雌激素通过SphK1/S1P信号促大电导钙激活钾通道表达在抑制结肠收缩中的作用

It is reported that estrogen was closely related to constipation，and could inhibit colonic smooth muscle contraction, but the mechanism is not yet fully understood. Our previous study found that estradiol (E2) can cause calcium decrease in colonic SMC, and membrane large conductance calcium-activated potassium channels (BK, involved in membrane hypermethylation, inhibited calcium influx and SMC contraction) upregulation, however, there is no estrogen response element in promoter region of BK coding gene; Sphingosine-1-phosphate (S1P) also promoted SMC BK expression. In addition, studies have shown that E2 increases the S1P by activating and promoting Sphk1 expression. We hypothesized that: E2 promote BK expression through the SphK1/S1P signal, decrease calcium influx, and inhibit colonic smooth muscle contraction. Therefore, this study propose to: ①detect E2 level in serum and the levels of SphK1, S1P and BK in colonic smooth muscle of patients with constipation and control group, to obtain relevant clinical evidence of E2 in constipation, and probable by affecting BK expression through SphK1/S1P; ②use SphK1 knockout mice and wild mice to confirm the effect of E2 on BK expression and systolic function in rat colonic smooth muscle through SphK1/S1P signal; ③further clarify the molecular mechanism of E2 regulates BK expression via SphK1/S1P and affects calcium concentration in colon SMC, so as to provide a potential target for clinical intervention in chronic constipation.

雌激素与便秘发病相关，抑制结肠平滑肌收缩，但机制尚不清楚。我们前期研究发现雌二醇(E2)干预致结肠平滑肌细胞(SMC)内钙浓度下降、胞膜大电导钙激活钾通道(BK，参与胞膜超级化，抑制钙内流及SMC收缩)表达上调，但BK编码基因启动子区域无雌激素反应元件；1-磷酸鞘氨醇(S1P)可促SMC BK表达。文献：E2作用SphK1使胞膜S1P增加。我们设想：E2通过SphK1/S1P促BK表达，抑制钙内流、结肠平滑肌收缩。本项目拟通过：①检测便秘患者血清E2和结肠组织SphK1、S1P、BK，获得E2参与便秘及可能通过SphK1/S1P影响BK表达临床依据；②利用SphK1基因敲除小鼠及野生鼠在体实验，明确SphK1/S1P介导E2对BK表达、结肠平滑肌收缩影响；③细胞实验进一步明确E2通过SphK1/S1P调节BK表达影响结肠SMC内钙浓度的分子机制，为寻找更安全有效治疗便秘的靶点提供理论依据。

雌激素（雌二醇E2是雌激素的主要成分）与便秘发病相关，研究显示其可抑制结肠平滑肌收缩，但机制不明。大电导钙激活钾通道（BK）在平滑肌细胞（SMC）中广泛表达，参与SMC的超极化和舒张机制。研究已知1-磷酸鞘氨醇（S1P）可促BK表达，E2作用鞘氨醇激酶1（SphK1）使胞膜S1P增加。本研究旨在阐明BK和SphK1/S1P可能介导E2诱导的结肠运动障碍及其潜在分子机制。.我们的研究显示：.1. 在育龄期女性中，便秘患者的血清E2水平较健康对照增高，且E2水平与便秘的严重程度具有相关性。.2. 在卵巢去势大鼠中，对其进行BSA-E2、E2和雌激素受体拮抗剂（ICI）干预研究发现：E2组表现出结肠平滑肌的微弱收缩和延迟的结肠转运，且其结肠BK、SphK1、S1PR1和S1PR2的表达增加；ICI干预可抑制以上E2的作用；在SphK1敲除小鼠中，E2干预后的这些影响基本消失。.3. 在体外细胞实验中，对大鼠结肠SMC的干预研究显示：E2可显著上调SMC中BK、SphK1、S1PR1和S1PR2的表达，减弱钙动员。抑制BK、SphK1、 S1PR1和S1PR2的表达可减弱E2对SMC钙动员的影响。并且E2可增加S1P释放，进而可能激活了S1PR1和S1PR2。SphK1缺失减弱了E2对S1PR1和S1PR2表达上调的影响。.这些结果表明，E2通过上调SphK1、释放S1P激活或上调BK表达，抑制SMC钙动员，从而抑制结肠平滑肌收缩。在E2诱导的BK上调过程中，S1PR1和S1PR2也可能参与其中。这些结果可能为便秘的治疗靶点和治疗方法选择提供理论依据。