| BackgroundBladder outlet obstruction(BOO)refers to the increase in urine outflow resistance caused by various diseases of the bladder neck and/or urethra.BOO is common in clinic and mainly caused by obstruction induced by benign prostatic hyperplasia,bladder neck sclerosis,urethral valve or urethral stricture,and generally behaved as partial bladder outlet obstruction(pBOO).pBOO can lead to abnormal bladder con-traction and changed compliance.It is a common cause of lower urinary tract symp-toms(LUTS).The impairment of bladder secondary to pBOO comes gradually and deteriorates cumulatively.Improper management for extended periods of time will cause severe damages to the bladder structure and further aggravate the deterioration of bladder function.Under normal circumstances,removing the obstruction helps to alleviate the LUTS caused by pBOO.However,the fact is that there remian a consid-erable number of patients suffered severe bladder dysfunction and refractory LUTS after the obstruction is removed,which suggests irreversible bladder dysfunction sec-ondary to pBOO.This is a point of the treatment of pBOO.However,there remains no ideal method to ameliorate the damage of bladder efficiently enough for the treat-ment of pBOO.Therefore,it is necessary to explore the structural and functional al-terations of the bladder after pBOO and to clarify the underlying mechanismAlteration in function and structure of the bladder secondary to pBOO occurs as a gradual and cumulative process.Previous studies have found that after the occurrence of pBOO,increased intravesical pressure induced local inflammation,leading to de-trusor hyperplasia,and edema and thickening of urothelium.At the same time,com-pression of blood vessels in bladder wall resulted in focal ischemia.Metabolic chang-es would promote angiogenesis and increase blood supplying for the bladder,which helps to compensate for bladder dysfunction to a certain extent.However,if the ob-struction existed chronically,damages caused by metabolic disorders in the bladder accumulated and the bladder function would eventually turn into a decompensated condition,leading to bladder fibrosis manifested as loss of detrusor and increased collagen deposition in bladder.This might be the reason for the irreversible bladder dysfunction secondary to pBOO.Adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK)is a key regulator of energy homeostasis and metabolic regulation,which could be activated in response to a variety of stresses and is proved to be involved with various diseases.pBOO is accompanied with a series of changes in energy metabolism and oxidative stress in bladder.However,the alterations and effects of AMPK in the process of bladder injury after pBOO are not completely clear.We speculate that AMPKa may play important roles in pBOO-related bladder dysfunction.With a rat model of pBOO,our research focused on the alterations in AMPKα in the bladder tissue after pBOO,explored the role of AMPKa in bladder dysfunction and bladder remodeling second-ary to pBOO,and tried to ameliorated the structural and functional impairments of the bladder after pBOO by intervening in this pathway.PurposeWe aimed to explore the change of activation of AMPK during the process of pBOO-induced structural and functional alterations in bladder,clarify the role of AMPK pathway in the pBOO-relevant bladder dysfunction and bladder remodeling,and explore the possibility of alleviating the structural and functional damages of the bladder secondary to pBOO through the intervention of the AMPK pathway.Method1.Establishment of the rat model of pBOOFemale Sprague-Dawley rats aged 7 weeks were anesthetized and disinfected in suprapubic area.The sterile catheter with an outer diameter of 0.9 mm was lubricated and inserted through the urethra.The urethra was carefully dissected and ligated with 5-0 silk suture,avoiding damages of bladder neck region.The catheter was removed after knotting.The rats in sham group were underwent the same procedure without knotting,which were compared as control.2.Evaluation of the bladder function of the rat modelCystometry were performed under anesthesia in this study.The urodynamic pa-rameters evaluated in this study were maximum pressure,baseline pressure,intercon-traction interval,voiding volume,residual volume,capacity(micturition volume+residual volume),compliance(voiding volume/(maximum pressure-baseline)),voiding efficiency(voiding volume/capacity),frequency and amplitude of non-voiding contractions(NVCs)during micturition cycles.3.Evaluation of changes in bladder structureThe mass of the bladder was weighted;HE staining was performed to observe the changes in bladder structure;Masson’s staining was performed to evaluate the level of bladder remodeling based on the content of muscle fibers and collagen fibers in the bladder tissue;RT-qPCR,Western Blotting were conducted to detect the expression of pro-fibrotic proteins like collagen I,collagen Ⅲ.4.Exploration of the structural and functional characteristics of bladders of pBOO rats in the early and late stages after obstruction separatelyAfter establishment of the model of pBOO rat,2-week and 9-week after the oper-ation were taken as the early and late stages of obstruction separately.Changes in structure and function of the bladder were measured according to the methods men-tioned above in 2 and 3.At the same time,cytokines in bladder including IL-6,IL-10,TGF-β1,TNF-α,IL-1β,HIF-la were measured.And indicators of inflammation and oxidative stress like Evens blue,MDA,SOD,CAT,GSH-Px were also detected.5.Exploration of the expression and activation of AMPK in pBOO ratsAfter the establishment of the pBOO rat model,2 weeks and 9 weeks after the operation were regarded as the early and late stages of obstruction respectively.Changes in the expression of AMPKa and p-AMPKα(Thr183/172),activated form of AMPKα,were detected in the bladder tissue6.Exploration of the effects of AMPK agonist on structure and function ofthe bladder in the early and late stages of pBOOThe AMPK agonist metformin was administrated to pBOO rats by gavage to de-tect its effects on alteration of bladder structure,function,inflammation,and oxidative stress secondary to pBOO.7.Exploration of mechanisms of AMPK in inhibiting bladder remodeling secondary to pBOOa.Isolate and culture primary bladder fibroblasts and bladder smooth muscle cells,and detect the effects of AMPK agonists on the cells;b.Explore the role of AMPK/Smad3 pathway on the pro-fibrotic characteristics of primary bladder fibroblasts:detect the effects of AMPK agonists like metformin,acadesine(AICAR)on Smad3-mediated collagen synthesis;c.Explore the role of AMPK/autophagy pathway on pro-fibrotic characteristics of primary bladder fibroblasts:detect the effects of AMPK agonists including metformin,AICAR on autophagy and the expression of collagen;Clarify the role of autophagy on synthesis and secretion of collagen with late-autophagy inhibitors like Bafilomycin Al,Ca-5f,and siRNAs including siAMPKα,siLC3B,in bladder fibroblasts.Results1.pBOO rats showed different urodynamic characteristics in early and late stages of pBOOUrodynamics showed that compared with sham rats,the 2-week pBOO rats showed increased maximum intravesical pressure,increased baseline pressure,pro-longed intercontraction interval,increased residual volume,increased bladder capacity,and decreased voiding efficiency.However,the bladder compliance did not change significantly and the non-voiding contractions(NVCs)was not obvious,suggesting that the bladder function was partially compensated.In 9-week pBOO rats,the uro-dynamics showed worse bladder function.In addition to the abnormal bladder func-tion already appeared in 2-week pBOO rats,decreased bladder compliance and fre-quent NVCs appeared.2.Bladders were manifested as inflammation in early stage after pBOO,and fibrosis in late stageFor 2-week pBOO rats,HE staining showed thickened urothelium and increased infiltration of inflammatory cells in bladder.Evens blue Extravasation also provided evidence for inflammation;Masson’s.staining showed detrusor hyperplasia without significant changes in contents of collagen fibers.The bladder remained basic struc-ture despite of the increased weight of the bladder.The histological features of this stage are characterized as inflammation and detrusor hyperplasia in the bladder tissue.For 9-week pBOO rats,western blots and Masson’s staining showed that more collagen was synthesized and deposited in the bladder wall.But it this stage,the in-flammation in the bladder was no longer obvious evidenced by the results of Evens blue Extravasation and HE staining.3.The activation of AMPK increased in early stage after pBOO,while de-creased in late stageIn bladders of 2-week pBOO rats,the expression of AMPKa and p-AMPKa(Thr183/172)did not decrease.And the expression of p-AMPKα(Thr183/172)even in-creased slightly.In bladders of 9-week pBOO rats,the expression of p-AMPKa(Thr183/172)was significantly reduced.4.Treatment of AMPK agonist alleviated inflammation and oxidative stress in bladder in early stage after pBOOpBOO rats were treated with AMPK agonist metformin or an equal volume of drinking water by gavage from the 3rd day after the operationFor 2-week pBOO rats,HE staining and Evans blue Extravasation showed allevi-ated inflammation in bladders of metformin-treated group compared to those of drinking-water-treated rats.The content of MDA was lower in the bladder tissues of metformin-treated rats.With the treatment of metformin,activities of total SOD,GSH-Px and catalase in bladder tissues were also rescued to a certain extent in pBOO rats;And the expression of IL-6,IL-β,HIF-α,TNF-α in bladder tissues was also suppressed at both mRNA and protein levels.western blots showed that compared to pBOO rats that did not receive drug treatment,the expression of NLRP3,IL-1β,cleaved caspase-1,and p-NF-κB p65(Ser536)was relatively lower in the bladders of pBOO rats treated with metformin.5.AMPK agonist treatment did not change bladder function significantly in early stage after pBOOThe baseline intravesical pressure was lower in AMPK agonist metformin-treated pBOO rats than that of dinking-water-treated pBOO rats.In addition,the maximum intravesical pressure,baseline pressure,intercontraction interval,residual volume,bladder capacity,voiding efficiency and bladder compliance did not differ signifi-cantly between pBOO and pBOO+metformin groups.6.Long-term treatment with AMPK agonist alleviated bladder dysfunction in pBOO ratspBOO rats were received treatment of metformin or an equal volume of drinking water by gavage from the 3rd day after the operation.Relevant experiments were performed 9 weeks after the operation.Compared with pBOO rats without drug treatment,pBOO rats treated with metformin behaved as lower maximum intravesical pressure,lower baseline pressure,less residual volume,less bladder capacity,lower frequency and amplitude of NVCs,and improved bladder compliance and voiding efficiency.7.Long-term treatment with AMPK agonist alleviated bladder remodeling secondary to pBOOpBOO rats were treated with AMPK agonist metformin or an equal volume of drinking water by gavage from the 3rd day after the operation.Related experiments were performed 9 weeks after the operation.Masson staining showed that compared to dinking-water-treated pBOO rats,metformin-treated pBOO rats behaved as milder bladder remodeling.Western blots and RT-qPCR also confirmed that less collagen was deposited in the bladders of metformin-treated pBOO rats compared to that in bladders of drinking-water-treated pBOO rats.8.Activation of AMPK inhibited the pro-fibrotic characteristics of isolated bladder fibroblasts in vitroAdministrations of AMPK agonists like metformin and AICAR inhibited the synthesis of collagen in primary bladder fibroblasts.The treatment also exhibited a significant inhibitory effect on upregulated synthesis of collagen induced by TGF-β.Interfering AMPKα with siRNA increased the accumulation of collagen in bladder fibroblasts.9.AMPK regulated the expression of collagen in bladder fibroblasts via TGF-β/Smad pathway and autophagy pathwayAMPKa agonists including metformin and AICAR inhibited the expression of p-Smad3(Ser423/425)and reduced the synthesis of collagen in bladder fibroblasts.The treatment of AMPK agonist also promoted the degradation of collagen by activating autophagy pathway.And late-stage autophagy inhibitors increased the accumulation of collagen in bladder fibroblasts.However,AMPK agonist metformin showed no significant inhibitory effect on the expression of collagen in fibroblasts when inter-fered with si AMPKa and siLC3B.Conclusion and significanceWith the use of the rat model of pBOO,we found that the structure and function of bladder changed gradually after pBOO;And pBOO rats showed different structural and functional characteristics in early and late stages after pBOO.In the early stage after the obstruction(2 weeks after modeling),the bladder was mainly characterized as evident inflammation and oxidative stress.At this time,the activity of AMPK in-creased as a certain degree of compensatory,and the compliance of the bladder was also compensated to a certain extent.In the late stage after obstruction(9 weeks after modeling),the inflammatory characteristics in bladder were no longer significant de-spite that fibrotic feature was obvious.At this time,the activation of AMPK was de-compensated,and the bladder function was further deteriorated.The bladde r compli-ance was decompensated accompanied with frequent involuntary bladder contraction.Further experiments in pBOO rats showed that rats treated with AMPK agonist met-formin had milder inflammatory appearance in early stage after pBOO.And long-term treatment of metformin also ameliorated bladder fibrosis and protected the bladder compliance to a certain extent in 9-week pBOO rats,which suggests that the activa-tion of AMPK has protective effects on ameliorating structural and functional im-pairments to the bladder secondary to pBOO.pBOO-induced bladder fibrosis comes as a gradual and cumulative result of bladder injury secondary to obstruction,which acts as an important reason for the irreversible impairment to bladder function after pBOO.Bladder fibrosis is mainly manifested as a large amount of collagen fibers de-posited in the bladder wall.The deposited collagen fibers arise from the excessively released collagen from bladder fibroblasts.Further in vitro experiments confirmed that activating AMPK could inhibit the synthesis and release of collagen by in vitro cultured bladder fibroblasts,which was achieved by inhibiting the synthesis of colla-gen via inhibition of TGF-β/Smad3 pathway as well as promoting degradation of col-lagen via activation of autophagy.Our research confirmed the important role of AMPK pathway in the process of bladder structural and functional deterioration secondary to pBOO.We confirmed that the activation of AMPK could inhibit the inflammation in the bladder in early stage after obstruction,and inhibit the collagen synthesis and deposition for long-term treatment via inhibiting TGF-β/Smad3 pathway-induced collagen production and via promoting autophagy-relevant degradation of collagen in bladder fibroblasts,which thereby delayed bladder remodeling and bladder dysfunction after pBOO.Our re-search provides a potential therapeutic target for pBOO patients to delay the progres-sive bladder dysfunction triggered by obstruction.Innovations and shortcomings of this researchIn our study,rat models of pBOO were used to observe the different manifesta-tions of bladders in the early and late stages after obstruction,respectively.The results confirmed the evolution from inflammation to fibrosis of the bladder secondary to pBOO.It was found that the activation of AMPK pathway might act as a compensa-tory mechanism to reduce inflammation,oxidative stress in bladder,and to protect the structure and function of the bladder in the early stage after pBOO.However,with the prolongation of the obstruction time,this mechanism would be decompensated,which was closely related to the aggravation of bladder disorders in structure and function.In addition,our data showed that activation of AMPK pathway inhibited the synthesis and release of collagen in in vitro cultured bladder fibroblasts,which was achieved via inhibiting the TGF-β/Smad3 pathway-mediated synthesis of collagen,and via promoting the autophagy of collagen.Our results suggest that AMPK agonists might be potential drugs to alleviate the malformation and dysfunction of the bladder sec-ondary to BOOIt is necessary to point out that limited by the clinical reality,the research on the alterations of bladder structure and function secondary to pBOO cannot be carried out in clinical patients,and researches in this topic are generally carried out in animal models.Despite that the model we adopted has been widely used in studies of pBOO,the pathogenesis in this model may not be exactly the same as that of pBOO patients in clinical,which is a shortcoming of this experiment. |
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