CN102657649A - Application of inhibitor SB (Sodium Butyrate) 203580 of p (phosphor) -p38 - Google Patents
Application of inhibitor SB (Sodium Butyrate) 203580 of p (phosphor) -p38 Download PDFInfo
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- CN102657649A CN102657649A CN2012101388814A CN201210138881A CN102657649A CN 102657649 A CN102657649 A CN 102657649A CN 2012101388814 A CN2012101388814 A CN 2012101388814A CN 201210138881 A CN201210138881 A CN 201210138881A CN 102657649 A CN102657649 A CN 102657649A
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Abstract
The invention aims to provide application of an inhibitor SB (Sodium Butyrate) 203580 of p (phosphor)-p38. Activation of phosphor-p38 mediated microglial cells contributes to pain correlated responses of a pain model of BmKI (Biomedical Knowledge Integration); the specific inhibitor SB 203580 of the p-p38 can inhibit the microglial cell and thus relieving the pain correlated responses in the pain model of the BmKI. The invention provides a theoretical basis for pain central sensitization induced by peripheral damages, a study carrier for studying cellular and molecular mechanisms and treatment strategies of nociception and an experimental basis for studying novel drugs for relieving the pain on clinic are provided. The SB 203580 has a prospect of becoming a tool medicine applied in studying correlated pain mechanism of p38 signal path and then has a potential prospect of developing drugs for treating and relieving pathological pain and maintaining corresponding drugs on clinic.
Description
Technical field
The present invention relates to the application of p-p38 inhibitor SB203580.
Technical background
Scorpio (Scorpion Buthus martensi Karsch, the venom that BmK) discharges during insect bite can cause have an intense pain, hydroderma and burning sensation.As the main neurotoxic polypeptide composition of Scorpio venom, BmK I is considered to the principal element that the BmK venom brings out the behavior of inflammation pain.Vola injection BmK I can bring out rat hindleg edema, the spontaneous pain that continues two hours, keeping the quick and injection foot of 7 days bilateral machinery pain, to keep 3 days burning pain quick.
Glial cell be considered at first the central nervous system play the support effect, but the exchanges and dialogues between more and more evidences hint glial cell and the neuron is played an important role in the generating process of the development of dissimilar nervous system disease and pain.Research recently shows the generation of some pain intensive conditions and keeps the activation that relates to non-neuronal cell (microglia and astrocyte) in the spinal cord.Moreover research shows that the inhibitor and the modulator of glial cell can improve some unusual pain sensation sensitization.
Activated spinal cord microglia shows variation (branch's chap), the intragentic change of Expression of born of the same parents, phagocytic function, cell proliferation of cellular morphology etc. in neurogenic and struvite animal pain model.Activated glial cell can discharge multiple inflammation, signaling molecule, and wherein some signaling molecule has been brought into play important effect in the evolution of modulation chronic pain.Yet, how activated also unknownly under pain status be about microglia.
(the mitogen-activated protein kinases of MAPK family; MAPKs) as one type of protein serine/threonine family; Comprise extracellular signal-regulated kinase 1/2 (extracellular signal-regulated kinase1/2; ERK 1/2), the terminal kinases of p38, c-Jun N-and ERK5, be the signaling molecule in g protein coupled receptor downstream, can multiple born of the same parents' external stimulus be converted in various born of the same parents and reply.The activation of MAPK signal path appears in a variety of pain intensive conditions in the spinal cord.P38 is as a member of MAPK family, and multiple extracellular signal is integrated through this signal path and is delivered in kytoplasm and the nuclear.No matter be under inflammation or nerve injury condition, more and more evidences show the development that is modulated at pain of p38 in the spinal cord glial cell and keep in work.In addition, also there are some researches show the generation of p38/MAPK signal path mediation inflammatory cytokine (like IL-1 and TNF).Yet under scorpion stung the pain pathological conditions, what kind of contact p38 signal path and microglial activation had also fuzzy.In conjunction with existing research, can't help guessing whether the activation of p38 in the microglia plays a role in the maincenter sensitization.In order to verify such guess, need to answer following problem: whether the spinal cord glial cell plays a role in the pain corelation behaviour that BmK I brings out; Whether vola injection BmK I can cause the activation of p38, its celluar localization what state; The activation of p38 is that the lasting machinery pain that how to cause being brought out by BmK I is quick quick with burning pain.
The chemical compound that the specific inhibitor SB203580 of p38 is the pyridine imidazoles, as a kind of reliable pharmaceutical research instrument, it is widely used in the analysis and research of p38 signal path effect in various pathology processes (inflammation pain etc.).
Summary of the invention
One of the object of the invention is to change through the time-histories of spinal cord inner cell level and molecular level under the analysis pain pathological conditions; Tectology and behavioural pharmacological experimental result before and after the contrast p-p38 inhibitor SB203580 administration; On the nociception central mechanism basis of research p38 mediation, a kind of p-p38 inhibitor SB203580 is provided the application in preparation treatment scorpion stings the pain medicine.
Two of the object of the invention is to provide the application of p-p38 inhibitor SB203580 in the alleviation scorpion stings the sensitization of inductive spinal cord microglia.
The present invention is at first making up the clinical scorpion of the simulation pain model of stinging.Result of study shows that p38 demonstrates the phosphorylation time-histories synchronous with the microglial activation time-histories in spinal cord; The two mark of fluorescence experimental result shows that p-p38 almost all marks with the OX-42 of labelling microglia altogether, and the activation of having pointed out microglia is that the phosphorylation by p38 mediates.Further the means of utilization p-p38 specific inhibitor SB203580 pharmacological intervention are carried out the follow-up study discovery, and the time-histories of microglia activates and suppressed by significance.Meanwhile, behavioristics's mensuration result shows that the machinery pain that characterizes BmK I pain model modeling index is quick quick by the inhibition of intrathecal injection SB203580 dose dependent with burning pain.In sum, the activation of the microglia of p38 phosphorylation mediation contributes to the pain correlated response of this pain model of BmK I, thereby the specific inhibitor SB203580 of p-p38 can suppress the pain correlated response in the BmK I pain model of alleviating of microglia.
The present invention provides theoretical basis for the pain sensation maincenter sensitization of being brought out by periphery injury, for the cellular elements mechanism and the therapeutic strategy of research nociception provides the research carrier, for lenitive newtype drug research clinically provides experimental basis.
SB203580 has becomes the prospect of a kind of tool drugs in the ache related Mechanism Study of p38 signal path, has development then and treats the potential prospect of alleviating the pathological pain generation and keeping relative medicine clinically.
Description of drawings
Fig. 1 is a microglial activation time-histories in the BmK I pain model rat L4-L5 spinal cord.A-H:L4-L5 spinal cord homonymy; A-h:L4-L5 spinal cord offside.
Fig. 2 is a microglial activation time-histories column statistics in the BmK I pain model rat L4-L5 spinal cord.
Fig. 3 is the phosphorylation time-histories of the interior p38 of rat L4-L5 spinal cord in the BmK I pain model.A-H:L4-L5 spinal cord homonymy; A-h:L4-L5 spinal cord offside.
Fig. 4 is the phosphorylation time-histories column statistics of the interior p38 of rat L4-L5 spinal cord in the BmK I pain model
Fig. 5 is that three types the celluar localization .B figure of p-p38 is astrocyte label GFAP, and E figure is microglia label OX-42, and H figure is neuron label NeuN; C, F and I mark the result altogether and show that p-p38 almost all is expressed in microglia, and other two kinds of cell types are not seen location significantly altogether.
Fig. 6 is the activation time-histories of the interior microglia of L4-L5 spinal cord after the SB203580 pretreatment.A-H:L4-L5 spinal cord homonymy; A-h:L4-L5 spinal cord offside.
Fig. 7 is the activation time-histories column statistics of the interior microglia of L4-L5 spinal cord after the SB203580 pretreatment.
Fig. 8 is the contrast of the activation time-histories left and right sides of microglia inhibition situation in the L4-L5 spinal cord after the SB203580 pretreatment.Wherein A is the contrast of left side microglial activation; B is the contrast of right side microglial activation.
Fig. 9 is the dose-dependent inhibition of SB203580 pharmacological intervention to the pain correlated response.A figure is quick to homonymy machinery pain, and B figure is that offside machinery pain is quick, and C figure is that the homonymy burning pain is quick, and D figure is that the offside burning pain is quick.
The specific embodiment
Embodiment one: the activation time-histories of microglia in the rat L4-L5 spinal cord in the BmK I pain model
Behind the subcutaneous injection BmK I of rat vola, with perfusion behind pentobarbital sodium (60mg/kg) deep anaesthesia.At first pour into the normal saline of 200ml from ascending aorta, change then 4% the paraformaldehyde of being 400ml (the PB buffer dissolving of 0.1M, pH=7.4).The lumbar vertebra spinal cord of getting corresponding time point continued fixing 12 hours with above formalin, used 20% sucrose PB solution tissue dewatering (tissue sinks to the bottom and get final product) afterwards, at last with 30% sucrose PB repetition tissue dewatering.With tissue freezing section (thickness 14um), be affixed on plectrum subsequently for experiment.The single mark of immunity adopts " avidin-biotin-peroxidase (ABC) " method; At first (0.01M pH=7.4) soaks into, and carries out anti-(48 h of hatching then with the PBS buffer in section; 4 ℃), two anti-(2 h of hatching; Room temperature) and ABC mixed liquor (1:200) hatch (2 h, room temperature), use glucose oxidase-diaminobenzidine-nickel (DAB) method to develop the color at last.More than hatching needs between the process to carry out rinsing three times with the PBS buffer, each 10 minutes.After dehydration, transparence and mounting, section can microexamination.Immunocompetence data (positive area or cell number) are analyzed with computer auxiliary software for data processing (Image-Pro Plus etc.).Used one anti-has in this case study on implementation: anti-OX-42 mice resource monoclonal antibody (CD11b, microglia label, 1:50, Santa Cruz company), two used anti-sheep anti mouses two anti-(1:200) for biotinylation.Referring to Fig. 1, and Fig. 2.
Above result shows, in spinal cord, microglia 8 hours after BmK I pain model is set up reach activated peak (comprising increasing and the branched overstriking of cell space of cell number) and fall after rise during at 1 day.Microglia in the above results suggest spinal cord contributes to BmK I pain model.
Embodiment two: the expression time-histories of p-p38 in the L4-L5 spinal cord in the BmK I pain model.
Section preparation and interpretation of result method are seen introduction among the embodiment one.Annotate: positive findings is added up in this case study on implementation is the number of p-p38 positive cell.Used one anti-is two used anti-goat-anti rabbits two anti-(1:200) for biotinylation of anti-p-p38 rabbit polyclonal antibody (1:200, CST company) in this case study on implementation.Referring to Fig. 3 and Fig. 4.
Experimental result shows in the spinal cord of L4-L5 sections, and what the number of p-p38 positive cell was followed BmK I pain model establishes the activation of time-histories property, peaks at 8 hours.The activation time-histories of this and microglia shows high correlation.The activated celluar localization of above results suggest p-p38 possibly is related with microglia.
Embodiment three: the celluar localization of p-p38 in spinal cord
Referring to Fig. 5.Section is prepared with reference to the method in the case study on implementation one.In the two mark tests of fluorescence, at first (0.01M pH=7.4) soaks into, and carries out first kind of one anti-hatch (24 h, 4 ℃) then and carries out second kind of one hatching and two resisting and hatch of resisting afterwards with two anti-hatch (2 h, room temperatures) with the PBS buffer in section.More than hatching needs between the process to carry out rinsing three times with the PBS buffer, each 10 minutes.Section is with observing down at microscope (Leica) behind the glycerol sheet.Immunofluorescence photograph is synthetic with Image J image processing software.Used one anti-has in this case study on implementation: anti-OX-42 mice resource monoclonal antibody (CD11b, microglia label, 1:50; Santa Cruz company), anti-GFAP mice resource monoclonal antibody (GFAP, astrocyte label, 1:300; CST company), anti-NeuN mouse monoclonal antibody (NeuN; The neuron label is 1:500) with anti-p-p38 rabbit polyclonal antibody (1:200, CST company); The sheep anti mouse two anti-(FITC) that two used anti-goat-anti rabbits that engage with indole carbon cyanines for fluorescein two anti-(CY3) and fluorescein engage with isothiocyanic acid, 1:200.
The result shows that the p-p38 positive findings almost completely carries out common mark with the male microglia of OX-42, and does not significantly locate altogether with male neuron of NeuN and the male astrocyte of GFAP.P-p38 almost all is expressed in the microglia in the results suggest BmK I pain model, and promptly the phosphorylation of p38 contributes to this pain model in the microglia.The activation of our microglia of The above results prompting and the phosphorylation of p38, the relation between the two how.
Embodiment 4:SB203580 is to the pharmacological intervention of p-p38 expression
Preparation, result treatment analysis and the used antibody of section are shown in embodiment one.Used SB203580 is the pyridine imidazoles man group inhibitor that one type of specificity suppresses p-p38 in the present case.The function that is widely used in studying p38 as a kind of pharmacological tool.The dmso solution of SB203580 (Sigma company) with 25%, preceding 30 minutes capable intrathecal injection 10 um (2 ug, 10ug and 50ug) of injection BmK I in the vola.Rat is at first used the shallow fiber crops of ether before the intrathecal injection, then with microsyringe with drug injection between L5-L6, inject successfully and swings as a token of with rat instantaneous whipping or serpentine.Referring to Fig. 6, Fig. 7 and Fig. 8.
Above result shows that the microglia time-histories in the BmK I pain model activates and can be suppressed and SB203580 pharmacological intervention and suppressing by the p-p38 specificity.The contrast left and right sides all has significant difference.This results suggest, the activation of microglia depends on p38 phosphorylation in the born of the same parents in the BmK I pain model.The activation that is the dependent microglia of p-p38 plays a role in this pain model.
Embodiment 5:SB203580 is to the pharmacological intervention of pain corelation behaviour in the BmK I pain model
For whether the phosphorylation of verifying p38 in the spinal cord plays a role in the pain sensation sensitization in BmK I pain model, behind intrathecal injection SB203580 pharmacological intervention, the test mechanical pain is quick quick with burning pain.In the concrete experiment, choose after the modeling 4 hours and test with 8 hours behavioristicss.Earlier rat is placed wire gauze (1 cm before the quick test of machinery pain
2Grid) on, and with resin testing cassete (20*20*30 cm
3) rat is isolated adaptive testing environment 30 minutes.The quick test of machinery pain is carried out the 2-3 stimulation of second to the rat biped respectively with 10 standardized von Frey fibers that pressure from 0.6 to 26 restrains, each stimulus intervals 10 seconds.Positive reaction be rat tested sufficient contract fast foot with shrink back, can cause in stimulating for ten times that positive reaction more than five times then writes down the pressure value of this test fiber.Choose the threshold value of minimum numerical value as this time point rat machinery pain.Earlier rat is placed on the glass plate (2 mm) before the burning pain test, and with resin testing cassete (20*20*30 cm
3) rat is isolated adaptive testing environment 30 minutes.The radiant heat light source shines in the rat vola under glass plate; Produce the hot spot of about 3 mm of diameter; Positive reaction be the tested foot of rat contract fast foot with shrink back; Each heat radiation 5 minutes at interval for the first time shines numerical value not as record, and the meansigma methods of getting 5 foot times that contract subsequently is designated as rat in the quick threshold value of this time point burning pain.Annotate: the longest heat radiation must not stimulate greater than 25 seconds.In the mensuration of the previous day rat being carried out pain sensation baseline of BmK I pain model rat being carried out behavioristics's test.Referring to Fig. 9.
Above result shows that after the SB203580 of the different meterings of intrathecal injection carried out pretreatment, BmK I pain mould pain corelation behaviour can significantly be suppressed by dose dependent ground.In sum, the microglial activation that p-p38 mediated contributes to the pain correlated response in the BmK I pain model.Thereby utilization p-p38 specificity inhibition and SB203580 can suppress the activation of microglia in the BmK I pain model alleviates pain correlated response in this model.The present invention provides theoretical basis for the pain sensation maincenter sensitization of being brought out by periphery injury, for the cellular elements mechanism and the therapeutic strategy of research nociception provides the research carrier, for lenitive newtype drug research clinically provides experimental basis.
Claims (2)
1. the application of p-p38 inhibitor SB203580 in preparation treatment scorpion stings the pain medicine.
2.p-p38 the effect of inhibitor SB203580 in the alleviation scorpion stings the sensitization of inductive spinal cord microglia.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109310768A (en) * | 2015-12-29 | 2019-02-05 | 得克萨斯大学体系董事会 | The inhibition of P38 MAPK for treating cancer |
| CN109700808A (en) * | 2019-02-19 | 2019-05-03 | 北京艾瑟尔生物医学研究有限公司 | Application in the drug of SB203580 altitude sickness caused by preparation prevention and/or treatment Acute Exposed Altitude |
| CN116617224A (en) * | 2023-05-04 | 2023-08-22 | 上海交通大学医学院附属瑞金医院 | Application of OPN and p38MAPK signal pathway targeting regulator in preparation of neurodegenerative disease drugs |
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| CN101647999A (en) * | 2009-06-26 | 2010-02-17 | 上海大学 | Application of sodium channel modulator BmK1 in building induction pain model |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101647999A (en) * | 2009-06-26 | 2010-02-17 | 上海大学 | Application of sodium channel modulator BmK1 in building induction pain model |
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| 龚琴等: "鞘内注射p38MAPK抑制剂SB203580对大鼠切口疼痛的影响", 《医学临床研究》, vol. 24, no. 04, 30 April 2007 (2007-04-30), pages 538 - 541 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109310768A (en) * | 2015-12-29 | 2019-02-05 | 得克萨斯大学体系董事会 | The inhibition of P38 MAPK for treating cancer |
| CN109700808A (en) * | 2019-02-19 | 2019-05-03 | 北京艾瑟尔生物医学研究有限公司 | Application in the drug of SB203580 altitude sickness caused by preparation prevention and/or treatment Acute Exposed Altitude |
| CN109700808B (en) * | 2019-02-19 | 2021-07-27 | 中国人民解放军总医院第七医学中心 | Application of SB203580 in preparation of medicine for preventing and/or treating altitude disease caused by acute altitude advance |
| CN116617224A (en) * | 2023-05-04 | 2023-08-22 | 上海交通大学医学院附属瑞金医院 | Application of OPN and p38MAPK signal pathway targeting regulator in preparation of neurodegenerative disease drugs |
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Application publication date: 20120912 |