The Role And Mechanisms Of ATP And P2X3 Receptor In Endometriosis Pain

Endometriosis is defined by the presence of functional endometrial tissue outside the uterine cavity.It's a common disease in reproductive age women,and seriously affects patients' life quality with the main clinical symptoms of infertility and pain.Endometriosis pain includes dysmenorrhea,dyspareunia,dysuria,defecation pain and chronic pelvic pain.Studies have shown that endometriosis is an estrogen-dependent chronic inflammatory disease.However,anti-inflammatory drugs are often not effective.Though estrogen-reduction treatments such as GnRH-a are effective in the treatment of endometriosis pain,the pain symptoms relapse after drug withdrawl due to the severe side-effects of low estrogen level.Surgical removal of endometriotic lesions is also not effective in eliminating pain symptoms in women with endometriosis.Therefore,it is necessary and urgent to explore the mechanism of endometriosis pain as well as new treatment methods.In recent years,studies have found that abnormal distribution of sensory nerve fibers and elevated expression of specific nociceptor receptors(such as transient receptor potential vanilloid 1,TPRV1)in endometriotic lesions are positively correlated with pain symptoms in patients with endometriosis.In women with deep infiltrating endometriosis(DIE),the distribution of sensory nerve fibers in endometriotic lesions is more abundant compared with other types of endometriosis.In terms of the severity of pelvic pain symptoms in women with endometriosis,it seems that endometriosis pain may be associated with infiltration of nerve fibers in endometriotic lesions.Pro-inflammatory factors(such as interleukin(IL)-1?,tumor necrosis factor-a)and sensory nerve terminal release(such as substance P)in the endometriotic lesions can stimulate sensory nerve fibers to cause nerve impulses,thereby sensitizing peripheral nerves,through the dorsal root ganglia(DRG)afferent into the dorsal horn of the spinal cord,resulting in pain symptoms of the patients.Therefore,endometriosis pain can be considered as a kind of inflammatory neuropathic pain.Adenosine triphosphate(ATP)is the most important energy source in human body.However,ATP and its nucleotide analogues are also extracellular signaling molecules.In physiological and pathological conditions,ATP and nucleotide analogues can be secreted by different cells,acting on their receptors that are widely distributed in various organs and tissues,producing the corresponding biological effects.According to the structure and function of ATP receptors,they can be divided into PI family(A1,A2A,A2B,A3),P2X family(P2X1-7)and P2Y family(P2Y1,P2Y2,P2Y4,P2Y6,P2Y11-14).Among them,P2X3 receptor(purinergic receptor P2X ligand-gated ion channel 3)is specifically distributed in epithelial cells and sensory neurons.Elevated expression and activation of P2X3 receptors often lead to hyperalgesia in animal models of chronic pain,suggesting than P2X3 receptor may play a role in nerve sensitization and signal transmission of neuropathic pain.However,whether and how P2X3 receptor participates in endometriosis pain is remains unclear.In this study,luciferin-luciferase assay showed that ATP content in endometriotic lesions were significantly higher than in eutopic endometrial tissues from endometriosis women and control endometrium from non-endometriosis women.Then,RT-qPCR,Western Blot and immunohistochemistry analysis showed that the expression levels of P2X3 receptor in ectopic endometrial lesions and eutopic endometrial tissues of endometriosis women were significantly higher than that in non-endometriosis endometrium,and were positively correlated with the severity of pain in patients with endometriosis.Purification and culture of ectopic stromal cells in vitro revealed that IL-1? can stimulate ATP release in endometriotic stromal cells,and both IL-1? and ATP intervention can activate the MAPK/ERK/CREB pathway,increasing mRNA and protein expression levels of P2X3 receptor.Afterwards,a rat model of endometriosis was established.It was found that the release of ATP and the expression levels of P2X3 receptor in ectopic endometrial lesions and DRG tissues in endometriosis rats were significantly increased,and both positively correlated with the severity of hyperalgesia induced by endometriosis.In vitro,ADP intervention led to activation of the activating transcription factor 3(ATF3)/AP-1 pathway and increased expression levels of the P2X3 receptor in a rat DRG cell line F11,which could be inhibited by ATF3 siRNA.Finally,we designed a chitosan oligosaccharide stearic acid(CSOSA)/liposomes(LPs)delivery system to package SP600125,a specific inhibitor of ATF3/AP-1 pathway.CSOSA/LPs/SP600125 effectively inhibited ADP-mediated up-regulation of P2X3 receptor in DRG cells and alleviated endometriosis-induced hyperalgesia as well as up-regulated expression levels of P2X3 receptor in endometriotic lesions and DRG tissues in a rat model of endometriosis.These results demonstrated that ATP and P2X3 receptor participated in endometriosis-associated hyperalgesia,and targeting P2X3 receptor may be used as a novel therapeutic way in treatment of endometriosis pain.Part I:ATP contents and P2X3 receptor expression levels in ectopic endometrial lesions and their relationship with pain symptomsObjective:To investigate the difference of ATP content and P2X3 receptors expression levels in ectopic endometrial lesions,eutopic endometrial tissues of endometriosis women and non-endometriosis endometrium,and to analyze their correlation with the severity of pain symptoms exhibited by patients.Methods:According to the enrollment criteria,different kinds of fresh endometrial tissues were collected;ATP content in tissues was tested by luciferin-luciferase assay.The expression levels of P2X3 receptor were detected by RT-qPCR,Western Blot and immunohistochemistry,respectively;Co-expression of CGRP and P2X3 receptors in ectopic endometrial lesions was observed by immunofluorescence double staining.Results:1.ATP content in ectopic endometrial lesions was significantly higher than that in eutopic endometrium from endometriosis women and non-endometriosis women.Moreover,ATP content in ectopic endometrial lesions of patients suffered with endometriosis pain was higher.2.P2X3 receptor was mainly expressed in glandular epithelial and superficial epithelial cells of ectopic lesions,and could also be expressed in endometrial stromal cells.The mRNA and protein expression levels of P2X3 receptor in ectopic endometrial lesions and eutopic endometrium of endometriosis women were significantly higher than eutopic endometrium of non-endometriosis women,and were positively correlated with the severity of endometriosis pain.3.Co-expression of CGRP and P2X3 receptors was observed in ectopic endometrial lesions.Conclusions:1.Elevated ATP content and P2X3 receptor expression levels may be involved in the pathology of endometriosis pain.2.The identification of P2X3 receptor on CGRP-stained sensory nerve fibers in ectopic endometrial lesions indicates the peripheral sensitization of women with endometriosis.Part ?:ATP and IL-1? upregulate P2X3 receptor expression levels in endometriotic stromal cells via MAPK/ERK/CREB pathwayObjective:To investigate the difference of ATP content and P2X3 receptor expression levels in ectopic endometrioitc stromal cells,eutopic endometrial stromal cells from endometriosis women and eutopic endometrial stromal cells from non-endometriosis women,and to explore the corresponding regulation mechanism of ATP content and P2X3 receptor expression in endometriotic stromal cells.Methods:Different types of fresh endometrial tissues were collected and endometrial stromal cells were purified and cultured in vitro;ATP content in different types of endometrial stromal cells was detected using luciferin-luciferase assay.The mRNA and protein expression levels of P2X3 receptor in endometrial stromal cells were detected by RT-qPCR and Western Blot,respectively;The ectopic endometrial stromal cells were stimulated with IL-1?,and the concentration of release ATP was detected;The activation of MAPK/ERK/CREB pathway as well as the expression levels of P2X3 receptor in ectopic endometrial stromal cells were tested after IL-1? or ATP intervention,with or without the existence of specific inhibitor PD98059.Results:1.The ATP content,mRNA and protein expression levels of P2X3 receptor in primary ectopic endometrial stromal cells were all significantly higher than those of eutopic endometrial stromal cells from endometriosis and non-endometriosis women.2.IL-1? intervention promoted ATP release from ectopic endometrial stromal cells.3.IL-1? and ATP intervention induced up-regulation of P2X3 receptor expression levels in ectopic endometrial stromal cells via activation of MAPK/ERK/CREB pathway,which were blocked by pre-treatment of specific inhibitor,PD98059.Conclusions:1.IL-1? and ATP can up-regulate P2X3 receptor expression levels in ectopic endometrial stromal cells via activation of MAPK/ERK/CREB pathway in vitro.2.IL-1? may participate in pathology of endometriosis pain by stimulating ATP release from ectopic endometrial stromal cells and up-regulating the expression levels of P2X3 receptor in ectopic endometrial stromal cells.Part ?:The relationship between ATP content/P2X3 receptors expression levels in ectopic endometrial lesions/DRG tissues and endometriosis-induced hyperalgesia in rats.Objective:To explore the relationship between ATP content/P2X3 receptor expression levels in ectopic endometrial lesions/DRG tissues and the severity of hyperalgesia in rats induced by endometriosis.Methods:A rat model of endometriosis was established by autologous transplantation,and the related sensitizations to heat and mechanical stimulation were detected weekly before and after surgery;Luciferin-luciferase assay,RT-qPCR,Western Blot And immunohistochemistry analysis were used to detect the secretion of ATP and the expression levels of P2X3 receptor in different types of endometrial tissues and DRG tissues of rats;Correlations of ATP secretion/P2X3 receptor expression levels and the severity of hyperalgesia in rats were analyzed.Results:1.Endometriosis rats developed thermal and mechanical hyperalgesia at 3 weeks after surgery compared with controls,and continued until six weeks after surgery;The severities of thermal and mechanical hyperalgesia were positively correlated in endometriosis rats.2.P2X3 receptor was mainly expressed on epithelial cells in endometrial tissues and small-medium sized neurons in DRG tissues;Secretion of ATP,as well as P2X3 receptor expression levels in ectopic endometrial lesions and DRG tissues from endometriosis rats were all significantly higher than those from controls,and were positively correlated to the severity of hyperalgesia.Conclusions:1.The up-regulated secretion of ATP and P2X3 receptor expression levels in the ectopic endometrial lesions from endometriosis rats are consistent with those from endometriosis patients.2.The up-regulated secretion of ATP and P2X3 receptor expression levels in DRG tissues from endometriosis rats indicate that ATP and P2X3 receptor are involved in the peripheral sensitization of endometriosis pain.Part IV:ADP up-regulates P2X3 receptor expression levels in DRG cells via ATF3/AP-1 pathwayObjective:To explore the regulation mechanism and the corresponding molecular signal transduction pathway of ATP and other nucleotide analogues on P2X3 receptor in DRG cells in vitro.Methods:The DRG cell line was cultured in vitro;The mRNA and protein expression levels of P2X3,ATF3,FOS and JUN were detected after ADP intervention,and their intracellular localization were observed by cellular immunofluorescence analysis;ATF3 overexpression plasmid and ATF3 specific siRNA were used measure the role of ATF3 in ADP-induced P2X3 receptor up-regulation;The sequence of P2X3 receptor promoter was synthesized and inserted into luciferase plasmid to test the activity of the P2X3 promoter induced by ADP intervention.Finally,chromatin immunoprecipitation(CHIP)was used to detect the binding of ATF3 on promoter of P2X3 receptor.Results:1.P2X3 receptor was expressed on the cell membrane,and ATP/ADP intervention increased the mRNA and protein expression levels of P2X3 receptor in DRG cells.2.ADP intervention activated the ATF3/AP-1 signaling pathway in DRG cells and up-regulated the expression of ATF3,FOS and JUN in the nucleus.3.Silencing ATF3 using ATF siRNA decreased ATF3 expression levels in nucleus and significantly inhibited ADP-induced up-regulation of P2X3 receptor on the membrane.4.ADP intervention increased the promoter activity of the P2X3 receptor.5.ADP intervention increased ATF3 binding on P2X3 receptor promoter.Conclusions:1.ADP intervention can up-regulate the expression levels of the P2X3 receptor in DRG cells.2.ADP up-regulates the P2X3 receptor by activating the ATF3/AP-1 signaling pathway to increase ATF3 binding on P2X3 promoter.FOS and JUN in the nucleus.3.Silencing ATF3 using ATF siRNA decreasedPart ?:CSOSA/LPs/SP600125 effectively inhibits ADP-mediated P2X3 receptor up-regulation in vitro and endometriosis-induced hyperalgesia in vivoObjective:To package SP600125 with chitosan oligosaccharide stearic acid(CSOSA)/liposomes(LPs),and to explore the effects of CSOSA/LPs/SP600125 on ADP-induced P2X3 receptor up-regulation in vitro and endometriosis-induced hyperalgesia as well as P2X3 receptor up-regulation in rats.Methods:The ATF3/AP-1 pathway specific inhibitor SP600125 was coated by nano-chitosan liposome CSOSA/LPs,and particle size,Zeta potential,and released rate were measured;The uptake of CSOSA/LPs/SP600125 and its effects on ADP-mediated activation of ATF3/AP-1 pathway and up-regulation of P2X3 receptor were detected in DRG cells in vitro;The distribution of CSOSA/LPs/SP600125 in rats and the inhibition effect of CSOSA/LPs/SP600125 on endometriosis-induced P2X3 receptor up-regulation and hyperalgesia were tested in vivo.Results:1.CSOSA/LPs could effectively encapsulate SP600125 and slow its release rate.2.CSOSA/LPs/SP600125 could be taken up by the DRG cells,and effectively inhibited the activation of the ATF3/AP-1 pathway and the up-regulation of the P2X3 receptor caused by ADP intervention.3.CSOSA/LPs/SP600125 could effectively enrich in the rat ectopic endometrial lesions,and effectively alleviated the up-regulation of ATF3 and P2X3 receptor in lesions and DRG tissues as well as hyperalgesia caused by endometriosis.Conclusions:1.The CSOSA/LPs carrier can effectively encapsulate SP600125.2.CSOSA/LPs/SP600125 inhibits ADP-mediated up-regulation of P2X3 receptor by inhibiting the ATF3/AP-1 pathway.3.CSOSA/LPs can be used as an effective delivery system to enrich the drug with poor water solubility in ectopic endometrial lesions.4.Targeting P2X3 receptor through inhibition of ATF3/AP-1 pathway may be considered as a novel way to treat endometriosis pain.