CN103599112B

CN103599112B - Application of bufanolide diene compound in preparation of medicines for treating sepsis immune paralysis

Info

Publication number: CN103599112B
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: 2015-02-18
Anticipated expiration: 2033-12-05
Also published as: CN103599112A

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 diolefinic compounds in preparation treatment sepsis immunological paralysis medicine

Technical field

The present invention relates to the novelty teabag of bufanolide diolefinic compounds for the preparation for the treatment of sepsis immunological paralysis medicine.

Background technology

Sepsis rates is high, and the whole world has more than 1,800 ten thousand severe sepsis cases every year, and the U.S. has 750,000 routine sepsis patients every year, and this numeral also rises with the speed of annual 1.5% ~ 8.0%.The pyemic state of an illness is dangerous, and case fatality rate is high, and whole world people's every day about 14000 dies from its complication, and the U.S. every year about 21.5 ten thousand people is dead.Learn investigation display according to Foreign Epidemic disease, pyemic case fatality rate exceedes myocardial infarction, becomes the main cause of non-cardiac patient death in intensive care unit(ICU).In recent years, although anti-infective therapy and multiple organ support therapy technology achieve 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 quality of life of the mankind, causes grave danger to human health.

Mononuclear cell, macrophage and neutrophilic granulocyte be infected and non-infectious stimulation time discharge 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 bring out sepsis, SIRS and septic shock.The early stage systemic inflammatory response of sepsis is the result Immunomodulatory therapies in sepsis.Intensive care medicine26 of inflammatory cytokine especially TNF-α, IL-6, IL-1 and IFN-γ effect, S124-S128, Kox et al., 2000).After people recognize caused by TNF-α, IL-6 and IL-1 inflammatory reaction and take part in the pathogenic course of SIRS, sepsis and multiple organ dysfunction syndrome (MODS), just devise the anti-inflammatory treatment of the various allogenic immune preparation for these inflammatory mediators.These anti-inflammatory substances comprise the specificity substance that antibody, soluble recepter, receptor antagonist etc. prevent the Cytokines such as TNF-α, IL-1, and the non-specific anti-inflammatory such as nonsteroidal antiinflammatory drug, glucocorticoid thing.Although application anti-inflammatory drug treatment animal sepsis achieves good effect, but the pyemic clinical anti-inflammatory treatment test of relevant human body is not succeeded, case fatality rate is even made to 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 lacks accurate immunosurveillance to pyemic evolution.Research shows, can there is strong anti-inflammatory response rapidly in body after proinflammatory reaction occurs sepsis in early days, simultaneously with the release of a large amount of Anti-inflammatory mediators, and some sepsis patient shows obvious immunosuppressive condition (Sepsis and immuneresponse.World J Emerg Med2,88-92., Chen et al., 2011).Kox etc. also confirm that and the later stage early stage at sepsis exists two kinds of immune states, and namely 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, occurred at the early stage of sepsis inflammatory reaction the development that compensatory inflammatory response syndrome (CARS) reacts with inflammation-inhibiting, this reaction is occupied an leading position and induced 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 developing sepsis process, mononuclear cell human leucocyte differentiation antigen DR (HLA-DR) expression reduces, the antigenspecific T lymphocyte activity that mononuclear cell is induced reduces, the change of mononuclear cell release cytokine profiles ability and polymorphonuclear leukocyte anergy (Schultz et al., 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 obtains the support of clinical research to a certain extent.The reports such as Volk in 1996, CD14 ⁺monocyte HLA-DR level reliably can differentiate sepsis immunosuppressive condition (Monocyte deactivation-rationale for a new therapeutic strategy in sepsis.Intensive caremedicine22, S474-S481, Volk et al., 1996).With the HLA-DR/CD14 of 30% ⁺as threshold value, patient's prognosis is obviously different.In addition, effectively immune state can be improved, at HLA-DR/CD14 with immunostimulant gamma interferon (IFN-γ) treatment ⁺while promoting, also observe proinflammatory cytokine TNF-α and discharge increase (Interferongamma-1b in the treatment of compensatory anti-inflammatory response syndrome:a newapproach: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, improving survival rate, is one of feasible strategy based on the immune system that reactivates of cytokine as GM-CSF, IFN-γ, TNF-α.

Colony stimulating factor CSF is a kind of cytokine regulating 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, neutrophil apoptosis can be postponed, raise the expression of CD11b and CD14 molecule, enhance and stick chemotaxis and Phagocytosis power.Clinical research has proved that G-CSF and GM-CSF can improve the monocyte function defect of severe postoperative the infected, increase the expression of TNF-α secretion and HLA-DR, and increase the immunocompetence etc. of lymphocyte quantity, regulatory T-cell proliferation and differentiation, enhancing Th1, thus reverse immunological paralysis.Orozco etc. report 58 routine atraumatic Abdominal pyemia patients, the treatment of application molgramostim (GM-CSF) combined with antibiotic, Infective morbidity incidence rate can be reduced, reduce hospital stays and expense (Molgramostim (GM-CSF) associated with antibiotic treatment in nontraumatic abdominal sepsis:arandomized, 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, TNF-alpha levels and monocyte HLA-DR in blood plasma increase, thus improves the immune state of sepsis patient, improves its survival rate ¹⁶⁴.IFN-γ is as immunoactivator, and administration time has strict control, because any immune modulating treatment all should based on the supervision of immunologic function.When the HLA-DR Expression of Monocytes of patient is lower, when showing antiinflammatory response, the IFN-γ of low dosage treats safe and effective; Being in the patient of the proinflammatory stage of reaction at those uses IFN-γ to be then harmful.

In addition, animal experiment study shows, at sepsis immunological paralysis phase administration restructuring TNF-α, significantly to increase the survival rate of animal, and strengthens animal liver, spleen removing pathogenic microorganism ability, shows that TNF-α has positive effect to the treatment of sepsis immunological paralysis.But, the therapeutical effect of restructuring TNF-α is greatly limited, depend on the type of animal institute pathogenic infection microorganism, ability that animal body self produces TNF-α and restructuring TNF-α administration time (Treatment of experimentalsepsis-induced immunoparalysis with TNF.Immunobiology208,381-389., Echtenacher etal., 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 existed in body.Thymosin alpha 1 can regulate the level of the cytokines such as TNF-α, IL-6 and IL-10 of severe infections patient, alleviates inflammatory reaction, improves the immunologic function of patient.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 reduced inflammation caused by medium, anti-inflammatory factors IL-10 level can be improved simultaneously, promote that anti-inflammatory factors and proinflammatory factor keep balance.Although Thymosin alpha 1 has positive role to treatment of sepsis, not all effective in pyemic any state administration.Along with going deep into of understanding sepsis immune state, consider that GM-CSF, IFN-γ or TNF-alpha protein drug half-life is short, side effect large, finding the small-molecule drug with immunoregulation effect is the promising research direction for the treatment of of sepsis simultaneously.

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

Summary of the invention

Object of the present invention is to provide the purposes of bufanolide diolefinic compounds for the preparation for the treatment of septicopyemia blood immunological paralysis medicine.Adopt bufanolide diolefinic compounds of the present invention effectively can reverse immunological paralysis mouse death rate as cinobufacin and spleen, lymph node CD4+T cell decline, and improve the expression of clinical severe sepsis patient mononuclear cell HLA-II antigen and immuno-stimulator, reverse person monocytic cell's Endotoxin Tolerance caused by lipopolysaccharide LPS two-hit.When shortage effectively treats the means of sepsis immunological paralysis clinically at present, application prospect is fine.

Technical scheme of the present invention is:

The application of bufanolide diolefinic compounds in preparation treatment sepsis immunological paralysis medicine.

The general formula of described bufanolide diolefinic compounds is:

R1: suberoyl, succinyl, oxalyl, heptanedioyl arginine

R2:-CH3,-CHO。

Bufanolide diolefinic compounds 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, and be the dry secretions of Bufonidae animal as Bufo siccus Bufo bufo gargarizans Cantor or Bufo melanostictus Bufo melanostictusSchneider, its general formula is shown in Fig. 1.

Think that bufanolide diolefinic compounds is reversing the biological activity can in sepsis immunological paralysis with identical (or similar) with cinobufacin according to pharmaceutically known structure activity relationship.

Bufanolide diolefinic compounds of the present invention is used for the treatment of the medicine of sepsis immunological paralysis, can through injection or infusion solutions 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 diolefinic compounds is known by pharmaceutical field obtains from Amphibian Bufo siccus, and the technical staff in pharmaceutical field also can be understood bufanolide diolefinic compounds and also can be obtained by the method for chemosynthesis.

The cinobufacin that the present inventor adopts Amphibian Bufo siccus to originate first, our experiments show that, this compound obviously can reverse the simulation clinical sepsis immunological paralysis mouse survival rate caused by 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 immuno-stimulator such as TNF α, GM-CSF, IFN γ.On the caused normal person's mononuclear cell Endotoxin Tolerance model of lipopolysaccharide (LPS), cinobufacin effectively can reverse the reduction that secondary LPS hits caused mononuclear cell TNF alpha expression ability.Lacking the medicine of effectively treating septic immunological paralysis at present clinically, especially when small-molecule drug, bufanolide diolefinic compounds has good Development volue and application prospect.

Accompanying drawing explanation

Fig. 1: the chemical structural formula of cinobufacin

Fig. 2: mice CLP perform the operation 48 hours after start to occur immunological paralysis state, to detect 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 is compared with matched group.

In Fig. 3: CLP Post operation different time points mice PBMC, TNF-α mRNA and IL-10mRNA expresses change (n=6).

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.Salmonella superinfection is after 48 hours, the ratio of CD4+ and CD8+T cell in FCM analysis different disposal group mouse spleen.

Fig. 6: cinobufacin reverses the HLA-DR α/β mRNA that in Normal human peripheral's blood monocyte, LPS causes and lowers. ^*p<0.01, ^{* *}p<0.001 and ouabain untreated fish group is compared.

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 there is no significant difference, ^*p<0.01 is compared 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 is compared with matched group.

Figure 10: cinobufacin reverses Endotoxin Tolerance.

Detailed description of the invention

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

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 animal experimental center.Experiment grouping: cecal ligation and perforation (cecal ligation and puncture, CLP) group (experimental group) and sham operated rats (sham group), CLP group is again according to ligation position and the perforation number of times foundation sepsis in various degree of caecum; Sham group is only carried out out 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 Blunt dissection tweezer is separated caecum and is held out by abdominal cavity, avoids the caecum branch damaging mesenteric, particularly arteria ileocaeca.Carry out ligation by the position of the pre-designed order of severity, guarantee that ligation ileocecal valve is not to keep the seriality of small intestinal.Penetrate (penetrate number of times and also determine sepsis degree) at the centre position holostrome of Cecal Ligation with 5ml syringe needle, and gently extrude a little feces to ensure the opening of perforation place.Caecum also receives abdominal cavity, sews up continuously and closes abdomen.With the Sterile Saline subcutaneous injection recovery animal (1mL/ only) being preheating to 37 DEG C after 6 hours, increase the portal hypertension state that animal sepsis is early stage.All animal surgeries completed within 10 minutes, keep consistency (Immunodesign of experimental sepsis by cecal ligation andpuncture.Nature protocols4,31-36, Rittirsch et al., 2008).

1.1.2 mouse peripheral blood mononuclearcell (PBMC) is separated

Eyeball of mouse gets 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 400g(about 1500 revs/min) centrifugal 15 minutes; Collect second layer ring-type milky mononuclearcell, use 4-5ml cell washing solution to wash, and with 500g(about 1800 revs/min) centrifugal 20 minutes, supernatant discarded, stay sedimentation cell, and add 1ml Trizol in cell, treat subsequent experimental research.

1.1.3 apoptosis measures

After cell harvesting, adopt the two dye of Annexin V/PI, flow cytomery Annexin V (+)/PI (-) cell is defined as apoptotic cell.

1.1.4 reverse transcription PCR measures

1.1.4.1 Total RNAs extraction

All article are all in advance with DEPC process also sterilizing.Add the chloroform/ml Trizol of 200 μ l in cell or tissue, vibrate and place 5min after 15 seconds, the centrifugal 15min of 12000g, 4 DEG C.Draw upper strata 400-500 μ l, transfer in clean eppendorf pipe, add 500 μ l isopropyl alcohols (simultaneously adding 50 μ l3M sodium acetates and 20 μ g glycogens when cell concentration is few), room temperature places the centrifugal 10min of 10min, 12000g, 4 DEG C.Visible RNA precipitation.Supernatant discarded, adds 1ml70% washing with alcohol precipitation, and centrifugal 5min, the eppendorf pipe of 7500g is positioned over the other RNA that dries of alcohol burner and precipitates, and add the DDW of 30 μ l DEPC process, 55 DEG C 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 getting equivalent does template and does pcr amplification, and adopt 20 μ l PCR reaction systems, PCR condition is 95 DEG C of 5min, 95 DEG C 30 seconds, 55-65 DEG C 30 seconds, the 72 DEG C of 30-90 seconds of 20-35 circulations altogether, then 72 DEG C extend 10min, and PCR primer 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 SYBR Green is 10 μ L to dilution 20-100, Forward primer(10 μM) 1 μ L, Reverse primer (10 μMs) 1 μ L, cDNA template (after dilution) 8 μ L, each sample pipetting volume 3 holes.PCR condition is 95 DEG C of 10min, 95 DEG C 30 seconds, 60 DEG C of totally 40 circulations in 60 seconds.

1.2. experimental result

Literature research shows that mice cecal ligation and perforation sepsis model effectively can simulate the immune state of clinical septic, CLP modeling is after 48 hours, the reduction of level of ability is produced (comprise proinflammatory and press down inflammatory cytokines) along with cytokine, 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 activation etc. of the drift of TH2 cytokine, Treg cell, by setting up CLP model, and investigate model foundation 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 is to peaking, then declines gradually; And IL-10mRNA level rose rapidly in 24 hours in PBMC, until 48 hours slightly decline (Fig. 3).

Be easy to occur the pathological state of superinfection in order to simulate clinical sepsis immunological paralysis state patient further, mice CLP basis is carried out the experiment of Salmonella two-hit.After CLP48 hour, the phenomenons such as immuno-suppressing cytokine 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 quick death in a short period of time, is unfavorable for that subsequent experimental is observed, therefore the cfu of two-hit S.tm. is decided to be 2 × 10 ³, Impulse time is decided to be CLP Post operation 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 the survival rate of immunological paralysis mice, bacteria clearance and CD4+ and CD8+T cell quantity

2.1 experimental technique

2.1.1 the foundation of sepsis superinfection model

Set up CLP sepsis model, within 48 hours, pneumoretroperitoneum injects 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 in conjunction with log-rank inspection and compare.

2.1.2 antibacterial coated plate counting experiments

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

2.1.3 FCM analysis CD4+ and CD8+T cell proportion

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

2.2 experimental result

The cinobufacin (0.01-0.1mg/kg) of low concentration scope effectively can improve the survival rate of immunological paralysis mice after CLP.As shown in Figure 4, after mice accepted S.tm. two-hit in CLP48 hour, 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, T percentage of lymphocyte obviously declines and illustrates that splenic T lymphocyte there occurs excessive Apoptosis, and the generation of this and immunity of organism palsy is closely related.This research finds the minimizing (Fig. 5) of CD4+ and CD8+T cell proportion in the spleen that cinobufacin can suppress CLP to cause 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 mouse 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 cases is collected

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

3.1.2 reagent and material

LPS and ouabain purchased from American Sigma company; People and mice mononuclearcell separating medium are purchased from Tianjin Hao ocean 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 kit purchased from American R & D company; Trizol purchased from American Invitrogen company; FastStartUniversal SYBR Green Master (Rox) is purchased from Roche company of Switzerland; Acrylamide, methylene diacrylamide, Ammonium Persulfate 98.5, TEMED purchased from American Ameresco company; PVDF(polyvinylidene fluoride) film purchased from American Pall company; EDTA-K2 anticoagulation sterile tube purchased from American BD company; Fluidic cell erythrocyte cracked liquid purchased from American BD company; Reverse Transcriptase 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.1 × PBS is diluted to before using.

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

3.1.4 instrument is used

20/20 ⁿluminometer fluorescence detector purchased from American Turner Biosystems Instrument company; FACSCalibur flow cytometer purchased from American BD company; Multi-functional microplate reader purchased from American Bao Te (Bio-Tek) company; Real-time PCR purchased from American 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 is managed, and is respectively No. 1 pipe (blank pipe), No. 2 pipes (CD14 is mono-marks pipe), No. 3 pipes (CD14/ Isotype control two mark pipe) and No. 4 pipes (the two mark pipe of CD14/HLA-DR); The anticoagulation that 100 μ l mix is added respectively in each pipe; Then, CD14 (APC) 5 μ l is added in pipe 2, the each 5 μ l of CD14 (APC) and isotype control Ab MouseIgG1 PerCP-Cy5.5 are added in pipe 3, the each 5 μ l of CD14 (APC) and HLA-DR (PE-Cy5) are added in pipe 4, mix gently, room temperature lucifuge reaction 15 ~ 30min; Then, in each test tube, add the special erythrocyte cracked liquid of fluidic cell (1 ×) 2ml, put upside down mixing, room temperature lucifuge places 10min; Under room temperature condition, the centrifugal 5min of 300g, abandons supernatant, then adds 2ml PBS and wash, and put upside down mixing, 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 the angtigen presentation through engulfing process to t helper cell, then activated T cell, B cell and phagocyte are in the activity of other interior immunocyte, the proinflammatory cytokines such as secretion IL-12, TNF-α, activate congenital and specific immune response, the invasion of opposing pathogenic microorganism.Mononuclear cell antigen presentation capability embodies by the equimolecular expression of cell surface HLA-DR, HLA-DP, HLA-DQ, CD80/86, wherein most importantly HLA-DR, enough HLA-DR express to maintenance specificity and non-specific immunity all very important.Research shows, the monocytic secretion capacity of septic and antigen presentation capability are obviously suppressed, and close with patient's Prognostic significance, and the research most that this aspect equals to start for 1996 with Volk is representative.They organ transplantation is merged in pyemic research propose onthe surface of monocytes HLA-DR and express lower than 30%(relative to normal person's group) can as the index of immunological paralysis clinically.Kox etc. use immuno-stimulator IFN-γ to treat the septic of immunological paralysis, and found that HLA-DR expresses and obviously recover, Plasma TNF-α and IL-6 level obviously increase.Visible HLA-DR can as the judge index of immune state, gives according to HLA-DR the improvement that certain immunostimulant is conducive to immunologic balance.

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

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

4.1 experimental technique

4.1.1. the separation of person monocytic cell

Get normal person or septic peripheral blood, EDTA anticoagulant, first within centrifugal 20 minutes, discard blood plasma with 200g.Then, to abandoning in plasmapheretic hemocyte the whole blood and tissue homogenate diluent careful mixing that add and double Plasma volumes.Mixed liquor is carefully superimposed on (mixed liquor: separating medium=2:1) on the liquid level of cell separation liquid, 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, fully after mixing, with 500g centrifugal 10 minutes, supernatant discarded stays sedimentation cell, has more again hanged cell, and namely 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 the mononuclearcell of separation with 4 × 10 ⁶/ ml number is inclined in the plastic culture plate of aseptic, puts 37 DEG C, 5%CO ₂spend the night in incubator, suck suspension gently with capillary pipette, then wash twice with appropriate PBS, adherent is mononuclear cell.

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 kit 96 orifice plate, every hole 100 μ l, seals 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 and detects antibody, seal 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 hatches 30min; Finally, in each hole, add 50 μ l stop buffers, use microplate reader to measure 450nm absorbance, if 540nm is 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 to confirm that magnificent bufotoxin is by cytokine TNF-α activating immune system further, thus reverse immunological paralysis, the present invention have collected the blood sample of ICU septic, by the expression of FCM analysis peripheral blood CD14+ onthe surface of monocytes HLA-DR, choose HLA-DR express lower than 30% blood sample, 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, can the expression of more 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. the separation of person monocytic cell

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 resistant cells model

End user's peripheral blood lymphocytes sets up Endotoxin Tolerance cell model: 1 μ g/ml LPS first time process cell is after 16 hours, PBS washed cell twice, then 1 μ g/ml LPS after-treatment cell is used, now cells show is LPS tolerance (LaRue and McCall, 1994) stimulated 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 establishes Endotoxin Tolerance model and carries out in vitro study, because sepsis patient all has the feature of Endotoxin Tolerance, and this model can effective immunological paralysis state in analogue body.Stimulate mononuclear cell after 16 hours at first time 1 μ g/ml LPS, then use same concentration LPS to carry out two-hit, find that now TNF-α mrna expression no longer raises, thus show that model is successfully established.Find in this research: while first time LPS stimulates, add 50nM China bufotoxin, and co-treatment is after 16 hours, TNF-α mRNA renewal amount is higher than alone LPS stimulating group, and it is responsive to hit performance to second time LPS, 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 equivalent to replace, in the middle of the protection domain that all should be encompassed in claim of the present invention.

Claims

1. the application of cinobufacin in preparation treatment sepsis immunological paralysis medicine.

2. application according to claim 1, is characterized in that: the medicine prepared with cinobufacin is with injection or infusion solution administration.