AGEs抑制结肠平滑肌细胞Ca2+转运在糖尿病结肠动力障碍中的作用和机制研究

Ca2+ is an important signaling messenger during smooth muscle cells (SMC) contractile process. There are two key points in SMC contractile mechanism: an increasing level of cytoplasmic Ca2+ and activation of myosin light chain kinase (MLC).According to documents: high level advanced glycation end products (AGEs) could impair Ca2+ transition in several kinds of cells in diabetic metabolism (DM).Our previous study have found that: AGEs levels in DM rats' serum and colonic tissue were increased, phosperalation level of MLC in DM rats' colonic muscle layer was decreased; AGEs also decreased level of cytoplasmic Ca2+ in colonic SMC and inhibited circular smooth muscle strips contraction. But the mechanisms of all above phenomenon are unknown. Then we hypothesized that: high level AGEs inhibit increasing level of Ca2+ in SMC by impair Ca2+ releasing from endoplasmic reticulum and Ca2+ transition by voltage-dependent Ca2+ channel on cell membrane; AGEs also reduce Ca2+ sensitivity of MLC by inhibit ROCK/CPI-17/MLCP signaling, which finally impair smooth muscle contractility and cause colonic dysmotility. In this study animal model and cell cultural technique will be used to investigate the effect of AGEs on SMC contractile function related protein expression and activation. Therefore, our study provides a potential target for clinical intervention in diabetic gastrointestinal motility disorders.

平滑肌细胞（SMC）胞浆Ca2+介导的下游分子肌球蛋白轻链（MLC，也称钙信号敏感蛋白）磷酸化是SMC收缩关键机制，糖尿病（DM）时糖基化终末产物（AGEs）可影响多种细胞Ca2+转运。我们前期研究发现：DM大鼠血清及结肠组织中AGEs水平升高；DM大鼠结肠环肌p-MLC表达减少；AGEs使结肠SMC胞浆Ca2+浓度减低、结肠平滑肌条收缩减弱，机制不详。本课题提出：升高的AGEs可影响：①SMC内质网释放Ca2+；②胞膜电压依赖Ca2+通道功能，抑制SMC胞浆Ca2+升高；此外，③AGEs还可降低MLC对Ca2+敏感性：借助ROCK/CPI-17/MLCP信号、使p-MLC表达减少、SMC收缩功能受损、结肠动力障碍。拟采用：DM动物模型及细胞实验，探讨AGEs对SMC的Ca2+转运相关通路蛋白表达及活化的作用和机制；为DM胃肠动力障碍的防治提出新的调控靶点。

胃肠动力障碍存在于30%～50%糖尿病患者。平滑肌是胃肠运动的执行者，其损伤必然导致胃肠动力障碍发生。但目前糖尿病（DM）结肠平滑肌病变的相关研究甚少。前期研究发现，糖基化终末产物（AGEs）参与了结肠平滑肌收缩障碍，可能是DM大鼠结肠转运功能和环肌收缩力下降的上游因素。本研究探讨了AGEs抑制DM结肠平滑肌Ca2+动员和Ca2+敏感的作用及其机制。主要研究结果：①DM模型大鼠结肠肌层中Ca2+动员相关蛋白L-a1c、IP3R、MLCK表达量无明显变化，IP3R磷酸化水平显著升高，MLC磷酸化水平显著降低；Ca2+敏感相关蛋白RhoA/ROCK活性降低，MYPT1、CPI17表达量无明显变化，MYPT1磷酸化水平显著降低；cAMP/PKA活性显著增加。但是DM大鼠接受了抗AGEs干预后，上述这些变化均可被改善或逆转。说明AGEs是影响DM结肠平滑肌Ca2+动员和Ca2+敏感的上游因素，cAMP/PKA信号可能参与AGEs的作用。②体外培养正常大鼠结肠平滑肌细胞（SMCs），将AGEs作用于SMCs，发现AGEs呈浓度依赖性抑制MLC磷酸化，降低乙酰胆碱（Ach）刺激下细胞内[Ca2+]i；将SMCs置于无Ca2+溶液中，AGEs显著降低Ach刺激下内质网释放Ca2+，同时增加IP3R磷酸化水平、抑制IP3R活性；利用胡萝卜内酯和新霉素耗竭胞内Ca2+，AGEs对胞外Ca2+内流无显著影响。说明AGEs通过磷酸化IP3R使其失活，进而降低内质网释放Ca2+。③体外实验中，AGEs降低RhoA/ROCK激酶活性，降低MYPT1磷酸化水平，而对CPI17活性无显著影响。提示AGEs通过抑制RhoA/ROCK/MYPT1通路降低SMCs的Ca2+敏感性。④AGEs呈浓度依赖性增加cAMP/PKA通路活性，PKA抑制剂H-89可逆转AGEs对IP3R及RhoA/ROCK/MYPT1通路的影响。⑤AGEs受体RAGE的中和抗体anti-RAGE可逆转AGEs对cAMP/PKA通路、Ca2+动员、Ca2+敏感性的影响。说明AGEs通过与SMCs上的RAGE受体结合，激活cAMP/PKA通路，抑制Ca2+动员和Ca2+敏感，进而影响SMCs收缩，参与DM结肠动力障碍的发生。本研究以Ca2+动员及Ca2+敏感性为靶点，进一步阐明了AGEs在DM结肠平滑肌病变中的作用及其机制。