Mechanism Of Verapamil Ameliorating Fibrosis In Urethral Stricture Through The Regulation Of The CAMKII/PI3K/AKT/mTOR Axis-mediated Autophagy

Background:The incidence rate of urethral stricture in male is increasing yearly,with higher incidence rate of local restenosis and adverse events,as well as multi-stage surgical revision,especially in patients with long segment,multiple,obliterative urethral stricture.The adjuvant intralesional injection therapy for urethral stricture has been reported effective in some clinical trials.Verapamil has been shown to have antifibrotic effects in various fibrotic diseases,including urethral stricture.However,the specific mechanisms in formation and progression of urethral fibrosis and pharmacological anti-fibrosis of verapamil remain unclear.Materials and Methods:1.We explored the clinicopathological role of Ca²⁺/calmodulin-dependent protein kinase Ⅱ（CAMKⅡ）and autophagy in the fibrosis of urethral stricture by verifying the expression level of phosphorylated CAMKⅡ and aberrant autophagy in human urethral stricture tissue.2.We constructed a cellular model of myofibroblasts which was transmitted from rat primary urethral fibroblasts,and intervened the fibroblasts with different concentrations of verapamil to determine the levels of autophagy and fibrosis related gene transcription and protein expression.3.We detected the autophagy and fibrosis levels,as well as cell migration ability,after the block of autophagy and overexpression of CAMKⅡ in rat primary urethral fibroblasts under the application of verapamil.4.We constructed a novel urethral stricture model in male Sprague Dawley rats and intervened the injured urethra with intralesional injection of verapamil and explore the role of verapamil on autophagy and fibrosis in vivo experiments.Results1.Collagen fiber deposition,inflammatory infiltration,and increased stiffness were observed in scar tissue of patients with urethral stricture,while the level of autophagy was significantly decreased.2.Transforming Growth Factor Beta（TGF-β）induced the transmission of rat primary urethral fibroblasts into myofibroblasts in a dose-and time-dependent manner.Verapamil activated autophagy in urethral fibrosis in a dose-dependent way.3.After hyperactivation of CAMKⅡ and blockade of autophagy flow,verapamil-induced anti-fibrosis effect was significantly antagonized by reduced autophagy.4.Autophagic flux in urethral scar tissue was significantly suppressed in male rat models of urethral stricture,and verapamil induced autophagy in local urethral tissue after trauma and inhibited the progression of fibrosis.Conclusion:1.The inhibition of autophagic flux mediated by phosphorylation of CAMKⅡ promotes urethral fibrosis.2.Verapamil activated autophagy by de-regulating the CAMKⅡ/The phosphatidylinositol-3-kinase（PI3K）/Protein kinase B（AKT）/The mammalian target of rapamycin（m TOR）axis and suppressed the progression of urethral fibrosis.