靶向调控RNA结合蛋白HuR在肥胖相关性勃起功能障碍中的治疗作用及其机制研究

The prevalence of obesity-associated erectile dysfunction is high, and the pathogenesis has not yet been well elucidated. Patients with OAED often have a poor response to first-line PDE5 inhibitors. Exploring the mechanisms and developing new treatment modalities is the main direction of current researches. Previous studies by applicants have shown that abnormal lipid accumulation in cavernosal smooth muscle plays a vital role in the development of ED. Excessive lipids can cause smooth muscle cell damage and diastolic dysfunction leading to ED. HuR is one of the most widely studied RNA-binding proteins. HuR is involved in the process of relaxing blood vessels and regulating lipid metabolism. Our preliminary results showed that HuR expression was decreased in obese ED rats, and the downstream smooth muscle relaxation and lipid metabolism pathway were impaired. Up-regulation of its expression promoted smooth muscle relaxation and improved lipid metabolism. The current project intends to use the all-in-one CRISPR/CjCas9 system carried by AAV, a more efficient and convenient in vivo treatment, to locally up-regulate the HuR gene through the injection into the penis, thereby activating the sGC-cGMP pathway and regulating lipid metabolism. In order to improve smooth muscle relaxation while reducing intracellular lipid deposition. This could be a long-term therapy for OAED. At the same time, we aim to explore the possible mechanisms underlining therapeutic method and provide theoretical bases for the development of obesity-associated ED drug targets.

肥胖相关性勃起功能障碍发病率高，发病机制尚未阐明，药物效果欠佳，探索发病机制、开发新的治疗方法是目前研究的主要方向。申请者先前研究表明海绵体平滑肌中脂质异常沉积在ED发病中起重要作用，过多的脂质可导致平滑肌细胞损伤、舒张功能下降而导致ED。HuR是目前研究最为广泛的RNA结合蛋白之一，文献报道HuR参与舒张血管、调节脂质代谢等过程。我们的预实验结果表明肥胖性ED大鼠HuR表达降低，其下游平滑肌舒张、脂质代谢通路受损，通过上调其表达可促进平滑肌舒张，改善脂质代谢。本项目拟利用AAV搭载的一体化CRISPR/CjCas9体系——这一更加高效便捷的在体治疗手段，通过阴茎局部注射，靶向上调HuR基因，从而激活sGC-cGMP通路及调控脂质代谢过程中的关键基因，改善平滑肌舒张功能的同时减少细胞内脂质沉积，以达到长效治疗肥胖性ED的目的。同时探讨其中可能的机制，为开发肥胖ED药物靶点提供理论基础。

海绵体平滑肌中脂质异常沉积在ED发病中起重要作用，过多的脂质可导致平滑肌细胞损伤、舒张功能下降而导致ED。项目组构建了肥胖性ED大鼠模型，通过海绵体测压评估大鼠的勃起功能，并应用免疫荧光、Western Blot、Realtime-PCR等方法验证了在肥胖型ED大鼠模型阴茎组织中，HuR表达降低，且下游平滑肌舒张、脂质代谢通路受损。随后，项目组设计了一套慢病毒搭载的一体化CRISPR-Cas9体系，使用WB、PCR等方法检测靶向上调阴茎海绵体平滑肌HuR对sGC-cGMP和CPT1B-FAO相关通路sGC各个亚基，CPT1B关键分子表达量的改变。再次构建肥胖型ED大鼠模型，通过海绵体测压证实造模成功后，将过表达HuR的病毒注射于阴茎局部。实验证实，靶向上调HuR基因可激活sGC-cGMP通路及调控脂质代谢过程中的关键基因，改善平滑肌舒张功能的同时能够减少阴茎内脂质沉积。本项目初步探讨了靶向调控HuR在治疗ED中的作用机制，为开发肥胖相关性ED药物靶点提供理论基础。