【作者】 肖凯文；

【导师】 李虹；

【作者基本信息】 四川大学 ， 外科学（泌尿）， 2023， 博士

【摘要】 目的:膀胱出口部分梗阻（Partial bladder outlet obstruction,PBOO）是造成尿频、尿急等下尿路症状（Lower urinary tract symptoms,LUTS）常见的原因之一,其可对膀胱的形态和功能造成严重损害。在临床上,PBOO主要由良性前列腺增生（Benign prostatic hyperplasia,BPH）、膀胱颈硬化症、尿道畸形或尿道狭窄引起。一般情况下,LUTS可随着梗阻解除而缓解,然而还有部分患者在梗阻切除后仍存在严重的膀胱功能障碍和难治性LUTS,该现象提示这些患者发生了PBOO继发的不可逆膀胱功能障碍。到目前为止,尚无有效的治疗方法来逆转PBOO继发的膀胱功能损害。因此,研究新的保护和治疗策略至关重要。PBOO继发的膀胱内压力升高、膀胱组织过度拉伸和缺氧/再灌注损伤,可对膀胱壁的结构与功能造成不可逆损伤,即膀胱重塑。膀胱重塑被认为是一个复杂的进程,从早期的炎症反应逐渐进展至膀胱纤维化,伴随膀胱尿路上皮层损伤与平滑肌层肥厚等。膀胱承受的病理性压力可导致炎症反应,其中多种生物合成或生物降解相关信号通路被激活,继而干扰膀胱功能。由于PBOO持续存在,这些生物学途径可能存在异常,导致膀胱壁炎症和细胞外基质（Extracellular matrix,ECM）过多沉积,引起膀胱结构紊乱并最终造成难治性膀胱功能障碍,抑制膀胱组织的炎症反应理论上可以延缓膀胱重塑的病理进程,改善LUTS。细胞焦亡是一种与炎症反应高度相关的细胞死亡方式,最初其是在沙门氏菌感染巨噬细胞时被发现,以依赖天冬氨酸半胱氨酸蛋白酶（Cysteinyl aspartate specific proteinase,Caspases）中Caspase-1活化和大量炎性细胞因子产生为特征。与细胞凋亡类似的是,细胞焦亡也可出现如细胞核凝集、染色质碎裂、原位末端转移酶标记（Terminal deoxynucleotidyl transferase-mediated d UTP nick-end Labeling,Tunel）染色阳性、Annexin V染色阳性等表现,但与细胞调亡不同的是,细胞焦亡的发生伴随细胞膜表面孔道形成,并造成胞质内容物释放至胞外和细胞外液的渗入,最终导致细胞膜失去其完整性和物质进出调节能力、细胞肿胀裂解、诱发炎症反应。细胞焦亡涉及多条信号通路,包括以炎症小体如NOD样受体热蛋白结构域相关蛋白3（NOD-like receptor thermal protein domain associated protein 3,NLRP3）为代表和Caspase-1活化为特征的经典细胞焦亡通路;以如脂多糖（Lipopolysaccharide,LPS）等外源性刺激诱导,以Caspase-4/5/11活化为特征的非经典细胞焦亡通路和近期发现的具有Caspase-3或Caspase-8依赖性的细胞焦亡通路等。尽管使动因子和信号通路存在差异,但细胞焦亡最终多是由具备在细胞膜上形成孔道的蛋白家族成员（Gasdermins,GSDMs）完成。一定程度的细胞焦亡有助于对抗病原体和炎症因子侵入,维持组织内环境稳定,但过度的细胞焦亡则在病理性免疫反应的激活中发挥重要作用。目前研究证实细胞焦亡参与多组织或器官感染性疾病、自身免疫性疾病、代谢性疾病和无菌性炎症性疾病的进程。作为潜在的重要治疗靶点,探究细胞焦亡在具体疾病中的调控机制有助于为该类型疾病的治疗提供新的思路。PBOO导致的膀胱重塑主要是由增高的膀胱内应力引发,这些力学变化包括病理性静水压力、牵张力和流体剪切力等,但力学刺激通过何种途径传递至细胞并转变为生物学信号仍是需要进一步探究的问题。一些研究已证实,组织器官能通过机械力敏感通道感受细胞膜机械力的变化,导致机械敏感蛋白形变,离子通道开放,进而将机械力学信号转化为电信号或化学信号,最终在多种机械力感知信号传导过程中发挥重要作用。机械力敏感蛋白如Yes-associated protein（YAP）可以通过感知力学刺激参与调控组织器官发育、生长、肿瘤的诱导和抑制、上皮细胞增殖和炎症等生物学过程。膀胱作为始终受力学影响的器官,从抑制由力学刺激诱导膀胱炎症的角度出发,结合细胞焦亡与炎症反应间的紧密关联,我们推测PBOO可能通过诱导膀胱细胞焦亡进而参与膀胱炎症反应,而机械力敏感蛋白YAP可能在其中发挥调控作用。目前力学刺激和细胞焦亡在PBOO诱导膀胱炎症中的作用和机制尚缺乏相关研究探索。因此,本研究着眼于该疾病治疗难题,希望通过PBOO模型的构建和体外应力病理性静水压模型,探索细胞焦亡在力学刺激诱导膀胱炎症中的作用,进一步深入地探讨YAP在细胞焦亡中的作用机制,并为未来的临床治疗和研究提供新的思路。材料和方法:1.构建C57BL/6雄性小鼠PBOO模型探究力学刺激诱导膀胱组织细胞焦亡发生:PBOO建模后通过小动物泌尿系彩超（评估膀胱充盈情况）、尿动力参数（最大排尿压及膀胱排尿间期）检测、苏木精-伊红染色（Hematoxylin-eosin staining,HE）、Masson染色（评估膀胱细胞形态和胶原纤维比例）及膀胱湿重/体重百分比验证模型效能并选择适宜的建模时间。通过转录组测序对比PBOO建模组小鼠膀胱组织与假手术组小鼠膀胱组织中炎症相关信号通路富集情况及细胞焦亡相关指标m RNA水平表达变化。免疫组化（Immumohistochemical,IHC）染色检测经典及非经典细胞焦亡信号通路靶标及下游炎性细胞因子在膀胱组织中的表达变化,明确焦亡在膀胱炎症中的主要作用部位是膀胱尿路上皮细胞。2.体外模型探究病理性力学刺激诱导人尿路上皮细胞（Human urothelial cells,HUCs）焦亡:在团队前期研究基础上,我们选择100 cm H₂O作为体外模拟PBOO的力学刺激参数（对照组:0 cm H₂O）,对HUCs施加不同时间梯度（0小时、1小时、3小时、6小时）静水压刺激,通过定量聚合酶链反应（Quantitative-polymerase chain reaction,q-PCR）检测经典及非经典细胞焦亡通路靶标及下游炎性细胞因子表达改变;制备细胞爬片,经扫描电镜观察细胞膜表面是否发生具有细胞焦亡特征性的形态学变化;选择最适宜的时间梯度（6小时）,Western blot检测病理性静水压刺激后HUCs中焦亡相关指标蛋白水平表达变化情况,并通过免疫荧光染色对Western blot结果进行验证。在上述研究的基础上,通过转录组测序探究病理性静水压干预下HUCs生物学功能变化。3.力学敏感蛋白YAP在病理性静水压诱导HUCs焦亡中的作用探究:综合体内、体外两种力学刺激模型的转录组测序结果及近期研究报道,选择力学敏感蛋白YAP作为力学刺激诱导膀胱尿路上皮细胞焦亡的潜在调控靶点。IHC检测PBOO建模后小鼠膀胱组织中YAP的表达变化。依据YAP的作用方式,在体外模型中,我们通过Western blot检测100 cm H₂O 6小时干预后HUCs中总YAP和磷酸化YAP表达变化,并通过免疫荧光染色对Western blot结果进行验证。构建腺病毒对YAP进行敲减和过表达,Western blot检测病理性静水压干预后HUCs中细胞焦亡相关指标表达变化,相同条件下通过免疫荧光染色检测病理性静水压干预后HUCs中细胞焦亡相关指标表达变化,对Western blot结果进行验证。YAP作为Hippo通路的关键下游环节,为进一步探究其在力学条件下可能的调控机制,通过Western blot对紧邻YAP的Hippo通路两级调控分子哺乳动物不育系20样蛋白激酶1（Mammalian STE20-like protein kinase1,MST1）和大肿瘤抑制因子1/2（Large tumor suppressor homologue 1/2,LATS1/2）进行检测,明确YAP是否经Hippo信号通路或是直接感受力学刺激而发挥调控作用。为了进一步探究YAP通过何种机制调控HUCs焦亡,从模拟病理性静水压条件下YAP表达升高的角度出发,我们对HUCs中的YAP进行过表达,再对其施加病理性静水压,行转录组测序并通过对转录组学数据的生物信息学分析探索在该生物学过程中可能存在调控作用的信号通路,Western blot检测上述信号通路标志蛋白在病理性静水压下及敲减和过表达YAP后再施加病理性静水压干预后表达变化。4.C57BL/6雄性小鼠PBOO模型中关于力学刺激通过YAP诱导膀胱尿路上皮焦亡相关机制研究:动物实验分为3组:假手术组、PBOO建模组、PBOO建模+YAP特异性抑制剂Verteporfin组。通过膀胱彩超、尿动力参数检测、膀胱湿重/体重百分比、HE染色及Masson染色探究功能学相关指标变化。采用IHC检测细胞焦亡相关指标的表达变化,明确YAP在膀胱出口梗阻诱导膀胱炎症的动物模型中调控细胞焦亡的作用机制。结果:1.小鼠PBOO模型构建成功,转录组学检测提示细胞焦亡可能在PBOO诱导膀胱炎症的进程中具有重要作用:通过部分结扎C57BL/6雄性小鼠膀胱颈的方式构建PBOO模型,建模后2周对PBOO组小鼠及假手术组小鼠行膀胱彩超发现PBOO建模后小鼠膀胱处于持续充盈状态;尿流动力学检测发现假手术组小鼠排尿压力曲线收缩间隔及压力差值稳定,PBOO建模组小鼠最大排尿压升高,排尿间隔时间有所减少;PBOO造模组小鼠的膀胱重量及小鼠膀胱重量/小鼠体重比例显著高于假手术组,HE染色和Masson染色结果提示PBOO组小鼠膀胱与假手术组相比,膀胱上皮细胞显著增生,胶原纤维分布比例也明显升高。对建模2周的PBOO组和假手术组小鼠膀胱组织进行m RNA转录组学测序,GO富集分析表明PBOO建模后对力学刺激反应、组织器官生长、固有免疫反应、炎症反应等生物学过程相关信号通路富集,GSEA分析说明PBOO建模后炎症相关信号通路在膀胱组织中富集,同时我们发现PBOO建模后经典细胞焦亡通路标志物Caspase-1和非经典细胞焦亡通路标志物Caspase-4均显著升高,提示PBOO建模中,力学刺激可能经Caspase-1相关经典细胞焦亡通路和Caspase-4相关非经典焦亡通路共同作用诱导膀胱细胞焦亡参与PBOO诱导膀胱炎症反应。IHC染色结果发现在PBOO建模后膀胱上皮组织中经典细胞焦亡通路标志物NLRP3、Caspase-1、非经典细胞焦亡通路标志物Caspase-4/11、细胞焦亡底物蛋白GSDMD和炎症因子IL-1β均在膀胱上皮组织中表达且呈显著升高趋势。以上结果提示尿路上皮组织可能在PBOO诱导膀胱细胞焦亡-膀胱炎症反应中发挥重要作用。2.病理性力学刺激诱导人尿路上皮细胞焦亡:完成HUCs的培养及病理性静水压体外模型构建后,q-PCR结果提示100 cm H₂O静水压干预3小时、6小时,相较无压力干预组,Caspase-1、Caspase-4、NLRP3、GSDMD和IL-1βmRNA表达水平显著增加,此外炎症细胞因子IL-18在各梯度组别中m RNA水平的表达变化均不明显,提示在静水压刺激诱导HUCs焦亡中可能IL-1β而非IL-18是主要作用的炎性因子。扫描电镜结果提示:100 cm H₂O 6小时处理组中,可见典型细胞焦亡形态,细胞膜表面孔道显著增多,以上结果说明100 cm H₂O 6小时可能是体外研究比较适宜的参数。Western blot实验表明100 cm H₂O干预6小时较无压力组可显著增加经典及非经典细胞焦亡通路相关标志蛋白在HUCs中的表达,免疫荧光实验同样支持这一结果。转录学测序结果表明随着静水压刺激的施加,HUCs生物学特性变化主要表现在炎症、组织器官发育再生、机械力刺激反应等方面。3.力学敏感蛋白YAP在病理性静水压诱导尿路上皮细胞焦亡中的作用:综合体内、体外两种力学刺激模型转录学结果及近期研究报道,我们推测力学敏感蛋白YAP可能在病理性静水压诱导尿路上皮细胞焦亡中存在调控作用。IHC染色结果提示PBOO模型构建后,膀胱上皮组织中YAP表达明显升高。Western blot结果显示100 cm H₂O 6小时静水压干预后HUCs中YAP总蛋白表达明显上升,但磷酸化YAP则显著下降。对照组和100 cm H₂O 6小时静水压组HUCs免疫荧光结果与Western blot结果一致。构建腺病毒对YAP进行敲减和过表达实验,病理性静水压诱导HUCs中经典细胞焦亡通路相关蛋白（NLRP3、Caspase-1、GSDMD和IL-1β）表达增加,但敲减YAP后可明显抑制该过程,而过表达YAP可能进一步促进上述蛋白的活化。免疫荧光验证结果与Western blot一致。YAP作为Hippo信号通路的关键下游环节,但其在力学刺激诱导HUCs焦亡的生物学过程中可能不依赖Hippo通路而是独立感受力学刺激发挥作用。通过对HUCs中YAP过表达再施加病理性静水压干预后的m RNA转录组学测序,我们发现MAPK信号通路、TNF信号通路和NF-κB信号通路富集,这些信号通路与细胞焦亡均存在显著关联。Western blot结果提示病理性静水压可诱导上述通路活化,而敲减和过表达YAP可能经上述通路调控病理性力学刺激诱导的尿路上皮细胞焦亡。4.小鼠膀胱PBOO模型中YAP通过Caspase-1相关经典细胞焦亡通路调控尿路上皮细胞焦亡:特异性抑制YAP能够改善PBOO建模引起的膀胱持续充盈,明显改善尿流动力学指标,如最大排尿压明显下降,排尿间隔明显延长。敲减YAP能明显减轻由于PBOO建模造成的膀胱湿重/体重百分比升高。HE染色及Masson染色结果提示PBOO+YAP抑制剂给药组与PBOO组小鼠膀胱组织相比,膀胱上皮细胞增生明显缓解,上皮层下及肌间胶原纤维比例有所下降。IHC染色结果提示敲减YAP能够抑制PBOO引起的膀胱上皮内经典焦亡通路相关标志物（NLRP3、Caspase-1、GSDMD和IL-1β）升高。结论:1.病理性力学刺激通过Caspase-1相关经典细胞焦亡通路和Caspase-4相关非经典细胞焦亡通路共同作用介导PBOO后膀胱尿路上皮的炎症进程。2.病理性力学刺激下,YAP通过Caspase-1相关经典焦亡通路诱导尿路上皮细胞焦亡。3.病理性力学刺激下,YAP不依赖经典的Hippo通路诱导尿路上皮细胞焦亡,而是可能通过激活TNF/MAPK-ERK/NF-κB信号通路发挥生物学效应。更多还原

【Abstract】 Objective:Partial bladder outlet obstruction(PBOO)is one of the most common causes of lower urinary tract symptoms(LUTS),which can cause serious impairment of bladder morphology and function.Clinically,PBOO is caused by benign prostatic hyperplasia(BPH),bladder neck sclerosis,urethral malformation,or urethral stenosis.In general,LUTS can be relieved as the obstruction is relieved.However,a significant number of patients still have severe bladder dysfunction and refractory LUTS after obstruction resection,suggesting that these patients have irreversible bladder dysfunction secondary to PBOO.Until now,there has been no effective treatment to mitigate PBOO’s secondary impairment of bladder function.Therefore,research on new,protective and therapeutic strategies are necessary.It is clear that the elevated bladder pressure,bladder tissue overstretching,and hypoxia/reperfusion injury after PBOO may gradually disrupt the physical structure and function of the bladder.Persistent bladder dysfunction is thought to be a complex sequential but overlapping process,from damage to the urothelial cells in the bladder to smooth muscle cell hypertrophy,resulting in inflammation and bladder fibrosis progression.Pathological pressure on the bladder can cause an inflammatory response that interferes with bladder function.Multiple biosynthetic or biodegradation pathways are activated.As stress persists,these biological pathways may be abnormal,with excessive deposits of pro-fibrotic extracellular matrix(ECM)proteins on the bladder wall.Structural disturbance of the bladder eventually leads to refractory bladder dysfunction.Inhibiting the inflammatory response of bladder tissue can theoretically delay the pathological process of bladder remodeling and relief LUTS.Pyroptosis,a type of cell death highly associated with inflammation,was first identified when salmonella infected macrophages,It is characterized by the activation of Cysteinyl aspartate specific proteinase-1(Caspase-1)and the production of a large number of inflammatory cytokines.Similar to cell apoptosis,pyroptosis can also appear,such as nuclear agglutination,chromatin fragmentation,terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)staining positivity,and Annexin V staining positivity,but unlike cell apoptosis,pyroptosis occurs with the formation of cell membrane surface pores,resulting in the release of cytoplasmic contents into the extracellular space and infiltration of extracellular fluid.Eventually,the cell membrane loses its integrity and its ability to regulate the flow of substances in and out of the cell,causing swelling and lysis of the cell and inducing the inflammatory response.Pyroptosis involves several pathways,including the classic pyroptosis pathway characterized by the activation of inflammasomes and Caspase-1.Nonclassical pyroptosis pathways are characterized by Caspase-4/5/11 activation induced by exogenous stimuli such as lipopolysaccharide(LPS),and recently discovered Caspase-3-or Caspase-8-dependent pyroptosis pathways.Despite differences in motility and signaling pathways,pyroptosis is ultimately accomplished by members of the Gasdermins(GSDMs)family,which have the ability to form pores in the cell membrane.A certain degree of pyroptosis is helpful to resist the invasion of pathogens and inflammatory factors and to maintain the stability of tissue environments,but excessive pyroptosis plays an important role in the activation of pathological immune responses.Current studies have found that pyroptosis is involved in the progression of multiple tissue or organ infectious diseases,autoimmune diseases,metabolic diseases,and aseptic inflammatory diseases.As an important potential therapeutic target,exploring the regulatory mechanism of pyroptosis in specific diseases is helpful to provide new ideas for the treatment of this disease.The bladder remodeling induced by PBOO is mainly due to increased stress in the bladder.These mechanical changes include pathological hydrostatic pressure,mechanical stretch and fluid shear stress.However,how mechanical stimulation is transmitted to cells and transformed into biological signals still needs to be further explored.Some studies have confirmed that tissues and organs can sense changes in cell membrane mechanical forces through mechanosensitive channels,leading to the deformation of mechanically sensitive proteins,opening of ion channels,and finally converting mechanical signals into electrical or chemical signals,which play an important role in various mechanical force sensing signal transduction processes.Meanwhile,some mechanosensitive proteins,for instance,Yes-associated protein(YAP)have also been found to be involved in regulating biological processes such as tissue and organ development,growth,tumor induction and inhibition,epithelial cell proliferation,and inflammation by sensing mechanical stimuli.The bladder is an organ that is always affected by mechanics,from the perspective of inhibiting bladder inflammation induced by mechanical stimulation,combined with the close correlation between cell pyroptosis and inflammatory response,we speculated that PBOO may participate in bladder inflammation by inducing bladder cell pyroptosis,and the mechanosensitive protein may play a regulatory role in this process.Currently,the role and mechanism of mechanical stimulation and cell pyroptosis in PBOO-induced bladder inflammation is still unclear.Hence,the current study mainly focuses on the problem of this disease and hopes to explore the role of pyroptosis in inducing bladder inflammation by mechanical stimulation through the construction of a PBOO model and the application of a pathological hydrostatic pressure in vitro stress device to simulate in vivo PBOO conditions,to further explore the detailed mechanism of pyroptosis and to provide new ideas for future clinical treatment and research.Materials and methods:1.PBOO model was constructed by partial ligation of the bladder neck in male C57BL/6 mice.After modeling,the efficacy of the model was verified by urinary color ultrasound(bladder filling condition examination),urine dynamic parameter detection(maximum voiding pressure and bladder voiding interval),hematoxylin-eosin(HE)staining,Masson staining(bladder cell morphology and collagen fiber proportion evaluation)and percentage of wet weight of the bladder,and the appropriate modeling time was selected.Transcriptome sequencing was applied to compare the enrichment of inflammation-related signaling pathways and the m RNA expression changes of pyroptosis-related indicators in the bladder tissues of mice modeled by PBOO and those in the sham operation group.Immunohistochemistry(IHC)was used to detect changes in classical and nonclassical pyroptosis signal pathway targets and downstream inflammatory cytokines in bladder tissue to determine the main interaction position of pyroptosis in bladder inflammation.2.In vitro model to investigate whether pathological mechanical stimulation can induce pyroptosis of human urothelial cells(HUCs):On the basis of our previous research,we selected 100 cm H2O as the mechanical stimulation parameter of simulated PBOO in vitro and subjected HUCs to hydrostatic pressure stimulation with different time gradients(0 h,1 h,3 h,6 h).The expression changes of classical and non-classical pyroptosis pathway targets and downstream inflammatory cytokines were assessed by quantitative-polymerase chain reaction(q-PCR).We planted HUCs on glass coverslips,and the morphology of the cell membrane surface was observed by scanning electron microscopy.The most appropriate time gradient(6 hours)was selected,and Western blotting was used to detect the changes in the expression levels of pyroptosis-related indicator proteins after pathological hydrostatic pressure stimulation.The results were verified by immunofluorescence staining.On the basis of the above studies,transcriptome sequencing was used to detect pathological hydrostatic pressure interfering with the biological function of HUCs.3.To explore the role of the mechanically sensitive protein YAP in pathological hydrostatic pressure-induced pyroptosis:Based on the transcriptome sequencing results of two mechanical stimulation models in vivo and in vitro and recent studies,the mechanically sensitive protein YAP was selected as a potential regulatory target for mechanical stimulation-induced pyroptosis of bladder urothelial cells.IHC staining was used to detect changes in YAP expression in the mouse bladder after PBOO modeling.According to the mode of action of YAP,in the in vitro model,we measured the expression of total YAP and phosphorylated YAP in HUCs treated with 100 cm H2O for 6 hours by Western blotting and verified the Western blot results by immunofluorescence staining.An adeno-associated virus was constructed to knock down and overexpress YAP.Western blotting was used to detect the expression changes in pyroptosis-related indicators in HUCs interfered with by pathological hydrostatic pressure.Under the same conditions,immunofluorescence was used to detect the expression changes of pyroptosis-related indicators in HUCs that were interfered with by pathological hydrostatic pressure.Western blot results were verified.YAP is a key downstream link of the Hippo pathway.To further explore the possible regulatory mechanism of YAP under mechanical conditions,the two-stage regulatory molecules MST1(Mammalian STE20-like protein kinase1)and LATS1/2(Large tumor suppresser homolog1/2)of the Hippo pathway adjacent to YAP were detected by Western blot,and it was found that YAP may directly sense mechanical stimuli without the Hippo pathway.To further explore the mechanism by which YAP regulates pyroptosis of HUCs,transcriptome sequencing was performed after YAP overexpression in HUCs under the intervention of pathological hydrostatic pressure,and then bioinformatics analysis of transcriptomic data was conducted to explore the signaling pathways that may participate in these biological processes.The expression changes of these signaling pathway markers were detected by Western blot analysis under pathologic hydrostatic pressure and after knockdown and overexpression of YAP.4.To explore the mechanism of bladder urothelial cells pyroptosis induced by mechanical stimulation through the construction of a bladder inflammatory response model in mice with PBOO.Mice were divided into three groups:the sham operation group,the PBOO modeling group,and the PBOO modeling+YAP-specific inhibitor verteporfin group.The changes of functional indicators were investigated by color ultrasound,urine dynamic parameters,wet weight percentage of bladder,HE staining and Masson staining.IHC staining was used to detect the expression changes of related indicators of pyroptosis and to elucidate the mechanism of YAP in regulating cell pyroptosis in the animal model of bladder inflammation induced by bladder outlet obstruction.Results:1.The PBOO model was constructed by partial ligation of the bladder neck of male C57BL/6 mice.Color Doppler ultrasonography was performed on the mice in the PBOO group and mice in the sham operation group at 2 weeks after modeling,and it was found that the bladders of mice were continuously full after PBOO modeling.In the sham operation group,the contractile interval and the pressure difference were stable,while in the PBOO modeling group,the contractile interval was not stable,the maximum voiding pressure was increased,and the time between urination was decreased.The mice were killed,and the weights of the bladders and mice were recorded.The results showed that the percentage of bladder weight(%of bladder weight/body weight)in the PBOO group was significantly higher than that in the sham operation group.Masson staining indicated that the bladder urothelial cells in the PBOO group were significantly proliferated and that the proportion of collagen fibers was also significantly increased compared with that in the sham operation group.Transcriptome sequencing was performed on the bladder tissues of mice in the PBOO group and the sham operation group after modeling for 2 weeks.GO enrichment indicated that PBOO modeling enriched signaling pathways related to biological processes such as mechanical stimulation response,tissue and organ growth,innate immune response,and inflammatory response.The results of gene set enrichment analyses(GSEA)indicated that inflammation-related signaling pathways were enriched in bladder tissues after PBOO modeling.At the same time,we found that the classical pyroptosis pathway marker Caspase-1 and the nonclassical pyroptosis pathway marker Caspase-4 were significantly increased after PBOO modeling,suggesting that in PBOO modeling,mechanical stimulation may induce bladder cells pyroptosis to further participate in PBOO-induced bladder inflammation through the combination of the Caspase-1-associated classical pyroptosis pathway and the Caspase-4-associated nonclassical pyroptosis pathway.After PBOO modeling,IHC staining showed that the expression of the classical pyroptosis pathway marker Caspase-1,nonclassical pyroptosis pathway marker Caspase-4/11,inflammatory cytokine IL-1βand cytokinesis effect protein GSDMD were all significantly increased in bladder epithelial tissues.These results suggest that the urothelial cells may play an important role in PBOO-induced pyroptosis and bladder inflammation.2.The culture and identification of HUCs and the construction of a pathological hydrostatic pressure model in vitro.The q-PCR results suggested that the m RNA expression levels of Caspase-1,Caspase-4,NLRP3,GSDMD and IL-1βwere significantly increased after intervention with 100 cm H2O hydrostatic pressure for 3h and 6 h compared with the non-pressure group.In addition,there was no significant change in the expression of the inflammatory cytokine IL-18 in all gradient groups,suggesting that IL-1βrather than IL-18 may be the main inflammatory cytokine in hydrostatic pressure-induced pyroptosis of HUCs.The results of scanning electron microscopy indicated that the typical pyroptosis morphology of cells was observed in the 100 cm H2O 6 h treatment group,and the pores on the surface of cell membranes increased significantly.The above results indicated that 100 cm H2O for 6 h may be a suitable parameter for in vitro study.Western blot assays showed that 100 cm H2O intervention for 6 h could significantly increase the expression of classical and nonclassical pyroptosis pathway-related marker proteins in HUCs compared with the non-pressure group,and immunofluorescence assays also supported the results.The results of transcriptional sequencing showed that with the increase in hydrostatic pressure,the biological characteristics of human urothelial cells were mainly manifested in inflammation,tissue and organ development and regeneration,and mechanical stimulation response.3.Based on the transcriptome sequencing results of in vitro and in vivo mechanical stimulation models and recent published studies,we speculate that the mechanical receptor YAP may play a regulatory role in pathologic hydrostatic pressure-induced pyroptosis of HUCs.IHC staining indicated that YAP expression increased significantly in bladder urothelial cells after PBOO model construction.Western blot results showed that the total protein expression of YAP increased significantly after 6 h of intervention with 100 cm H2O,but phosphorylated YAP decreased significantly.The immunofluorescence results were consistent with the Western blot results.An adeno-associated virus was constructed for YAP knockdown and overexpression experiments.Pathological hydrostatic pressure induced increased expression of classic pyroptosis pathway-related proteins(NLRP3,Caspase-1,GSDMD,and IL-1β)in HUCs,but knockdown of YAP significantly inhibited this process.Overexpression of YAP may further promote the activation of the above proteins.The results of immunofluorescence staining were consistent with the Western blot results.As a key downstream link of the Hippo pathway,YAP may not go through the Hippo pathway in the biological process of mechanical stimulus-induced urothelial cell pyroptosis but may play a role independently in sensing mechanical stimulation.Through transcriptome sequencing of HUCs after YAP overexpression under the intervention of pathologic hydrostatic pressure,we found that significant correlations between HUCs pyroptosis and enrichment of the MAPK signaling pathway,TNF signaling pathway and NF-κB signaling pathway probably exist.Western blot results suggested that pathologic hydrostatic pressure could induce the activation of the above pathways,while knockdown and overexpression of YAP might regulate the above pathways in the process of HUCs pyroptosis.4.The mechanism related to bladder urothelial cells in the construction of the bladder inflammatory response model of mice with PBOO:specific inhibition of YAP can relief the continuous bladder filling caused by PBOO and significantly improve urinary flow dynamics parameters,such as the maximum voiding pressure decreasing significantly and the voiding interval prolonging significantly.The knockdown of YAP significantly reduced the increase in the wet weight of the bladder due to PBOO modeling.The results of HE staining and Masson staining suggested that the proliferation of bladder urothelial cells and the proportion of collagen fibers decreased in the PBOO+YAP inhibitor group compared with the PBOO group.The results of IHC staining suggested that knockdown of YAP inhibited the increase in classical markers of pyroptosis(NLRP3,Caspase-1,GSDMD,and IL-1β)in the mice bladder urothelial cells due to PBOO.These results suggest that YAP may regulate the pyroptosis of bladder urothelial cells by activating the Caspase-1 related classical pyroptosis pathway in mice PBOO models of bladder inflammation.Conclusion:1.Mechanical stimulation may induce bladder urothelial cells pyroptosis to participate in the process of bladder inflammation through the combined action of the Caspase-1-related classical pyroptosis pathway and the Caspase-4-related nonclassical pyroptosis pathway.2.The mechanically sensitive protein YAP may be involved in the process of mechanical stimulation-induced urothelial cells pyroptosis through the Caspase-1-related classical pyroptosis pathway.3.YAP may not promote mechanical stress-induced pyroptosis of HUCs by the classical Hippo pathway but by activating the TNF/MAPK-ERK/NF-κB signaling pathways.更多还原