Estrogen Reduces Bladder Excitability In Rats With Cyclophosphamide Induced Chronic Cystitis And Its Mechanism

Background and purposeThe therapeutic effect of estrogen on interstitial cystitis/bladder pain syndrome(IC/BPS)is poor,which seriously affects the daily life and mental health of patients.Since the epidemiological results of IC/BPS show a high incidence in women and the average age at the onset of symptoms is around 45 years old,coincide with the perimenopausal period,suggesting that sex hormones may play an important role in the prevalence of IC/BPS in women.However,the use of estrogen in the treatment of IC/BPS is still controversial,mainly due to the inconsistent or even completely opposite results on researches containing estrogen therapy of IC/BPS.The potential mechanisms need further research.It has been reported that only estrogen receptor β(ERβ)is expressed in the bladder of human and rat,and estrogen plays a role in bladder function through ERβ.Activation of ERβreduces the expression of the P2X3 receptor(P2X3R)in trigeminal ganglia(TG)and dorsal root ganglion(DRG)neurons and is involved in pain transduction.The bladder micturition reflex also involves the afference of bladder sensation.Whether ERβ in the bladder inhibits bladder excitability by reducing afferent nerve excitability needs further research.In view of those mentioned above,we aimed to explore whether estrogen affects bladder excitability by affecting the expression of P2X3 R in the bladder in rats with cyclophosphamide-induced cystitis.MethodsPart Ⅰ: Estrogen reduces bladder excitability in rats with cyclophosphamide induced chronic cystitis1 Chronic cystitis model was established in rats by intraperitoneal injection of cyclophosphamide(CYP),and bladder excitability was compared between cystitis rats and normal rats by in vivo cystometry.2 The effects of estrogen on bladder excitability of cystitis rats were observed by in vivo cystometry after estrogen deprivation and ERβ agonist replacement.Part Ⅱ: Effects of estrogen on P2X3 R expression in rat bladder1 The effects of estrogen deprivation and ERβ agonist replacement on bladder P2X3 R expression were detected by immunohistochemistry,RT-PCR and Western blot.2 The expression,distribution and co-location of ERβ and P2X3 R in bladder were detected by immunohistochemistry and immunofluorescence.Part Ⅲ: Inhibition of bladder epithelial P2X3 R significantly reduces bladder excitability1 The bladder epithelium of rats was corroded by protamine sulfate solution incubation in the bladder,and then the damage of the bladder epithelium was observed by HE staining.2 The impaired urothelium cystitis rat group and the intact urothelium cystitis rats in the control group were infused with P2X3 R antagonist AF-353,respectively,and the effect of P2X3 R on the excitability of the bladder was observed by in vivo cystometry.Part Ⅳ: The effect of ERβ on the expression of P2X3 R in bladder epithelial cells1 Rat bladder epithelial cells were isolated and cultured.Human epithelial cell line SV-HUC-1 cells were cultured in a complete medium supplemented with 10% carbon adsorbed fetal bovine serum to eliminate the effect of endogenous estrogen on the epithelial cells.2 The expression of P2X3 R in rat bladder epithelial cells and SV-HC-1 cell lines was determined by RT-PCR and Western blot after treated with different concentrations of17β-estrogen(E2)and ERβ agonist(DPN).3 The expression of P2X3 R in rat bladder epithelial cells was determined by RT-PCR and Western blot after the rats were treated with ERβ agonist(DPN)and ERβ antagonist(PHTPP).Part Ⅴ: Inhibition of P2X3 R reduces stretch-released ATP in human urothelial cells1 SV-HUC-1 cell lines were treated with P2X3 receptor antagonist AF-353 and A-317491.After 48 h stretch culture,the concentration of ATP in cell culture medium was determined.2 Urothelial cells were precultured with ERβ agonist DPN or E2 for 72 h and then stretched for 48 h to determine the concentration of ATP in cell culture medium.3 Urothelial cells were precultured with ERβ inhibitor PHTPP and DPN(or E2)for 72 h,and then stretched for 48 h to determine the concentration of ATP in cell culture medium.ResultsPart Ⅰ: Estrogen reduces bladder excitability in rats with cyclophosphamide induced chronic cystitis1 Chronic cystitis model of rats was successfully established by intraperitoneal injection of cyclophosphamide(75mg/kg)every other day for consecutive seven days,and the rats showed stable bladder hyperexcitability;2 Estrogen deprivation can aggravate the urinary frequency in IC/BPS rats,and ERβagonist can reduce the urinary frequency in IC/BPS rats,suggesting that estrogen plays an important role in the bladder excitability of IC/BPS rats.Part Ⅱ: Effects of estrogen on P2X3 R expression in rat bladder1 Estrogen deprivation up-regulated bladder P2X3 R expression in IC/BPS rats,while supplementation with ERβ agonist DPN could down-regulate the up-regulated P2X3 R expression in IC/BPS rats.2 P2X3 R was mainly expressed in the bladder epithelium and subepithelial mesenchymal cells,while ERβ was only expressed in the bladder epithelium and hardly expressed in the subepithelial mesenchymal cells.3 ERβ and P2X3 R co-locate in epithelial cells,and ERβ has the structural basis of influencing P2X3 R expression.Part Ⅲ: Inhibition of bladder epithelial P2X3 R significantly reduces bladder excitability1 In cystitis rats with intact uroepithelium,intravesical infusion of P2X3 R inhibitor AF-353 significantly reduced intercontractile interval(ICI)and bladder peak pressure(BPP);2 In cystitis rats with impaired uroepithelium,intravesical infusion of P2X3 R inhibitor AF-353 had no significant effect on ICI and BPP;3 P2X3 R in uroepithelium has significant influence on ICI and BPP.Part Ⅳ: The effect of ERβ on the expression of P2X3 R in urothelial cells1 The expression of P2X3 R in human urothelial cell line SV-HC-1 is affected by DPN and E2 and reversed by ERβ antagonist PHTPP.2 The effects of DPN and E2 on the expression of P2X3 R in human urothelial cell line SV-HC-1 varied with concentrations.Low concentrations of DPN and E2 inhibited the expression of P2X3 R in epithelial cells,while high concentrations of DPN and E2 upregulated the expression of P2X3 R.Part Ⅴ: Inhibition of P2X3 R reduces stretch-released ATP in human urothelial cells1 Stretching increased ATP release of cultured urothelial cells in vitro.2 P2X3 R inhibitors AF-353 and A-317491 can reduce stretch-released ATP from urothelial cells.3 Urothelial cells were precultured with ERβ agonist DPN or E2 for 72 h,and then stretch culture significantly reduced the stretch-released ATP.Urothelial cells were precultured with the ERβ inhibitor PHTPP with DPN(or E2)for 72 h,and then stretch culture partially reversed the reduced stretch-released ATP by preculture with DPN or E2.ConclusionsEstrogen has a direct effect on the regulation of bladder overactivity by downregulating the expression of bladder epithelial P2X3 R through its nuclear receptor ERβ and reducing the ATP released from urothelium during bladder filling,thereby inhibiting the generation of micturition reflex.Our present research provides evidence for the clinical application of estrogen to treat IC/BPS and other disease with bladder hyperexcitability.In addition,we demonstrated that ERβ may be a more selective therapeutic target of bladder overactivity diseases.However,our findings are based on the in vitro experiments,further in vivo studies are needed to illustrate the usefulness of clinical therapy.