CN101391100A - Use of recombined human source thioredoxin in preparing medicine for treating endotoxemia - Google Patents
Use of recombined human source thioredoxin in preparing medicine for treating endotoxemia Download PDFInfo
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- CN101391100A CN101391100A CNA2008101511564A CN200810151156A CN101391100A CN 101391100 A CN101391100 A CN 101391100A CN A2008101511564 A CNA2008101511564 A CN A2008101511564A CN 200810151156 A CN200810151156 A CN 200810151156A CN 101391100 A CN101391100 A CN 101391100A
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Abstract
The invention discloses the application of recombined human-derived thioredoxin for preparing medicine for treating endotoxemia. Experiments show that the thioredoxin can directly combined with endotoxin in vivo or vitro and carry out glycosylation reaction so as to play a role of neutralizing the endotoxin and protecting important organs such as liver and the like. The invention is suitable for patients having symptoms such as endotoxemia with liver injure. The invention is simple and practical without special contraindications, and can be widely used in hospitals, cliniques, and the like.
Description
Technical field
The present invention relates to use small protein Drug therapy infectious disease field; more particularly; relate to the application that recombined human source thioredoxin is used to prepare treatment endotoxemia medicine, the affirmation recombined human source thioredoxin is treated application and the mechanism thereof in the endotoxemia at the performance organ protection.
Background technology
Endotoxemia be since in the blood antibacterial or intralesional antibacterial discharge a large amount of endotoxins to blood, or import the liquid of a large amount of contaminated with endotoxins and cause.Bacterial endotoxin has very complicated biologic activity, endotoxemia can appear in the multiple disease of multisystem, can cause a series of pathophysiological changes, comprise sepsis, shock, disseminated inravascular coagulation (DIC), multiple organ dysfunction syndrome (MODS) etc., case fatality rate is high.Therefore, anti-endotoxin and blocking-up or the damage that weakens its mediation have important clinical significance.Lipopolysaccharide (LPS) is the ingredient of gram-negative bacteria cell wall, comprises lipid A (Lipid A), core polysaccharide, three compositions of specific polysaccharide.Be about to LPS traditionally and be called endotoxin, wherein by lipid A performance activity of endotoxin, oxidative stress and subsequent lipid peroxidation are proved to be the lethal major reason of LPS.Up to the present, the treatment of endotoxemia lacks active drug.Polymyxin B (polymyxin B, though PMB) can the deactivation endotoxin and suppress gram-negative bacteria, because it is to the kidney toxic side effect, and is difficult to remove from blood, so blood is restricted to PMB treatment endotoxemia.Therefore, need the blank that exploitation one class is reliable for effect, side reaction is less, the simple medicine of application is filled up this field badly.
Thioredoxin (Trx) system, comprise Trx, Trx reductase (TrxR), NADPH and Trx peroxidase (TrxP), be control cellular oxidation/reduction (redox) state and a cell proliferation/existence, in cell the oxidoreduction enzyme system of wide expression.The conserved sequence that contains the cystine composition of two oxidoreduction sensitivities at the catalytic center of Trx: cystine-glycine-proline-cystine, the i.e. active center of Trx.Trx can reducing condition (sulfur hydrogen bond) or the state of oxidation (disulfide bond) exist, but the inverse conversion of this active center sulfur hydrogen bond and disulfide bond participates in intracellular oxidation/reduction process.Therefore, the Trx system is considered to another endogenous antioxidant system except that glutathion (GSH) and superoxide dismutase (SOD) system.Except the activity of oxidation/reductase, Trx can bring into play antiinflammatory action by regulating neutrophilic granulocyte activity and migration.Recombined human source thioredoxin (rhTrx) can be treated some diseases with leukocyte infiltration, comprises the inductive injury of lung of cytokine or cancer therapy drug, of short duration polarization cerebral ischemia, autoimmune myocarditis etc.Yet rhTrx could be used for the treatment of endotoxemia and but rarely have report at home and abroad, does not more know the mechanism of its anti-endotoxin.
Summary of the invention
The objective of the invention is to, provide and confirm that recombined human source thioredoxin (rhTrx) is used to prepare the application of treatment endotoxemia medicine, and confirm that rhTrx is to the curative effect of endotoxemia, especially it is to the effect of the organ injury that takes place in the endotoxemia, and explain its concrete mechanism of action, in the hope of being application small protein Drug therapy infectious disease field, as endotoxemia, provide direct foundation.
The applicant studies show that recombined human source thioredoxin (rhTrx) molecular weight is little, and biologic activity is strong, and the administration afterreaction is rapid, and bioavailability is good, and the good clinical application prospect is arranged.
The applicant finds under study for action:
1, the exogenous animal LPS that gives handles, and can make the active reduction of liver Trx, also liver has been caused obvious impairment simultaneously.
2, aspect machine-processed, the applicant finds first that in the world LPS can combine generation fructosamine (Fructosamin with Trx fast by glycosylation, FA), thereafter, the core polysaccharide of one of LPS component can significantly suppress the Trx activity, and this effect presents dosage and dependent characteristics of time.
3, on the other hand, the exogenous rhTrx that gives treats, and can reverse the damage of LPS to body and liver, but give glycosylated Trx then not this effect.
In a word, above-mentioned studies confirm that, thioredoxin can be by in the glycosylation and endotoxin, thereby brings into play organ protection in endotoxemia, and prompting rhTrx is at endotoxemia, potential therapeutic value when especially organ injury taking place.
Description of drawings
Fig. 1 is that the exogenous animal LPS that gives handles, and can make the active reduction of liver Trx.A. high dose LPS (20 μ g/g) but handle time dependence ground inhibition liver Trx activity; B.LPS handled after 12 hours can suppress liver Trx activity; C.LPS handles and can make liver Trx express increase.Experimental data represents with mean ± standard deviation after learning processing by statistics,
*Expression is compared with the Sham sham operated rats, and promptly there is the significant difference of highly significant p＜0.01.
Fig. 2 gives LPS to handle, and liver is damaged; The exogenous rhTrx (4 μ g/g) that gives treats, and can reverse the damage of LPS to body and liver, but give glycosylation Trx then not this effect.Experimental data represents with mean ± standard deviation after learning processing by statistics,
*Expression is compared with the Sham sham operated rats, p＜0.01;
Show with the LPS group and compare p＜0.01;
##Expression is compared p＜0.01 with LPS/Trx group sham operated rats.
But Fig. 3 gives LPS to handle the blood sugar lowering level; The exogenous rhTrx (4 μ g/g) that gives treats, and can partly recover blood glucose, but give glycosylation Trx then not this effect.Experimental data represents with mean ± standard deviation after learning processing by statistics,
*Expression is compared with the Sham sham operated rats, p＜0.01;
Show with the LPS group and compare that promptly there is significant significant difference p＜0.05.
Fig. 4 gives the LPS processing can increase liver Caspase-3 activity, promptly promotes hepatocellular apoptosis; Exogenously give then not this effect of rhTrx or glycosylation Trx treatment.Experimental data represents with mean ± standard deviation after learning processing by statistics,
*Expression is compared with the Sham sham operated rats, p＜0.01.
Fig. 5 gives LPS to handle, and can make the active reduction of liver Trx, and the active reduction of Trx does not rely on the NOx level or thioredoxin conjugated protein (TXNIP regulates the body redox reaction by interacting with Trx) is expressed.A.LPS handles back blood plasma NOx level; B.LPS handles back liver NOx level; C.LPS handles back liver TXNIP expression.
Fig. 6 gives LPS to handle, and can make the active reduction of Trx in stripped level.A.LPS (37 ℃, 2H LPS=1mg/ml) can suppress the Trx activity; The core polysaccharide of one of B.LPS component can significantly suppress the Trx activity, and the inhibition effect of its another component lipid A is weak relatively; But C.LPS dose dependent ground suppresses the Trx activity; But D.LPS time dependence ground suppresses the Trx activity.
Fig. 7 is that LPS can combine with Trx, and its molecular weight is increased.A. detect the Westernblot result of Trx level, the Trx level after 1:LPS (1mg/ml, 37 ℃ of 2H) handles, 2: matched group (37 ℃ of 2H) Trx level, 3: untreated fish group Trx level; B. detect the Westernblot result with the bonded LPS level of Trx, 1: untreated fish group (Trx), 2:Trx+LPS (1mg/ml, 37 ℃ of 2H) processed group, 3:LPS (1mg/ml) group; What C. this figure and B figure was used is same film, and what added is Trx antibody, and it is stronger to can be observed LPS processing back Trx band.
Fig. 8 is that LPS combines with Trx by glycosylation.A. in stripped level, Trx is by the LPS glycosylation, M: molecular weight standard, P: positive control, C: negative control, 1:hTrx (10 μ g), 2:hTrx (10 μ g)+LPS (5mg/ml), 3:LPS (5mg/ml), 4:hTrx (10 μ g), 5:hTrx (10 μ g)+LPS (10mg/ml), 6:LPS (10mg/ml); B.LPS handles back Trx molecular weight to be increased, and grouping is identical with figure A; C.Trx by the LPS glycosylation generate the initial product fructosamine (Fructosamin, FA).
Fig. 9 is that the glycosylation process testing result of standard shows, handles Trx with monosaccharide, can make it that significant glycosylation takes place.A. three kinds of different monosaccharide make Trx that glycosylation take place; B. back Trx activity is handled in the standard glycosylation; C. standard sugar glycosylation reaction dosage and time dependency ground increase the Trx molecular weight; D. standard sugar glycosylation reaction dosage and time dependency ground suppress the Trx activity.
The present invention is described in further detail for experiment that provides below in conjunction with accompanying drawing and inventor and result thereof.
The specific embodiment
1, set up animal endotoxemia model and research approach:
Handle adult mice with lipopolysaccharide (LPS, 20 μ g/g) lumbar injection, detect the Trx activity of a plurality of organs of body, and observe the hepar damnification situation.
1) handles at LPS and gave Trx or glycosylation Trx (4 μ g/g) in preceding 10 minutes, detect Trx activity and hepar damnification situation.
2) hatch jointly with LPS and recombined human source thioredoxin (hTRX), detect LPS different component, LPS variable concentrations, different time points the active influence of Trx.Wherein, hTRX is cloned and expressed to recombined human source thioredoxin (hTRX) method routinely in escherichia coli, purification rhTRX albumen, and the apoptosis index adopts Caspase-3 activity and FACS method to detect, and mtt assay detects cell proliferation.
3) with the combine situation of Westernblot detection LPS, detect research Trx glycosylation with PAS dyeing and fructosamine with Trx.
4) detect Trx glycosylation and effect with the standard glycosylation process.
2. result of study:
1) the exogenous animal LPS that gives handles, and can make the active reduction of liver Trx, increases but it is expressed; The active reduction of Trx do not rely on nitration, nitrozation reaction or thioredoxin conjugated protein (TXNIP regulates the body redox reaction by interacting with Trx) expression.Simultaneously, LPS has caused obvious impairment to liver.
2) aspect machine-processed, the applicant finds first that in the world LPS can combine with Trx, and its molecular weight is increased; Afterwards, the core polysaccharide of one of LPS component can significantly suppress the Trx activity, and the inhibition effect of its another component lipid A is weak relatively.The applicant is by further discovering, LPS its essence is a kind of glycosylation with combining of Trx, this response speed is very fast, generate initial glycation product------fructosamine (Fructosamin in conjunction with the back, FA), by this reaction, but LPS dose dependent and time dependency ground suppress the Trx activity.
3) be the above-mentioned discovery of checking, the applicant detects the sensitivity of Trx to glycosylation with the glycosylation process of standard, and the result shows, handles Trx with monosaccharide, can make it that significant glycosylation takes place, and this effect also presents dosage and dependent characteristics of time.
4) on the other hand, the exogenous rhTrx that gives treats, and can reverse the damage of LPS to body and liver, but give glycosylated Trx then not this effect.
In sum, the applicant utilizes the scale-model investigation of animal endotoxemia to find, this model mice liver Trx is active to be reduced, and liver damages, its machine-processed and endotoxin by glycosylation and Trx in conjunction with relevant; And give the rhTrx lumbar injection the active reduction of its hepar damnification degree, while Trx activity are recovered to some extent, thereby obviously be alleviated the endotoxemia symptom, potential clinical value is arranged.
Endotoxemia can appear in the multiple disease of multisystem, and incidence rate is also higher relatively: acute hepatitis 37-64%, explosive hepatitis 58-100%, hepatitis C 61.54%, cholelithiasis companion acute obstruction pyogenic infection 85%, burn 85%, septicemia 70%, multiple organ dysfunction syndrome 100%, acute pancreatitis 90%, skin soft-tissue infection 70-81.1%, abdominal cavity infection 72-84%, urinary tract infection 70-80% (nephritis), cancer 70%, pneumonia 100%, on feel 100%.Except that causing lethal infection shock, multiple organ dysfunction syndrome, disseminated inravascular coagulation etc., endotoxemia also can directly or indirectly damage liver usually, causes carbohydrate metabolism disturbance and zymetology, proteometabolic change, and case fatality rate is high.One of main Therapeutic Principle of endotoxemia reduces endotoxic generation and absorption, the applicant finds first in the world that then thioredoxin can pass through in the glycosylation and endotoxin, thereby alleviate hepatocyte injury, necrosis, promote liver function to reply, in endotoxemia, bring into play organ protection; And humanized's thioredoxin molecular weight of recombinating is little, and bioavailability is good; Than animal derived preparation, immunogenicity is little; Lumbar injection, simple and convenient.Therefore, it is having good prospects for application aspect the treatment of endotoxemia.
Use of the present invention to as if endotoxemia takes place, especially also with the patient of symptoms such as hepar damnification.The present invention is simple, and does not have special contraindication, can be extensive use of at large hospital, Branch Clinic etc.
Claims (3)
1. recombined human source thioredoxin is used to prepare the application of treatment endotoxemia medicine.
2. application as claimed in claim 1 is characterized in that, the medicine of described recombined human source thioredoxin preparation is lumbar injection preparation or intravenous formulations.
3. application as claimed in claim 1; it is characterized in that; described medicine is used for the patient of the concurrent important organ depletion of serious endotoxemia, by exogenous administration and glycosylation takes place and in and endotoxin, to alleviate patient's toxemia reaction and protection body important organ.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586202A (en) * | 2012-03-13 | 2012-07-18 | 中国科学院海洋研究所 | Thioredoxin and preparation method and application thereof |
| CN115128283A (en) * | 2022-07-20 | 2022-09-30 | 郑州大学第一附属医院 | Marker for severe acute pancreatitis and application thereof |
-
2008
- 2008-09-28 CN CNA2008101511564A patent/CN101391100A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586202A (en) * | 2012-03-13 | 2012-07-18 | 中国科学院海洋研究所 | Thioredoxin and preparation method and application thereof |
| CN115128283A (en) * | 2022-07-20 | 2022-09-30 | 郑州大学第一附属医院 | Marker for severe acute pancreatitis and application thereof |
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Open date: 20090325 |