell immunosuppressive Activity assay
1. Materials:
MTT (sigma chemicals, usa); mouse cytokine detection ELISA kits (Boster Biotech, wuhan Co., ltd.); anti-CD3/CD28 mAbs and FITC-Anti-CD3, FITC-Anti-CD4, PE-Anti-CD4 and PE-Anti-CD69, PE-Anti-CD25 (BD Biotech, inc., U.S.A.). RPMI 1640 medium and fetal calf serum (Hangzhou Siji Green technologies Co., ltd.); magniSort TM A Mouse CD3 positive sorting kit,
Untouched TM mouse CD4 cell isolation kit, trizol, RNase-free DNase, cyclosporine A (CsA) (lnvitrogen, USA). ERK1/2, p-ERK1/2, JNK, p-JNK, akt, p-Akt, STAT5, p-STAT5 antibodies (CST, inc., U.S.A.).
Experimental animals: balb/c mice (male and female, weight 18-20g, beijing Sibefu laboratory animal science and technology Co., ltd., quality certificate number SCXK (Jing) 2011-0004).
2. Method for producing a composite material
(1) Spleen separation
After the mouse is killed after the neck is broken, soaking the mouse in 75% ethanol for 3min, placing the mouse on an ultra-clean workbench, exposing the abdomen, extracting the spleen under an aseptic condition, and placing the spleen in a culture dish containing 10ml 1640 culture medium; sucking 4-5ml 1640 culture medium with 5ml sterile syringe, repeatedly washing spleen with the syringe to obtain single cell suspension, and adjusting cell density to 5 × 10 6 Each/ml is ready for use;
(2)CD3 + magnetic bead sorting of cells
Centrifuging 300g of the spleen single cell suspension for 5min;
add 4ml of sorting buffer (PBS buffer containing 3% FBS and 10mM EDTA) to resuspend the cells;
centrifuging at 300g for 5min, and washing twice;
the cell pellet was resuspended in sorting buffer and adjusted to a density of 1X 10 8 Per ml;
add 20. Mu.L of MagniSort per 100ul of cell suspension TM Mixing the Positive Selection Antibody uniformly, and reacting at room temperature for 10min;
washing the cells with 4ml of sorting buffer 2 times, and centrifuging for 5min at 300g each time;
discarding the supernatant, and resuspending the cell pellet with a sorting buffer solution, wherein the volume of the added sorting buffer solution is 1.2.4;
add 20. Mu.L MagniSort per 100ul cell suspension TM Uniformly mixing the Positive Selection Beads, reacting at room temperature for 10min, and using the magnetic Beads after shaking uniformly;
supplementing the sorting buffer solution to 2.5m, and mixing uniformly by using a 1ml pipettor up and down for 3 times;
placing the centrifuge tube on a magnetic frame, and standing at room temperature for 5min;
the supernatant was aspirated and the two steps were repeated 3 times;
the supernatant was aspirated off, and the cell density was adjusted to 2X 10 using growth medium (1640 medium containing 10% FBS) 6 Per ml;
(3) Cell model
Sorting the CD3 + The cell suspensions were plated in 6-well cell culture plates, 1500 ul/well, respectively, where cell proliferation was detected for CD3 + Cells were pre-labeled with CFSE for 2 hours;
the method comprises the following groups: normal cultured cells, anti-CD3/CD28 mAbs (5. Mu.g/ml) stimulated T cells, anti-CD3/CD28 mAbs + 48.1. Mu.M Compound 1, anti-CD3/CD28 mAbs + 9.5. Mu.M Compound 1, anti-CD3/CD28 mAbs + 1.8. Mu.M Compound 1, anti-CD3/CD28 mAbs + 0.25. Mu.M CsA, for 5 days;
(4) Flow assay
Centrifuging the cells for 5min at 300g after cell model;
discard the supernatant and resuspend with 1000. Mu.l PBS, wash twice, centrifuge 5min at 300g each time:
adding PBS to resuspend the cells and adjusting the cell density to 1X 10 6 One for each ml, 100. Mu.l of cell suspension was aspirated for each flow combination, and the assay combinations were CD3/CD4/CD25/CD69 and CD3/CFSE, respectively
Extracellular antibody labeling: respectively adding 2.5 μ l of CD3, 5 μ l of CD4, 5ul of CD25 and 5ul of CD69, and reacting at room temperature in a dark place for 30min;
centrifuging at 300g for 5min, discarding the supernatant, resuspending with 1000 μ l PBS, washing twice, and centrifuging at 300g for 5min;
adding 500ul cell fixing agent, reacting for 10min at room temperature;
centrifuging at 300g for 5min, discarding the supernatant, resuspending with 1000 μ l PBS, washing twice, and centrifuging at 300g for 5min;
adding 400 mul PBS, mixing, and detecting on a machine;
data analysis was performed by CellQuest software.
3. Results of the experiment
After 5 days of administration, compound 1 significantly inhibited CD3 induction by anti-CD3/CD28 mAbs dose-dependently + Proliferation of T cells, calculated IC 50 The value was 9.5. Mu.M. Flow detection results show that anti-CD3/CD28 mAbs significantly increase CD3 in mouse skin cells + Percent of T cells, compound 1 significantly reduced CD3 at 9.5 and 48.1. Mu.M concentrations + Percentage of T cells, compound 1 high dose group (48.1 μ M), showed activity comparable to that of the positive drug CsA (figure 2 of the specification).
Flow assay experiments showed that 5 days after administration, anti-CD3/CD28 mAbs significantly increased CD69 and CD25 expression in T cells compared to the control group, but compound 1 significantly inhibited CD69 and CD25 increase compared to the anti-CD3/CD28 mAbs group (fig. 3 of the specification).
anti-CD3/CD28 mAbs significantly increased CD4 in mouse skin cells 5 days after administration + And CD8 + Percent T cell, CD3 + The content of T cells did not change significantly. Compound 1 can significantly reduce CD4 + While significantly increasing CD8 + Amount of T, for CD3 + T cells had no significant effect, and this resulted in CD4 + /CD8 + A significant reduction (figure 4 of the description).
Example 3: CD4 of (20S, 24R) -20, 24-epoxy-9, 19-cycloartane-3 beta, 12 beta, 16 beta, 25-tetrol-3-O-beta-D-xylopyranoside + T cell immunosuppressive Activity assay
1. Material
The same as in example 2.
2. Method of producing a composite material
(1)CD4 + Magnetic bead sorting of cells
Centrifuging 300g of the spleen single cell suspension for 5min;
add 4ml of sorting buffer (PBS buffer containing 3% FBS and 10mM EDTA) to resuspend the cells;
centrifuging at 300g for 5min, and washing twice;
the cell pellet was resuspended in sorting buffer and adjusted to a density of 1X 10 8 Per ml;
50. Mu.L of IMag per 100ul of cell suspension was added TM Mixing anti-mouse CD4 Magnetic Particles uniformly, and reacting at room temperature for 10min;
reacting for 30min at the temperature of 6-12 ℃;
placing the reaction centrifugal tube on a magnetic frame, and placing for 2-4min at room temperature;
removing supernatant, supplementing sorting buffer solution to 1ml, and mixing up and down for 3 times by using a 1ml pipettor;
repeating the two steps;
the supernatant was aspirated off, and the cell density was adjusted to 2X 10 using growth medium (1640 medium containing 10% FBS) 6 Each/ml.
(2)CD4 + Cells were divided into the following subgroups:
normal cultured cells, anti-CD3/CD28 mAbs (5. Mu.g/ml) stimulated T cells, anti-CD3/CD28 mAbs + 48.1. Mu.M Compound 1, anti-CD3/CD28 mAbs + 9.5. Mu.M Compound 1, anti-CD3/CD28 mAbs + 1.8. Mu.M Compound 1, anti-CD3/CD28 mAbs + 0.25. Mu.M CsA, for 5 days;
cell culture supernatants for ELISA detection;
the standard substance diluting kit is used after balancing the room temperature;
the concentration of the standard substance is as follows: IL-2, IL-4 and, IL-10
IFN-γ：1200、800、400、200、100ng/L；
IL-2：480、320、160、80、40ng/L；
IL-4：180、120、60、30、15pg/ml；
IL-10：480、320、160、80、40pg/ml；
IL-17：180、120、60、30、15pg/ml
Simultaneously, blank holes are arranged, 40ul of sample diluent is added into the sample holes, and 10ul of samples to be detected are added respectively;
adding 100ul enzyme-labeled antibody into each hole, adding no blank hole, and reacting at 37 ℃ for 30min;
washing: washing the ELISA plate with a washing solution for 5 times, and patting dry;
color development: adding TMB color development solution, 100 ul/hole, and reacting at 37 deg.C in dark for 15min;
and (4) terminating: adding stop solution at 50 ul/hole;
reading: the absorbance was read at 450nm, a standard curve (4-PL) was plotted with the standard concentration and the absorbance, and the contents of IL-2, IFN-. Gamma., IL-4 and IL-17, IL-10 in the sample were calculated from the standard curve.
(3) WB detection
Protein extraction
Precooling an RIPA protein extraction reagent, adding a protease inhibitor, adding 0.1M PMSF mother liquor before the protein extraction is started, wherein the final concentration of PMSF is 1mM, and simultaneously adding the protease inhibitor;
cell pellet at 1X 10 7 1ml of lysis solution was added, the cells were suspended sufficiently by blowing with a gun, and after completion, the cells were incubated on ice for 20min, centrifuged at 4 ℃,13000rpm, and then for 20min. And taking the supernatant after centrifugation, subpackaging and storing, and detecting.
Protein quantification by BCA method
Preparing a BCA working solution A: b liquid = 50: 1, and each extracted BSA standard substance is diluted;
diluting the sample by 5-10 times with PBS, adding 150ul BCA working solution, mixing uniformly, incubating at 37 ℃ for 30min or incubating at room temperature for 60min, and reading OD value by a 570nm wavelength filter of an enzyme-labeling instrument;
adjusting the protein concentration: RIPA adjusted protein concentration, adding 4 × reduced sample buffer solution to make sample final concentration 2mg/ml, boiling for denaturation for 5min. WB detection experiment for target protein
Loading amount of the protein sample to be detected; upper 13 ug/well;
electrophoresis conditions: selecting electrophoresis buffer solution according to the size of the detected protein, selecting an MOPS buffer system when the size of the protein is larger than 25KD, and selecting an MES buffer system when the size of the protein is smaller than 25 KD;
the constant voltage is 90V, the constant voltage is 120V after about 20min, and the electrophoresis stopping time is determined by prestained protein marker;
wet transfer method, transfer film conditions: the membrane switching time is 1h, wherein the membrane switching time is prolonged to 2h when the molecular weight of the protein is more than 220 KD;
and (3) sealing: immersing the membrane in 5% skimmed milk powder-TBS completely, and shaking gently at room temperature for 30min;
primary antibody incubation: diluting primary antibody with 5% skimmed milk powder-TBS, incubating at room temperature for 10min, and standing at 4 deg.C overnight;
antibody dilution summary Table
| Numbering | First antibody name | Protein | Dilution ratio | |
| 1 | ERK | 42/ |
1∶500 | |
| 2 | p-ERK | 42/ |
1∶500 | |
| 3 | | 80KD | 1∶1000 | |
| 4 | p- | 80KD | 1∶1000 | |
| 5 | | 60KD | 1∶500 | |
| 6 | p- | 60KD | 1∶500 | |
| 7 | | 70KD | 1∶500 | |
| 8 | p- | 70KD | 1∶500 | |
| 9 | | 120KD | 1∶500 | |
| 10 | p- | 120KD | 1∶500 |
The next day the membranes were removed from 4 degrees, incubated at room temperature for 30min, washed: washing membrane with TBST for 5 times, each time for 3min;
and (3) secondary antibody incubation: diluting the secondary antibody with 5% skimmed milk powder-TBS, goat anti-rabbit IgG (H + L) HRP, 1: 20000, gentle shaking at room temperature for 40min, washing membrane: washing membrane with TBST for 6 times, each time for 3min;
ECL was added to the film and reacted for 3-5min, film exposure: 10s-5min (the exposure time is adjusted along with different light intensities), developing for 2min, and fixing;
official experiment of reference protein WB
Washing membrane with striping Buffer at 37 deg.C for 30min (if the molecular weight difference between the target protein and the reference protein is above 10K, the striping Buffer can be omitted);
washing the membrane: washing the membrane with deionized water for 3 times;
washing the membrane: washing the membrane for 3 times by TBST, 3min each time;
immersing the membrane completely in 5% skimmed milk powder-TBS, and shaking gently at room temperature for 30min;
incubation internal reference: adding GAPDH mouse monoclonal antibody, diluting with 5% skimmed milk powder-TBS, diluting at ratio of 1: 10000, and incubating at room temperature for 40min;
washing the membrane: washing membrane with TBST for 5 times, each time for 3min;
and (3) secondary antibody incubation: diluting the secondary antibody with 5% skimmed milk powder-TBS, goat anti-mouse IgG (H + L) HRP, 1: 10000, and gently shaking at room temperature for 40min;
washing the membrane: washing membrane with TBST for 5 times, each time for 3min;
ECL is added on the membrane and reacts for 3-5min, and the film is exposed: 10s-5min, developing for 2min, and fixing;
analyzing the gray value of each WB detection strip by using Image J software;
3. results of the experiment
The anti-CD3/CD28 mAbs can significantly increase CD4 compared to the control group + The levels of IFN-gamma, IL-2, IL-4, IL-10 and IL-17 in T cells. Compared with the anti-CD3/CD28 mAbs group, the compound 1 can significantly reduce the content of IFN-gamma and IL-10, and has good dose dependence, and the high dose of the compound 1 even shows the activity similar to that of a positive drug. Although compound 1 can also significantly reduce the levels of IL-2 and IL-4, it does not exhibit dose dependence. The experimental results show thatThe compound 1 can obviously inhibit the secretion of Th1 type cell factors, th2 type cell factors and Th17 type cell factors (figure 5 in the specification).
WB experimental structure shows that anti-CD3/CD28 mAbs can significantly increase CD4 compared with the control group + The ratio of the p-ERK/ERK, p-JNK/JNK, p-Akt/Akt and p-STAT5/STAT5 protein contents in T cells. Compared with the anti-CD3/CD28 mAbs group, the compound 1 can obviously reduce the values of p-ERK/ERK, p-JNK/JNK, p-Akt/Akt and p-STAT5/STAT5, and has good dose dependence, and the high-dose compound 1 even shows similar activity compared with the positive drug. The experimental result shows that the compound 1 can inhibit CD4 induced by anti-CD3/CD28 mAbs through ERK, JNK, PI3K/Akt channels + T cell proliferation (FIG. 6 of the specification).
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples. Any other variations which do not depart from the spirit and principle of the invention are deemed to be within the scope of the invention.
Claims (9)
1. The application of a compound shown as the following in preparing a medicinal composition of a T cell immunosuppressant.
2. The use according to claim 1, wherein the T cell immunosuppressive agent is for inhibiting T cell proliferation, inhibiting T cell activation, and inhibiting cytokine secretion.
3. The use according to claim 2, wherein the cytokines are Th1 type cytokines, th2 type cytokines and Th17 type cytokines.
4. The use according to claim 3, wherein the Th1 type cytokine is IL-2, TNF- α, IFN- γ, the Th2 type cytokine is IL-4, IL-6, IL-10, and the Th17 type cytokine is IL-17.
5. Use according to claim 1, for the preparation of a medicament for the treatment of autoimmune diseases.
6. The autoimmune disease of claim 5 is autoimmune hepatitis, rheumatoid arthritis, insulin Dependent Diabetes Mellitus (IDDM), ulcerative colitis, multiple Sclerosis (MS), scleroderma, myasthenia gravis, polymyositis/dermatomyositis, lymphomatous goiter, autoimmune cytopenia, sjogren's syndrome, vasculitis syndrome, or systemic lupus erythematosus.
7. The use according to claim 1, wherein the medicament for treating autoimmune disease is a pharmaceutical composition comprising the triterpene saponin compound according to claim 1 and a pharmaceutical excipient.
8. The use according to claim 1, wherein conventional flavoring agents, disintegrating agents, preservatives, lubricants, wetting agents, binders, solvents, thickeners, solubilizing agents and pharmaceutical excipients are added to prepare any one of the clinically acceptable formulations.
9. The use according to claim 8, selected from the group consisting of tablets, capsules, granules, oral liquid preparations, injections.
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