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IL-1β for Protease-activated Receptor-4Expre-Ssed in Primary Sensory Neurons

Author: ZhangRui
Tutor: WangZhaoJin
School: Taishan Medical College
Course: Human Anatomy and Embryology
Keywords: pain IL-1β discogenic Dorsal root ganglion Protease activated receptor4 Sensory neurons Protein Kinase C
CLC: R338
Type: Master's thesis
Year: 2013
Downloads: 9
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ObjectiveIn cultured rat primary sensory neurons and dorsal root ganglia (dorsal root ganglion,DRG) for the research model to research interleukin-1β (Interleukin-1β, IL-1β) onprotease-activated receptor-4(PAR4) in DRG primary sensory neurons in rats. Observedthe intracellular mechanisms of PAR4receptors, so as to further clarify the PAR4involvedin the regulation of peripheral pain and provide experimental basis for its treatment.Methods1. Primary sensory neurons separation and trainingMale Wistar rats, weight of about200g, After abdominal cavity anesthesia by0.4%pentobarbital sodium with2ml, acute separation DRG, take bilateral DRG, digestion bypancreatic enzyme, percussion, made into cell suspension, kind of in six holes trainingboard,training primary sensory neurons.2. Incubation of IL-1βThe experiment was divided into four groups: control group (IL-1β,0ng/ml), IL-1β(25ng/ml) group, IL-1β (50ng/ml) group, IL-1β (100ng/ml) group. Training DRG neuronsfor24hours, different concentrations of IL-1β (IL-1β final concentrations of0ng/ml,25ng/ml,50ng/ml,100ng/ml) incubation、cultured rat DRG sensory neurons for fourhours. Trizol cracking extraction of the training of the sensory neurons DRG total RNA,with RT-PCR detection the expression of PAR4gene and the influence of PAR4expression induced by IL-1β3. Male Wistar rats, weight of about200g, After abdominal cavity anesthesia by0.4%pentobarbital sodium with2ml, injection IL-1β (0.5ml,50ng/ml) at paraspinal muscles、paraspinal subcutaneous and L5/6Intervertebral disc, after4hours, acute separation L4,L5, L6ganglia, fixed with4%paraformaldehyde, frozen sections8μm,immunofluorescence method combines with confocal laser scanning microscopy to observe the expression of PAR4in rat DRG sensory neurons.4. PKC agonists(phorbol12-myristate13-acetate,PMA325nm), PKC inhibitor(chelerythrine chloride,5nm),, pre-incubated for1hour, and then apply IL-1β (50ng/ml)acting on the DRG sensory neurons cultured for4hours, Trizol cracking extraction of thetraining of the sensory neurons DRG total RNA, with RT-PCR detection expression ofPAR4gene and PAR4agonists and inhibitor on IL-1βregulation PAR4gene expression.5. PKC agonists(0.5ml,20ng/mL) or inhibitors(0.5ml,20ng/mL) were injectedto the rats paraspinal muscles, paravertebral subcutaneous and L5/6discfor1hour, andthen injected to the paravertebral muscle IL-1β (0.5ml,50ng/ml), paravertebralsubcutaneous and L5/6disc for4hours. The experiment was divided into four groups:control group(IL-1β,0ng/ml;PKC agonists and inhibitors0ng/ml), IL-1β+PKC agonistgroup, IL-1β group, IL-1β+PKC inhibitor group. Acutely isolated L4, L5, L6ganglion,fixed with4%paraformaldehyde, frozen sections8μm, immunofluorescence methodcombines with confocal laser scanning microscopy to observe the expression of PAR4DRG sensory neurons in the rat, analysis the expression of PAR4applications after of PKCagonists and inhibitors of induced by interleukin.Results1. the distribution of PAR4in DRG neuronsThe results of Immunofluorescence histochemistry showed that the expression ofPAR4in not applied IL-1β rat dorsal root ganglion neurons, positive cells are mostly smalland medium sized cells, some large neurons also showed PAR4expression.27.31±0.49%positive neurons were PAR4, were round or ovoid, the positive markers mainly appears inthe cell membrane, cytoplasm, nucleus did not see tags.2. The change of DRG neurons expression after injection of IL-1βApplication of IL-1β intervention4hours, acute separation L4, L5, L6dorsal rootganglion neurons, using immunohistochemical methods, we can see a lot of sensoryneurons expressing PAR4positive.63.39±0.54%positive neurons were PAR4positive(positive cell counts were used drug injection disc group sliced observation and analysis),PAR4positive cells were significantly increased than normal dorsal root ganglion cells, thedate was statistically significan compared with the control group(P <0.05). Some cellsPAR4enhanced fluorescence, positive cells are mostly small and medium sized cells, largeneurons also showed PAR4positive, were round or ovoid, the positive markers were seenin the cell membrane, cytoplasm and nucleus did not see tags.. In some neurons fibers ofganglion also PAR4positive. 3. The influence of IL-1βon PAR4mRNA in primary sensory neurons expressionThe results of RT-PCR showed that: different concentrations of IL-1β makePAR4mRNA expression was significantly increased, and with the increase of theconcentration of IL-1β, its expression was increased, there was a positive correlationcoefficient increases, respectively was1.04times,1.18times,1.24times compared withthe control group. Different concentrations of IL-1β group compared with the controlgroup were statistically significant (P<0.05), with different concentrations of IL-1β group,there was statistically significant (P <0.05).4. The regulatory role on the expression of PAR4mRNA induced with IL-1βbyPKC agonists and inhibitorsThe result of RT-PCR showed that: IL-1β and PKC agonist group can enhance theexpression of PAR4mRNA expression, and the expression of PKC agonist group wasobviously increased than IL-1β group, PKC agonist+IL-1β group is1.11times higherthan pure IL-1β group, there was statistically significant (P <0.05); while PKC inhibitorcan reduce the expression PAR4mRNA with the inhibitor of IL-1β,the expression wasobviously lower compared with pure IL-1β group, its0.88times, there was statisticallysignificant (P <0.05).5. PKC on the changes of PAR4Immunoreactive cells after injection of IL-1βRespectively injected PKC agonist (0.5ml,20ng/mL)and inhibitors (0.5ml,20ng/mL)in subcutaneous、paraspinal muscles and intervertebral disc of rat,1hourlater, then injecting IL-1β (0.5ml,50ng/mL) for4hours. The result of Immunofluorescenceshowed that:PAR4positive cells was significantly changed in L4-L6DRG, PKC agonistscan increased the PAR4expression positive cells, the PAR4positive cells in PKC agonist+IL-1βgroup is71.21±0.57%(this group cell counts were used drug injection disc groupsliced observation and analysis), there was statistically significant(P<0.05), Which is stillexpress in small and medium sized cells,some large neurons express; PKC inhibitors canreduced the PAR4expression positive cells, the PAR4positive cells in PKC inhibitors+IL-1βgroup is21.93±0.33%(this group cell counts were used drug injection disc groupsliced observation and analysis), there was statistically significan(tP<0.05), Some positivecells fluorescent display brightness diminished.Conclusion1. The result of Immunofluorescence showed that some small primary sensoryneurons in rats DRG expression PAR42.In the rats were injected with IL-1βin paraspinal muscles, subcutaneous and intervertebral disc,4hours later, PAR4positive neurons were increased than those withoutIL-1β injection group, mainly some small and medium sized cells, some large cells alsoexist expression3. Different concentrations of IL-1β acts on vitro rat DRG primary sensory neurons,the expression of PAR4was obviously increased, with the increased of IL-1β,theexpression of PAR4mRNA was increased, were positively related. Explanation thatIL-1βcan regulate the expression of PAR4mRNA.4. PKC agonists can enhance the regulation of IL-1β on PAR4mRNA, on the contrary,PKC inhibitors can reduce regulation of IL-1β on PAR4mRNA; in rats,paraspinal muscles,subcutaneous and intervertebral disc injection PKC agonists or inhibitors, can increase ordecrease the PAR4positive neurons in DRG.Demonstrated that PKC signaling involved inthe regulatory role on the expression of PAR4induced with IL-1βin DRG primary sensoryneurons.

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