内脏脂肪素经结肠平滑肌KATP通道参与糖尿病结肠动力障碍的机制研究

Impaired smooth muscle contractility plays an important role in diabetic gastrointestinal motility disorders. ATP-sensitive potassium channels (KATP) participate in many parts of the smooth muscle relaxation mechanism. Activation of KATP inhibits cell membrane depolarization, subsequently decreasing Ca2+ influx into the cytoplasm. Visfatin is a novel adipokine that is mainly produced by perivascular adipose tissue. Increased production of visfatin in diabetes mellitus (DM) might impair vascular smooth muscle and myometrial contractility. Our previous research found that visfatin levels in the serum of DM rats were increased. In addition, visfatin upregulated KATP expression in colonic smooth muscle cells (SMCs) and inhibited the contraction of colonic circular smooth muscle strips. However, the mechanisms of these phenomena are unknown. We hypothesized that during diabetes development, accumulated visfatin upregulates KATP expression and inhibits the increasing level of Ca2+ in colonic SMCs, leading to colonic dysmotility. NADPH oxidase and ROS-dependent NF-κB activation are involved in this upregulation effect of visfatin on KATP expression. We would use tissue samples from type ⅡDM patients and an animal model, and visfatin knockout mice, to investigate the effect of visfatin on colonic motility and KATP expression. We also plan to perform in vitro cell experiments to detect the role of NADPH/ROS/NF-κB pathways in the upregulation of KATP expression by visfatin. Therefore, our study will identify a potential target for clinical intervention in diabetic gastrointestinal motility disorders.

胃肠平滑肌细胞(SMC)收缩障碍是糖尿病（DM）胃肠动力异常的主要原因，与ATP敏感性钾通道（KATP）激活后减少胞外Ca2+内流密切相关。作为新发现的脂肪因子，DM患者血清内脏脂肪素（VF）含量显著升高，并能抑制血管及子宫平滑肌收缩，但机制不明。我们已发现：VF能够抑制结肠平滑肌收缩，并上调结肠SMC胞膜KATP表达。故提出假说①：VF可由KATP途径抑制胞外Ca2+内流，降低结肠SMC收缩力。VF多通过激活NADPH及ROS依赖的NF-kB信号影响基因转录。故提出假说②：VF可激活NADPH/ROS/NF-kB上调KATP表达。本课题拟结合DM患者及动物，探讨VF与结肠动力障碍的相关性；并应用VF敲基因小鼠，进一步验证VF、KATP与DM结肠动力障碍的关系；同时提取大鼠结肠SMC，以免疫荧光、干扰RNA等技术，探究VF上调KATP表达的机制。为DM结肠动力障碍的防治提出新的调控靶点。

背景和目的：糖尿病相关胃肠动力障碍的机制尚不清楚。本研究拟探讨促炎因子内脏脂肪素（visfatin, VF）在糖尿病大鼠结肠平滑肌收缩功能障碍中的作用及其机制。.方法：将20只Sprague-Dawley大鼠随机分为对照组和2型糖尿病（type 2 diabetes, T2DM）组。检测血清和结肠肌组织中的VF水平，测量结肠排空时间和平滑肌条的收缩性，并分析结肠肌组织中ATP敏感性钾通道（ATP-sensitive potassium, KATP)）的表达。在体外，我们测试了VF对结肠平滑肌细胞（colonic smooth muscle cells, CSMC）中细胞内活性氧（reactive oxygen species, ROS）水平，NF-κB核转录，KATP通道Kir6.1亚基表达，细胞内Ca2 +浓度和肌球蛋白轻链（myosin light chain， MLC）磷酸化的影响。还测试了NAC（ROS抑制剂）和BAY 11-7082（NF-κB抑制剂）对Kir6.1表达的影响。.结果：T2DM大鼠血清和结肠肌肉组织中的VF水平升高，远端结肠排空时间延迟，结肠平滑肌条的收缩力减弱，并且在结肠肌肉组织中KATP通道表达增加。 体外研究结果提示VF显着抑制正常大鼠结肠平滑肌肌条的收缩力。在原代CSMC中，VF引起ROS超负荷，增加NF-κB核转录活性并增加Kir6.1的表达，最终降低细胞内Ca2+水平和MLC磷酸化。 NAC和BAY 11-7082抑制了VF诱导的Kir6.1上调。.结论：VF可能通过ROS /NF-κB途径上调KATP通道的Kir6.1亚基表达并干扰Ca2 +信号传导，从而引起CSMC的收缩力下降。