The Autophagy Of Detrusor Myocytes Is Involved In Interstitial Cystitis And Cyclophosphamide-induced Cystitis

Background and Aims:Macroautophagy (hereafter,“autophagy”), an evolutionarily conserved cellularprocess，By isolating intracellular organelles and protein aggregates and delivering them tolysosomes for clearance, acts as housekeeping function in regulation of the quality andquantity intracellular biological function.Currently, more studies suggest that autophagypathway and/or proteins play a crucial role for controlling immune responses andinflammation. Dysregulated autophagy may contribute to the pathogenesis and progressionof a variety of human inflammatory diseases and autoimmune diseases.,including Crohn’sdisease, cystic fibrosis lung disease, diabetes, liver diseases and intestinal inflammation, etc.Bladder pain syndrome/interstitial cystitis (BPS/IC) is a urological bladderinflammatory diseases which characterized by urinary frequency, urgency, pelvic pain andother discomfort. Though numerous potential pathogenesis, including infection,autoimmune disorder, toxic substances in the urine, urothelial dysfunction and neurogenicinflammation have been studied, the exact pathogenic mechanisms of BPS/IC have not beenwell clarified. But, the inflammation and immune disorders paly a key role in thepathogenesis of BPS/IC.Autophagy in smooth muscle was confirmed in blood vessels, airway and corpuscavernosum, suggesting its important role for the tissue protection. However, little isknown about the role and function of autophagy in the detrusor smooth muscle cell and itsrole in the pathogenesis of BPS/IC. Cyclophosphamide (CYP), a chemothera-peutic drug which is effective in the treatment of neoplastic diseases, has been used toinduce cystitis in rodents through its toxic metabolite, acrolein and has been commonly usedto investigate the effects of inflammation and immunity to urinary bladder dysfunction(including BPS/IC). This study aims to investigate the existence and efficacy of theautophagy in the detrusor myocytes in human interstitial cysititis (IC) and cyclophosphamide-induced cystitis animal model.Methods:In the first part, we explored that autophagy exists in human and rat detrusor myocytes,First, the bladder tissue from human and rat was harvested.Second, transmission electronmicroscope(TEM) and double-labeled immunofluorescence were respectively useddetection detrusor myocytes Autophagosomes and Co-location of LC3and α-SMCA. Third,western blot and RT-PCR were used Confirmed the expresion of LC3proteins and genes.In the second part, We investigate autophagy changes between control human bladderand BPS/IC bladder as well as pathological changes. First, the bladder tissue from BPS/IChuman was harvested.Second, transmission electron microscope(TEM) and double-labeledimmunofluorescence were used detection the changes of detrusor myocytesautophagosomes and LC3fluorescent spots. Third, western blot were used Confirmed theexpression of LC3proteins. Fourth, HE and toluidine blue staining were applied evaluationthe histological score and mast cell count.In the third part, We investigate the efficacy of the autophagy in the detrusor myocytesin cyclophosphamide-induced cystitis animal model. First, Groups classification and animalmodels construction. Second, transmission electron microscope(TEM) and double-labeledimmunofluorescence were used detection the changes of detrusor myocytesautophagosomes and LC3fluorescent spots in each groups. Third, western blot were usedConfirmed the expression of LC3, p-p70s6k and SOD2proteins in each groups. Fourth,HE and toluidine blue staining were applied evaluation the histological score and mast cellcount in each groups. Fifth, ELISA were used measurement the expression of IL-1β, IL-6,IL-8, MDA and GSH in each groups. Sixth, the bladder function were evaluate usingcystometry in each groups.Main result1.detrusor myocytes autophagosomes and Autophagy-related marker proteins and gensLC3were confirmed exist in human and rat detrusor myocytes with electron microscope,western blotting, RT-PCR and double-labeled immunofluorescence.2.Compared with the control group, bladder tissue from patients with IC indicatedthinning and edema in the epithelium with inflammatory infiltration in the lamina propriaand muscle layer. Moreover, quantitative assessment showed a significant increase in mast cell number in the muscular layer compared to the control group. Using electron microscopy,double-labeled immunofluorescence and western blot, we found autophagy in detrusormyocytes was significantly increased in IC patients compared to the control group,butwithout increased function,Under electron microscope, we found injured organelles,including mitochondria had not been swallowed by the autophagosome in IC patientsdutrusor myocytes.3. Autophagy activation was detected in the detrusor myocytes fromcyclophosphamide-treated rats as evidenced by increased LC3, p-p70s6k expression andautophagosomes. Enhanced inflammation, oxidative stress and overactived detrusor incyclophosphamide-treated group suggesting autogphagy of detrusor myocytes may beinsufficient activated. With pre-treated rapamycin, an autophagy agonist, the inflammationand oxidative stress were significantly decreased, and the bladder histology and micturitionfunction were significantly improved. In contrast, with pre-treated chloroquine, anautophagy antagonist, the inflammation and oxidative stress were dramatically increased,and the bladder histology and function were seriously injured.Conclusions:1. We preferentially confirmed autophagy in human and rat detrusor myocytes withelectron microscope, western blotting and double-labeled immunofluorescence.2. Compare with control human, we found that the number of autophagy in PBS/ICpatients dutrusor myocytes increased, but without increased function. Under electronmicroscope, we found injured organelles, including mitochondria had not been swallowedby the autophagosome in IC patients dutrusor myocytes. these result indicated that thefunction of autophagy disorders, This might be one of the reasons that the bladderinflammation continued.3. The present study demonstration that autophagy exist in rat detrusor cells andenhanced the detrusor cells autophagy expression via rapamycin significant decreasedinflammation, oxidative stress, and improved bladder function.besides，rapamycin，aeffective pharmacological agent, which is already clinically available, could be applied inIC.