LIM激酶在TGF-β1介导的膀胱出口梗阻致膀胱过度活动中的作用和机制研究

An increasing number of patients are suffering from lower urinary tract symptoms (LUTS) in recent years, among which, overactive bladder (OAB) suggestive of bladder outlet obstruction (BOO) induced by benign prostate hyperplasia is the most bothersome symptom for these patients. The current strategies, muscarinic receptor antagonist, or β3 receptor agonist, however, show low efficacy, and may increase the risk of acute urinary retention. In our preliminary investigation, increased expression level of TGF--β1 was detected in urine from rat with OAB suggestive of BOO, increased contractions of both smooth muscle tissue and smooth muscle cells induced by TGF--β1 were also detected in vitro, which may involve in LIM kinases (LIMK), theses contractile effects of TGF--β1 can be inhibited by LIMK inhibitors, which indicates that TGF--β1 may enhance the smooth muscle contraction by mediating LIMK. Additional evidence also demonstrates, that LIMK inhibitors show inhibiting effect on smooth muscle contraction by decreasing the level of phospho-cofilin in vitro. Therefore, it is hypothesized that TGF--β1 may involve in the development of OAB suggestive of BOO by inducing increased expression levels of LIMK and the subsequent increased phospho-cofilin level. The present project aims to explore this possible mechanism, meanwhile, this project will verify the key role of LIMK in bladder smooth muscle contraction, and therefore, to provide the evidence that LIMK may be a potential new pharmacological target for the treatment of LUTS.

下尿路症状（LUTS）发病率逐年升高，其中因前列腺增生症梗阻膀胱出口后导致的膀胱过度活动症是最困扰患者的症状。目前临床多采用M受体阻滞剂或β3受体激动剂治疗，但效果有限且存在尿潴留风险。我们前期研究发现：TGF-β1在膀胱出口梗阻致膀胱过度活动的雄性大鼠尿液中表达升高，并可以增强膀胱平滑肌的收缩，而抑制LIM激酶（LIMK）表达或活性后可以显著削弱TGF-β1的促收缩效应；此外，LIMK介导的丝切蛋白(cofilin)磷酸化是控制平滑肌收缩关键信号分子。因此，我们推测TGF-β1可能通过促进LIMK介导的丝切蛋白磷酸化，从而增强膀胱平滑肌收缩并促进了膀胱出口梗阻后膀胱过度活动症的发生发展。本项目拟从细胞、组织及动物三个层面探究并进一步验证LIMK在TGF-β1诱导的膀胱出口梗阻致膀胱过度活动中的关键作用，并为研究以LIMK为靶点的LUTS药物治疗提供理论依据。

膀胱过度活动症（OAB）是下尿路症状（LUTS）中最困扰患者的症状，但药物治疗效果一般。我们前期发现OAB膀胱尿液中TGF-β1表达升高，其可能通过LIMK调控膀胱平滑肌的收缩与增殖。在本项目中，我们通过细胞、组织和动物三个层面，利用组织浴槽实验、细胞组织免疫染色、蛋白免疫印迹、RNA干扰、OAB大鼠造模及体内膀胱形态学研究、临床样本分析等实验手段，验证了TGF-β1在OAB中促平滑肌收缩和增殖的新功能，同时阐明了TGF-β1调控LIMK介导cofilin磷酸化这一信号通路的调控关系。并且，我们重点验证了LIMK抑制剂SR7826和LIMKi3抑制cofilin磷酸化从而抑制膀胱平滑肌收缩和增殖等作用的特异性。此项目一方面进一步阐明了OAB的发病机制，另一方面，也为开发以LIMK为靶点的小分子化合物抑制剂（SR7826和LIMKi3）的临床转化提供一定的实验基础和理论依据。