Role And Mechanism Of Hydrogen Sulfide Pathway In Protease Activated Receptor 4(PAR4)-related Bladder Pain

IntroductionBladder pain is the most common visceral pain in urology,a typical manifestation of interstitial cystitis/bladder pain syndrome(IC/BPS).Interstitial cystitis/bl adder pain syndrome is a chronic,non-bacterial,inflammatory,refractory bladder disease,with lower urinary tract symptoms(frequent urination or urgent urination),without significant pathological changes of bladder.It seriously affects the quality of life of patients,especially in women,and the incidence of IC/BPS increases with ages.The etiology of IC/BPS is not fully clear.At present,there are three sorts of views:(1)bladder epithelial dysfunction,resulting in increase of bladder epithelial permeability or sensitivity;(2)bladder and neurogenic inflammation,and the typical bladder pathological manifestation is named hunner ulcer;(3)neuropathic pain,including increased amounts of afferent nerves,changes of brain structure and function,and abnormalities of endogenous inhibition pathway.Because of the unclear pathogenesis and individual differences,there is no effective treatment for IC/BPS patients.Nowadays,treatments are mainly limited to conservative treatment,including behavioral therapy,oral or intravesical perfusion therapy,bladder hydration and dilatation therapy,but these treatments often fail or can only improve symptoms in the short-term.The rest of the treatments has no valid clinical evidence and can only be used as an alternative treatment.A large number of studies have shown that the increased sensitivity of bladder afferent neurons is associated with increased pain or sensory hypersensitivity.The pelvic and lower abdomen/lumbar visceral nerves A? and C-fibers that govern the bladder are not only sensitive to mechanical stimulation,but also sensitive to both chemical stimulation and temperature stimulation,especially when sensitized.These nerves are distributed in the serous layer,the muscle layer and urothelium.A variety of mediators including substance P(SP),nerve stimulating peptide A,Calcitonin gene-related protein(CGRP),vasoactive intestinal peptide,and endorphins are able to regulate afferent pathways.However,the study in local nerve endings-related pathological pain of bladder is unclear,and the chemical mediators involved in bladder pain need to be further explored.H2S is now considered as third type of gas molecule followed by NO and CO,playing important roles in both physiological and pathological conditions.In mammalian tissues,H2S is derived from L-cysteine,catalyzed by the glutathione hydroxyethyl beta synthase(CBS)and the glutathione gama lyase(CSE).H2S-producing enzymes are widely expressed in human and mammalian bladders,with many physiological functions,such as cell protection,inhibition of tumor development,regulation of inflammatory process,involvement of visceral pain regulation,alteration of smooth muscle motor movement and so on.H2S can relax the urinary bladder detrusor,improving the urinary tract symptoms in human and animal models;the role of H2S in the inflammatory process is contradictory;H2S can enhance the behavior of bladder pain induced by cyclophosphamide.Although H2S can mediate bladder pain,but how H2S affects bladder pain and interstitial cystitis is still to be studied.For now,there are several mouse models to simulate one or more of the symptoms of IC/BPS,we intravesically instilled protease activated receptor 4-activated peptide(PAR4-AP)to build a mouse pain model,because protease-activated receptor 4 can obviously trigger bladder pain and significantly increased levels of protease was found in the urine of IC/BPS patients.This study contains the following two parts:(1)to investigate the hydrogen sulfide signaling pathway mediating PAR4-related bladder pain via macrophage migration factor(MIF);(2)to explore the role of the hydrogen sulfide signaling pathway on inflammation and micturition behaviour of PAR4-related bladder pain.Part 1:Hydrogen sulfide signaling pathway sensitizes PAR4-induced bladder painObjectiveThe bladder pain model was induced by intravesical perfusion of PAR4-AP,pretreated by the H2S-related drugs.Detecting the changes of bladder pain in mice,and the changes of H2S and-related enzymes in the bladder of mice to investigate the role of hydrogen sulfide signaling pathway in altering protease activated receptor 4(PAR4)-induced bladder pain.Method1.Stimulation protocols10-13 weeks-old healthy female C57BL/6J mice were divided into 12 groups,the first 10 groups randomly assigned 24 mice,the last 2 groups randomly assigned 6 mice.Anaesthetized by pentobarbital sodium,urine tubes(PE10,11mm)were inserted into the bladder of mice.Pressing the abdomen to discharge urine completely.Among them,In the first 10 groups,18 mice of each group were cut off bilateral ureter surgically before treatment,in order to prevent the urine from kidneys affecting the results.The bladder perfusion was collected 1 hour later and mice were killed.Urine was stored in the-80?refrigerator.The remaining 6 mice of each group and the last 2 groups were not operated and given only bladder perfusion.24 hours later,the mice were tested for pain and voiding behavior.SV-HUC-1 cells were incubated in the 24-well plate,with F12K medium containing 10%fetal bovine serum.1 hour before the experiment,replacing the original medium with a fresh one.Cells were divided into 16 groups and repeated 3 times for each treatment group.2.Experiment SetupStimulating the lower abdomen of mice with four different Von Frey fibers 0.008g,0.020g,0.040g,0.070g respectively,from weakest to strongest,recording responses of mechanical allodynia of mice 24 before and after the administration of drugs.Using Western Blot to detect the expression of H2S-producing enzymes CBS and CSE in bladder tissues.Detecting the location of CBS and CSE in bladder tissues by immunohistochemical staining(IHC).Testing the expression of cbs and cse by real-time qPCR.Applying H2S release assay to detect the activity of H2S-producing enzymes in human,mouse models and cells.By means of ELISA,the expression of MIF in different groups of cell homogenate,bladder tissues and bladder perfusion were quantified.3.Data analysisAll statistical analysis was performed using Sigmaplot software(SPSS).The data were analyzed using Student's t-test or one-way ANOVA,and results are presented as the mean ± SEM.At least six mice were randomly assigned to each group.Cells were repeated 3 times for each group.Exact number of mice used in each group was indicated in the figure legends.A P-value<0.05 was considered statistically significant.Result1.Hydrogen sulfide signaling system exists in mouse bladder mucosa and SV-HUC-1 cellsReal-time qPCR and Western blot showed the expression of CBS and CSE in the mouse bladders and SV-HUC-1 cells,and the immunohistochemical staining showed that CBS and CSE were widely expressed in the cell membrane and nucleus of the mouse bladder mucosa.In order to detect whether H2S can be produced in the bladder tissue and human bladder SV-HUC-1 cells in mice,we collected bladder perfusion 1 hour after treatment and SV-HUC-1 cell homogenate to perform the release assay of hydrogen sulfide,and the results showed that H2S can be detected in the bladder perfusion fluid of mice,and the H2S level of bladder perfusion is decreased when treated by AOAA and PAG respectively,and the H2S level of bladder perfusion is increased when treated by NaHS.The same results can be seen in SV-HUC-1 cell homogenate.2.Upregulation of hydrogen sulfide signaling system activated by protease activated receptor 4(PAR4)Real-time qPCR and Western blot were used to detect the expression of CBS and CSE in the SV-HUC-1 cells and mouse bladders treated by PAR4-AP or control peptide.However,there was no significant difference between CBS and CSE protein expression.H2S release test showed that H2S were significantly increased in SV-HUC-1 cells,mouse bladder and human bladder tissues treated by PAR4-AP compared with treated by control peptide.3.H2S mediates PAR4-induced mechanical hyperalgesiaAfter different treatments,we stimulated the abdominal and perineal areas of the mice with Von Frey filaments and recorded the number of positive reactions.Interestingly,pretreatment with AOAA completely inhibited mechanical hyperalgesia,and pretreatment with NaHS greatly enhanced PAR4-induced hypersensitivity.In contrast,pretreatment with SAM or PAG only minimally altered PAR4-induced hypersensitivity.Notably,PAG,AOAA,SAM and NaHS did not obviously alter the pain threshold if PAR4 was not activated.Next,we further investigated H2S production with above treatments.We found that pretreated with PAG or AOAA markedly decreased H2S production.Moreover,the inhibitory effect of AOAA on H2S production was more obvious than that of PAG.A new CBS inhibitor,Benserazide,was reported in 2017.Our results showed Benserazide didn't significantly change the pain threshold when PAR4 is not activated;when PAR4 activated,Benserazide can only partially inhibited the mouse pain and hypersensitivity.4.H2S probably mediates PAR4-induced bladder pain through MIFHomogenates from cells pretreated with the CSE inhibitors PAG(10 mM)and SAM(1 mM)showed no changes in MIF protein levels;however,cells pretreated with the CBS inhibitor AOAA(1 mM or 5 mM),the H2S donor NaHS(1 mM or 5 mM)or the CBS activator SAM(5 mM)showed obviously reduced or increased protein levels of MIF.Similarly,bladder homogenates pretreated with the CBS inhibitor AOAA(50 mg/kg,i.p.)had increased protein levels of MIF,while MIF protein levels remained unchanged in homogenates pretreated with the CSE inhibitor,PAG(100 mg/kg,i.p.).MIF protein levels were elevated in bladder homogenates pretreated with the H2S donor,NaHS(50 nM per mouse),but were not elevated in homogenates pretreated with the CBS activator,SAM(100 mg/kg,i.p.).These results were in agreement with those obtained for perfusion samples.Conclusion1.CBS and CSE were expressed in SV-HUC-1 cells as well as in mouse bladder tissues and could play a role in endogenous generation of H2S.We firstly proved the existence of CBS in the mouse bladder.2.Protease activated receptor 4 can improve the activity of hydrogen sulfide synthase in SV-HUC-1 cells and mice bladder tissues and increase H2S production in SV-HUC-1 cells and bladder tissues of mice.Above is firstly proved by us.3.PAR4 can cause bladder pain hypersensitivity,which can be enhanced by H2S.4.H2S may mediate the bladder pain model of PAR4 mice through MIF.Part 2:Role of hydrogen sulfide signaling pathway in the bladder inflammation and micturition behavior of PAR4-induced bladder painObjectiveEstablishing the model of bladder pain intravesically instilled PAR4-AP,treating with different H2S drugs,detecting the changes of bladder inflammation in mice,and observing the urinary behavior of mice,aiming to explore the effect of hydrogen sulfide signaling pathway on inflammation and voiding function in the bladder pain model induced by PAR4.Method1.Stimulation protocolsEstablishment of the mouse interstitial cystitis/bladder pain syndrome model induced by protease-activated receptor 4 is seen as the first part.Establishment of the mouse interstitial cystitis/bladder pain syndrome model induced by cyclophosphamide in mice is as follows.10-13 weeks old healthy female C57BL/6J mice were divided into 4 groups,and each group were randomly assigned 6 mice.(1)cyclophosphamide(i.p.200mg/kg),killed 4 hours later;(2)normal saline(i.p.),killed 4 hours later;(3)Cyclophosphamide(i.p.75mg/kg)were injected on the 1th,4th,7th day,and killed on the 8th day;(4)The normal saline(i.p.)were injected on the 1th,4th,7th day,and killed on the 8th day.2.Experiment SetupWeighing mouse bladder and body weight,to judge the degree of bladder edema according to the ratio;the inflammation degree of bladder edema and neutrophil infiltration in mice was detected by H&E staining;the urinary amount and micturition frequency were detected by using noninvasive mouse urine detection method(VSA)in mice;the expression of TNFa,IL4 was detected by ELISA.3.Data analysisAll statistical analysis was performed using Sigmaplot software(SPSS).The data were analyzed using Student's t-test or one-way ANOVA,and results are presented as the mean ± SEM.At least six mice were randomly assigned to each group.Exact number of mice used in each group was indicated in the figure legends.A P-value<0.05 was considered statistically significant.Result1.hydrogen sulfide mediated PAR4 bladder pain model without causing bladder edemaWe weighed the mouse's bladder and body weight,compared with the control group,and there was no statistically significant difference in the ratio between the two groups,but the model of interstitial cystitis/bladder pain syndrome induced by cyclophosphamide could cause obvious bladder edema and the ratio of bladder to body weight in mice.H2S itself would not cause bladder edema and would not cause bladder edema in PAR4-induced bladder pain models.2.Hydrogen sulfide mediated PAR4 bladder pain model without triggering bladder inflammationPAR4-induced bladder pain model didn't cause significant bladder inflammatory changes,the model of interstitial cystitis.induced by cyclophosphamide in mice could cause obvious bladder inflammation,manifested as neutrophil infiltration,mucous membrane edema,H2S itself didn't cause bladder edema,and didn't cause bladder inflammation in the PAR4-induced model.3.hydrogen sulfide mediated PAR4 bladder pain model without altering micturition behaviorUpon activation of PAR4 and treatment of H2S drugs,there was no statistically significant difference in the number of urine plaque,total area of urine plaque,number of main urine plaque,total area of main urinary plaque and total area of main.urine plaque/total area of urinary plaque.4.Role of hydrogen sulfide in the PAR4-related inflammatory factorsThe changes of Th1 transcription factor T-bet and Th2 transcription factor GATA-3 after activation of PAR4 were detected in real-time qPCR,and we found that T-bet did not change,but GATA-3 significantly increased in the treatment group.ELISA showed no changes of TNFa expression,but the expressions of IL4 were significantly increased when treated with PAR4.Interestingly,H2S reduced all IL4 when PAR4 activated.Discussion1.H2S mediates PAR4-related bladder pain without overt evidence of bladder injury or inflammation.2.H2S mediates PAR4-related bladder pain without altering micturition behavior.3.PAR4-related bladder pain affects the expression of Th2-type cells and IL4.4.Overall,PAR4-induced bladder pain model is a new type of interstitial cystitis/bladder pain syndrome mouse model,which can cause bladder hypersensitivity,but without obvious bladder inflammatory changes and voiding behavior changes.The abnormal activity of H2S synthase and the abnormality of H2S production may be closely related to the change of bladder pain behavior after activation of PAR4.H2S/CBS/CSE signaling pathways may play an important role in interstitial cystitis/bladder pain syndrome,and our research provides new ideas for the diagnosis and treatment of interstitial cystitis/bladder pain syndrome.