基于VEGF-STAT3/Myocardin-VEGF反馈环路探索前列通窍胶囊对缺氧所致膀胱平滑肌细胞表型转化的调控机制

Bladder hypoxia plays a crucial part in the pathophysiology of partial bladder outlet obstruction（pBOO）-induced bladder dysfunction, which also leads to angiogenesis in bladder muscle bundle companied with augmented secretion of vascular endothelial growth factor（VEGF）. As is known, bladder smooth muscle cells’（BSMCs）phenotypic transformation is the basis of variation of bladder structure and function at the cellular level. It has been indicated in the previous study that Qianlietongqiao capsules can improve detrusor dysfunction markedly. However, whether Qianlietongqiao capsules functions through regulating BSMCs’ phenotypic conversion induced by hypoxia is unclear. For further exploration , we will establish pBOO rat models through the ligation of prostatic urethra using polypropylene suture and these models will be treated with drugs by gavage. Intravesical pressure is determined by bladder inserting. And the hypoxia model of cells will be realized by using hypoxic chamber. Additionally, VEGF-treated BSMCs will be synchronously treated or not with siRNA STAT3, plasmid Myocardin and Qianlietongqiao capsules-containing serum, respectively. On the whole, molecular biological techniques will be utilized to detect the expression of HIF-1α、VEGF、VEGFR、phenotypic switch-related and signal pathway-related factors in the bladder or BSMCs. Through the research，it aims to elucidate how VEGF regulates hypoxia-induced BSMCs’ phenotypic conversion and Qianlietongqiao capsules’ targets, also, it will lays a theoretical basis for the further develop of new Chinese patent medicine.

膀胱缺氧是膀胱出口部分梗阻（pBOO）所致膀胱功能障碍的重要病理环节，缺氧还可导致膀胱肌束内血管新生及血管内皮生长因子（VEGF）分泌量增多，而膀胱平滑肌细胞（BSMCs）表型转化是膀胱结构与功能改变的细胞学基础。前期研究证实前列通窍胶囊可显著改善膀胱逼尿肌功能，为进一步探索该药是否通过对缺氧所致BSMCs表型转化的调控作用来改善膀胱功能的，该研究首先采用聚丙烯缝线部分结扎SD大鼠前列腺部尿道构建pBOO动物模型后灌胃给药，然后膀胱插管检测膀胱内压，并利用缺氧小室建立细胞缺氧模型，用VEGF处理BSMCs，用siRNA STAT3、质粒过表达Myocardin及前列通窍胶囊含药血清干预，采用分子生物技术手段检测HIF-1α、VEGF、VEGFR、表型转化和信号通路相关分子在膀胱及BSMCs中的表达，以此揭示VEGF对BSMCs表型转化的调控机理及该药的作用靶点，为开发中药新药提供理论依据。

膀胱缺氧是膀胱出口部分梗阻（pBOO）所致膀胱功能障碍的重要病理环节，缺氧还可导致膀胱肌束内血管新生及血管内皮生长因子（VEGF）分泌量增多，而膀胱平滑肌细胞（BSMCs）表型转化是膀胱结构与功能改变的细胞学基础。前期研究证实前列通窍胶囊可显著改善膀胱逼尿肌功能，本研究拟进一步以“VEGF介导的表型转化”为切入点探讨前列通窍胶囊改善pBOO所致膀胱功能障碍以及VEGF调控表型转化的作用机制。体内实验表明，前列通窍胶囊可明显提升pBOO大鼠膀胱血流，抑制膀胱湿重、膀胱指数的增加，改善纤维化、炎症等病理改变，具有抗膀胱组织缺氧的潜能，并可有效抑制膀胱平滑肌表型转化。体外实验表明，低氧环境下可激活膀胱平滑肌细胞（BSMCs）中HIF-1α/VEGF信号，而前列通窍胶囊含药血清具有抑制作用；低氧环境下BSMCs、血管内皮细胞（ECs）、BSMCs-ECs共培养模型中VEGF分泌量显著增加；外源性VEGF可介导BSMCs表型转化、STAT3磷酸化水平升高并抑制Myocardin的表达，而前列通窍胶囊含药血清可抑制外源性VEGF介导的BSMCs表型转化；敲减STAT3或过表达Myocardin可削弱VEGF所致的BSMCs表型转化作用，而过表达STAT3或Myocardin均可促进BSMCs中VEGF的分泌。最后，还利用网络药理学和分子对接技术对前列通窍胶囊治疗膀胱出口部分梗阻的作用机制进行了探讨。综上，前列通窍胶囊可能通过抑制缺氧微环境下VEGF介导的表型转化改善pBOO所致膀胱损伤，而VEGF可通过STAT3/Myocardin调控表型转化，且VEGF与STAT3/Myocardin形成反馈回路。该研究为进一步将前列通窍胶囊开发为治疗梗阻（如：良性前列腺增生）所致膀胱结构与功能损害的中药新药奠定了部分理论基础。