CN103599112A

CN103599112A - Application of bufanolide diene compound in preparation of medicines for treating sepsis immune paralysis

Info

Publication number: CN103599112A
Application number: CN201310653592.2A
Authority: CN
Inventors: 殷武
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2013-12-05
Filing date: 2013-12-05
Publication date: 2014-02-26
Anticipated expiration: 2033-12-05
Also published as: CN103599112B

Abstract

The invention relates to an application of a compound with a bufanolide diene structure mother nucleus in the preparation of medicines for treating sepsis immune paralysis. Cinobufagin derived from an amphibian toad is adopted. Experiments prove that the compound is capable of obviously reversing the survival rate of a mouse with the clinically simulated sepsis immune paralysis which is caused by the cecal ligature and puncture of a mouse and secondary salmonella hit and inhibiting the reduction of CD4+ and CD8+ cells of the spleen and the lymph gland of the mouse with the immune paralysis. On blood mononuclear cells of a clinical severe sepsis patient, the cinobufagin can reverse the reduction of the expression level of TH1 immune stimulating factors such as TNF (tumor necrosis factor)-alpha, GM-CSF (granulocyte-macrophage colony-stimulating factor) and IFN (interferon)-gamma which are caused by sepsis. On a normal human mononuclear cell endotoxin tolerance model which is caused by lipopolysaccharide (LPS), the cinobufagin can effectively reverse the reduction of expression capacity of mononuclear cell TNF alpha caused by secondary lipopolysaccharide hit. The compound has very high development value and application prospect.

Description

The purposes of bufanolide dienes compound in preparation treatment sepsis immunological paralysis medicine

Technical field

The present invention relates to the new purposes for the preparation for the treatment of sepsis immunological paralysis medicine by bufanolide dienes compound.

Background technology

Sepsis incidence rate is high, and the whole world has every year and surpasses 1,800 ten thousand severe sepsis cases, and the U.S. has 750,000 routine sepsis patients every year, and this numeral is also with annual 1.5%～8.0% speed rising.The pyemic state of an illness is dangerous, and case fatality rate is high, and whole world people's every days approximately 14000 dies from its complication, and annual approximately 21.5 ten thousand people are dead in the U.S..According to sick learning of Foreign Epidemic, investigate demonstration, pyemic case fatality rate has surpassed myocardial infarction, becomes non-heart patient's main causes of death in intensive care unit(ICU).In recent years, although anti-infective therapy and organ dysfunction supporting technology have been obtained significant progress, pyemic case fatality rate is still up to 20%～80%.Treatment of sepsis cost is high, and medical resource consumption is large, has a strong impact on the mankind's quality of life, human health is caused to grave danger.

Mononuclear cell, macrophage and neutrophilic granulocyte are infected and discharge during non-infectious stimulation the inflammatory mediators such as a large amount of cytokines, participate in promoting that the proinflammatory stage of reaction of development occurs sepsis, the excessive release of these inflammatory mediators can be brought out sepsis, SIRS and septic shock.The early stage systemic inflammatory response of sepsis is the especially result Immunomodulatory therapies in sepsis.Intensive care medicine26 of TNF-α, IL-6, IL-1 and IFN-γ effect of inflammatory cytokine, S124-S128, Kox et al., 2000).When recognizing the inflammatory reaction due to TNF-α, IL-6 and IL-1, people participated in, after the pathogenic course of SIRS, sepsis and multiple organ dysfunction syndrome (MODS), just having designed the anti-inflammatory treatment of the various exogenous immune formulations for these inflammatory mediators.These anti-inflammatory substances comprise that antibody, soluble recepter, receptor antagonist etc. prevent the specificity substance of the Cytokines such as TNF-α, IL-1, and the non-specific anti-inflammatory drug such as nonsteroidal antiinflammatory drug, glucocorticoid.Although application anti-inflammatory drug treatment animal sepsis has been obtained good effect, but the pyemic clinical anti-inflammatory treatment test of relevant human body is not succeeded, even make case fatality rate increase (Immunoparalysis as a cause for invasive aspergillosis Intensive care medicine29,2068-2071., Hartemink et al., 2003).

Someone thinks that one of the reason of sepsis anti-inflammatory treatment failure is that pyemic evolution is lacked to accurate immunity monitoring.Research shows, can there is rapidly strong anti-inflammatory response in body after the proinflammatory reaction of the early stage generation of sepsis, while is with the release of a large amount of Anti-inflammatory mediators, and some sepsis patient shows obvious immunosuppressive condition (Sepsis and immune response.World J Emerg Med2,88-92., Chen et al., 2011).Kox etc. also confirm and later stage existence two kind immune states early stage at sepsis, and early stage high inflammatory conditions, by the anti-inflammatory mechanisms of body institute antagonism, causes the low inflammatory conditions of secondary.Bone etc. also emphasize, at sepsis inflammatory reaction early stage, have occurred that the development of compensatory inflammatory response syndrome (CARS) with inflammation-inhibiting reaction, this reaction occupy an leading position and induce the appearance of " immunological paralysis " in some patients.Immunological paralysis is a kind of acquired immunodeficiency state, be found in the sepsis patients such as major operation, burn, multiple injuries, main manifestations is that in sepsis evolution, mononuclear cell human leucocyte differentiation antigen DR (HLA-DR) expression reduces, the antigenspecific T lymphocyte activity decreased that mononuclear cell is induced, mononuclear cell discharges change and polymorphonuclear leukocyte anergy (the Schultz et al. of cytokine profiles ability, 2000), its harm is to bring out Secondary cases nosocomial infection, makes patient's prognosis mala.CARS hypothesis seems to explain the reason of anti-inflammatory treatment " failure ", and has obtained to a certain extent the support of clinical research.The reports such as Volk in 1996, CD14 ⁺monocyte HLA-DR level can be differentiated sepsis immunosuppressive condition (Monocyte deactivation-rationale for a new therapeutic strategy in sepsis.Intensive care medicine22 reliably, S474-S481, Volk et al., 1996).HLA-DR/CD14 with 30% ⁺as threshold value, patient's prognosis is obviously different.In addition, with immunostimulant gamma interferon (IFN-γ) treatment, immune state can effectively be improved, at HLA-DR/CD14 ⁺when being raised, also observe proinflammatory cytokine TNF-α and discharge increase (Interferon gamma-1b in the treatment of compensatory anti-inflammatory response syndrome:a new approach:proof of principle.Archives of internal medicine157,389., Kox et al., 1997).Obviously, this research is the strongest evidence of CARS hypothesis.

In order to reverse sepsis immunological paralysis, improve survival rate, based on cytokine as the immune system that reactivates of GM-CSF, IFN-γ, TNF-α be one of feasible strategy.

Colony stimulating factor CSF is a kind of cytokine that regulates hematopoietic stem cell to copy, breed, break up, wherein G-CSF and GM-CSF centering granulocyte, mononuclear phagocyte, lymphocyte have Pasitive Regulation Effect of Genseng, can postpone neutrophil apoptosis, the expression of raising CD11b and CD14 molecule, has strengthened and has sticked chemotaxis and Phagocytosis power.Clinical research has proved that G-CSF and GM-CSF can improve severe postoperative the infected's monocyte function defect, increase the expression of TNF-α secretion and HLA-DR, and increase lymphocyte quantity, regulate T cell proliferation and differentiation, strengthen the immunocompetence of Th1 etc., thereby reverse immunological paralysis.Orozco etc. report 58 routine atraumatic Abdominal pyemia patients, the treatment of application molgramostim (GM-CSF) combined with antibiotic, can reduce and infect complication rate, minimizing hospital stays and expense (Molgramostim (GM-CSF) associated with antibiotic treatment in nontraumatic abdominal sepsis:a randomized, double-blind, placebo-controlled clinical trial.Archives of surgery141,150.Orozco et al., 2006).

Clinical research shows that IFN-γ can make the expression of IL-6 in blood plasma, TNF-alpha levels and monocyte HLA-DR increase, thereby improves the immune state of sepsis patient, improves its survival rate ¹⁶⁴.IFN-γ is as immunoactivator, and administration time has strict control, because the supervision that any immune modulating treatment all should be based on immunologic function.When patient's HLA-DR Expression of Monocytes is lower, when showing antiinflammatory and replying, the IFN-γ treatment of low dosage is safe and effective; In patient at those in the proinflammatory stage of reaction, use IFN-γ to be harmful to.

In addition, animal experiment study shows the α at sepsis immunological paralysis phase administration restructuring TNF-, can significantly increase the survival rate of animal, and strengthens animal liver, spleen removing pathogenic microorganism ability, shows that treatment has positive effect to TNF-α to sepsis immunological paralysis.Yet, the therapeutical effect of restructuring TNF-α is greatly limited, depend on the type of animal institute pathogenic infection microorganism, ability and restructuring TNF-α administration time (the Treatment of experimental sepsis-induced immunoparalysis with TNF.Immunobiology208 that animal body self produces TNF-α, 381-389., Echtenacher et al., 2003).

At present, also have report to use Thymosin alpha 1 treatment sepsis, it is a kind of protein polypeptide hormone, belongs to the physiologically substance existing in body.Thymosin alpha 1 can regulate the level of the cytokines such as severe infections patient's TNF-α, IL-6 and IL-10, alleviates inflammatory reaction, improves patient's immunologic function.Thymosin alpha 1 can delay the proinflammatory cytokine ascendant trends such as TNF-α, IL-6 as immunomodulator under the autoimmune prerequisite of adjustment patient, the injury response reducing inflammation due to medium, can improve anti-inflammatory factors IL-10 level, promote anti-inflammatory factors and proinflammatory factor to keep balance simultaneously.Although Thymosin alpha 1 has positive role to treatment of sepsis, not all effective in pyemic any state administration.Along with going deep into that sepsis immune state is understood, consider GM-CSF, IFN-γ or TNF-alpha protein drug half-life is short, side effect is large simultaneously, finding the small-molecule drug with immunoregulation effect is the promising research direction for the treatment of of sepsis.

Bufanolide dienes compound is the very strong micromolecular compound of a class activity, and its structural core is a cyclopentanoperhydro-phenanthrene, and 17 of cyclopentanoperhydro-phenanthrene are replaced by hexa-atomic pyrone ring, main skin and separated obtaining neck gland from Bufo siccus.In recent years, in animal body, find endogenous bufanolide dienes compound, mainly comprise endogenic dihydroxy toadpoison dienoic acid lactone (bufalin), marinobufagin (marinobufagenin) etc., further research shows, bufanolide dienes compound in body is mainly synthetic by adrenal gland and hypothalamus secretion, its precursor substance is cholesterol and Progesterone, is subject to the hormone regulating and controllings such as feritin, angiotensin, Endothelin and epinephrine.

Summary of the invention

Object of the present invention is to provide bufanolide dienes compound for the preparation of the purposes for the treatment of septicopyemia blood immunological paralysis medicine.Adopt bufanolide dienes compound of the present invention as cinobufacin can effectively reverse immunological paralysis mouse death rate and spleen, lymph node CD4+T cell declines, and improve the expression of clinical severe sepsis patient mononuclear cell HLA-II antigen and the immunostimulation factor, reverse person monocytic cell's Endotoxin Tolerance due to lipopolysaccharide LPS two-hit.In the situation that lack clinically at present the effectively means for the treatment of sepsis immunological paralysis, application prospect is fine.

Technical scheme of the present invention is:

The application of bufanolide dienes compound in preparation treatment sepsis immunological paralysis medicine.

The general formula of described bufanolide dienes compound is:

R1: suberoyl, succinyl, oxalyl, heptanedioyl arginine

R2:-CH3,-CHO。

Bufanolide dienes compound of the present invention is as cinobufacin.

Cinobufacin of the present invention, its molecular formula is C ₂₆h ₃₄o ₆, molecular weight is 442.54, English name cinobufagin, be Bufonidae animal as the dry secretions of Bufo siccus Bufo bufo gargarizans Cantor or Bufo melanostictus Bufo melanostictus Schneider, its general formula is shown in Fig. 1.

According to the structure activity relationship pharmaceutically, think that bufanolide dienes compound can have the biological activity of identical (or similar) with cinobufacin aspect reverse sepsis immunological paralysis.

Bufanolide dienes compound of the present invention is used for the treatment of the medicine of sepsis immunological paralysis, can be through injection or transfusion form administration, and dosage is had nothing in common with each other because medicine is different,

The natural drug extraction separation method that above-mentioned bufanolide dienes compound can be known by pharmaceutical field obtains from Amphibian Bufo siccus, and the technical staff in pharmaceutical field also can understand bufanolide dienes compound and also can obtain by the method for chemosynthesis.

The inventor adopts the cinobufacin in Amphibian Bufo siccus source first, our experiments show that, this compound can obviously reverse the clinical sepsis immunological paralysis of the simulation mouse survival rate due to mice cecal ligation and perforation+Salmonella two-hit, the minimizing of Immunosuppression paralysis mouse spleen and lymph node CD4+ and CD8+ cell.On clinical severe sepsis patient blood mononuclear cell, cinobufacin can reverse the existing ability of mononuclear cell angtigen presentation that sepsis causes and reduce the reduction with the TH1 immunostimulation factor such as TNF α, GM-CSF, IFN γ.On normal person's mononuclear cell Endotoxin Tolerance model due to lipopolysaccharide (LPS), cinobufacin can effectively reverse secondary LPS hit due to the reduction of mononuclear cell TNF alpha expression ability.Lacking clinically in the situation of medicine, the especially small-molecule drug of effectively treating septic immunological paralysis at present, bufanolide dienes compound has good exploitation value and application prospect.

Accompanying drawing explanation

Fig. 1: the chemical structural formula of cinobufacin

Fig. 2: mice CLP operation started to occur immunological paralysis state after 48 hours, detects after CLP CD4+ and CD8+T apoptosis situation (n=5) in different time points mouse spleen; ^*p<0.05, ^*p<0.01, ^{* *}p<0.001 compares with matched group.

TNF-α mRNA and IL-10mRNA expression variation (n=6) in different time points mice PBMC after Fig. 3: CLP operation.

Fig. 4: the phase administration of cinobufacin immunological paralysis improves the survival rate of sepsis mice

Fig. 5: the phase administration of cinobufacin immunological paralysis suppresses the minimizing of spleen CD4+ and CD8+T cell quantity.After Salmonella superinfection 48 hours, the ratio of CD4+ and CD8+T cell in fluidic cell detection different disposal group mouse spleen.

Fig. 6: cinobufacin reverses the HLA-DR α/β mRNA that in normal person's peripheral blood lymphocytes, LPS causes and lowers. ^*p<0.01, ^{* *}p<0.001 compares with ouabain untreated fish group.

Fig. 7: septic mononuclear cell stimulates more responsive to cinobufacin.Under 50nM cinobufacin stimulates, septic mononuclear cell TNF-α mrna expression is higher than Normal group; N.S. represent not have significant difference, ^*p<0.01 compares with matched group.

Fig. 8: cinobufacin stimulates septic mononuclear cell to produce GM-CSF mRNA

Fig. 9: cinobufacin stimulates septic mononuclear cell to produce interferon γ mRNA, ^*p<0.05, ^*p<0.01 compares with matched group.

Figure 10: cinobufacin reverses Endotoxin Tolerance.

The specific embodiment

The following examples can explain the present invention, but do not limit in any form the present invention.

Embodiment 1: the foundation of immunological paralysis mouse model

1.1 experimental technique

1.1.1 mice cecal ligation and perforation sepsis model is set up

6-8 C57/b16 male mice in age in week, derives from Yangzhou University's zoopery center.Experiment grouping: cecal ligation and perforation (cecal ligation and puncture, CLP) group (experimental group) and sham operated rats (sham group), CLP group is set up sepsis in various degree according to the ligation position of caecum and perforation number of times again; Sham group is only opened abdomen, is closed abdomen program.Anesthesia adopts pentobarbital sodium (1%, 200-250 μ l) lumbar injection, and abdominal part median line open abdomen enters abdominal cavity, and the separated caecum of passivity dissecting forceps is also held out by abdominal cavity, avoids damaging the caecum branch of mesenteric, particularly arteria ileocaeca.By the position of the pre-designed order of severity, carry out ligation, guarantee that ligation ileocecal valve is not to keep the seriality of small intestinal.With 5ml syringe needle, at the centre position of caecum ligation holostrome, penetrate (penetrate number of times and also determine sepsis degree), and gently extrude a little feces to guarantee the opening of perforation place.Caecum is also received abdominal cavity, sews up continuously and closes abdomen.After 6 hours, with the Sterile Saline subcutaneous injection recovery animal (1mL/ only) that is preheating to 37 ℃, increase the early stage portal hypertension state of animal sepsis.All animal surgeries completed within 10 minutes, (Immunodesign of experimental sepsis by cecal ligation and puncture.Nature protocols4 keeps consistency, 31-36, Rittirsch et al., 2008).

1.1.2 mouse peripheral blood mononuclearcell (PBMC) separation

Eyeball of mouse is got blood, collects blood in EDTA-K2 anticoagulation sterile tube, and mixes with organizing diluent 1:1; Mixed liquor is placed on isopyknic cell separation liquid liquid level, with approximately 1500 revs/min of 400g() centrifugal 15 minutes; Collect second layer ring-type milky mononuclearcell, use 4-5ml cell washing liquid to wash, and with approximately 1800 revs/min of 500g() centrifugal 20 minutes, supernatant discarded, stay sedimentation cell, and add 1ml Trizol in cell, treat subsequent experimental research.

1.1.3 apoptosis is measured

After cell harvesting, adopt Annexin V/PI couple to dye, flow cytometer detects Annexin V (+)/PI (-) cell and is defined as apoptotic cell.

1.1.4 reverse transcription PCR is measured

1.1.4.1 total RNA extracts

All article are all processed and sterilizing with DEPC in advance.Chloroform/ml the Trizol that adds 200 μ l in cell or tissue, places 5min after vibrating 15 seconds, the centrifugal 15min of 12000g, 4 ℃.Draw upper strata 400-500 μ l, transfer in clean eppendorf pipe, add 500 μ l isopropyl alcohols (cell concentration adds 50 μ l3M sodium acetates and 20 μ g glycogens simultaneously when few), room temperature is placed 10min, the centrifugal 10min of 12000g, 4 ℃.Visible RNA precipitation.Supernatant discarded, adds 1ml70% washing with alcohol precipitation, the centrifugal 5min of 7500g, and eppendorf pipe is positioned over the other RNA precipitation of drying of alcohol burner, the DDW that adds 30 μ l DEPC to process, 55 ℃ dissolve 10min.

1.1.4.2.RNA reverse transcription

Reverse transcription reaction adopts TaKaRa company test kit

1.1.4.3RT-PCR reaction

The cDNA that gets equivalent does template and does pcr amplification, adopts 20 μ l PCR reaction systems, and PCR condition is 95 ℃ of 5min, 95 ℃ 30 seconds, 55-65 ℃ 30 seconds, the 72 ℃ of 30-90 seconds of 20-35 circulations altogether, then 72 ℃ are extended 10min, and PCR product is in 1% agarose gel electrophoresis, and EB develops the color.

1.1.4.4q-PCR reaction

Get the cDNA of reverse transcription as template, doubly, q-PCR reaction system is that SYBR Green is 10 μ L to dilution 20-100, Forward primer(10 μ M) 1 μ L, Reverse primer (10 μ M) 1 μ L, cDNA template (after dilution) 8 μ L, 3 holes of each sample pipetting volume.PCR condition is 95 ℃ of 10min, 95 ℃ 30 seconds, 60 ℃ of totally 40 circulations in 60 seconds.

1.2. experimental result

Literature research shows that mice cecal ligation and perforation sepsis model can effectively simulate the immune state of clinical septic, after CLP modeling 48 hours, be accompanied by the reduction that cytokine (comprising proinflammatory and but inflammatory cytokines) produces level of ability, similar immunological paralysis state starts to manifest, and CLP mice is more responsive to antibacterial superinfection.

Immunocyte apoptosis, TH1 cytokine are immunological paralysis key characters to the drift of TH2 cytokine, the activation of Treg cell etc., by setting up CLP model, and investigate model and set up spleen CD4+ and CD8+T apoptosis situation after 24,48,72 hours afterwards, find that CLP24 hour two kinds of apoptosis are obvious, along with time apoptosis ratio increases (Fig. 2) gradually.Meanwhile, CLP modeling is after 6 hours, and in mouse peripheral blood PBMC, the expression of TNF-α mRNA, to peaking, then declines gradually; And IL-10mRNA level rose rapidly in 24 hours in PBMC, until within 48 hours, slightly decline (Fig. 3).

In order further to simulate the pathological state that clinical sepsis immunological paralysis state patient is easy to occur superinfection, on mice CLP basis, carry out the experiment of Salmonella two-hit.After CLP48 hour, the phenomenons such as immunosuppressant cell factor great expression and a large amount of apoptosis of T cell show that mice has entered the immunological paralysis phase, therefore set the Salmonella (S.tm.) that this time carries out a series of cfu of lumbar injection and carry out two-hit, result shows that S.tm. two-hit accelerates the death (Fig. 4) of CLP mice.And injection is higher than 2 * 10 ³the Salmonella of cfu can make mice dead fast in a short period of time, is unfavorable for subsequent experimental observation, therefore the cfu of two-hit S.tm. is decided to be to 2 * 10 ³, Impulse time is decided to be CLP and performs the operation latter 48 hours.The follow-up study that the success of this immunological paralysis model is established as sepsis immunological paralysis lays the foundation.

Embodiment 2: magnificent bufotoxin improves survival rate, bacteria clearance and CD4+ and the CD8+T cell quantity of immunological paralysis mice

2.1 experimental technique

2.1.1 the foundation of sepsis superinfection model

Set up CLP sepsis model, within 48 hours, pneumoretroperitoneum is injected 200 μ l containing 2 * 10 ³salmonella (S.tm., ATCC14028s) the bacterium liquid of CFU carries out superinfection.Use the survival curve of GraphPad Prism5.0 to analyze, and carry out significance comparison in conjunction with log-rank check.

2.1.2 antibacterial coated plate counting experiment

The anticoagulation 5 μ l and the spleen homogenate diluent 25 μ l(that get matched group and CLP superinfection mice are used normal saline to carry out gradient dilution) carry out coated plate, culture medium is tryptose soya agar culture medium, incubated overnight, and carry out count of bacteria.

2.1.3 fluidic cell detects CD4+ and CD8+T cell proportion

Take out the spleen of matched group and CLP group mice and lymph node (comprise under mice oxter, hip, mesenteric lymph node) in the vessel that contain 2ml pre-cooling PBS, spleen and lymph node are placed on microscope slide hair side, the mutual friction of two microscope slide phases, cell is extruded into singlely as far as possible, then is filtered into individual cells suspension through filter (70 μ m aperture).The individual cells suspension of spleen and lymph node is carried out to packing, sample of every 100 μ l; Spleen suspension needs erythrocyte cracked liquid to process, and adds 1ml erythrocyte cracked liquid, room temperature cracking 5min, the centrifugal 2000rpm5min of room temperature; With the resuspended spleen cell of 1ml pre-cooling PBS, and lymph node suspension is supplied to 1ml with PBS, 2000rpm4 ℃ of centrifugal 5min; Then with the coated FITC-CD4 of 1%BSA and PE-Cy5-CD8a antibody (following steps are all wanted lucifuge operation), place 20-30min on ice; 2000rpm4 ℃ of centrifugal 5min, abandons supernatant, 500 μ l PBS re-suspended cells; Cell is placed in to streaming Special test tube, and up flow type machine detects.

2.2 experimental result

The cinobufacin of low concentration scope (0.01-0.1mg/kg) can effectively improve the survival rate of immunological paralysis mice after CLP.As shown in Figure 4, mice was accepted after S.tm. two-hit in CLP48 hour, and the 54th hour and 78 hours twice intraperitoneal administration cinobufacin 0.01 and 0.1mg/kg find that cinobufacin can significantly improve the survival rate of mice.These data show that cinobufacin specificity could improve the survival rate of CLP mice in the administration of immunological paralysis phase.

In spleen there is excessive Apoptosis in the T percentage of lymphocyte explanation splenic T lymphocyte that obviously declines, and the generation of this and immunity of organism palsy is closely related.This research finds that cinobufacin can suppress the minimizing (Fig. 5) of CD4+ and CD8+T cell proportion in spleen that CLP causes in conjunction with S.tm. two-hit in the administration of immunological paralysis phase.Result shows that magnificent bufotoxin may suppressor T cell apoptosis in the mice sepsis immunological paralysis phase.

Embodiment 3: magnificent bufotoxin improves sepsis immunological paralysis patient mononuclear cell HLA-II antigen

Experimental technique:

3.1 experiment material

3.1.1 sepsis case is collected

Patient hospitalizes the patient who is diagnosed as severe sepsis in the first center ICU of Anesthesia Department of Affiliated Hospital of The 2nd Army Medical College during from JIUYUE, 2012～2013 year April.This test is examined by the first Ethics Committee of Affiliated Hospital of The 2nd Army Medical College, and patient or family numbers of patients obtain and fully inform, all signed Informed Consent Form.

3.1.2 reagent and material

LPS and ouabain are purchased from U.S. Sigma company; People and mice mononuclearcell separating medium are purchased from Tianjin Hao Yang biotech firm; Pancreas peptone soybean broth culture medium (TSB) and tryptose soya agar culture medium (TSA) are purchased from Qingdao Hai Bo biotech firm; TNF-α ELISA test kit is purchased from U.S. R & D company; Trizol is purchased from American I nvitrogen company; FastStart Universal SYBR Green Master (Rox) is purchased from Switzerland Roche company; Acrylamide, methylene diacrylamide, Ammonium Persulfate 98.5, TEMED are purchased from U.S. Ameresco company; PVDF(polyvinylidene fluoride) film is purchased from U.S. Pall company; EDTA-K2 anticoagulation sterile tube is purchased from U.S. company BD; Fluidic cell uses erythrocyte cracked liquid purchased from U.S. company BD; Reverse transcription test kit PrimeScript RT Reagent Kit, PrimeStar high-fidelity DNA synzyme, Taq enzyme, restricted enzyme, T4DNA ligase are purchased from Japanese Takara company; Glue reclaims test kit purchased from Shanghai Shen Neng lottery industry biotech firm.

3.1.3 buffer

10 * PBS:2.294g NaH ₂pO ₄h ₂o, 28.96g Na ₂hPO ₄12H ₂o, 85g NaCl is dissolved in 1L DDW, is settled to 1L.Before use, be diluted to 1 * PBS.

10 * PBST:2.294g NaH ₂pO ₄h ₂o, 28.96g Na ₂hPO ₄12H ₂o, 85g NaCl is dissolved in 1L DDW, adds 5 ‰ Tween20.Before use, be diluted to 1 * PBST.

3.1.4 use instrument

20/20 ⁿluminometer fluorescence detector is purchased from U.S. Turner Biosystems Instrument company; FACS Calibur flow cytometer is purchased from U.S. company BD; Multi-functional microplate reader is purchased from U.S. Bao Te (Bio-Tek) company; Real-time PCR is purchased from U.S. BioRad company.

3.1.5CD14+ the detection of onthe surface of monocytes HLA-DR

Collect mononuclear cell in severe sepsis patient blood, get flow cytometer Special test tube, and carry out labelling and corresponding serial number, every part of specimen four pipes, are respectively No. 1 pipe (blank pipe), No. 2 pipes (the mono-mark pipe of CD14), No. 3 pipes (the two mark pipes of CD14/ homotype contrast) and No. 4 pipes (the two mark pipes of CD14/HLA-DR); The anticoagulation that adds respectively 100 μ l to mix in each pipe; Then, in pipe 2, add CD14 (APC) 5 μ l, in pipe 3, add each 5 μ l of CD14 (APC) and homotype control antibodies Mouse IgG1 PerCP-Cy5.5, in pipe 4, add each 5 μ l of CD14 (APC) and HLA-DR (PE-Cy5), mix gently room temperature lucifuge reaction 15～30min; Then, add special-purpose erythrocyte cracked liquid (1 *) 2ml of fluidic cell in each test tube, put upside down and mix, room temperature lucifuge is placed 10min; Under room temperature condition, the centrifugal 5min of 300g, abandons supernatant, then adds 2ml PBS washing, puts upside down and mixes, and the centrifugal 5min of 300g, abandons supernatant; Finally, 500 μ l PBS re-suspended cells, can carry out FACS detection immediately.

3.1.6 reverse transcription PCR (RT-PCR) and real-time quantitative PCR (q-PCR)

The same 1.1.4 of method

Table 1.PCR primer sequence is as follows

3.2 experimental result

In innate immune response, CD14+ mononuclear cell can be the angtigen presentation through engulfing processing to t helper cell, then activated T cell, B cell and phagocyte are in the activity of other interior immunocyte, the proinflammatory factors such as secretion IL-12, TNF-α, activate congenital and specific immune response, the invasion of opposing pathogenic microorganism.Mononuclear cell antigen presentation ability can embody by cell surface HLA-DR, HLA-DP, the equimolecular expression of HLA-DQ, CD80/86, HLA-DR most importantly wherein, enough HLA-DR express all very important to maintaining specificity and non-specific immunity.Research shows, the monocytic secretion capacity of septic and antigen presentation ability are obviously suppressed, and close with patient's Prognostic significance, and it is the most representative that this aspect equals with Volk the research that starts for 1996.They merge and in pyemic research, propose onthe surface of monocytes HLA-DR and express lower than 30%(with respect to normal person's group organ transplantation) can be used as the index of immunological paralysis clinically.Kox etc. are used the septic of immunostimulation factor IFN-γ treatment immunological paralysis, found that HLA-DR expresses obviously recovery, and Plasma TNF-α and IL-6 level obviously increase.Visible HLA-DR can be used as the judge index of immune state, according to HLA-DR, gives the improvement that certain immunostimulant is conducive to immunologic balance.

The present invention's discovery is in normal person's mononuclear cell, and coupling cinobufacin and LPS process can significantly reverse by the alone HLA-DR α/β mrna expression causing of LPS and lower (Fig. 6).

The magnificent bufotoxin recuspine of embodiment 4 swashs septic monocytes TNF-α, GM-CSF and interferon γ

4.1 experimental technique

4.1.1. person monocytic cell's separation

Get normal person or septic peripheral blood, EDTA anticoagulant, first discards blood plasma for centrifugal 20 minutes with 200g.Then, to abandoning in plasmapheretic hemocyte, add and double whole blood and the tissue homogenate diluent of blood plasma volume and carefully mix.Mixed liquor is carefully superimposed on the liquid level of cell separation liquid (mixed liquor: separating medium=2:1), then with 600g centrifugal 15 minutes.Collect second layer ring-type milky cell and put into centrifuge tube, and add the RPMI-1640 culture fluid of 4 times of volumes, after fully mixing, with 500g centrifugal 10 minutes, supernatant discarded is stayed sedimentation cell, has more again hanged cell, and repeated washing obtains PERIPHERAL BLOOD MONONUCLEAR CELL 2 times.Use platform to expect that blue dyestuff carries out cell counting, living cells percentage rate is more than 95%.By separated mononuclearcell with 4 * 10 ⁶/ ml number is inclined in the plastic culture plate of aseptic cleaning, puts 37 ℃, 5%CO ₂in incubator, spend the night, with capillary pipette, suck gently suspension, then use appropriate PBS washed twice, the adherent mononuclear cell that is.

4.1.2ELISA detect TNF-alpha content

The TNF-α standard substance of variable concentrations and laboratory sample (comprising cells and supernatant and blood plasma) are joined in R & D ELISA test kit 96 orifice plates, and every hole 100 μ l, seal reacting hole with shrouding gummed paper, incubated at room 2h; Every hole adds cleaning mixture 300 μ l, washs 4 times, then adds 200 μ l enzyme marks to detect antibody, seals reacting hole, incubated at room 2h with shrouding gummed paper; Wash 4 times, and in each hole, add 200 μ l chromogenic substrates, room temperature lucifuge is hatched 30min; Finally, in each hole, add 50 μ l stop buffers, use microplate reader to measure 450nm absorbance, establish 540nm as tuning wavelength.By the content of TNF-α in standard curve conversion laboratory sample.

4.1.3RT-PCR method detects TNF-α, GM-CSF, interferon γ mRNA

The same 1.1.4 of method

Table 2.PCR primer sequence

4.2 experimental result

In order further to confirm that magnificent bufotoxin is by cytokine TNF-α activating immune system, thereby reverse immunological paralysis, the present invention has collected the blood sample of ICU septic, by fluidic cell, detect the expression of peripheral blood CD14+ onthe surface of monocytes HLA-DR, choose HLA-DR and express the blood sample lower than 30%, separation obtains mononuclear cell, and In vitro culture also carries out medicine irritation.

Found that septic mononuclear cell is more responsive to 50nM cinobufacin than normal person mononuclear cell, show as the multiple compared with normal mononuclear cell high (Fig. 7) that TNF-α mRNA is increased, and now septic mononuclear cell stimulates sensitivity obviously to reduce to LPS, similarly, magnificent bufotoxin also can stimulate septic mononuclear cell GM-CSF(Fig. 8) and interferon γ formula mrna expression (Fig. 9).Show that magnificent bufotoxin is under immunological paralysis state, the more expression of effective stimulus mononuclear cell immune activation cytokine, Promote immunity functional rehabilitation.

The magnificent bufotoxin of embodiment 5. reverses the Endotoxin Tolerance of LPS induction

5.1 experimental technique

5.1.1. person monocytic cell's separation

The same 4.1.1 of method

5.1.2RT-PCR method detects TNF-α

The same 4.1.3 of method

5.1.3 the foundation of human peripheral blood mononuclear cell's separation and LPS tolerance cell model

End user's peripheral blood lymphocytes is set up Endotoxin Tolerance cell model: 1 μ g/ml LPS processes cell after 16 hours for the first time, twice of PBS washed cell, then use 1 μ g/ml LPS after-treatment cell, now cell shows as the LPS tolerance (LaRue and McCall, 1994) to stimulating for the second time.

5.2 experimental result

In order to continue to verify that magnificent bufotoxin reverses immunological paralysis by TNF-α, end user's peripheral blood lymphocytes has been set up Endotoxin Tolerance model and has been carried out in vitro study, because sepsis patient all has the feature of Endotoxin Tolerance, and the effective immunological paralysis state in analogue body of this model.At 1 μ g/ml LPS for the first time, stimulate mononuclear cell after 16 hours, then use same concentration LPS to carry out two-hit, find that now TNF-α mrna expression no longer raises, thereby show that model is successfully established.In this research, find: in LPS stimulation for the first time, add 50nM China bufotoxin, and co-treatment is after 16 hours, TNF-α mRNA renewal amount is higher than alone LPS stimulating group, and responsive to the performance of LPS strike for the second time, and now TNF-α mrna expression also raises (Figure 10) to some extent.Data show that magnificent bufotoxin also can suppress Endotoxin Tolerance at cellular level.

The foregoing is only preferred embodiment of the present invention; not be used for limiting practical range of the present invention; if do not depart from the spirit and scope of the present invention, the present invention is modified or is equal to replacement, all should be encompassed in the middle of the protection domain of claim of the present invention.

Claims

1. the application of bufanolide dienes compound in preparation treatment sepsis immunological paralysis medicine.

2. application according to claim 1, is characterized in that: the general formula of described bufanolide dienes compound is:

R1: suberoyl, succinyl, oxalyl, heptanedioyl arginine

R2:-CH3,-CHO。

3. application according to claim 2, is characterized in that described bufanolide dienes compound is cinobufacin.

4. application according to claim 1, is characterized in that: the medicine of preparing with bufanolide dienes compound is with injection or infusion solution administration.