Chronic Administration Of Exogenous H₂S Improves Erectile Function In BCNI Rat Though Inhibiting Phenotypic Modulation Of Corpus Cavernosum Smooth Muscle Cell

BACKGROUND&OBJECTIVERadical prostatectomy(RP)is the primary treatment for patients with localized prostate cancer in clinc.However,the incidence of erectile dysfunction(ED)post-operation remains as high as 30 to 87%.It may be related to intraoperative cavernous nerve injury,but the specific mechanism is unknown.Phenotype modulation of smooth muscle cells is defined as the ability to transform from the contractile phenotype into synthetic one reversibly.Failure of CCSMCs(corpous cavernosum smooth muscle cells)to acquire and maintain the contractile phenotype plays a crucial role in ED.Our previous studies confirmed that the CCSMCs underwent phenotypic modulation in rat penile with BCNI(bilateral cavernous nerve injury),which is closely related to EDH2S is a gasotransmitter discovered in recent years.H2S can improve erectile function in diabetic ED or aging ED rats.However,there are limited reports on the effect of exogenous H2S supplementation on ED induced by cavernous nerve injury and its mechanism.The purpose of this study was to study the effect of chronic exogenous H2S supplementation on erectile function in rats with BCNI and to determine whether the improvement of erectile function by exogenous H2S is related to the inhibition of Phenotype modulation of CCSMC.METHODS1.The BCNI-ED rat model was established by bilateral cavernous nerve injury.One day after surgery,the rat that divided in NaHS treatment group(BCNI+NaHS group)encountered intraperitioneal injection of NaHS solution(100(Mμ/kg)for 4 weeks.The rats were divided into sham operation group(sham group),BCNI injected with NaHS treatment group(BCNI+NaHS group)and BCNI injected with normal saline model group(BCNI+NS group).Four weeks later,electrostimulation and intracavermous pressure(ICP)\mean arterial pressure(MAP)were performed to evaluate penis erectile function.Hematoxylin and eosin(H&E),Masson trichrome staining,immunohistochemistry(IHC),real time qPCR and Western blot(WB)were detected.2.The smooth muscle cells of corpus cavernosum were obtained by tissue explant adherent method.To ensure the reliability of subsequent experiments the SM a actin of smooth muscle markers and contraction related factors were detected by immunofluorescence assay to identify CCSMC.3.The effect of NaHS to CCSMCs at different concentrations(0,100,200,400,800,1600,3200)was detected by flow cytometry and CCK-8 assay.4.The cells treated with PDGF-BB(platelet-derived growth factor)were used as phenotype modulation model,the cells pretreated with or without NaHS for 30 min and then treated with or without PDGF-BB for 24 h were divided into 4 group,including normal control group,NaHS group,NaHS+PDGF-BB group and PDGF-BB model group.The expression levels of a-sma,calponin,OPN mRNA and protein were detected,and RhoA,ROCK1 protein were detected as well.RESULTS1.Chronic exogenous H2S can improve BCNI rat erectile function.ICP/MAP in BCNI+NS group was significantly lower than that in Sham group,that is,ED.Four weeks after intraperitoneal injection of NaHS,the ratio of ICP/MAP,the ratio of MASSON smooth muscle to collagen,the expression of a-SMA,Calponin and CBS,CSE protein were higher than those in BCNI+NS group,while the expression of OPN protein was lower than that in BCNI+NS group.2.Expression of endogenous H2S-producing enzymes in BCNI-ED Rats were Decreased.Compared with Sham group,the expression of CBS,CSE in BCNI+NS group was significantly decreased,while the expression of-CBS,CSE in BCNI+NaHS group was higher than that of BCNI+NS group.3.CCSMC culture results.Through immunofluorescence observation,it was found that the positive rate of a-SMA was much high.The results suggested that tissue explant adherent method isolated CCSMC culture from rat corpus is efficinet and proper.4.Toxicity Test of exogenous H2S to CCSMC in different concentrations of NaHS.The results of flow cytometry and CCK-8 assay showed that the concentration of NaHS from 0-3200 μ M was not significantly different from each other group.The apoptosis did not increase with the increase of NaHS concentration.CCSMC had good tolerance to exogenous H,S.5.Exogenous H,S can inhibit the phenotype modulation of CCSMC induced by PDGF-BB.In PDGF-BB group,the expression levels of a-SMA,Calponin mRNA and protein are significantly decreased and the expression of OPN mRNA and protein is significantly increased.NaHS pretreatment group could significantly inhibit this change.CONCLUSIONChronic administration of exogenous H2S can improve erectile function and increase the expression of endogenous H2S producing enzymes CBS and CSE in BCNI rats.High purity cavernosum smooth muscle cells can be obtained by tissue explant adherent method.CCSMC has good tolerance to in NaHS-containing medium H2S may inhibit the phenotypic transformation of CCSMC by down-regulating RhoA/ROCKl pathway,thus improving erectile dysfunction in rats with bilateral cavernous nerve injury.