小电导钙激活钾通道3在雌二醇抑制结肠平滑肌收缩中的作用及机制

It’s reported that estrogen could inhibit colonic smooth muscle contraction, and was closely related to constipation. Small conductance calcium activated potassium channel 3 (SK3) participated in many parts of smooth muscle relaxation mechanism. Our previous study found that SK3 expressed in colon smooth muscle cells (SMCs), and 17 beta estradiol (E2) up-regulated SK3 expression through nuclear receptors, but the mechanism is unknown. Cell depolarization cause extracellular Ca2+ influx, and induce smooth muscle contraction. Sk3 are involved in the generation of slow after-hyperpolarization and the inhibition of cell depolarization. We hypothesis that E2 regulate SK3 protein expression by regulating of transcription factor expression through nuclear receptors, and E2 promote SK3 expression of colonic SMCs, which decreased SMCs Ca2+ influx, thereby inhibiting the contraction. Therefore, this study propose to use animal experiment to observe the effect of E2 on SK3 expression in rat colonic smooth muscle and contract function. We also plan to do in vitro cell experiment to observe when E2 interference and overexpression of SK3 performed, the intracellular Ca2+ concentration changes in the SMCs of rat colon, and distinguish the E2 nuclear receptor type participated in, and explore the post receptor signal pathway as well. Therefore, our study provides a potential target for clinical intervention in chronic constipation.

雌激素与便秘发病相关，有抑制结肠平滑肌收缩作用，小电导钙激活钾通道3（SK3）参与多部位平滑肌舒张机制，我们前期研究证实结肠平滑肌细胞（SMC）有SK3表达， 17β雌二醇（E2）通过核受体促SK3表达，但机制不明。细胞去极化使胞膜外Ca2+内流，诱发平滑肌收缩，而SK3的功能是参与形成慢后超极化电位、抑制细胞去极化。我们设想：E2结合核受体，通过调节转录因子表达，作用于SK3基因转录及蛋白表达；E2促结肠SMC SK3表达，减弱SMC的Ca2+内流，从而抑制收缩。因此本项目拟进行：①动物实验观察E2对大鼠结肠平滑肌SK3表达及收缩功能的影响；②细胞实验观察E2干预及过表达SK3时，大鼠结肠SMC胞内Ca2+浓度变化，鉴别参与促SK3表达的核受体类型，探讨受体后信号通路。为寻找更安全有效治疗便秘的药物提供理论依据。

慢性便秘是一种全球性的常见病和慢性病，严重影响人们身心健康和生活质量。育龄期和妊娠期妇女发病率更高。雌激素与便秘发病相关，有抑制结肠平滑肌收缩作用，但机制不明。小电导钙激活钾通道3（SK3）参与多部位平滑肌舒张机制，我们前期研究证实结肠平滑肌细胞（SMC）有SK3表达，17β雌二醇（E2）促SK3表达，但机制不明。细胞去极化使胞膜外Ca2+内流，诱发平滑肌收缩，而SK3的功能是参与形成慢后超极化电位、抑制细胞去极化。本研究探讨了E2促SD大鼠结肠SMC SK3表达、并抑制结肠收缩的作用及机制。主要研究成果：①通过对雄性SD大鼠的分组干预，发现超生理浓度的E2促结肠平滑肌条SK3表达，并抑制结肠收缩，但生理浓度E2无此作用，说明可能与雄性大鼠雌激素受体（ER）数量、敏感性低有关。②通过对雌性SD大鼠分组干预，发现生理浓度的E2可促结肠平滑肌条SK3表达，不能通过细胞膜的BSA-E2无作用，说明E2促SK3表达作用由核受体介导；肌条实验发现，与对照比较，E2干预的卵巢去势大鼠结肠收缩减弱，说明E2抑制结肠收缩。③离体细胞干预鉴定了介导E2促SK3表达的ER亚型为ERα，并初步探讨了受体后途径：可能通过SK3编码基因KCNN3转录调节因子Sp1和Sp3发挥作用；在结肠SMC，E2干预、质粒转染过表达SK3后检测细胞内Ca2+荧光浓度发现，E2及过表达SK3均可抑制SMC内Ca2+动员、抑制平滑肌收缩。本研究首次阐明了在大鼠结肠平滑肌，E2由ERα介导、促SK3表达、抑制SMC内Ca2+动员，从而抑制平滑肌收缩的机制，并初步探讨了该作用的ER受体后信号通路。从而为寻找更安全有效治疗女性便秘提供靶点及理论依据。