CN105078975A - Application of Necrostatin-1 as neutrophile granulocyte apoptosis accelerant - Google Patents
Application of Necrostatin-1 as neutrophile granulocyte apoptosis accelerant Download PDFInfo
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Abstract
The invention discloses an application of Necrostatin-1 as a neutrophile granulocyte apoptosis accelerant. Study on compound 5-(1H-benzpyrole-3-ylmethyl)-3-methyl-2-thioketone-4thioketone conducted by the inventor shows that except for the known function of inhibiting programmed necrosis, 5-(1H-benzpyrole-3-ylmethyl)-3-methyl-2-thioketone-4thioketone also has the effects of promoting the apoptosis of the neutrophile granulocyte, and treating neutrophile granulocyte apoptosis abnormity diseases by promoting the apoptosis of the neutrophile granulocyte, especially the acute lung injury caused by LPS. Molecules of 5-(1H-benzpyrole-3-ylmethyl)-3-methyl-2-thioketone-4thioketone are small, can be easily accepted by the mechanism, also can specifically act on neutrophile granulocyte to promote the apoptosis of the neutrophile granulocyte, and have no influences on the apoptosis of other cells.
Description
Technical field
The present invention relates to the new opplication of compound, particularly Necrostatin-1 is as the application of neutrophil apoptosis promoter.
Background technology
Neutrophilic granulocyte (polymorphonuclearneutrophil, PMN) be a kind of cell that in hemocyte, the life-span is the shortest, for terminally differentiated cells, it breaks up in red bone marrow, ripe, be released into subsequently in blood circulation, in peripheral blood, mean half-life is 4-10 hour, although its life span is ofer short duration, but in inflammatory reaction, but play very important effect, it is the first line of defence of immunity of organism system of defense on the one hand, if on the other hand it in inflammation stove by excessive activation, postpone to remove or secondary necrosis, react and damaging surrounding tissue by discharging oxygen-derived free radicals and proteolytic enzyme and other harmful content thing exacerbate inflammation, inflammation is even made to delay and chronicity, thus cause various diseases to produce as rheumatoid arthritis, pyemia, systemic inflammatory response syndrome, acute coronary syndrome, the diseases such as acute lung injury.
Thus, moderately and at the right moment remove PMN can control inflammation generation, develop and lapse to.The effective way that PMN apoptosis is safety, quick, non-inflammatory removes PMN.The PMN of apoptosis not only retting conditions, the miopragia such as to engulf, and more easily by phagocyte identification and removing, the phagocyte that intake of apoptosis PMN also can be secreted transforming growth factor-β (TGF-β) and prostacyclin PGE-2 etc. and be pressed down scorching medium.
Current research, for promoting the medicine of neutral grain apoptosis less, mainly contains following several.
1, cell cycle protein dependent kinase (CDK) inhibitor.Research shows human neutrophil express cell cyclin-dependent kinases CDK1, CDK2 and CDK5, cyclin enzyme inhibitor R-roscovitine, NG7, Hymenialdisine are by suppressing the activity of CDK, promote neutrophil apoptosis, can be used for the disease for the treatment of neutrophil apoptosis exception as acute lung injury, pleuritis, arthritis etc.
2, lipoxin (lipoxins, LXs), disappear element (resolvins, Rvs).Research shows that they can be stoped by a kind of non-inflammatory form and reduce the infiltration of neutrophilic granulocyte at inflammation part, promotes that macrophage is to apoptosis centriole cell (PMNs) and microorganism panning and removing.
3, the inhibitor of NF-κ B: NF-κ B is the important transcription factor of regulation and control PMN apoptosis, and it is relevant with the activation of NF-κ B that inflammatory mediator causes PMN Apoptosis delay; Research shows that multiple NF-kB inhibitor (gliotoxin, SN-50, PDTC) can promote the apoptosis of neutrophilic granulocyte.
4, death receptor Fas agonist: death receptor Fas agonist can by improving the expression of Fas, and suppress the effects such as the expression of anti-apoptotic proteins Bcl-2 and Mcl-1 albumen to promote the apoptosis of neutrophilic granulocyte.
5, IL-10: research shows that IL-10 can strengthen the removing of chmice acute injury of lung lungs neutrophilic granulocyte, and can the granulocytic Apoptosis inhibitor effect of antagonism LPS centering.
But these medicines above have obvious defect in promotion and application, cell cycle protein dependent kinase (CDK) inhibitor, death receptor Fas agonist not only can induce the apoptosis of neutrophilic granulocyte, also can cause other Normocellular apoptosis, how while promoting neutrophil apoptosis and don't the apoptosis affecting other cells be difficult to solve.The inhibitor of same lipoxin, the element that disappears, NF-κ B, the target spot of IL-10 also often participate in multiple links of inflammation, be difficult to ensure do not cause other side effect while promotion neutrophil apoptosis, and the price of synthesis higher be difficult to be applied to clinical.Therefore develop a kind of can the medicine of specific induction neutrophil apoptosis significant.
5-(1H-indol-3-yl methyl)-3-methyl-2-thioketone-4-imidazolidinone (Necrostatin-1) is that find recently a kind of can the medicine of the procedural necrosis of specific suppression, molecular weight is 259.3D, belongs to alkaloids substance.Research previously finds it for apoptosis and autophagy without any inhibitory action, does not have direct antioxidation, for Normocellular form, breeds, to stick and ATP, calcium and oxygen free radicals etc. all do not make significant difference.
Summary of the invention
The object of the present invention is to provide Necrostatin-1 as the application of neutrophil apoptosis promoter.
Modern medicine study shows, neutrophilic granulocyte but plays very important effect in inflammatory reaction, it is the first line of defence of immunity of organism system of defense on the one hand, if it is removed or secondary necrosis by excessive activation, delay in inflammation stove on the other hand, can exacerbate inflammation reaction and damaging surrounding tissue and cause various diseases, moderately and at the right moment removing neutrophilic granulocyte can control inflammation generation, develop and lapse to.The effective way that neutrophil apoptosis is safety, quick, non-inflammatory removes PMN.Based on above-mentioned thought, inventor studies compound 5-(1H-indol-3-yl methyl)-3-methyl-2-thioketone-4-imidazolidinone, find that 5-(1H-indol-3-yl methyl)-3-methyl-2-thioketone-4-imidazolidinone is except the effect of the procedural necrosis of known suppression, also there is the effect promoting neutrophil apoptosis, and the acute lung injury caused by promoting the apoptosis of neutrophilic granulocyte to treat neutrophil apoptosis abnormal diseases, particularly LPS.
The molecule of 5-(1H-indol-3-yl methyl)-3-methyl-2-thioketone-4-imidazolidinone is little, not only easily absorbed by body, and specificly can act on neutrophilic granulocyte, promote the apoptosis of neutrophilic granulocyte, and on the apoptosis of other cells without impact.
Accompanying drawing explanation
Fig. 1 is each group of neutrophil apoptosis rate, and the Annexin-V positive is apoptotic cell;
Fig. 2 is that each group of neutrophil apoptosis rate compares, (n>=3,
#p≤0.05,
##p≤0.01 is compared with blank group);
Fig. 3 shows with the apoptosis characteristic of the neutrophilic granulocyte of Nec-1 Dual culture, and result has shown the performance of obvious neutrophil apoptosis characteristic, namely neutrophilic granulocyte by leaflet core to occurring and concentration phenomena;
Fig. 4 is the neutrophil apoptosis rate testing result after neutrophilic granulocyte 100 μMs of Nec-1 Dual culture 3h, 6h, 12h, 18h,
##compared with blank group, P<0.05;
Fig. 5 is the PBMC apoptosis rate after PBMC and 20 μM, 50 μMs, 100 μMs Nec-1 Dual culture 20h, compared with blank group, and P > 0.05;
Fig. 6 is the human neutrophil (5 × 10 after being separated
6/ ml) detect the apoptosis situation of each group of neutrophilic granulocyte respectively with after Nec-1 (100 μMs), LPS (100ng/ml), GM-CSF (20ng/ml), dbcAMP (0.1mM) Dual culture 20h;
Fig. 7 is the impact of Nec-1 centering granulocyte Bax, Mcl-1, cleavedcaspas-3 protein expression;
Fig. 8: a: total cell number in each group mice bronchoalveolar lavage fluid; B: neutrophilic granulocyte number in each group mice bronchoalveolar lavage fluid; C: total protein concentration in each group mice bronchoalveolar lavage fluid; D: the vigor of MPO in each group mouse lung tissue; E: each group mouse lung tissue dry-wet weight ratios;
##p≤0.01 compared with blank group,
*p≤0.01 is compared with LPS group;
Fig. 9: a-c is cytokine situation of change in each group of bronchoalveolar lavage fluid, and d-e is cytokine change in each group of lung tissue,
##p≤0.01 compared with blank group,
*p≤0.01 is compared with LPS group;
Figure 10: each group lungs pathological change situation;
Figure 11: the a-b apoptosis rate respectively organizing neutrophilic granulocyte in mice bronchoalveolar lavage fluid;
##p≤0.01 compared with blank group,
*p≤0.01 is compared with LPS group;
Figure 12: immunofluorescence sends out the apoptosis situation detecting each group of mouse lung tissue neutrophilic granulocyte, adopts MPO antibody labeling neutrophilic granulocyte, Cleavedcaspase3 antibody labeling apoptotic cell, and the two positive of MPO and Cleavedcaspase3 is apoptosis neutrophilic granulocyte.
Detailed description of the invention
For convenience of description, below by compound 5-(1H-indol-3-yl methyl)-3-methyl-2-thioketone-4-imidazolidinone (Necrostatin-1) referred to as Nec-1.
Below in conjunction with experiment, further illustrate technical scheme of the present invention.
Experiment one, Nec-1 promote human neutrophil apoptosis
1. experiment material
1.1 neutrophilic granulocyte
The Third Affiliated Hospital of Southern Medical University recruits healthy volunteer 30, informs volunteer's experiment content, and signature Informed Consent Form, ethics scheme is audited by Ethics Committee of Nanfang Medical Univ and passed through.Extract volunteer's peripheral blood 10ml, Separation of Neutral granulocyte.
Test medicine
Nec-1 adds DMSO and dissolves, and be mixed with the solution that concentration is 10mg/ml, after 0.22mm filtering with microporous membrane sterilization treatment ,-20 DEG C of preservations are stand-by.
Main agents
1.4 key instrument
2. method
2.1 neutrophilic granulocytes are separated
EDTA anticoagulant blood-collecting pipe collector peripheral blood 10ml; Respectively 5ml peripheral blood is added in 5ml human lymphocyte separating medium and (carry out in 15ml centrifuge tube); Attention action softly slowly adds; 500g is centrifugal, 30-35 minute, room temperature; Collected after centrifugation low strap neutrophilic granulocyte, divide two-layer, ground floor is PBMC, and the second layer is neutrophilic granulocyte; Being diluted by RP1640 pure water is that 50%RP1640 is used for resuspended neutrophilic granulocyte, recovers cytoactive (5-10 minute); Centrifugal 10 minutes of 400g, removes supernatant, collects neutrophilic granulocyte, is resuspended in RP1640, and CD15 magnetic bead sorting obtains human neutrophil.
The impact of centering granulocyte apoptosis
Human neutrophil (5 × 10 after separation
6/ ml) with Nec-1 (20 μMs, 50 μMs, 100 μMs) the Dual culture 20h of variable concentrations after detect the apoptosis situation of each group of neutrophilic granulocyte; Human neutrophil (5 × 10 after separation
6/ ml) detect neutrophil apoptosis situation with Nec-1 (100 μMs) Dual culture respectively at 3h, 6h, 12h and 18h; Apoptosis detects and adopts Annexin-V/PI apoptosis detection kit flow cytomery neutrophil apoptosis.
The impact of Cytokines in Peripheral Blood Mononuclear apoptosis
Adopt human peripheral blood single nucleus cell (PBMC) separating medium to be separated and obtain PERIPHERAL BLOOD MONONUCLEAR CELL, the PERIPHERAL BLOOD MONONUCLEAR CELL (5 × 10 after separation
6/ ml) with Nec-1 (20 μMs, 50 μMs, 100 μMs) the Dual culture 20h of variable concentrations after detect the apoptosis situation of each group of cell.
Can the effect of the multiple neutrophil apoptosis inhibitive factor of antagonism
In acute with chronic inflammatory process; the apoptosis of neutrophilic granulocyte usually can be blocked as LPS, GM-CSF, dbcAMP (dibutyryl-cAMP) by the product of microorganism or some risk factor; and the abnormal apoptosis of neutrophilic granulocyte usually can cause lasting and don't controllable inflammation and tissue damage; therefore we detect Nec-1 whether can these factors of antagonism, make the apoptosis of neutrophilic granulocyte recover normal.Human neutrophil (5 × 10 after separation
6/ ml) detect the apoptosis situation of each group of neutrophilic granulocyte respectively with after Nec-1 (100 μMs), LPS (100ng/ml), GM-CSF (20ng/ml), dbcAMP (0.1mM) Dual culture 20h.
The impact of centering granulocyte Bax, Mcl-1, cleavedcaspas-3 protein expression
Apoptosis and Bax, Mcl-1, cleavedcaspas-3 albumen of neutrophilic granulocyte are closely related, research shows that the high expressed of Bax, cleavedcaspas-3 obviously promotes the apoptosis of neutrophilic granulocyte, and Mcl-1 albumen has suppression neutrophil apoptosis, promote the effect of neutrophilic granulocyte survival.GM-CSF is the cytokine of usually high expressed in a kind of inflammation, and is a kind of neutrophil apoptosis inhibitor of classics, can be used as the positive control medicine that neutral grain apoptosis is limited.Human neutrophil (5 × 10 after separation
6/ ml) respectively with Nec-1 (100 μMs), GM-CSF (20ng/ml) Dual culture 20h after, cell is cleaned 3 times by phosphate buffered solution, add the cell pyrolysis liquid of pre-cooling, under several with rifle piping and druming, lysate is fully contacted with cell, takes out lysate, centrifugal 10 minutes of 12000g, get supernatant, for detecting each group of neutrophilic granulocyte Bax, Mcl-1, cleavedcaspas-3 protein expression situation.
Statistical method applied statistics software SPSS13.0 carries out statistical disposition analysis.
Result
3.1Nec-1 induces neutrophil apoptosis
Result of study shows: compared with matched group neutrophil apoptosis rate (63.15 ± 5.15%), 70.44 ± 4.43% are respectively with the neutrophil apoptosis rate after 20 μMs, 50 μMs, 100 μMs Nec-1 Dual culture 20h, 75.19 ± 4.28%, 84.78 ± 8.36% (P<0.05), in table 1, Fig. 1-2.Nec-1 induces neutrophil apoptosis to have concentration dependent, increases with drug level, and the apoptosis of induction neutrophilic granulocyte is more obvious.And compared with matched group, occurred that obvious neutrophil apoptosis characteristic shows with the neutrophilic granulocyte of Nec-1 Dual culture, namely neutrophilic granulocyte is by leaflet core to appearance and concentration phenomena, sees Fig. 3.And Nec-1 induces neutrophil apoptosis to have time dependence, and the effect of the drug-induced neutrophil apoptosis of the increase along with the time is more obvious, in table 2, Fig. 4.
Table 1 variable concentrations Nec-1 centering granulocyte apoptosis rate affects
Note: the neutrophil apoptosis rate after neutrophilic granulocyte and 20 μMs, 50 μMs, 100 μMs Nec-1 Dual culture 20h detects,
▲compared with blank group, P<0.05.
Table 2 more than time point Nec-1 centering granulocyte apoptosis rate affects
Note: the neutrophil apoptosis rate after neutrophilic granulocyte 100 μMs of Nec-1 Dual culture 3h, 6h, 12h, 18h detects,
▲compared with blank group, P<0.05.
Cytokines in Peripheral Blood Mononuclear apoptosis is without impact
Result of study shows: induce neutrophil apoptosis different from Nec-1, after Nec-1 and peripheral blood mononuclear cells Dual culture 20h, the each dosage group of Nec-1 20 μMs, 50 μMs, 100 μMs all can not induce the apoptosis (P<0.05) of PBMC, in table 3, Fig. 5.Nec-1 can not induce the apoptosis of PBMC, and prompting Nec-1 may specific effect and neutrophilic granulocyte, the apoptosis of induction neutrophilic granulocyte, and on the apoptosis of other cells without impact.
Table 3 variable concentrations Nec-1 affects PBMC apoptosis rate
PBMC apoptosis rate after note: PBMC and 20 μM, 50 μMs, 100 μMs Nec-1 Dual culture 20h detects,
▲compared with blank group, P > 0.05.
Can the effect of the multiple neutrophil apoptosis inhibitive factor of antagonism
Human neutrophil (5 × 10 after separation
6/ ml) detect the apoptosis situation of each group of neutrophilic granulocyte respectively with after Nec-1 (100 μMs), LPS (100ng/ml), GM-CSF (20ng/ml), dbcAMP (0.1mM) Dual culture 20h.Testing result finds, the apoptosis rate that LPS (100ng/ml), GM-CSF (20ng/ml), dbcAMP (0.1mM) respectively organize neutrophilic granulocyte is starkly lower than matched group neutrophilic granulocyte (P<0.05), and three groups add with Nec-1 (100 μMs) afterwards the apoptosis rate of neutrophilic granulocyte obviously rise (P<0.05), prompting Nec-1 can the effect of the multiple neutrophil apoptosis inhibitive factor of antagonism, promotes the apoptosis of neutrophilic granulocyte.See (table 4-6, Fig. 6 a-d).
Table 4Nec-1 antagonism LPS suppresses neutrophil apoptosis effect
Note: each group of neutral grain apoptosis rate after neutrophilic granulocyte and LPS, Nec-1 Dual culture 20h,
▲compared with LPS, P < 0.05.
Table 5Nec-1 antagonism GM-CSF suppresses neutrophil apoptosis effect
Note: each group of neutral grain apoptosis rate after neutrophilic granulocyte and GM-CSF, Nec-1 Dual culture 20h,
▲compared with LPS, P < 0.05.
Table 6Nec-1 antagonism dbcAMP suppresses neutrophil apoptosis effect
Note: each group of neutral grain apoptosis rate after neutrophilic granulocyte and dbcAMP, Nec-1 Dual culture 20h,
▲compared with LPS, P < 0.05.
The impact of centering granulocyte Bax, Mcl-1, cleavedcaspas-3 protein expression
Result of study finds: the human neutrophil (5 × 10 after separation
6/ ml) respectively with Nec-1 (100 μMs), GM-CSF (20ng/ml) Dual culture 20h after, detect each group of neutrophilic granulocyte Bax, Mcl-1, cleavedcaspas-3 protein expression situation.Find that the expression of Bax, cleavedcaspas-3 of GM-CSF group neutrophilic granulocyte is starkly lower than normal group, Mcl-1 albumen apparently higher than normal group, and adds the expression with promoting Bax, cleavedcaspas-3 after Nec-1, suppresses the expression of Mcl-1 albumen.Prompting Nec-1 is by promoting Bax, suppressing the path of the expression of Mcl-1 to promote the apoptosis of neutrophilic granulocyte.See Fig. 7.
Inventor studies and finds that micromolecular compound Nec-1 can induce the apoptosis of human neutrophil, and with concentration and the apoptosis-induced effect of the increase of time more obvious; In addition, Nec-1 can the effect of the multiple neutrophil apoptosis inhibitive factor of antagonism, makes the apoptosis of neutrophilic granulocyte recover normal.In acute with chronic inflammatory process; the apoptosis of neutrophilic granulocyte usually can be blocked product as microorganism or some risk factor (LPS, GM-CSF, dbcAMP) by some factors, and the abnormal apoptosis of neutrophilic granulocyte usually can cause lasting and don't controllable inflammation and tissue damage.This apoptosis-promoting effect prompting of Nec-1, Nec-1 can be used for the treatment of the limited relevant disease of multiple neutrophil apoptosis.
Further research shows that Nec-1 is by promoting Bax albumen, suppressing the path of the expression of Mcl-1 albumen to promote the apoptosis of neutrophilic granulocyte.
Experiment two, Nec-1 are to the protective effect of mice with acute lung injury
Acute lung injury (ALI) is the clinical syndrome increasing to main manifestations with pneumonia and permeability occurred after infection, wound.When body is hit, Different types of etiopathogenises (to comprise in lung/lung outside, infection/non-infection) a large amount of neutrophilic granulocyte (PMN) can be caused directly or indirectly to enter pulmonary parenchyma rapidly, and play an important role in the ALI occurred thereafter, except release oxygen-derived free radicals and protein resolvase, PMN also discharges some inflammatory mediators as IL-1, TNF-α, IL-8 etc., can attract further, activate PMN, and extending PMN life cycle, exacerbate inflammation is reacted.Promote that neutrophil apoptosis is the important channel that in inflammatory reaction stove, PMN removes.
Experiment material
1.1 laboratory animals: healthy male BALB/C mice, body weight 18-22 gram, purchased from Nanfang Medical Univ's Experimental Animal Center, tests in advance and conforms for 1 week, freely drink water and search for food, and circulates every day to dark with 12h illumination/12h.Animal feeding room also keeps temperature 25 ± 1 DEG C under controlled conditions, and humidity is 40-80%.All operations and experiment stream observe " management of laboratory animal regulations ".
Main agents
1.3 key instrument
2. method
The configuration of 2.1 main agents:
(1) Nec-1 medicinal liquid: Nec-1 is suspended in DMSO with the concentration of 10mg/ml before use, and afterwards according to corresponding dosage solvent dilution pneumoretroperitoneum drug administration by injection, dosage is 5mg/kg;
(2) LPS solution: be configured to 1mg/ml storing solution with normal saline, in-20 DEG C of preservations after subpackage, faces the used time with normal saline dilution to 100 μ g/ml.
Grouping and ALI modeling
BALB/C mice is divided into 3 groups at random, is respectively blank group, LPS model group, LPS+Nec-1 group, often organizes 12 (6 for bronchoalveolar lavage fluid group Indexs measure, 6 for each Indexs measure of lung tissue).After mice fasting one evening, LPS+Nec-1 group gives Nec-1 by 5mg/kg dosage gavage, and normal saline group and LPS group then give the solvent of same volume.Administration after 6 hours after, mice is anaesthetized through the Nembutal sodium solution (60mg/kg) of lumbar injection 10%.Naive mice collunarium gives 50 μ l normal saline, and all the other are organized equal collunarium and give 50 μ lLPS solution (100 μ g/ml).Then normal water, feed.Modeling is after 4 hours, and mouse peritoneal injection overdose of sodium pentobarbital is anaesthetized and sacrificed by exsanguination.
The collection of bronchoalveolar lavage fluid (BALF) and cell counting
After sacrificed by exsanguination mice, cut off skin of neck, be separated trachea, penetrate tracheal strips with venous indwelling meter sleeve pipe along centripetal direction, and fix with silk thread, connect 1ml syringe bronchoalveolar lavage 3 times, each PBS injecting 0.5ml pre-cooling.Collect irrigating solution (BALF) about 1.4ml, get wherein 0.1ml for total cell count; Remaining at 500g, centrifugal 10min under 4 DEG C of conditions, after getting supernatant subpackage ,-80 DEG C save backup; Get the dry rear Wright-Giemsa dyeing of cell precipitation smear ， Liao again, at oily Microscopic observation 200 cells, to PMNs classified counting, finally draw the content of PMNs in BALF.
Bronchoalveolar lavage fluid total protein detects
In bronchoalveolar lavage fluid, total protein concentration detects and adopts Bradford method protein quantification to detect, and detects by Bradford protein quantification test kit (green skies company) description method.
The collection of lung tissue sample
Mice, after putting to death, is opened thoracic cavity, lung tissue is separated rapidly, rinsing slightly in pre-cooling PBS, and use filter paper wiped clean.Getting left lung is soaked in the paraformaldehyde of 4%, for pathology detection and Immunofluorescence test; The residue lobe of the lung is used for the detection of the detection of lung tissue dry-wet weight ratios and lung tissue MPO (myeloperoxidase (MPO)) vigor.
In lung tissue, MPO detects
Ground with after liquid nitrogen freezing after precise lung tissue weight, be prepared into the tissue homogenate of 5%, Nanjing is adopted to build up company MPO testing cassete, utilize MPO to have to make the ability of hydrogen-peroxide reduction, when hydrogen donor one neighbour connects hydrogen supply amine hydrogen supply, 460nm place is by MPO content in colorimetric determination lung tissue.
Each group of lung tissue dry-wet weight ratios detects
Lung tissue is taken out immediately after sacrifice, with the blood of PBS liquid rinsing lung tissue, surface is blotted with filter paper after cleaning, remove lung tissue segment precise and be recorded as " weight in wet base ", subsequently lung tissue is put into 80 DEG C of baking ovens and toast 24h, claim weight is " dry weight ", ratio between two is dry-wet weight ratios.
The detection of cytokine in bronchoalveolar lavage fluid and lung tissue
Adopt ELISA method to detect the content of cytokine TNF-α, IL-6, MIP-2 in bronchoalveolar lavage fluid and lung tissue homogenate, detection method is with reference to description.
Lung tissue disease's Neo-Confucianism detects
After lung tissue sample is fixing in poly formic acid, draw materials, dehydration, section after stone Wrong embeds, makes lung stone Wrong tissue slice, the pathological change of light Microscopic observation lung tissue routinely.
In bronchoalveolar lavage fluid and lung tissue, neutrophil apoptosis rate detects
Adopt neutrophilic granulocyte in CD11b and Ly6G/Ly6C labelling mice bronchoalveolar lavage fluid, adopt Annexin-V/PI apoptosis detection kit flow cytomery neutrophil apoptosis; In lung tissue, neutrophil apoptosis rate detects and adopts immunofluorescence method to detect, and adopts MPO antibody labeling neutrophilic granulocyte, Cleavedcaspase3 antibody labeling apoptotic cell, and the two positive of MPO and Cleavedcaspase3 is apoptosis neutrophilic granulocyte.
Statistical method: applied statistics software SPSS13.0 carries out statistical disposition analysis.
Result
3.1Nec-1 the infiltration of inflammatory cell in minimizing injury of lung
Mice modeling detects inflammatory cell infiltration number in bronchoalveolar lavage fluid after 4 hours.Result shows, total cell number in naive mice bronchoalveolar lavage fluid and neutrophilic granulocyte number all less, and in LPS model group, total cell number and neutrophilic granulocyte number are all apparently higher than blank group (P<0.05), the total cell number of LPS+Nec-1 group and neutrophilic granulocyte number are starkly lower than LPS group (P<0.05), the inflammatory cell infiltration that Nec-1 inhibits LPS to induce.In table 7, Fig. 8 a, b.
Table 7Nec-1 reduces the infiltration of injury of lung inflammatory cell
Note: mice modeling detects inflammatory cell infiltration number in each group of bronchoalveolar lavage fluid after 4 hours,
*compared with normal group, P<0.05,
▲compared with LPS group, P<0.05.
Suppress total protein content in bronchoalveolar lavage fluid:
The concentration of total protein in mice bronchoalveolar lavage fluid is detected by Bradford method, result shows: the blank group (P<0.05) of LPS group lungs total protein content, Nec-1+LPS group total protein content is starkly lower than LPS group (P<0.05).Illustrate that pulmonary's albumen that Nec-1 can suppress LPS to induce oozes out.The results are shown in Table 8 and Fig. 8 c.
Table 8Nec-1 suppresses total protein content in bronchoalveolar lavage fluid
Note: mice modeling detects total protein content in each group of bronchoalveolar lavage fluid after 4 hours,
*compared with normal group, P<0.05,
▲compared with LPS group, P<0.05.
The MPO vigor suppressing LPS to cause increases
It is how many in the distribution of tissue that MPO vigor has reacted neutrophilic granulocyte, detects Nec-1 to the impact of MPO vigor in lung tissue.Result shows, and LPS group lungs MPO vigor is higher than blank group (P<0.05), and Nec-1+LPS group MPO vigor is starkly lower than LPS group (P<0.05).Illustrate that the MPO vigor that Nec-1 can suppress LPS to induce increases.Result is as shown in table 9 and Fig. 8 d.
The MPO vigor that table 9Nec-1 suppresses LPS to cause increases
Note: mice modeling detects MPO vigor in each group of lung tissue after 4 hours,
*compared with normal group, P<0.05,
▲compared with LPS group, P<0.05.
Suppress induced by LPS lung tissue water content, permeability raise
Lung tissue dry-wet weight ratios embodies the permeability situation of lungs, and lungs weight in wet base and dry weight are than larger, and illustrate that lungs water content is higher, the permeability of lungs is higher, and lung tissue inflammation damnification is more serious.Result shows: LPS group lungs do weight in wet base apparently higher than blank group (P<0.05), Nec-1+LPS group dry-wet weight ratios is starkly lower than LPS group (P<0.05), and result shows lung tissue water content that Nec-1 can suppress to be induced by LPS, permeability raises.In table 10 and Fig. 8 e.
The lung tissue water content that table 10Nec-1 suppresses to be induced by LPS, permeability rising
Note: mice modeling detects dry-wet weight ratios in each group of lung tissue after 4 hours,
*compared with normal group, P<0.05,
▲compared with LPS group, P<0.05.
The release of the inflammatory factor in the bronchoalveolar lavage fluid of being induced by LPS and lung tissue is suppressed to increase
Result shows: TNF-a, IL-6, MIP-2 content in naive mice bronchoalveolar lavage fluid and lung tissue is all little, the release of the cytokine in LPS model group is then significantly higher than blank group (P<0.05), the release of cytokines that Nec-1 can suppress LPS to induce, LPS+Nec-1 group cytokine content is starkly lower than LPS group (P<0.05).The release of the inflammatory factor that result shows in the bronchoalveolar lavage fluid that Nec-1 can suppress to be induced by LPS and lung tissue increases.The results are shown in Table 11-and Fig. 9.
Table 11Nec-1 suppresses inflammatory factor release in the bronchoalveolar lavage fluid of being induced by LPS
Note: mice modeling detects Inflammatory Factors Contents in each group of bronchoalveolar lavage fluid after 4 hours,
*compared with normal group, P<0.05,
▲compared with LPS group, P<0.05.
Table 12Nec-1 suppresses inflammatory factor release in the lung tissue of being induced by LPS
Note: mice modeling detects Inflammatory Factors Contents in each group of lung tissue after 4 hours,
*compared with normal group, P<0.05,
▲compared with LPS group, P<0.05.
Improve the lung pathologies change of LPS induction
After sacrifice, get the lobe of the lung fix, cut into slices after carry out HE dyeing.Observation by light microscope finds, control group mice lung tissue alveolar structure is complete, and alveolar wall is without edema, and pulmonary parenchyma is without obvious inflammatory cell infiltration; LPS model group mice alveolar wall then obvious edema thickens, and interstitial lung neutrophil infiltration a large amount of as seen, has significantly hemorrhage, edema, demonstrates serious pneumonia and pulmonary edema phenomenon.And Nec-1 significantly reduces the pulmonary lesion of LPS induction.The results are shown in Figure 10.
Promote the apoptosis of mice with acute lung injury neutrophilic granulocyte
In bronchoalveolar lavage fluid, neutrophil apoptosis rate detects and finds, the apoptosis rate of LPS group neutrophilic granulocyte is starkly lower than blank group neutrophilic granulocyte (P<0.05), and the apoptosis rate of LPS+Nec-1 group neutrophilic granulocyte is apparently higher than LPS group (P<0.05), prompting Nec-1 can the effect of antagonism LPS, promotes the apoptosis of neutrophilic granulocyte.In lung tissue, Immunofluorescence test finds that the apoptosis rate of LPS group neutrophilic granulocyte is lower than blank group equally, and the apoptosis rate of LPS+Nec-1 group neutrophilic granulocyte is apparently higher than LPS group.Result shows that Nec-1 promotes the apoptosis of mice with acute lung injury neutrophilic granulocyte.In table 13, Figure 11-12.
Table 13Nec-1 promotes the apoptosis of mice with acute lung injury neutrophilic granulocyte.
Note: in bronchoalveolar lavage fluid, neutrophil apoptosis rate detects,
*compared with normal group, P<0.05,
▲compared with LPS group, P<0.05.
Acute lung injury (ALI) is the clinical syndrome increasing to main manifestations with pneumonia and permeability occurred after infection, wound.When body is hit, Different types of etiopathogenises (to comprise in lung/lung outside, infection/non-infection) a large amount of neutrophilic granulocyte (PMN) can be caused directly or indirectly to enter pulmonary parenchyma rapidly, and play an important role in the ALI occurred thereafter, except release oxygen-derived free radicals and protein resolvase, PMN also discharges some inflammatory mediators as IL-1, TNF-α, IL-8 etc., can attract further, activate PMN, and extending PMN life cycle, exacerbate inflammation is reacted.Apoptosis is the main path that in inflammatory reaction stove, PMN removes.ALI clinical manifestation is the incorrigible hypoxemia of intractable, and its case fatality rate is very high, is the key factor causing Critical illness death.Because relevant medication effect is not good, finding safer effective class of medications is the modern clinical huge challenge faced.
Inventor's early-stage Study finds that micromolecular compound Nec-1 can induce the apoptosis of human neutrophil, and with concentration and the apoptosis-induced effect of the increase of time more obvious; Nec-1 can the effect of the multiple neutrophil apoptosis inhibitive factor of antagonism, makes the apoptosis of neutrophilic granulocyte recover normal.In acute with chronic inflammatory process; the apoptosis of neutrophilic granulocyte usually can be blocked product as microorganism or some risk factor (LPS, GM-CSF, dbcAMP) by some factors, and the abnormal apoptosis of neutrophilic granulocyte usually can cause lasting and don't controllable inflammation and tissue damage.This apoptosis-promoting effect prompting of Nec-1, Nec-1 can be used for the treatment of the limited relevant disease of multiple neutrophil apoptosis.
The acute lung injury that this experiment adopts Nec-1 treatment LPS to cause, experiment display, the mouse lung inflammatory cell infiltration that Nec-1 induces LPS, albumen ooze out and inflammatory cytokine release has obvious inhibitory action, and Nec-1 also significantly can reduce the vigor of MPO in lung tissue.These protective effects obtain further confirmation in lung pathologies section.Research also finds that Nec-1 is relevant with promoting the apoptosis of neutrophilic granulocyte to the therapeutical effect of the acute lung injury that LPS induces.These results of study are that the potential clinical practice of the abnormal relevant disease of Nec-1 treatment neutrophil apoptosis and Nec-1 antiinflammatory action provides experimental basis.
Claims (3)
1.Necrostatin-1 is as the application of neutrophil apoptosis promoter.
The application of 2.Necrostatin-1 in preparation treatment neutrophil apoptosis abnormal diseases.
3. application according to claim 2, is characterized in that: neutrophil apoptosis abnormal diseases is selected from acute lung injury, rheumatoid arthritis, pyemia, systemic inflammatory response syndrome, acute coronary syndrome.
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| CN108642005A (en) * | 2018-05-22 | 2018-10-12 | 广州迪澳医疗科技有限公司 | A kind of lymphocyte separation medium containing bridging agent |
| CN111808797A (en) * | 2020-07-28 | 2020-10-23 | 湖南赛诺生物科技股份有限公司 | Application of Necrostatin-1 and preparation for promoting maturation of islet cells of newborn pigs |
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| CN101674826A (en) * | 2005-12-20 | 2010-03-17 | 哈佛大学校长及研究员协会 | Chemical compound, screening and Therapeutic Method |
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| CN108642005A (en) * | 2018-05-22 | 2018-10-12 | 广州迪澳医疗科技有限公司 | A kind of lymphocyte separation medium containing bridging agent |
| CN108642005B (en) * | 2018-05-22 | 2021-07-27 | 广州迪澳医疗科技有限公司 | Lymphocyte separation liquid containing connecting agent |
| CN111808797A (en) * | 2020-07-28 | 2020-10-23 | 湖南赛诺生物科技股份有限公司 | Application of Necrostatin-1 and preparation for promoting maturation of islet cells of newborn pigs |
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