| Estrogen(E)is a steroid hormone,depending on the three kinds of Estrogen Receptor,including ER?,ER?,(G protein-coupled receptor)GPR30 through a variety of signaling pathways involved in the regulation of many important life activities,including reproduction.The study found that E playing an important role in suppressing the inflammatory reaction and neurodegenerative,promoting neural axon regeneration,affecting the release of neurotransmitters,protecting nerve function.In addition,epidemiological studies have found significant gender differences in pain.It was found that ER and GPR30 of colon in the human and mouse could inhibit the number of colonic contraction and pain induced behaviors,suggesting that E might play a regulatory role in the production or transmission of pain.As a pain receptor,P2X3 is also an ion channel receptor for ATP ligand gated.When combining with ATP,the cationic channels be opened,and the permeability of Ca2+ is the largest.Pain of Somato-visceral is mainly caused by small and medium-sized neurons in the Dorsal Root Ganglion(DRG).When the DRG neurons are stimulated,the changes of intracellular [Ca2+]i level affects the pain threshold directly.It was found that estrogen increased intracellular [Ca2+]i in vitro cultured hippocampal neurons through m ER,and activate the ERK pathway,show that E can adjust downstream signaling pathways by Ca2+ channel in nerve cells.So,how does the E influence ATP induced the changes of [Ca2+]i in sensory neurons? In this experiment the immunofluorescence double-standard technique was used to to study the distribution of ER,P2X3 and the co-expression of the two receptors in the DRG neurons of adult female rabbits and primary culture from newborn rabbits;The Ca2+ imaging technology of laser confocal microscopy was used to monitor the synergistic effect of different concentrations of 17?-E2,ATP on the DRG neurons.It was found that,(1)In adult female rabbits,three types of ER were distributed mainly in some DRG neurons,and an obvious "heterogeneity" was observed in the number,size type,degree of response and distribution in positive neurons;P2X3 were expressed in some small and medium sized neurons mainly,and also a certain "heterogeneous" on degree of expression;The co-expression of ER??GPR30 with P2X3 were found in some small and medium neurons mainly,and ER? with P2X3 were found in some medium neurons mainly.(2)In primary culture from newborn rabbits,three types of ER were distributed mainly in some DRG neurons,and an obvious "heterogeneity" was observed in the number,size type,degree of response and distribution in positive neurons;P2X3 were expressed in some small and medium sized neurons mainly,and also a certain "heterogeneous" on degree of expression;The co-expression of ER and P2X3 were found in some small and medium neurons mainly.And that were approximated similar to those in adult rabbits.(3)The changes of Ca2+ fluorescence intensity of DRG neurons in primary cultured induced by 17?-E2 of different concentrations were monitored by Ca2+ imaging technology of laser confocal microscope.DRG neurons were divided into 3 types,positive sensitivity neurons(19.28±0.698)%,negative sensitivity neurons(3.54±0.828)% and insensitive neurons(77.17±1.484)%,respectively.Positive sensitivity neurons were divided into strongly positive sensitivity neurons(0.195±0.118)and weakly positive sensitivity neurons(0.032±0.003)depending on the intensity of Ca2+ fluorescence changes.Interestingly,with the increase of 17?-E2,the proportion of strongly positive sensitivity neurons decreased,but no difference was detected in the proportion of total positive sensitivity neurons at each group.It indicated that with the increasing of 17?-E2,the increasement of [Ca2+]iwas inhibited in strongly positive sensitivity neurons.(4)The changes of Ca2+ fluorescence intensity of DRG neurons in primary cultured induced by ATP of different concentrations were monitored by Ca2+ imaging technology of laser confocal microscope,DRG neurons were divided into 4 types,including strongly positive sensitivity neurons,weakly positive sensitivity neurons,negative sensitivity neurons,insensitive neurons,and the proportion of each reaction type was dependent on concentration obviously.When the ATP was increased to 1000?M,the proportion of the each type neurons tended to be stable,strongly positive sensitivity(60.29%),weakly positive sensitivity(25.00%),and insensitive(14.71%),respectively.(5)The effect of 17?-E2 to the change of Ca2+ fluorescence intensity of DRG neurons in primary cultured induced by ATP(1000 ?M)were monitored by Ca2+ imaging technology of laser confocal microscope,Different concentrations 17?-E2 were both inhibitory to the Ca2+ in some strongly positive sensitivity neurons induced by 1000 ?M ATP,and it's related to concentration,the inhibition was greatest at 10 n M;The proportion of insensitive neurons increased slightly when applying 17?-E2(1n M,1000 n M),and decreased slightly when applying 17?-E2(10 n M,100 n M);The proportion of weakly positive sensitivity neurons did not change much when applying 17?-E2(1n M,1000 n M),but increased when applying 17?-E2(10 n M,100 n M).The inhibition mode of high concentration and low concentration is the same and different from that of middle concentration.The above results show that,first,the "heterogeneity" of ER and P2X3 in the distribution and co-expression of DRG suggests that estrogen may play a different role in different pain neurons.Second,The influence of different concentrations of 17?-E2 on the fluorescence intensity of Ca2+ indicates that Ca2+ pathway is one of the molecular mechanisms by which estrogen affects the activity of some sensory neurons.Among them,estrogen can play an inhibitive effect though inhibit the Ca2+ pathway effect of strongly positive sensitivity neurons.The third,Estrogen can reduce [Ca2+]i though inhibit Ca2+ channel effect in strong positive sensitivity nociceptive neurons.However,there is a difference in the mechanism of Ca2+ pathway effect in weakly positive sensitivity and insensitive neurons.In conclusion,17?-E2 has different effects on ATP-induced pain neurons [Ca2+]i,and has a degree of concentration dependence.It is suggested that the Ca2+ pathway is one of the molecular basis of the complex regulation mechanism of estrogen on pain. |
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