kallistatin基因在勃起功能中的多靶点调控作用及机制研究

Recently, target genes for erectile dysfunction (ED) treatment are still confined to single target gene, which has unsatisfactory curative effect on ED and fails to cure of it radically. Therefore, seeking a novel pleiotropic effective target seems to be of great importance for ED treatment. Previous studies showed that kallistatin had multiple biologic functions independent of the tissue kallikrein-kinin system, including vasculature relaxation, anti-oxidative stress, anti-inflammation, anti-fibrosis, anti-apoptosis and stimulation of neointima hyperplasia in cardiovascular protection. Our previous studies showed that the expression level of kallistatin gene was positively correlated with erectile function in aged rats and diabetic rats induced by administrating streptozocin (STZ). Human kallistatin gene mediated with recombinant adenovirus not only could greatly improve the erectile function, but also increase the cell content of cavernosum smooth muscle cells and endothelium cells in aged rats by intracavernous injection. These results suggest that kallistatin may function as a vasodilator and regulator in penile corpus cavernosum and erectile function independent of its binding to tissue kallikrein, which would provide new insights into the pathogenesis of essential ED and aid in the future design of therapeutic agents. In this project study, RNA interference and transgenic technology are used to explore the possible pleiotropic protective effects of kallistatin on corpus cavernosum relaxation, inhibiting oxidative stress, fibrosis, apoptosis and tissue repair following injury at cellular and molecular level in corpus cavernosum via KLF4-eNOS, PI3K-Akt-eNOS, p38 MAPK and p42/44 MAPK pathway in Vitro and in Vivo, which is expected to find a new multi-target with minimal side effects for the drug development and gene therapy of ED.

目前勃起功能障碍（ED）基因治疗仍局限于单基因靶点，其疗效有限且未能从病理生理学水平实现ED治疗目的。研究表明，kallistatin（Kal）基因通过多靶点作用在心血管中发挥舒血管、抗氧化、抗炎症、抗纤维化、抗凋亡及促平滑肌细胞增殖等多重生物学效应。申请者前期研究发现，老龄及糖尿病性ED大鼠海绵体中Kal表达水平显著降低且与勃起功能呈正相关，而腺病毒介导的人源Kal基因可显著增强老龄性ED大鼠勃起功能且增加海绵体平滑肌和内皮细胞含量。本项目拟利用RNA干扰和转基因技术，通过KLF4-eNOS、PI3K-Akt-eNOS、p38 MAPK和p42/44 MAPK信号通路研究Kal基因在生理和病理条件下对海绵体舒张、氧化应激、纤维化、细胞凋亡和组织损伤修复的影响，探索其对勃起功能的生理调控和改善机制，从而阐明Kal基因在勃起功能中的多靶点调控作用及机制，有望为ED治疗寻找到多靶点基因和药物。

目前勃起功能障碍（ED）的基因治疗仍局限于单基因靶点，其疗效有限且未能从病理生理学水平实现ED治疗目的。研究表明，kallistatin（kal）基因通过多靶点调控作用在心血管系统中发挥舒张血管、抗氧化应激、抗组织纤维化、抗细胞凋亡及促血管生成等多重生物学效应。阴茎海绵体作为机体血管系统的成员之一，其相关治疗药物和基因靶点与心血管系统具有先天的亲缘性，kal基因多靶点生物学功效亦能在海绵体组织及勃起功能调控中充分发挥甚至更佳。项目申请前课题组研究发现，老龄及糖尿病性ED大鼠海绵体中kal基因表达水平显著降低且与勃起功能呈正相关，而腺病毒介导的kal基因可显著改善老龄性ED大鼠勃起功能并增加海绵体组织内的平滑肌和内皮细胞含量。本项目在前期工作基础上，利用RNA干扰和过表达转基因技术，通过在生理条件下和病理条件下动物模型，从KLF4-eNOS、PI3K-Akt-eNOS、NO-cGMP、RhOA/ROCK1/LIMK2/cofilin、p38 MAPK和p42/44 MAPK等多条信号通路探索kallistatin基因在生理条件下调控勃起功能的生理机制及病理条件下改善勃起功能的治疗机制，从而为ED治疗寻找到新的多靶点基因和药物。本研究结果显示，正常生理条件下，敲低或上调kal基因表达在短期内对勃起功能和海绵体组织结构无显著影响，但可影响KLF4-eNOS通路改变细胞凋亡状态；在糖尿病性ED和老龄性ED模型中，局部给予kal基因治疗后，kal基因通过活化内皮细胞KLF4-eNOS通路增加eNOS表达，同时上调海绵体平滑肌中iNOS表达，NO浓度的增加一方面通过NO-cGMP通路增加cAMP和cGMP浓度，进而激活BKCa、MaxiK α和β1离子通道，促进海绵体平滑肌舒张，另一方面下调NAD(P)H氧化酶p22phox、p47phox和p67phox表达水平而降低组织氧化应激水平而减少海绵体细胞凋亡，同时上调的P42/44MAPK通路可发挥促海绵体平滑肌和内皮细胞增殖效应；在糖尿病性ED模型中，kal尚可通过P38和RhoA/ROCK1/LIMK2/cofilin通路降低海绵体组织纤维化水平，从而重塑海绵体组织结构和改善勃起功能。因此，kal基因有望成为从病理生理学水平实现ED治疗目的的多靶点基因和药物。