Local Synthesis Of Estrogen In The Rostral Ventromedial Medulla Is Involved In The Regulation Of Visceral Pain Via GPR30

Visceral pain is the most frequentsymptom encountered in the clinic.Visceral hypersensitive diseases(such as irritable bowel syndrome,interstitial cystitis,endometriosis,etc.)are very common and are more prevalent in the females.Research regarding gender differences in visceral pain has increased substantially in recent years.People look forward to better understand the mechanisms of pain and to improve the outcome of analgesic treatment through the study on the gender differences in visceral pain.It is widely believed that the gender differences in visceral pain suggest that estrogen plays an important role in the occurrence and development of visceral pain.The rostral ventromedial medulla(RVM)is an essential part of the endogenous pain modulation pathway,which project to the spinal cord to facilitate or inhibit nociceptive transmission in the dorsal horn.In addition,RVM is also one of the main functional areas where endogenous opioid peptides exert analgesic effect.It has been shown that dysfunction of the descending pain facilitation and inhibition pathways may be associated with the development and maintenance of visceral hyperalgesia.Studies conducted in our group found that local application of estrogen in the RVM caused a significant increase in colorectum distension(CRD)-induced viscero-motor response(VMR),suggesting that estrogen may affect the desending pain modulation pathways.Aromatase(estrogen synthase),the enzyme that catalyzes the transformation of androgens into estrogens,is a member of the P450 superfamily of cytochrome enzymes.Many areas of the CNS reportedly express aromatase and are capable of local de novo synthesis of estrogen and there has been substantial experimental evidence for the involvement of locally synthetized estrogen in the regulation of neuronal functions.We recently detected expression of aromatase in the RVM.Hence the first objective of this study is to reveal the role of locally synthetized estrogen in visceral hyperalgesia.Estrogen has long been known to interact with nuclear receptors ER? and ER? to regulate protein synthesis at the transcription level(ie,genomic effect).More recently,however,large number of studies have shown that estrogen may also act through the G-protein coupled receptor-GPR30 to produce rapid non-genomic effects.Through immunohistochemistry and Western blot,our group detected expression of GPR30 rather than ER?/ER? in RVM.Furthermore,similarly to the effects of estrogen,GPR30 selective agonist G1 applied to the RVM resulted in an increase in CRD-induced VMR,promoted phosphorylation of the ?-type opioid receptor(MOR)and attenuated the analgesic efficacy of morphine at this site.These results suggest that within RVM,estrogen acts via GPR30 and the downstream signaling involves the phosphorylation of MOR.Therefore,the second major objective of this study is to comfirm the role of GPR30 in the development and maintenance of colorectal hyperalgesia.The following are the main findings:1.Local synthesis of estrogen in RVM is involved in centralcolorectal hyperalgesia.1.1 Central colorectal hyperalgesia is assoicated with an upregulation of aromatase expression.Triple intermittent intraperitoneal injection of cyclophosphamide(CPM)induced apparent chronic cystitis in the adult rats.At day 10 following the third injection of CPM,rats were examined for colorectal pain behavior using the colorectal distension induced abdominal withdrawal reflex(AWR)score.CPM treated rats(CPM rats)had a significantly higher AWR score than the saline-treated rats and the AWR score of female CPM rats was significantly higher than that of the male CPM rats.Immunohistochemistry and Western blot analysis showed that the expression of aromatase was markedly increased in RVM of CPM-treated rats.1.2 Aromatase inhibitors ameliorate CPM-induced colorectal hyperalgesia.The colorectal hyperalgesia was attenuated by application of the aromatase inhibitors(Fadrozole and Letrozole)intercisternaly to inhibit estrogen synthesis,as was the phosphorylation of ?-type opioid receptor(MOR)in RVM of CPM-treated rats.Expression of MOR was increased by inhibition of estrogen synthesis in RVM of CPM-treated rats.However,FAD or LET did not affect the expression of aromatase and GPR30 in RVM of CPM-treated rats.2.GPR30 plays an essential role in CPM-induced colorectal hyperalgesia.2.1 GPR30-sh RNA prevents the development of CPM-induced colorectal hyperalgesia.Adeno-associated virus carrying GPR30-sh RNA(GPR30-sh RNA-AAV)was microinjected into RVM of CPM-treated rats to knockdown GPR30 expression,using virus carrying scrambled nucleotide sequence(Scr-AAV)as control.Efficacy of the sh RNA was confirmed by Western blot detection of GPR30 in RVM.Behavioral test showed that the average AWR score of CPM rats pretreated with GPR30-sh RNA-AAV was similar to the average AWR score of saline control rats and was significantly lower than that of the CPM-rats pretreated with Scr-AAV.Western blot analysis indicated that the phosphorylation of MOR was inhibited,while MOR expression was enhanced by knock-down of GPR30 expression in RVM of CPM-treated rats.2.2 GPR30 blocker ameliorates CPM-induced colorectal hyperalgesia.Similarly to the effect of GPR30-sh RNA-AAV,chronic continuous infusion of GPR30 blocker G15 into the cerebellomedullary cistern via the osmotic minipump also prevented the development of colorectal hyperalgesia and downregulated the expression of phosphorylated MOR in CPM rats.In summary,based on the previous work of our lab,we further clarified the role of local synthesis of estrogen in RVM in the regulation of colorectal hyperalgesia.We found that aromatase expression in RVM was apparently increased and the colorectal pain sensation was significantly enhanced in cyclophosphamide treated rats.Inhibition of local estrogen synthesis in RVM through intracisternum application of aromatase inhibitors ameliorated CPM-induced colorectal hyperalgesia and concomittantly caused a decrease in the phosphorylation of MOR.Sh RNA knock-down of GPR30 expression and pharmacological blockade of GPR30 in RVM both prevented the development of CPM-induced colorectal hyperalgesia with concomittant decrease in phosphorylation of MOR.These results provide strong support to our hypothesis that locally produced estrogen in the RVM is involved in the genesis and maintenance of chronic visceral hyperalgesia via activation of GPR30 and the downstream signaling may involve phosphorylation of MOR.