Expression Of PAR4in Primary Sensory Neurons And Its Effects On TRPV1

ObjectiveProtease activate receptors4(PAR4) for G protein coupling receptors, participate in multiple pathological physiology process. Recently found PAR4out weeks inflammation andpain in the regulation plays an important role.Train the rats dorsal root ganglion（DRG） as the research model, Research the expression of the PAR4in the primary sensory neurons Observe and confirm physiological andmolecular mechanism of PAR4involved in the visceral pain signal transduction and regulation, and what kind of contact between with different damage sexual stimulation and differrent ways and the relationship with TRPV1receptors, so as to further clarify PAR4participate in visceral pain in the process of signal transduction adjust action and peripheral mechanism.Methods1. Through the acute isolation and culture of the rats DRG sensory neurons usingimmune fluorescence observation the expression of PAR4and TRPV1in the sensoryneurons in rats DRG.2. Trizol cracking extraction of the training of the sensory neurons DRG total RNA,with RT-PCR detection PAR4gene expression and PAR4agonists, PAR4and TRPV1geneexp-ression of influence, and PKA signal channel to express and PAR4PAR4mediatedTRPV1gene expression regulatory role.3. RIPA cell lysate fluid extraction of the training of the DRG neurons total protein,PAR4agonists, PAR4detection of protein expression and the influence of TRPV1channelsPKA signal and to express and PAR4PAR4influence of protein expression TRPV1regulatory role.Results1. DRG separation and training Young male Wistar rats, weight of about100g, acute separation DRG, quickly takebilateral DRG digestion, percussion,made into cell suspension liquid,kind of in six holestraining board observation different period the number of cells,the size and the length ofthe bumps.Vaccination in six holes training board neural cell cultures4hours,the cartbefore the horse a microscope.Visible part of the nerve cells can beat ached to the wall,adherent nerve cells were round or oval,cell body surrounded by a halo,there is no protruding.Neurons in the nucleus in cells in the central or the side of the cell body, andprominent nucleoli.24hours after inoculation,the majority of the cells adherent, adherentcells to grow small protrusions, in addation to a single nerve cell spread,and often see se-veral of many nerve cells together, sent to the surrounding the dendrites culture3-4days ofnerve cells processes gradually extended thickening,you can see the synapses of nervecells intertwined to form a sparse net work structure.with prolonged incubation time,thetrunk and branches of the nerve cell processes are significantly prolonged and thickeningof the nerve cell processes to form the network structure becomes more dense, the nervecell body is gradually increasing. with prolonged incubation time,the number of cells willbe reduced.2. The expression of PAR4in DRG neuronsImmunofluorescence organization chemical results show that:the in vitro culture ratDRG of nerve cells， visible a large number of sensory neurons in the DRG displayedPAR4expression. Positive cells for some more small and medium-sized neurons,11-28microns in diameter, Also visible some large neurons express PAR4. Training after24hours, PAR4positive neurons grew protuberant appear, cultivate3to4days DRG neuronscan appear PAR4positive axons Positive markers mainly appear in cell membranes, cellplasma, the nucleus has not seen the mark.3. PAR4and TRPV1coexistence in DRG neuronsThe in vitro culture of rats of DRG nerve cells, cultivate1day TRPV1positive cellsand PAR4positive cells similar, Note the amount of the medium and small TRPV1positivebody of the neuron, round or oval in shape. TRPV1positive thing distribution and cellmembrane, cytoplasm, nuclear has not seen the mark. Two signs with fluorescent confocallaser results show that: the in vitro culture DRG neurons are visible PAR4/TRPV1doublemarking. The count of30hole on the cover glass DRG cells showed that there were65.3%-3.2%(162/248) PAR4positive neurons express TRPV1,Also, TRPV1positiveneurons PAR4expression. cell count showed that there were95±2.4%（162/171）%. 4. AYPGKF-NH2on PAR4expression in primary sensory neurons and the effect ofintracellular mechanisms（1）PAR4agonist on PAR4in primary sensory neurons expressing effect PAR4agonists,AYPGKF-NH2(10μm) to join the training of the nerve cells function1hour,Western blot showed that: PAR4agonists promote PAR4protein expression was increasedin plus PAR4agonist group and blank control group was statistically significant (P<0.05).RT-PCR results showed add PAR4agonists group and control group PAR4mRNAexpression blank compared increased1.2times PAR4agonist group and blank controlgroup was statistically signigicant (P<0.05).（2）PKA agonists and inhibitors on PAR4express influence will PKA agonists(forskolin1μM) and inbibitors (3μ M PHKI14-22) incubation of cultured neural cells, therole of30min, then add PAR4agonists were incubated for1hours, Western blot resultsshow: joins PKA agonists of PAR4protein expression decreased, join PKA inhibitors ofPAR4protein expression increased. significant(P<0.05). RT-PCR resules show that: joinPKA agonists of PAR4mRNA expression decreased,Add PKA inhibitors group and controlgroup PAR4mRNA expression blank compared increased1.17times Agonists andantagonists group and blank control group was statistically significant(P<0.05).5. PAR4on primary sensory neurons effects on TRPV1expression and intracellularmechanismsThe influence of PAR4activation to TRPV1expression,PAR4agonistsAYPGKF-NH2(10μm) to join the training of the nerve cells function1hour, Wsetern blotshowed that: DRG neurons TRPV1protein expression quantity and compared withcontrols blank, fell by58%(n=3), plus PAR4agonist group and blank control group wasstatistically significant(P<0.05).RT-PCR results showed that: PAR4agonists, makeTRPV1mRNA expression and reduction of the blank compared with controls, fell by20%(n=3) plus PAR4agonist group ang blank control group was statisticallysignificant(P<0.05).Conclusion1.The in vitro culture rat DRG primary sensory neurons expression PAR4.Many PAR4positive neurons and TRPV12.AYPGKF-NH2may raise PAR4protein and mRNA DRG primary sensory neuronsin the expression. PKA agonists, reduce AYPGKF-NH2PAR4protein and to express the regulation effects of mRNA, and PKA inhibitors increase AYPGKF-NH2PAR4proteinand mRNA expression of the regulation and function.3.PAR4activated TRPV1protein and mRNA cocoa cut in DRG primary sensory neurons expression. PKA agonists, increase AYPGKF-NH2and to TRPV1mRNA the regulation effects of expression, and PKA inhibitors reduce AYPGKF-NH2TRPV1protein and mRNA expression of the regulation and function.