PDGF Regulates The Gap Junctions Of Nerve Injuryed Corpus Cavernosum Smooth Muscle Through AKT/β-catenin Signaling Pathway And PNS Intervention

Objectives Erectile dysfunction is one of the common complications after radical prostatectomy,the inevitable injury of erectile nerve caused by the operation is the most important mechanism.Currently the first-line medicine phosphodiesterase inhibitors are the main treatment of neurogenic ED,but up to 35%of patients are ineffective.Therefore,in-depth study of pathophysiological changes and molecular mechanism of cavernous tissue after nerve injury has great clinical significance in prevention and treatment of ED after radical prostatectomy.Method:1.Cultured smooth muscle cells were isolated from corpus cavemosum of normal rats and ED rats by enzyme digestion method,for detecting the changes of the ERK,STAT3,AKT,Cx43 proteions,when the CCSMCs were treated with the growth factors or PDGF inhibitor.Western blot was used to detet the expression of P-catenin in the cytoplasm or in the nucleus,and the expression of ERK、STAT3、AKT、Cx43 proteins related to PDGF path way.Functional gap junctions were evaluated by scrape-loading dye transfer assay.The pathway of PDGF impacting the expression of Cx43 was illuminated by regulating the expression of STAT3 or AKT through the expression plasmid and siRNA.Promoter luciferase reporter assay and Chromatin immunoprecipitation assay(CHIP)were used to detect the interaction of Cx43 promoter and β-catenin.2.A method of resection of bilateral cavernous nerve of SD rats was used to establish ED models.After 12 weeks,the erection rate of ED rats were tested through injection APO at 100μg/kg in Corpus cavernosum tissue.And then rats were injected with growth factors(PDGF)at 200ng/kg/d.A successful erection was identified by the penis head expansion and distal penile body appearance.Then the rats were executed and the penile tissues were harvested and assessed for the Cx43 and PDGFR expression via Western Blot or IHC.3.Isolated penile smooth muscle cells of ED rats were treated with 3-MA,PNS and PNS associate with 3-MA,and Rapamycin,in order to explore related Beclin-1,LC3Ⅱ/Ⅰ and cleaved capase3,and the level changes in gap junction protein Cx43.The gap junction of smooth muscle cells were detected by immunofluorescence,and MDC staining was used to detect the formation of autophagosome.Results:1.PDGF could increase the expression of Cx43 and induced the accumulation ofβ-catenin in nucleus,and then improve the gap junction.Inhibitionof PDGFR activity decreased the phosphorylation of AKT and STAT3,and reduced the β-catenin accumulation in the nucleus and downregulated the Cx43.Meanwhile,the change of P-catenin in cytoplasm was not obevious.Also we found that knockdown of β-catenin could downregulate the expression of Cx43 correspondingly,while overexpression of β-catenin or treatment with GSK3 inhibitor could upregulate the expression of Cx43.Moreover,activation of beta-catenin induced theaccumulation of P-catenin in nucleus,which abolished the Cx43 downregulated effect induced by the PDGFR inhibitor.Furthermore,the promoter luciferase assayand CHIP assay showed that P-catenin regulated Cx43 transcription via interaction with the promoter of Cx43.2.The group of BCN showed the ED symptom,while the Erection rate reached 40%after treatment with PDGF.In vivo assay,western blot and IHC suggested that PDGF injection contributed to the erectile function via upregulation of nucleus β-catenin and Cx43.3.Both PNS and Rapamycin have increased autophagy and inhibited apoptosis.PNS group has improved gap junction by up-regulating Cx43,while Rapamycin has no similar effect.Conclusions:PDGF could induce p-catenin accumulation in nucleus,regulate the connexin43 expression via activation the PDGFR/AKT pathway,and then inprove the cavernosum smooth muscle cells gap junction,which may be a target to treat ED in clinical.PNS could improve the gap junction by promoting the autophagy of smooth muscle cells of the penis,inhibiting the apoptosis,and up-regulating Cx43.