Upregulation Of TRPM3 Channel In Bladder Afferents Is Involved In Chronic Pain In Cyclophosphamide-induced Cystitis And Its Regulatory Mechanisms

IntroductionInterstitial cystitis/bladder pain syndrome(IC/BPS)is a clinical syndrome characterized by chronic pain in the lower abdomen or perineum at the end of bladder filling,with or without frequency,urgency,and the presence of Hunner ulcer lesions.Chronic pain is the cardinal manifestation in patients with IC/BPS,and the suffering from pain can lead to the depression or even suicidal behavior.However,the pathophysiological mechanisms underlying bladder pain are not yet clear,also no effective treatment is available for IC/BPS patients.Thus,reveal of the underlying mechanisms for bladder pain and identification of the new therapeutic targets has been an important research topic in functional urology.The recognized mechanisms of IC/BPS pathogenesis include:urothelium damage,inflammation,mast cell accumulation and activation,autoimmune disorder,and psychological stress et al.Whatever the mechanism is,peripheral sensory sensitization is the common pathway in the bladder.Furthermore,peripheral sensory sensitization is also an important underlying mechanism for frequency or urgency,the two symptoms of overactive bladder(OAB).The bladder primary sensory afferents originate from the cell bodies located in the lumbosacral dorsal root ganglia(DRG).Its peripheral ends are the nerve terminals within the bladder wall,and the central ends project into the dorsal horn of the spinal cord.Molecular sensors,such as transient receptor potential(TRP)channels,acidsensitive channels or ATP channels expressed on sensory afferents are responsible for the detection of mechanical and chemical signal changes in the bladder wall,and convey this information to the central nervous system,leading to bladder sensation such as bladder filling and pain.Alterations in the expression of these channels has been implicated in bladder peripheral sensory sensitization.Studies have shown that upregulation of the expression and function of TRPV1,TRPA1,and TRPV4 are involved in the peripheral sensory sensitization and thus the hyperalgesia in IC/BPS patients or animal models of IC/BPS.After the recognition of TRPV1 and TRPA1,the two important pain channels expressed in sensory afferents,another pain-sensing channel TRPM3,have recently been identified.Like TRPV1 and TRPA1,TRPM3 channels are also expressed in sensory afferents.It is a calcium-permeable cation channel that can be activated by noxious heat(>40℃)or chemical ligands including the neurosteroid pregnenolone sulphate(PS)and the potent synthetic ligand CIM0216.Activation of TRPM3 by injection of PS in the plantar elicits an acute pain response in mice,whereas TRPM3 knockout mice lack of this acute pain response.To our knowledge,almost all studies on the roles of TRPM3 in nociception and chronic pain exclusively focus on somatic sensory neurons.Its roles in visceral nociception and chronic visceral pain have not been studied.In particular,no reports were found on the roles of TRPM3 in the development of chronic pain associated with IC/BPS.Mast cell recruitment and activation play an important role in peripheral sensory sensitization in IC/BPS conditions.Mast cell activation releases tryptase-like proteases(Tryptase)and trypsin,which can selectively act on the protease-activated receptor 2(PAR2),a Gq-type of the G protein-coupled family.Activation of PAR2 receptors have been shown to be involved in somatic pain through sensitization of TRPA1 and TRPV1 by Protein kinase A or Protein kinase C(PKA/PKC).However,whether PAR2 receptor activation could modulate TRPM3 channel activity and thus involved in chronic pain associated with IC/BPS has not been reported yet.The important role of various inflammatory mediators including nerve growth factor(NGF),bradykinin,histamine,and prostaglandin E2(PGE2)have been revealed in the development of chronic pain associated with IC/BPS.These inflammatory mediators could lead to peripheral sensory sensitization through upregulation of TRPA1 and TRPV1 in somatic chronic pain conditions such as neuropathic pain and inflammatory pain.There are reports showing that NGF and bradykinin but not histamine could enhance the intracellular Ca2+ increase induced by TRPM3 agonist PS in DRG neurons,suggesting the upregulation of TRPM3 function.Whereas,the role of PGE2 in TRPM3 channels modulation,another important inflammatory mediator in IC/BPS chronic pain,has not been revealed.In this study,we established a rat model of IC/BPS by intraperitoneal injection of cyclophosphamide(CYP).We aimed to(1)examine the role of TRPM3 in bladder nociception by injecting TRPM3 agonist into the bladder wall to activate TRPM3;(2)determine the role of TRPM3 in bladder hyperalgesia and voiding frequency in CYPinduced IC/BPS model rats;(3)examine the effects of PAR2 receptor activation and PGE2 on TRPM3 channel modulation in DRG neurons.This study is expected to provide a new theory for the underlying mechanisms of chronic pain and OAB associated with IC/BPS,and lay the foundation for TRPM3 as a pharmaceutical target for bladder pain treatment in IC/BPS patients.Part Ⅰ.Activation of TRPM3 channel in bladder sensory afferents evoked acute bladder painAimTo explore the expression of TRPM3 channels in bladder sensory afferents and the role of TRPM3 activation in acute bladder pain.Methods1.Immunofluorescence double staining was done to examine TRPM3 expression in bladder sensory afferents expressing calcitonin gene-related peptide(CGRP).2.DRG neurons innervating the bladder was retrogradely labeled with the dye DiI.Immunofluorescence was done to reveal the TRPM3 expression and its co-expression with TRPA1 and TRPV1 in DiI+DRG neurons.3.Calcium imaging was performed to examine TRPM3 functional expression and its co-expression with TRPA1 or TRPV1 in cultured bladder DRG neurons.4.Painful behavior assessments were done to observe the acute pain responses induced by injection of TRPM3 agonist PS/CIM0216 into the bladder wall,and the blocking effects of TRPM3 antagonist(primidone)or CGRP receptor antagonist BIBN.The rats were divided into the Vehicle control,TRPM3 agonist PS and CIM0216(CIM),CIM+Primidone(CIM+Prim),and CIM+BIBN(CIM+BIBN)group.Spontaneous pain behaviors were measured with two methods at 30 min after the drug injections:(1)observing and scoring of the eye movement or locomotion such as squinting behavior,limb stretching and body twisting behavior within 5 min;(2)open field experiments were used to record the total distance of movement and the central area distance within 5 min.5.Comparison of bladder size,CGRP release(ELISA),vascular permeability(Evans blue),bladder tissue morphology,inflammatory cell infiltration,and mast cell infiltration(HE staining and toluidine blue staining)in the five groups of rats.Results1.TRPM3 channel is expressed as well as co-expressed with TRPA1 and TRPV1 on bladder sensory nerves and in DRG neurons.2.Injection of TRPM3 agonists into the bladder wall of rats induced neurogenic inflammation,which are manifested by edematous changes,CGRP release,plasma extravasation and inflammatory cell accumulation in the bladder wall.These manifestations can be attenuated by co-injection with TRPM3 antagonist(Primidone)and CGRP receptor blocker(BIBN).3.Injection of TRPM3 agonists into the rat bladder wall evoked acute pain responses,which was blocked by co-injection with TRPM3 antagonist(Primidone)and CGRP receptor blocker(BIBN).ConclusionActivation of TRPM3 channels on bladder sensory afferents can lead to acute bladder pain.Moreover,activation of TRPM3 could induce neurogenic inflammation by triggering CGRP release from the sensory afferents,and thus exacerbates acute pain responses.Part Ⅱ.Upregulation of TRPM3 channel in bladder sensory afferents mediates chronic pain in IC/BPS model ratsAimTo investigate the role of TRPM3 channel in the development of chronic pain and OAB in CYP induced IC/BPS model rats.Methods1.Rats were injected with cyclophosphamide(CYP,i.p.75mg/Kg)for 3 times on day 1,4,and 7 to establish an IC/BPS model.2.DRG neurons innervating the bladder were retrogradely labeled with DiI.FISH technique was used to examine the mRNA expression of TRPM3,TRPA1,and TRPV1 in DRG neurons.3.Western Blot was performed to examine the protein expression of TRPM3,TRPA1 and TRPV1 in L6-S1 DRGs neurons.4.Calcium imaging was performed to examine the functional expression of TRPM3,TRPA1 and TRPV1 in cultured DRG neurons.5.Rats were divided into three groups:Control,CYP and CYP+Prim(Prim 2mg/Kg was injected 30 minutes before each CYP injection).The blocking effects of Primidone(Prim)on painful behaviors and voiding behaviors in CYP rats were examined.6.Knocking down TRPM3 expression with intrathecally injection of TRPM3 antisense oligonucleotide(Antisense),then to observe the changes of pain behaviors and voiding behaviors in CYP rats.Rats injected with mismatch oligonucleotide of TRPM3 were used as the control group.Rats were divided into Control,CYP,CYP+Mismatch and CYP+Antisense groups.7.Mechanical stimulation with Von Frey filaments in the lower abdominal area to observe the evoked painful behavior(mechanical)in different group of rats.8.Radiation heat stimulation in the lower abdominal area to observe the evoked painful behavior(noxious heat)in different group of rats.9.Open field experiments to observe the spontaneous pain behavior in different group of rats.10.HE staining and toluidine blue staining were done to observe the histomorphology and the mast cell infiltration in the bladder tissue,respectively,in different group of rats.11.Bladder pressure-volume measurements(Cystometrograms,CMG)and urine spot tests were performed to observe the voiding behavior in different group of rats.Results1.Compared to control rats,CYP rats showed increased sensitivity to mechanical and noxious heat stimulation and reduced locomotion.CYP rats also exhibit higher voiding frequency.These indicate that the IC/BPS model was successfully established.2.FISH results showed that the mRNA expression levels of TRPM3 and TRPA1 in the sensory neurons innervating the bladder(DiI+)were increased in CYP rats compared to control rats,while in DiI-neurons,only the mRNA expression of TRPM3 was increased.3.Western blot results showed that the protein level of TRPM3,TRPA1 and TRPV1 were all increased in L6-S1 DRGs of CYP rats compared to control rats.4.In CYP rats,the TRPM3 agonist PS induced a higher amplitude of intracellular Ca2+increase in both DiI+and DiI-DRG neurons,and the fraction of PS responsive neurons was also increased.No significant between-group differences were found in the fraction of AITC or capsaicin responsive neurons either in DiI+or DiI-neurons.However,there were significant increases in the amplitude of AITC(TRPA1 agonist)or capsaicin(TRPV1 agonist)induced Ca2+increase in DiI+ neurons in CYP rats compared to control rats.A significant increase in the amplitude of capsaicin induced Ca2+increase was also found in DiI-neurons in CYP rats.However,there was no between-group difference in the amplitude of AITC induced Ca2+increase in DiI-neurons.5.Application of TRPM3 blocker primidone(i.p)significantly reduced the painful behaviors and the voiding frequency in CYP rats.6.Knockdown TRPM3 expression in CYP rats significantly reduced the painful behaviors and the voiding frequency in CYP rats.7.CYP rats showed significant mucosa rupture and bladder edema and mast cell infiltration compared with control rats.These histological and inflammatory manifestations in CYP rats were significantly reduced with application of TRPM3 blocker or knockdown TRPM3 expression.ConclusionUpregulation of TRPM3 channels(both expression and function)in bladder sensory neurons are involved in the development of chronic pain and overactive bladder(OAB)in IC/BPS.Pharmacological block or knockdown TRPM3 expression alleviate chronic pain and OAB in IC/BPS,suggesting that TRPM3 channel may be a new pharmaceutical target for the treatment of IC/BPS.Part Ⅲ.Mechanisms underlying TRPM3 up-regulation in CYP rats-The role of PAR2 receptorAimTo explore the roles of PAR2 receptor activation in TRPM3 regulation in bladder primary sensory neurons and the possible intracellular signaling pathways.Methods1.Immunofluorescence tests were performed to see whether TRPM3 channel and PAR2 receptor are co-expressed in L6-S1 DRG neurons.2.Immunoprecipitation(CO-IP)was used to observe whether TRPM3 and PAR2 are structurally coupled.3.FISH was performed to observe the mRNA expression of TRPM3 and PAR2 between Control and CYP rats and their co-expression in DRG neurons.4.Western blot was used to observe the protein expression of TRPM3 and PAR2 in L6S1 DRGs between Control and CYP rats.5.Calcium imaging was used to examine the effect of PAR2 activation on PS-induced intracellular Ca2+ increase in L6-S1 DRG neurons from normal rats and to explore the role of the three downward signaling pathways of PAR2 activation:(1)PLC-DAG-PKC(2)PLC-IP3-Ca2+ and(3)cAMP-PKA.Results1.Immunofluorescence results showed that TRPM3 and PAR2 were co-expressed in L6-S1 DRG neurons and the bladder nerve endings.2.CO-IP results showed that TRPM3 and PAR2 were structurally coupled.3.FISH results showed that mRNA expression levels of TRPM3 and PAR2 were increased in both DiI+ and DiI-neurons in CYP rats compared to normal rats,and they are co-expressed in the same neuron at mRNA level.4.Western Blot showed that protein expression levels of TRPM3 and PAR2 were increased in L6-S1 DRG neurons in CYP rats compared to normal rats.5.Calcium imaging in L6-S1 DRG neurons from normal rat showed that application of PAR2 agonist Sligrl could induce an increase in intracellular Ca2+and also could increase the amplitude of PS-induced intracellular Ca2+as well as increased the proportion of PS responsive neurons.The PAR2 receptor blocker(FSLLRY)abolished the enhanced PS response induced by Sligrl pretreatment.Pretreatment with vesicular transport blockers EXO/END reduced the enhanced PS response induced by PAR2 activation.However,PKA or PKC blockers did not affect the effects of PAR2 activation.ConclusionTRPM3 channel and PAR2 receptor are structurally and functionally coupled in bladder sensory afferents.PAR2 activation could up-regulate TRPM3 expression and function by facilitating the trafficking of TRPM3 channel from the cytoplasm to the membrane.Activation of PAR2 receptors by tryptase released from the mast cells may be one of the important mechanisms for the involvement of mast cells in IC/BPS associated chronic pain.Part Ⅳ.Mechanisms underlying TRPM3 up-regulation in CYP rats-The role of PGE2AimTo explore the regulatory roles of PGE2 on TRPM3 channels in bladder DRG neurons and the possible involving intracellular signaling pathways.Methods1.ELISA assay was performed to measure the PGE2 level in bladder tissue,L6-S1 DRGs and urine from control and CYP rats.2.RT-qPCR and Western blot were done to determine the mRNA and protein expression levels of COX2(the key enzyme for PGE2 synthesis)and the four PGE2 receptorsEP1-EP4 in L6-S1 DRGs from control and CYP rats.3.Immunofluorescence double-staining was done to observe the co-expression of EP1 or EP4 receptors with TRPM3 in L6-S1 DRG neurons.4.Calcium imaging was performed to examine the effects of PGE2 on PS-induced intracellular Ca2+increase and the involvement of EP1 and EP4 receptors.Results1.ELISA results showed that the levels of PGE2 in bladder tissue,L6-S1 DRGs and urine were significantly increased in CYP rats.2.RT-qPCR results showed that the mRNA expression levels of COX2 and EP1,EP2 as well as EP4 receptors in L6-S1 DRGs of CYP rats were significantly increased.3.Western blot results showed that the protein expression levels of COX2,EP1,EP4 receptors in L6-S1 DRGs of CYP rats were significantly increased.4.Immunofluorescence double staining results showed that EP1 and EP4 were coexpressed with TRPM3 in L6-S1 DRG neurons from normal rat.5.Calcium imaging results showed that pretreatment with PGE2 enhanced the amplitude of PS-induced intracellular Ca2+ increase in DRG neurons,which was blocked by the non-selective EP receptor blocker(AH6809).EP1,but not EP4 receptorspecific agonists enhanced the PS-induced response.ConclusionPGE2 levels in bladder tissue,L6-S1 DRGs and urine were increased in CYP rats.PGE2 could upregulate TRPM3 function mainly through EP1 receptor activation.Upregulation of TRPM3 may be one of the important mechanisms for PGE2 involvement in IC/BPS associated chronic pain.